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About Google Book Search Google's mission is to organize the world's information and to make it universally accessible and useful. Google Book Search helps readers discover the world's books while helping authors and publishers reach new audiences. You can search through the full text of this book on the web at|http: //books .google .com/I •, * i . ■ ••• . >> I ANf Of^O UNlVMEDCm «"=>?? HO 1987 • rANI*ORO,CA043OS COLLECTED STUDIES ON I M M U N ITY BY PROFESSOR PAUL EHRLIGH Pthrf GoaaeUor tad Director of the Royal Inatitote for Experimeatal Frenkfurt, Germany AND BY HIS COLLABORATORS With Several New Contributions, including A CHAPTER WRITTEN EXPRESSLY FOR THIS EDITION BY PROFESSOR EHRLICH TRANSLATED BY Dr. CHARLES BOLDUAN Profeaaor of Baeteriology and Hygiene in Fordham Univertity, N. Y« Aaaittant Bacteriologist, Retearch Ljiboratory, Department of Healtk City of New York FIRST EDITION FIRST THOUSAND NEW YORK JOHN WILEY & SONS LONDON: CHAPMAN & HALL, LIMITED 1906 Copyright, 1906 BY CHARLES BOLDUAN KOBXRT DRUMMOND, PRXNTBS, NKW YORK To Dr. a L T H O F F Pbivy Councilor, Director is thb Prussian Ministry of Education, Berlin, etc The Able Friend and Promoter of Medical Science This Volume is Dedicated in Grateful Appreciation S^*^'^^ TRANSLATOR'S PREFACE. No apology is needed for presenting this translation of Ehrlieh'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. ill 1 1 i I Tbb preeent 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 immunity, 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 with a. Bumnmry of generalizations derived from an enormous mass of experi- mental data.' When Behring's great discovery of antitoxin opened new patha 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 results in mind, consists in bending all eSorts 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 underlying the same, for these in turn will aid practical progress. I By pursuing the latter method it has been found that the immunity I reaction is merely a repetition of certain processes of normal mcta- I holism, 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 bad I iny ooUeagueB, Dr. Morgenroth nod Prof. Neisaer, present the reault of their ex- I tensive technical experiences with ha^molytic and bacteriolytic test-tube czperi- ] roents. in two special chapters. (Chapters XXtX and XXX.) Bl 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 effects 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 haemolysins, 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 GERiMAN EDITION. vii metabolism, 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 Paltaufs 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 imder discussion are ; (1) the constitution of active cytotoxic sub- VUl PREFACE TO THE GEEIMAN EDITION. stances, whether or not they are made up of two p)arts possessing different functions; (2) the union of specific amboceptors with the complements; (3) the plurality of complements. I 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, n^y 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 arilen in titrating and standardizing diphtheria antitoxin might be overcon^e. To this kind and able friend I have therefore dedicated this volume as a token of my gratitude and esteem. Paul Ehrlich. Fbankfuet a. M., February 1904. CONTENTS. A .• CBAFTBR PAOB . I. CJoin'RIBUTIONS TO THE TbEORT Of Ltsin Action Ehrldch and Morgenroth, 1 • II. Concerning HiEMOLTsiNS. (Second Communication.) Ehrhch and Morgenroth, 1 1 • III. Studies on HiBMOLYsiNB. (Third Communication.) Ehrlich and Morgenroth, 23 IV. Contributions to the Studv or Immunity von Dungem, 36 New Experiments on the Side- chain Theory. Phagocytosis and Globulicidal Immuity. V. Contributions to the Study of Immunity von Dungem, 47 Receptors and the Formation of Antibodies. Milk Immune Serum. » VI. Studies on Hemolysins. (Fourth Communication.) Ehrlich and Morgenroth. 56 t' VII. Studies on Hemolysins. (Fifth Communication.) Ehrlich and Morgenroth. 71 ^ VIII. Studies on HiEMOLYsiNs. (Sixth Communication.) Ehrlich and Morgenroth. 88 IX. Concerning the Mode of Action OF Bactericidal Ser^ M. Neiaser, 120 X. Tbe Deflection of Complements IN Bactericidal Test-tube Ex- periments Lipstein. 132 XI. Active Immunity and Overneu- trauzbd Diphtheria Toxins Rehna. 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. CBAPTEB FAOB XIV. The Escape of Hemoglobin from Blood-cells Hardened with Corrosive Sublimate Sachs. 163 XV. A Contribution to the Study of the Poison of the Common Garden Spider Scuiha. 167 XVI. A Study of Toad Poison Prdscher. 175 XVII. Concerning Alexin Action Sachs. 181 XVIII. Concerning the Plurality of Complements of the Serum Ehrlich and Sachs. 195 XIX. Concerning the Mechanism of the Action of Amboceptors Ehrlvch and Sachs. 209 XX. Differentiating Complements by Means of a Partial Anticom- PLEMENT Marshall and Morgenroth, 222 XXI. Concerning the Complemento- PHiLE Groups of the Ambo- ceptors Ehrlich and Marshall. 226 • XXII. Concerning the Complementi- bility of the Amboceptors Morgenroth and Scichs. 233 XXIII. The Production of Hemolytic Amboceptors by Means of Serum Injections Morgenroth. 241 XXIV. The Quantitative Relations be- tween Amboceptor, Comple- ment, AND Anticomplement Morgtnroth and Sachs. 250 XXV. The Hemolytic Properties of Organ Extracts Korschun and Morgenroth. 267 XXVI. Review of Besredka's Study, " Les Antihemolysines Natu- RELLES " Marshall and Morgenroth. 283 XXVII. The Mode of Action of Cobra Venom Kyes. 291 XXVIII. Further Studies on the Dysen- tery Bacillus Shiga. 312 XXIX. Methods of Studying Hemoly- sins Morgenroth. 326 XXX. The Technique of Bactericidal Test-tube Experiments M. Xeisser. 348 XXXI. The Property of the Brain to Neutralize Tetanus Toxin Marx. 356 XXXII. The Protective Substances of THE Bix>od. Ehrlich. 364 XXXIII. The Receptor Apparatus of the Red Blood-cells. Ehrlich. 390 CONTENTS a CBAPTEB FAGB XXXI V. The Relations Existing between Chemical Constitution, Distri- bution, AND PhAKMACOLOOICAL Action Ekrlich. 404 XXXV. A Study op the Substances which Activate Cobra Venom Kyea and Sachs 443 XXXVI. The Isolation op Snake-venom Lecithids Kyea. 466 XXXV 1 1. The Constituents op Diphtheria Toxin Ehrlich 481 XXXVIII. Toxin and Antitoxin: A Reply to the Latest Attack of Gruber Ehrlich. 614 XXXIX. The Relations Existing between Toxin and Antitoxin and the Methods of their Study Ehrlich and Scxhs. 547 XL. The Mechanism op the Action op A NTT amboceptors Ehrlich and Sachs, 561 XLI. A General Review op 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. Moroexroth. One of the most important advances in the study of immunity is the discovery of Pfeififer'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 inmiune sera. As is well known, the phenomenon of bacteriolysis, first demon- strated by Pfeiflfer 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 bacilli together with a small amount of immune serum are intro- duced into the abdominal cavity of a nonnal guinea-pig. Subse- quently Metchnikoflf (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 (Annal. 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. PfeifTcr'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- tionof 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 bacilU, 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 treats 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 hapmolytic 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 OF LYSIN ACTION. 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 will be clear to the reader. Very likely, therefore, the mechanism of haemolysis and that of bacteriolysis are very similar. The study of haemolysis thus gains considerable theoretical significance. Being so fortunate sls 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 mixture 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 scrum 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 haemolysis, although this phenomenon was carefully looked for.^ * Th^; 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 noniial agglutinating action of sera on bacteria, was obser\'ed 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 TMMUNlTy. U the immune serum is heated to 56° C, it completely loses its solvent action. The addition of serum of normal animals to this ittactimted serum causes it to be reaUivaled. For this purpose one can use not only normal goat serum but also nonnal 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 tlie serum is kept on ice and protected agtunst light it very soon shows a diminution of its reactivating power. In uantitative experiments, therefore, the inactive (stable) immune serum should alwara be reactivated by a perfectly fresh normal scrum. In hasmolysis, as in Pfeiffer's bacteriolysis, we are therefore forced to assume the existence of two substances. One of these, spedSc 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 preparatorj' step neces- sary for the ha?molyiic action, as Bordet seems to assume. The specific agglutinin Las no relation whatever to the hemolytic immune body, yimilarly, according to the views of eminent bacte- riologists, the specific bacteriolytic substances have no relation tg the agglutinins. The lysins may exist independently of the agglu- tinins and these again independently of the bacterioloytic sulistances, The reader is reminded of the interesting observations of Pfciffer and Kolle. These investigators described an immune serum which was strongly bacteriolytic but which did not at all agglutinate (Cen- tralhlatt f. Ilakt., 1896. Vol. XX, Noa. 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, Comples rend. Soc. de Biol., XI. 27-97). Pfeiffer's original theorv' 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 wa CONTRIBUTIONS TO THE THEORY OP LYSIN ACTION. 5 irder to throw some light on these problems that Ehrlich devised side-chain theory. At first Ehrlich's theory was applied to the origin of the anti- toxins and to the chemical relation existing between the toxina and certain atomic groups of the protoplasmic molecule. Pfeiffer him- self applied the theory to the substances specifically bacteriolytic for cholera bacilli, and was able to demonstrate experimentally that the source of these bodies was in the spleen, the bone-marrow, and the lymph bodies (Heiffer and Marx, Zeitschr. f. Hyg., Vol. 37, 189S). Wassermann, who in his well-known tetanus experiments had fur- :hed the first demonstration of the soundness of the side-chain succeeded in showing the source of the specific typhoid iCteiiolysin. The study of these bacteriolytic processes brought up B number of important questions directly concerning the side-chain theory, aTid 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 possibihty is given for the formation of a corre- sponding antibody. The antilxKly, 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, luced in response to the invading substance (toxin, toxoid, etc), iust combine chemically with the latter. If the invading substance in soluble form, as. for example, the toxins, the neutralization in the solution. If, however, it is not directly soluble, ig originally an insoluble part of, say, a bacterial or blood cell, len the dissolved antibody in the blood will be abstracted from its solvent fluid and anchored by the cell particle. In the well-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 bj' the crushed brain cells. In order to maintain the analogy we should expect that in our experiment the immune body rfissoZj«rf in the goat senim would be anchored by the eryUtrocytea of sheep blood. The manner of procedure in this experiment is very simple and feonsists in the addition to sheep blood, or a dilution of the same, immune serum which has been heated to 56° C in order to destroy solvent properties. The mixture is then centrifuged to separate cells and the fluid. In case the immune body has been anchored 6 COLLECTED STUDIES IN IMlklUNITY. • 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 inunune 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 suffi- • cient amount of addiment. If a corresponding amount of inunune body has been anchored by the blood-cells, 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 immimized 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 aft^r 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 j 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 hlood-ceUs 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 mixtiu-e centrifuged, and the two por- tions tested separately, by the addition of inunune 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 wUh 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 wUl 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-cells will be found to have loaded themselves with inmiune body, leaving the addiment in the fluid. The experiment shows that under the conditions mentioned, addiment and inmiune body exist in the fluid entirely independent of one another. It still remained to determine the combining affinities 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 mixture. The centrifuged fluid does not, by itself, dissolve blood COLLECTED STUDIES IN IMMUNTT?? added to it, or it does so in only a very limited degree. When, hi ever, new immune body is added, the blood-cella are completely solved. From these experiments we conclude that the sediment thia contained both components, though not in equivalent proporti 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 excess of addiment. The explanation of these phenomena presents no difficulties, must be assumed that under certain circumstances the immune bod] and addiment enter into loose, readily dissociated chemical eombi- nation. Thia combination is hastened by heat and retarded by cold in entire conformity to the views previously expressed by Ehrhch (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 hapfophore groups, one v'ith a strong afjinily for the corre- sponding kaplophore 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 tiea 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 havhig the character of a digestive ferment. Morgen- roth, by the experiments in which by immunization he succeastully pipduced 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 preliminary analysis all the various phenomena are now readily explained. We assume that the immune body combuica with the small amount of digesting ferment 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 1 d i CONTRIBUTIONS TO THE THEORY OF LYSIN ACTION. V action over to these cells. From this we see alao why the digestive I actioQ becomea manifest only on the addition of immune body. This brings the fennent, present in the serum fluid in such sniaU 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 ia always one group which fits only to the cells or substances used to excite ita 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 dilhcult problem of the mode of origin of the lysins. According to Ehrlich'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 , Oi^anismus) Ehrlich had pointed out that the atomic groups thus bftnchored to the living substance were much more readily oxidized ■and that they therefore represent the nourishment (/car eSox^/y) of Vthe cell. . The study of immunity has considerably extended this view md taught us that the antilxxiy represents such thrust-off side- ; 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- will be differently constituted. If a side-chain is designed ) assimilate relatively simple substances, we may believe that the 1 of a single combining group will suffice. Verj' likely the ■nde-chains which anchor toxins are of this simple type. But it is ntirely different when a giant molecule (albumin molecule) is to be asimilated. 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 ])ro- ccsaes into smaller part.s. It will be particularly advantageous to I the cell it its "grasping arm" is at the same time a carrier of a fer- tnentaiive group which can at once be brought to bear on the anchored Uolecide. We see such well-atlapted contrivances (in which the pasping 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 pecuUar 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 immimizing process, one succeeds in having a surplus of these side-chains produced, they will be produced with both these fimctional groups and thrust off into the blood as immune body. This explains the wonderful contrivance whereby the injection of a bacterimn is followed by the production of a substance which de8tro3rB this bacterium by dissolving it. This phenomenon is nothing but the reproduction of a process of normal cell life. n. CONCERNING ILEMOLYSINS.^ Second Communication. By Professor Dr. P. Ehruch and Dr. J. Morqenroth. In a previous paper 2 we demonstrated the relations existing between the red blood-cells to be dissolved and the two components of a specific haemolysin produced by immimization. It will 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 erythrocytes 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 inmiune body. Proof was thus afforded that, in confonnity 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 d^ree 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 inmiunization (Nov. 12 * Reprinted from Berl. klia Wochenschr. 1899, No. 22. ' See pages 1-10 of this volume. 11 Etik^ st&BfsS f*i and Feb. 24) of two male goats tjy injecting them Bubcutaneously with increasing amounts ot defibrinated sheep blood. In a short time a strongly active senim was produced in both animals, and we were able to observe how, following the general laws of .inmu- 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 ph3^iological 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 fi 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 11, 0.15-0.2 cc. suf- ficed for complete solution. It should be mentioned that the serum of buck 11 even before immunization possessed a slight solvent effect on sheep blood. This, however, was so slight that 4.0 cc. o£ the senmi were not nearly able to dissolve 5 cc. of the 5% blood mixture, and 1.2 cc. serum produced only a just noticeable amount ot solution. Heating the serum to 57° C. for half an liour 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 immime body with the erj-throcytes of the sheep at 0° C. can be readily demonstrated, for at this temperature 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 ser& of a large number of normal goats one will find same sera which poaseaa this feeble solvent power for sheep blood. Thus the normal goat sera which we eraploired 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 bad already called attention to the great variability of the agglutinating property. CONCERNING HEMOLYSINS. 18 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 senun 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 immime 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 haemolytic power when heated for half an hour to 56® C. This has been shown by Buchner to be true of all normal haemolytic sera. The sera of our two bucks even when heated for three-quarters of an hour to 56^ C. showed only a scarcely appreciable diminution of their solvent action on sheep bloody 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 hsemolysins 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 all hsemolysins 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 haemolytic bodies normally present. In order, however, to analyze these phenomena completely, it was absolutely essential to obtain the two components of the complex * Thif) 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 haemolytic 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 inunune 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 staie 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 pre\dously 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 treat^xl 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 necessar}'^ 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 wnll be recalled that we had found the original addiment which fitted 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 niunber of goats examined by us. AMien the serum of these goats was heated for ^ to | hr. to. 56° C. and its normal hipmolytic CONCERNING H.EMOLYSINS. 16 properties for other blood-cells were entirely destroyed, it was still able to typically reactivate the particular immune body here con- cerned.^ 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, like the thermos tabile addiment, fitted the immune body. We must therefore conclude that the inunune 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 varying 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 uhis present in both cases, but that the serum of the goat first immunized con- tained only tJie thermolabile addiment, while the sera of the animals examined later contained also the thermostabile 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 (buck II) is of considerable interest. Having thus arrived at some understanding of the action of the haemolytic sera produced by immunization it seemed essential that we extend our investigations to the haemolytic properties of normal sera. These properties had long been known and had been studied particularly by Buchner and his pupils.^ The fact that the haemolytic action of normal serum is destroyed by moderate heat led us to believe that the normal ha^molysins 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 lurmoglobinuria originate through analogous ha?molysins. Many years ago Ehrlich showed that the hnemoglobi- 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 r6le in the convalescence of severe anaemias. Ifi COLLECTED STUDIES IN IMMUNITY. not of simple constitution; but the experimental solution of this problem was attended with great difficulties. The primary t^sts necessary to demonstrat* 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 hiemolytic. 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 luemolytic 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 experienre 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 addimerti, which we shall hereafter terra complemcni. 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 thm explained by a lack of interbody, and this is supplied by the addition of inactive senun. 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 ia extremely simple, the counter-demonstration, that this interbody has combined with the sixlimenteel 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 immimized goat b 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 circuHLilanees prevailing at the time, the affinity of the interbody's haptophore group for the blood-cells is greater than the affinity of ita haptophore group for the complement. A measure of the i CONCERNING HiEMOLYSINS. 17 relative affinity is found in the degree of temperature at which combination occurs. In the case of the lysin obtained by immimiza- tion, which has already been described, the combination of the blood- cells with the corresponding haptophore group of the immime 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 immune body and comple- ment in a free state, i.e. uncombined. In this case, of course, it is possible completely to abstract the immune body from this mixture by means of the red blood-cells. 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. Natxu^lly between these two extremes all 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 haemotropic group of the interbody is not much greater than that of the group fitting the addiment. In this case we are imable 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 still 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 r61e of com- plement. It was of course clear that such a combination of circum- stances would only be a fortunate chance occurrence, and that only 18 COLLECTED STUDIES IN IMMUNITY, by examining a large number of separate cases would such a favor- able combination be foimd. 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 senun of the same species from which the blood-cells are derived. This serum should 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 haemolytic action exhibited by a serum, normally or in response to immimizing procedures, is due, in the cases examined by us, to the combined action of /tw> substances. Now that we had at our command the interbody of the haemol3rsin 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 demonstratioas. 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 senun, introduced by Spronck, has not realized the expectations a priori held out for the procedure. CONCERNING HEMOLYSINS. 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, while 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 conditions: (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 foimd. 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 haBmol)rtic 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 h8emol)rsins 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: COLLECTED STl-DIES IN IMHTOTTY. (1) The isolated destruction of single lysins by means of thermic and ehemic influences. (2) The binding of the different lysins by means of corresponding species of blood, thus making their elective removal possible. With red blood-cella this procedure, to which we shall return in a sub- sequent article, offera many technical difficulties. On the other hand, with a different kind of specific constituent of the serum, namely, the agglufinins, this method is easily applied, 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 globulicidal eel serum, have obtained a serum wtuch neutralizes the action of this eel serum, in other words, one containing an antilysin, Kvidcntly this reactively formed anti- body thrusts itself into the h^motropic group of the interbody and thus deflects this from the erythrocyte. Our attempts, based on these premises, to produce an isolaled antibody for some of the lysias 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 hemolytic action of dog eenim 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 (hicmolysins, agglutinins, ferments, antiferraents) is very readily harmonized with Ehrlich's views. According to his conception all these sulistances 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, I Inat. I'asteiir. March 18!t9. ' Ebrliub, Saueratoffbedurfnias dea Organisnius. Berlin, 1885. J CONCERNING H.EMOLYSINS. A lai^e part of these side-chains may, under suitable circumstances, be thrust off and thus appear in the blood. Considering the large numiier 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 targe number of factors such aa 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 docs not do so at all. The action of rabbit serum a guinea-pig blood shows a special variability. A veiy mteresting example is afforded by lamprey senim, which, I as is well known, i>03sesses an extraordinarily toxic action for labora- t lory animals in general and also for reii blood-cells in vitro. Dr. r-Schonlein of Naples, whose recent death we lament, was kind enough I to experiment with this for us. His investigations showed that the h emim of a not inconsiderable number of lamprej's possesses no rtoxic action at all, so that it could be injected into rabbits intra^ I venously in amounts of 2 cc. without any damage whatever. It is clear that this extensive variability enormously increases the fliculties in investigating these sera. Thus on repeating the well- lown experiment of Buchner, whereby a mixture, in certain pro- joriions, of dog and rabbit sera loses its ha-molytie 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 hiemolytic effect was only more or less lost. I We beheve that all these invest igation.s support the view we have Lalready expressed regarding the nature of the complex poisons of the vblood-sera. v. Dungem (Muench. med. Wochenschr., 1899, No. 14), H^ing his action on some new experiments of hLs, has accepted our ^Kews. We can content ourselves, therefore, with merely mentioning BDOther view, recently expressed by Bordet ' He has confirmed the statements made by us regarding the fixation of the specific immune body hy means of the correspondine er>^throcyte, and he has ad- ' .\ntiai. dc l"in«tit. Tasteur, .4pril 18i)9. 22 COLLECTED STUDIES IN IMMUNITY. mitted that the fixation process is connected with the solvent process, but he believes that the nature of this connection requires a special . hypothesis : "On pourrait rapprocher, si une comparaison un peu grossi^re 6tait permise, la modification apport^ par la substance sensibila- trice [our immune body] sur le globule, de celle qui consisterait k changer la structure d'une serrure, de fa^on k y permettre Tintroduc- tion facile d'lme ou de plusieurs clefs qui n'y entraient pas auparavant ou n'y p6n6traient qu'avec difficult^. Deux clefs sufhsamment sem- blables enterontd^s lors indiff^rtment." One could therefore pictiu^ the mode of action of the two sub- stances as it is conceived by Bordet to be like a safety lock which re- quires two keys to open it, of which the first is necessary in order to make the main lock accessible. Against this mechanical conception it can be urged that the keys do not fly into the lock of their own accord, but that certain forces are necessary to effect this. Our theory supplies a very simple explanation for this ; the driving force is the chemical affinity between the fitting groups. The entire line of experiments made by us was designed to show whether the two substances, together, combined with the blood-cells at one place or whether, separately, at two different places. Our decision was determined by the demonstration that the addiment was in no way fixed by the red blood-cells. Had Bordet repeated not only one of our experiments, but the entire series, the inapplicability of his hypothesis would have become e\a- dent to him. If active immune senmi is treated with red blood-cells, at 0® C. as described in our first article, thus fixing the immune body, the lock, according to Bordet, is made accessible, i.e. the conditions are fulfilled whereby the addiment (Bordet's alexin) could pene- trate to the blood-cells. As a matter of fact, however, imder these circumstances the addiment does not do so. This, as well as the new facts mentioned in the present article, harmonize best with our theory. If, however, this mode of action of the lysins is accepted, it will be impossible not to hold the same views regarding the living pro- toplasm, and assume in this the presence of side-chains of pecxiUar. character which are designed to grasp highly complicated substances. It must further be assumed that these side-chains, beside their grasp- ing group, are endowed with a second group which, by fixation of peculiar ferments, effects a digestive action. m. STUDIES ON ILEMOLYSIS.i Third Communication ^ By Professor Dr. P. Ehrlich and Dr. J. Moroenroth. By injecting one animal with the cells of another, we can produce substances in the serum of the first, which have a specific damaging or destructive influence on these cells. This possibility has within a short time extended the theoretical doctrines of immunity in vari- ous directions. First Belfanti and Carbone showed that the serum of animals, after these had been treated with blood-cells of a differ- ent species, acquires a high degree of toxicity for just this species- Shortly afterward, Bordet was able to demonstrate that this toxicity in corpore corresponds to a specific haBmol3rsis in vitro. This was confirmed independently by von Dungeon and Landsteiner by expert, ments published somewhat later, and further by those of our own mentioned in previous communications. The result of the experi- ments is always, that, following the introduction of red blood-cells of one species into the organism of another, a hsemolysin is formed which so injures the blood-cells of the first species that their haemo- globin goes into solution. Bordet also showed that this haemolysis depends on the action of two substances in the haemolytic serum. The importance of this subject, due specially to the complete analogy between the h8Bmol3rtic and the bacteriolytic processes, led us to a detailed study of the mechanism of these processes. We were able to show that the substance produced by immunization, the immune body, possesses a maximum chemical afl^ty for the corre- sponding blood-cell. This aflSnity is due to the presence of a specific combining group in the molecule of the immune body, which fits to a corresponding group in the protoplasm of the erythrocyte. Beside this, the immune body possesses a second combining group * Reprint from the Berliner klin, Wochenschr. 1900, No. 21. 'See pages 1 and 11. 23 which fits to a group in a fermenl-like body of normal serum, namely, the complement (addimeutj. By virtue of these two baptophoie groups, tlie immune body functionates as a coupler or interbody. carrying the action of the complement over onto the red blood-c^lls, In order to facilitate expression, Ilia! combining group of the. pro- toplasmic motccuU: to which tli£ introduced group ia anchored will here- after be termed receptor. The side-chain, tor example, which com- bines with the tetanus toxin in the organism ia such a receptor. The tetanus antitoxin itself is nothing but the surplus of receptors thrust off into the blood. Similarly, that complex which latir functionates as immune body is a receptor bejore being thrust off. In the further course of these investigations it has been found that the function to produce peculiar antibodies analogous to immune bodieii ia not confined to bacteria and erythrocytes. Cells of the most varied kind, provided they are absorbed, excite the production of immime bodies, in conformity with the requirements of the side- chain theory. Lands teiner. Metchnikoff, and Moxter succeeded in producing an immune serum against spermatozoa; von Dungem, a specific serum which acted on ciliated epithelium; and Mecthni- koff, an immune serum against Ieiicoc\1es and kidney epithelium. Here also in the cases examined for this purpose (v. Dungem, Moxter) it could be shown that the sjjecific active substances are of complex nature, consisting of an inunune body and a corresponding comple- ment, and that the immune body possesses a specific aflinity for the corresponding cells. Tlie great theoretical significance of these investigations which open up a new field to the study of immimity is clearly apparent, but whether in the near future they will have any jiractical results remains to be seen, In the pursuit of these studies, we were led to extend our research^ into another direction which seemed to us of special importance in the understanding of pathological processes. The experimental investigatiorLs thus far made have dealt exclu- sively with the changes in the serum which occur when an animal is made to absorb foreign cell material. This mode of exjjeriment, however, is not limited in any way by the nature of the subject, but is dependent entirely on the will of the experimenter, and it there- fore lacks all physiological analogy. In pathology, the changes foremost to be considered ore thoso re=uhing from the absorption, by an organism, of Us own ceM mate- I STUDIES ON ILiJdOLYSIS. 25 ■rial. Such occasions are presented by many different diseases. Keeping to the blood, for example, if an individual suffers a con- siderable subcutaneous hemorrhage or one into a body-cavity, or if part of his blood-cori>uscles are destroyed and dissolved by certain blood-poisons, the essential conditions, just aa in an exijeriment, are given for the reactive formation of substances possessing specific injurious affinities for these biood-cells. The same, however, can apply to other tissues; for eveiy acute atrophy of an organ's paren- chyma can lead to the absorption of cell material and to its conse- quences. The conditions necessaiy for the development of specific cell poisons may !>e presented by various ch'c urns tan ces, thus, when, spontaneously or under the influence of araenic, large lymph-gland tumors are absorbed; when a slmina meJts and disappeare under 'Specific treatmnt; when the white blood-cells, owing to the action '■frf toxms or other substances, are caused to disintegrate; when, owing to certain raetabohc or infectious diseases, acute atrophy of the li\'er ensues, etc. We shall further have to assume that these conditions can be fulfilled, in a wider sense, when, under the influence of certain general diseases, there occurs active dissolution of oi^ ganizod material of any kind instead of atrophy of a single organ. It is therefore of the highest jiathological import.ance to determine whether the absorption of its oinn body material can excite reactive changes in the oi^nism, and what the nature of these changes is. The simplest conditions and thtwe most accessible to experimental study are those which arise on the alieorption of blood-cells. But here we face a ious dilemma. If an animal organism, when injected with blood- 'tdls of foreign species, always pnxiuces a specific hipmolysin for each of these species, it must surely be following a natural law; and it is improbable that this law which applies in any particular number of cases should be suspendefl in the case of blood-cells of the same individual. On the other hand, it is not to be denied that the forma- tion of such hipmolytic substances would appear drateolngical in the highest degree. For example, if, in an individual who has had an extensive hemorrhage into a body-cavity, the absorption of this caused the formation of a blood poison which destroyed the It of the blood-cells, this would be a phenomenon whose actual occurrence lacks any clinical evidence whatever and one which no one k'illing to accept. It cannot he doubted that the organism seeks a way out of this difficulty by means of certain regulating contrivances, whose deter- ' thos Bciirii ■-CeIIs Cexten; niood KfiBt C 26 COLLECTED STUDIES IN DIMCNITY. mination will be of the highest interest. To be sure the studj" of this question oSers coiujiderabte difficulties, difficulties through which previous experiments in this direction have been brought to naught. (Belfanti and Carbone, liordet.) We lia\-e from the beginning maintained that it is possible to gain an inaight into these processes, only when any changes occurring in the serum are determined by means of frequent and progressive examinations. Small laboratory animals, because of the amount of blood required tor these continuous examinations, are therefore unavailable, and hence we selected goats as being best adapted for these experiments. After it had been determined that a single injection of a large amount of blood sufficed to produce the 8])ecifiG hiemolytic sub- stances in the sermn, we usually injected our animals once with a lai^e amount of goat-blood. (SOO-900 cc. for a goat of 35-40 kg.) In order to overwhelm the body as rapidly as possible with the con- stituents of the blood-cells, we made use of intraperitoneal injectiona. For the same reason we thought it best not to inject intact blood- corpuscles, but to inject blood which had been made laky by the addition of water. We argued that blood-cells of the same species as the animal injected would be destroyed very slowly in the peri- toneal cavity of this animal, and that consequently the absorption would be so gradual as to prevent the occurrence of what may be termed an " ictus immunisatorius." From the second or third day on, we withdrew samples of scnim from the animals so treated, and tested the solvent action on the blood of numerous other goats. Our method generally was first to determine whether any indica- tions of hjDmolytic action were present. For this purpose a drop of normal goat blood was allowed to fall into undiluted serum of the treated goata, and the occurrence of any red coloration looked for. If this test was positive, we proceeded to test the htemolyain in the usual manner by adding decreasing amounts of this serum to tubes containing 1 co. of a 5% mixture of goat-blood in 0.85% salt solution. With these preliminary remarks we proceed to our first posi- tive test (February 16, 1900). The subject of this was a strong male goat, buck A. weighing 33.5 kg., into whom there were injected intraperitoneally 920 cc. goat-blood (mixed from the blood of goats 1, 2, and 3) made laky by the addition of 750 cc. water. From the second day on, small amounts of blood were withdrawn daily ft iyf|^ STUDIES ON HiEMOLYSIS. 27 the purpose of obtaining serum. This serum, as we had antici- pated, never showed a trace of hsemoglobin coloration. As early as the second day, a sUght solvent action for the blood of goats 4 and 5 was developed. A drop of the blood allowed to fall into the undi- luted senun of buck A suffered partial solution, so that after the blood-corpuscles had sedimented, the serum remained slightly tinged with red. By the fifth day the solvent property had increased considerably; 0.5 cc. senun completely dissolving 1.0 cc. of the 5% blood-mixture of goat No. 4. By the seventh day the action had reached its maximum. 0.3 cc. serum produced complete sola* tion (No. 4); 0.07 a just appreciable effect. As we now had at our disposal a sufficient amount of hsemolysin^ we sought to determine whether this haemolysin dissolved all goat blood-corpuscles without exception. We found that of nine goats which we examined, the majority were markedly sensitive to this haemolysin. Thus goats Nos. 1, 2, 4, 5, 6, and 9 were highly sen- sitive; two goats, Nos. 3 and 8, somewhat less so; and only one, No. 7, (which had previously been treated for some time with the expressed juice of eel muscle,) showed so slight a susceptibility that even imdiluted serum failed to cause strong solution. After noting these results it was important to determine the behavior of the blood-cells of this buck toward the haemolysin of his own serum. If a drop of blood was added to the serum, in vitro, not even a trace of solution occurred. These blood-cells then were entirely insusceptible to the haemolysin of their own serum, as had already been indicated by the absence of hsemoglobin coloration in the freshly drawn serum. If we designate the specific haemolysin developed by the injec- tion of blood of foreign species as heterolysiriy then we must designate the haemolysin due to the injection of blood of the same species as isolysin. In no case, however, and this is to be emphasized, are we here dealing with an autolysin, i.e. a lysin which dissolves the blood-cells of the animal in whose serum it circulates. However, such a condition is not at all a matter of course, and the question arises why the isolysin in this case does not also functionate as auto- lysin. The toxins as well as the haemolysins can act only when they are anchored by certain haptophore groups, the receptors, whereby the action of the poisons is concentrated on the cells possessing these receptors. If these groups are lacking, the poison has no point of attack. We have already demonstrated that a haemolv-sin, or rather its immune body, is anchored by the erythrocytes, and the solution of the aljove question therefore becomes very easy. To begin, we have determined that the isulysin behaves like a typical hfemoiysin of the well-known kind. It loses its action by being heated for half an hour to 55° C. (destruction of the complement) and is reac- tivated by the addition of a corresponding amount of normal goat Berura, Next we have determined that the immune body of the iaolysin is bound by the susceptible blood-cells in typical fashion; that the blood-cells of the immunized animal, however, take up only traces of the immune body in vitro, amounts far less than those taken up by the almost insensitive blond-cells of goat No. 7. This phenomenon can at once be ascribed to a slight mechanical absorption. We see, therefore, that the serum's own inseasilive blood-celts are incapable of anchoring the specific immune body of the isolysin. This result can be explained in either of two ways. It may be assumed that the blood-cells lack this receptor entirely, or that. although the cells possess the receptor, the affinity of this had already been satisfied by the immune body in the circulation. In the latter case, however, it is incomprehensible why the blood-cells were not dissolved by the complement also circulating in the blood. Further reasons against the latter assumption will be apparent laler, and so we shall at once discuss a series of facts which, according to our views, demonstrate that the insusceptibility of the blood-cells in this case is due to an ahsolvte lack of these receptors. Assuming that a given toxin, in an organism, finds receptors which anchor it, the injection of this toxin will be followed by the production of a corresponding aniibody. If, howev'er, an organism lack receptors for this poison, the firHt essential for the production of an antibody will be wanting. In the de\'elopment or non-develop- ment of antibodies we shall have an indication of the presence or absence of recejitors. Now the hicmolysins belong to the class of poisons which pro- duce antibodies. We om^elves have demonstrated that the normal hajmolysin.'j of dog's and goat's serum, when injected into a forcipii animal body, excite the production of anliha>molysins. The ques- tion was whether the isolysin when injected into the organism of other goats would be able to cause the production of an anti-isoli/sin. In order to save material we injected a young goat (No. 10), whoae STUDIES ON H.^3I0LYSIS. 29 blood-cells we had previously shown to be very sensitive to the iso- lysin, several times with considerable quantities of serum A. As a matter of fact an antibody was developed, so that 0.4 cc. of the serum thus obtained were able to protect 1 cc. of a 5% sensitive goat-blood-cell mixture against solution by isolysin A (0.5 cc). The blood-cells of this same goat No. 10, on the contrary, after they had been repeatedly washed with physiological salt solution to free them from serum, proved just as susceptible to the isolysin as before. Hence it follows that the isolysin here concerned, isolysin A, causes the production of antilysins in the body of the same species when it finds fitting receptors. From this we conclude that the insensitiveness of the red blood-cells can only be due to the lack of receptors for the isolysin. A further conclusion must be that these receptors are not present in any other tissue of buck A, that they are absent in the entire organism, for other- wise there should have been a formation of anti-isolysin. It goes without saying that we repeated these experiments on a large number of animals in order to exclude all accidental phenom- ena. In the course of these experiments we noted numerous and interesting variations in the reaction to isolysins. Of special interest is goat B, which had been treated exactly like buck A. At first it seemed as though the experiment with this animal would run an entirely different course, for during the first fourteen days we were unable to detect even a suggestion of an iso- lysin. The red cells, however, remained completely sensitive to the isolysin derived from buck A. Then suddenly on the fifteenth day after the blood injection a haemolysin made its appearance, one which acted on goat blood quite as strongly as the isolysin of buck A. The animal's own blood-cells were just as insensitive to this haemolysin as were those in the first experiment to theirs. Here also, then, we were dealing with an isolysin, not an autolysin. The sen- sitiveness of the blood toward isolysin A continued. We now examined the majority of our goats in order to determine their sen- sitiveness to this isolysin, and found that some animals which were highly sensitive to isolysin A were very slightly sensitive to isolysin B, and vice versa. The blood of buck A occupied a peculiar place. It was as completely insensitive to isolysin B as it was to that of its own serum. From the behavior of the blood of the various animals toward these two isolysins. it was clear that these isolysins were essentially 30 COLLECTED STUDIES IN IMMUOTTY. different. This was positively proven by the fact that the anti- isolysin A was entirely ineffectual against isolysin B. The difference between these two isolysins is further illustrated by the difference of the intervals between blood injection and isol3^in formation. In the one case this was only a few days and in thie other fourteen days. That the injection of the goat blood should result in the formation of two entirely distinct and easily differentiated icolysins was cer- tainly a remarkable phenomenon. And yet this did not exhaust the multiplicity of the isolysins. In a third goat, C, (injected on the same day as B and with sim- ilar amounts of the same blood,) a hemolysin C appeared on the seventh day which again differed from isolysins A and B. This, furthermore, proved itself an isolysin, for the blood-cells of the ani- mal were entirely insensitive to its action, though they were sensitive to isolysins A and B. This fact shows that isolysin C differed from isolysins A and B. It is specially noteworthy that, although the two goats B and C were injected at the same time with similar amounts of the same blood, they should develop different isolysins. This observation is particularly important because it shows that the constitution of the isolysin is dependent on the individuality of the animal in which it is developed. It is also very remarkable that these three isolysins, A, B, and C, were able to destroy not only goat blood-cells, but also those of sheep. The sheep erythrocytes therefore possess three different groups which are identical with those of these goat blood-cells, or at least are closely related to them. On the other hand still another isolysin, D, does not dissolve sheep blood-cells. After having observed three different isolysins in three different goats, we are in no wise to assume that this exhausts the possibilities.* On the contrary, it seems highly probable that by further experi- ments we shall come to know other isolysins. Nevertheless it must not be assumed that this variation of the isolvsins is unlimited. It is to be expected that a sufficient repetition of the experiments will finally lead us to recognize a certain cycle of constantly repeat- ing types. The attainment of this goal, however, is rendered very * Note on revision. — In the mean time we have obtained a fourth isolysin, D, which differs from isolysins B and C in the fact that it dissolves the blood- cells of B and C. Erythrocytes of A are not dissolved, but the isolysin differs from A in its behavior to various normal kinds of goat blood. The behavior of isolysin D toward sheep blood has already been mentioned. I vwiU ■in ™ STUDIES ON HAEMOLYSIS. tedious by the fact that in some casea in which the production cf an isolysin is attempted after the method already outlined, no iso- lysin is formed. We have records of a number of goats in which the injection of goat blood produced apparently no effect wliatever; among these ia one which was injected with its own blood. The ditlerence in the isolysins in their dependence on the injected' blood and on the individuality of the treated animal, the fact that there is formed always an isoli/sin, not an autolysin, the special con- ditions governing the formation of the anti-isolysins, the failure of the isolj'sin reaction in certain cases, — all these make the problems connected with the above facts appear very complicated, and make it necessarj' now to analyze these more closely. Every red blood-cell possesses a large number of side-chains with haplophore groups, each of which is able to combine in the animal body with fitting receptors. Let us, in our own case, designate such a group of the injected goat erythrocytes aa group a, and a corre- sponding receptor as receptor a. There will then be presented two possibilities. First ia the possibility that the « receptor b entirely absent in the organism of the goat into which the blood is injected. If this be the case, there ia lacking the &saential condition for the formation of any reactive product, and the result of the injection will be entirely negative. It, however, the second possibility obtains, and a receptors are lent in the lx)dy of the animal injected, there are again two ways In which the reaction may proceed: (1) the at receptors exduaively may be present; (2) besides these, the organism may contain the same group a which is present in the injected blood-cells. We shall study these two cases separately and begin with the simpler, in which only a roceptora are present. In this case the conditions for the formation of a hffimolysin are given and the bind- ing, hyper-regeneration, and final thrusting-ofT of the a receptors will follow. This newly formed immune body, in conjunction with the complement always nonnall>' present, will dissolve all those goat blood-ceib, and only those, which possess the group a. But as this group a, according to our assumption, is completely al)aent in the organism of the animal itself, the immune body fails here to find any point of attack. The immune body therefore will accu- mulate in the blood without hindrance and without causing the shghtest damage to the organism. This case is the one which appliea to the examples of isolysin formation described by us, for it ia the 32 COLLECTED STUDIES IN IMMUNITY. only one which fulfills the conditions necessary for a. permanent existence of a free hamnlysin. The course of the reaction, however, is entirely different in the second case, i.e., when the group a of the foreign blood-cells which fits into the receptor group is found also in the organism of the animal injected, being present in its blood-cells and tissues. In this case, groups fitting to one another would be present in the same organism. A pregnant example is seen in this, that both the rennin and the antirennin group may occur simultaneously in the Organism. In fact we believe that this simultaneous occurrence of such corre- sponding groups is a very frequent phenomenon in the economy of the organism, and that it occurs especially in those cases in which & certain cell is dependent for its nutrition on the products of a, dif- ferent kind of cell.' If this is the case, i.e., when group a is present in the organism beside the receptor group, the first phase will proceed just as in the first case. There will be a binding, regeneration, and thrusting-off of the receptor as immune body. The difference in the course of the reactions becomes manifest in a second phase in which these thrust-off receptors are taken up by group a. I'nder certain circumttances this might lead to serious injury, namely, when the thrusting-off of the receptors as immune bodies occurs so suddenly that the organism is overwhelmed, the red blood- cells anchoring the receptor group and being dissolved by the cvct- present complement. In this case, then, an autolysin could develop. But this result need not of necessity ensue. It can be prevented, tor example, if at first only small amounts of the liberated receptor ' In contrast to this we shall have to assume that tingiilar Imptophora groilpi occur wherever it is designed to catch hold of certain exogenous constituents of the nourishment. In immunizs.tion i(. is of eoiae consequence whether & aingular group funcdlonates as receptor, or one which corrcBponUs (o another. The former is probably the ease wilh the toxin, and this pertnits of an extraor- dinary increftse in the production of antitoxin, being limited by no regulating contrivance. If, howe\'er, tlie antigroup is present in the organism, owing to eeeondnry influennes. s regulatory production of now antigroups will occur. This might be the reason why it ia apparently impossible to increase the pro- duction of antirennin to any desired degree. The antirennin finds the cort«- aponding rennin group in the organism and causpB (ho production and thniBting off of This group. As a result of this series of chimges we find at one time th&t the nerum of an animal contains free antirennin, at another time that rennin Ib heing excreted by the urine. STUDIES UN H-E.M0LY8IS. 33 body) reach the tissues. Tliis would effect a production wid thrusting-off of the eorreaponding group a, which woiild then Birculate as an antiautolysin and serve to switch the autolysin there- ifter formed, away from the blood-ceils, lie this aa it may, whether the organism be injured as a result of an acute flooding with the liberated receptors, or whether this injurj' be prevented by the slow Bourse of the reaction, the end result in the second case will regularly be a development of an anliautolysin} The three possibilities, therefore, which present themselves on the injection of blood of the same species are: 1, ihe failure of any formation of heemolysin; 2, the formaiion of an isolysin; 3, the derelop- ment of an nnlumtolysin. Each haptophore group of the red blood-cells (and we have reason to assume a large number of different groups in each erythrocyte of everj- species) will have to react, in the animal body, according to the above scheme. This leads to a large number of posgibilities. If, for example, an injected blood-cell possesses three haptophore groups, a, ^, X, it will be possible for a to cause the development of an isolysin, ^ an antiautolysin, while ;• produces no eJTect whatever. This, of course, complicates the problem extraordinarily. A multiplicity of variations is presented whose complete investigation would require a great deal of time and labor. TTie three cases above- nentioned, howe\-er, amply suffice to explain all our observations Lhus far. The differences in the three isolysins previously described to be ascribed to the action of three different haptophore groups of the blood-cells; and the fact that the same blood injected into Iwo inimala causes the development of different isolysins is to be explained ay the individual differences in Ihe receptors. Finally, the failure of any isolysin reaction whatever would correspond to an absence of suitable receptors. ' The cases here discitssed are of general stgciificance for the question whether lolyeins exist at all. and they deterniino also the conditions under which hiMnolyeins of normal eenim are capable of existence <8ee also the second •ommunieation, pages 11-23). The fact that a normal hn'tnolysin dissolves the >lood-<»lIs ot foreign iiperies but spares its own hlood-eells, that, for example, log serum dissolvej) guinea-pig blood, rat blood, goat blood, eheep blood, etc., Mat not dog blood, is only a Btngle instance of the above-mentioned general that autolyttins are not capable of existence in an organism; for the presence jf receptore. which ts essential to the production of niitolysins, would, if the lutolysins should develop, soon result in a compeoeation by means of anti- lutolymn form at ion. 34 COLLECTED STUDIES IN IMMUTmT. Though the existence of the antiautolyain is theoretically pes- Bible, we have thus far been unable to demonstrate it. To do this it would first be necessary to get hold of an appropriate autolysin. The possibility of getting this, however, is only conceivable in such favorable cases where the autolysin might be produced critically and in large amounts. This certainly did not occur in the cases observed by us, and we were therefore compelled to try a different method to demonstrate such an antibody. We know of a number of hEemoIysina which dissolve goat-blood and which therefore fit to certain haptophore groups of the goat blood cells. It is con- ceivable that one of these haptophore groups is identical with that of the autolysin sought for, and that an antiautolysin fits this group.! With this end in view we have made a number of experiments and tested the action of our inactive goat serum on the goat^blood- disaolving action of dog serum, pig serum, and goose serum a d on the serum of a rabbit treated with goat blood. The results, however, were not positive. From this, of course, we are not to conclude that antiautolysins are not at all present in these cases. We shaU rather extend and vary our experiments in all possible directions ' until a lucky coincidence leads ua to find a fitting hiemolysin. Perhaps the most important of the questions thus presented is whether this deficiency of binding groups in the red cells b performed, or whether it is due to a new regulating power of the organism. In the latter case this power would be suited in the highest degree to protect the body even without the formation of an antiautolysin. In one case, to be sure (goat E), it seemed aa though the insen- sitivenesa was developed only in response to the blood injection. The blood-cells of this goat (the goat had been repeatedly injected) were primarily sensitive to isolysins A and B, After the injection there developed a complete insensitiveness to isolysin B. although the sensitiveness to A remained. In this case an isolysin was not developed, so that if accidental circumstances are excluded, it appears as if under the influence of this blood injection a direct change or destruction of the binding groups had taken place. We may perhaps also assume that the complete insensitiyeneas ' The multipliKity of the combining groups of the blood-cells is well illustrftted by the blood of buck A. This blood is insensitive to (he isolysins mentioned. Independently of this, however, it retains complete Bensitiveness to hipmolynna of a different origin, pig serum, goose serum, specific goose serum from rabbita. p STUDIES ON H/KMOLYSIS. 35 pf buck A to isolysin B is a secondary one, due to the treatment; for thus far, among the many normal goata examined, we liave failed to find a single one whose blood-ceils are completely insensitive to isolyains A or B. These phenomena require further and more extended investi- gation, and in this we are at present engaged. In closing we should like to point out that the difference between isolyains and autolysins emphasized by us makes several recent attempts directed to the solution of certain pathological processes, particularly those of autointoxication in man, appear questionable. It has frequently been ascertained that serum secretions and excre- tions of the diseased body are poisonous in animal experiments, and the conclusion has been drawn that the substances to which this poisonous action is due must exert an injurious effect on the organism of the patient. From the above analysis we see that this conclusion is not at all imperative. If, for example, the serum of a scarlet fever patient is especially toxic to guinea-pigs, it is possible that the same may be absolutely harmless to the patient himself. Even if one demonstrates that the serum of aniemic individuals dis- solves the blood-cells of other individuals, it does not prove that this property is of any significance for the origin of the amemia. In the contrary it is highly probable that this htemolysm is only isolysin and not an autolysin. The above experiments may suiRce to show how very complicated the conditions are when the mat-erial of its own body is absorbed by an organism. Drawing a general conclusion, however, we may say that such an absorption, which as already stated ext€nda to the greatest variety of cells and occurs in numerous instances, will not as a rule lead to permanent injury of the organism, owing to the formation of reaction products. Only when the internal regu- latuig contrivances are no longer intact can great dangers arise. ■In the explanation of many disease phenomena it will in the future to consider the possible failure of the internal regii- as well as the action of directly injurious exogenous or endog- lus substances. IV. CONTRIBUTIONS TO THE STUDY OF IMMUNITY.' By Dr, von DusaBRK, Univeraity of Freiburg, Germ&E^. A. New Experiments on tbe Side-chain Theory. The combining ejtperiments of Ehrlich and Morgenroth ^ showed conclusively that the two components of an immune serum necessary for hiEmolyais and first demonstrated by Bordet, namely the immune body which withstands heating to 56° C. and the complement (addi- ment) which is present even in normal serum, can under certain cir- cumatances exist in a serum side by side, uncombined. The immune body possessed a strong affinity to the blood-cells to which it spe- cifically belonged, being anchored by these cells at 0° C. and thus separated from the complement, which latter remained in the serum. The complement was abstracted from the serum by the erythrocjtea only at higher temperatures provided the immune body was present at the same time. 'When the latter was absent the blood-eells failed to combine with any complement whatever. The complement, therefore, because of its lack of affinity, was unable to act on the blood-cells, and likewise the mere anchoring of Ihe immune body by the blood-cells, without the presence of the complement, was unable to effect any hsEmolysis. The most plausible explanation for these facta was this, that solution is effected by the complement, but that this substance first requires the immune body to enable it to lay hold of the blood-cells. Bordet ' has assumed that the immune body, independently of the complement, combines with the substance of the erythrocyte and so changes this that it (the erythrocyte) now combines with the complement. Against this assumption must be urged that as a matter of fact there is a definite relation between immune body ' Reprinted from the Miinehener med. Wochensohrift, No. 20, 1900, 'See pages 1-23 of this volume. • Annalos de I'lusiitut Pasteur, 1899, No. 14. CONTRIBUTIONS TO THE STDDY OF IMMUNITY. 371 \ and complement of the same speoies. Aji immune serum inactivated ■ by heating to 5C° C. can always be reactivated by the addition of fresh blood serum from an animal belonging to the same species aa that from which the inmiune serum was derived. The eomplementa of other species of animal, however, reactivate this immune body i in the most divergent manner. The results of the combining experiments were readily harmonized with the requirements of the side-chain theory. The immune body is nothing but a side-chain with two haptophore groups, which has been produced in excess and thrust off into the blood. One of these haptophore groups possesses a strong chemical affinity for the corre- sponding group of the erythrocyte, and ordinarily it serves to anchor nutritive material possessing corresponding haptophore groups to the cells. The other haptophore group is able to combine more or less completely with complement present in the scrum. It is probably designed to collect from the blood plasma the ferment- like complement, which, by splitting up the nutritive substances, makes their assimilation possible. There is, however, another view to take of these phenomena. It comprehensible that the cell, aa such, produces the two compo- its necessary for haemolysis simultaneously and in relation with each other, in such fashion that in the assimilation of the substances anchored, it constantly produces the complement required by meam of ita own activity and docs not depend on the supply from with- out, from the blood plasma. The assumption of such a complex system — in which two members so intimately connected are yet so readily dissociated— offers difficulties which it is unnecessary to discuss further, especially because, as will be seen later, experi- ints have precluded this possibility. If, however, the side-chain theory is correct we shall axpect: 1. That immune body and. complement are not present in the imune serum in etpiivalent proportions, but that quantitatively itiiey may be independent of each other. 2. That the same group of the red blood-ceJls which in hemolysis combines with the immune body causes the production of the inv- mune body. 3. That cells which possess such form of complex side-chains are enabled by the presence of the eomplementophile groups to abstract complement from the blood serum. 1. The question whether in the immunity reaction only the inao- Kfis CI ^eact tive immune body is produced, wtiich then combines secondarily with the complement present in the blood, or whelher the two sulj- stances reach the circulation together, can under favorable con- ditions be answered by an exact quantitative analysis of the immune serum for immune body and complement. I have therefore treated a nmnber of rabbits with cattle blood, cow's milk, and tracheal epithelium of cattle, and examined the hemolytic immune sera thus obtained for their exact content in im- mune body and complement. Corresponding to the material injected, the erythrocytes of cattle were always used as a reagent. The method employed was the same in all cases ; decreasing amounts of Uie various blood sera were mixed, each with one-half cc. 5% cattle blood dilution (in 0,89c NaCl solution), the mixture was kept at 37° C. for two hours and tested for hiemolysis. It was then very readily proven that an equivalence between immime body and com- plement does not at all exist. If such an equivalence were present, the immune body of the fresh immune senim would be completely aatiu-ated with complement and would not become more active by the further addition of com- plement. The experiments demonstrated the contrarj', for in some cases the power of the immune sera was markedly incraseed by the addi- tion of normal rabbit serum, which, in the doses employed, was not itself able to effect the slightest solution of the cattle blood-cells, For example, if the fresh serum of a rabbit which had been treated with cattle blood was able to make ten times its volume of a 5% cattle blood mixture completely laky, the same serum on the addi- tion of a sufficient amount of complement was able to dissolve 320 times its volume. On comparing the various immune sera with each other, it is seen that this increase in the hemolytic action on the addition of complement is in direct proportion to the amount of im- mime body present. The- experimevls therefore prove that quantilatively (he immune body is entirely independent of the complemetU. We can, however, go further and determine quantitatively the exact amount of complement contained in the normal serum on the one hand and in the immune serum on the other. The amount of complement contained in the various normal sera was determined by always testing with the same amount of & blood immune body. In fixinij sHch a utandord serum it is only neces- sary to take as a measure the action of an immune body saturated mth CONTRIBUTIONS TO THE STUDY OF IMMUNITY. 39 complement, for equal amounts of immune body act differently with different amounts of complement. In all my tests on the amount of complement contained in a serum, I used so much inactivated blood immune serum that the inunune body, when saturated with com- plement, could dissolve sixteen times the amount of blood present. The experiments demonstrated that the amount of complement contained in normal rabbit serum is fairly constant, and even in different animals is not subject to great fluctuations. Proceeding as just described, it was found that complete solution took place in all cases on the addition of V40 to V20 cc. normal serum. Within definite limits therefore the complement in rabbit blood seems fixed. The amount of complement contained in immune serum could be determined by comparing the haemolytic action of the fresh serum with its action, after inactivation (by heating for twenty minutes to 56° C), on the addition of various amounts of normal rabbit serum, the complement content of which was known. The serum of the rabbits treated vnth cattle blood, serum which had been shown to contain such a large excess of immune body, was tested i, J^, 3, 4, iif and H days after the injection and failed in all of the numerous cases to show even a trace of increase in the amount of com- plement it contained, A peculiar state of affairs is thus presented. Since haemolytic action is dependent on the immune body so far as this can combine with the complement, we see that the haemolytic action of fresh immune serum can be increased only up to a certain point, determined by the amount of complement contained in the normal blood serum. All additional amounts of immune body formed in the course of the immunity reaction therefore remain latent, and manifest their action only when the immune body is brought into combination with greater amounts of complement. This can be done artificially, in test tube experiments, by the addi- tion of normal serum, or experimentally by injecting the immune body into a suitable animal body.^ Immune serum therefore differs from normal serum only in its con- tent of inactive immune body.^" Accordingly, in the immunity reaction, only inactive immune body is produced by the cells in excess. This > So also the earlier observations, as those of R. Pfeiffer, on cholera serum, my own on epithelial immune serum, and those of Moxter on antispermatozoa serum, in which the immune sera, in themselves little or not at all active, showed their full power when injected into fitting animal bodies, are to be explained by the relative poverty of these sera in preformed complement. COLLECTED STUDIES IN IMMUNir result is easily understood on the basis of the side-chain theory, if we assume that the production of the complement is enth^ly inde- pendent of the binding of the mjected substances by the side-chaina, and !8 probably referable to other cells. If the production and thrusting off of the particular side-chains exceeds a certain limit, these side-chains will fail to find in the blood serum any more complement whose haptophore group is still available. The disproportion between immune body and complement then sets in. This will be most marked in those cases in which the normal serum contains but little complement and in which a considerable production of immune body can be effected. 2. Certain experiments which I have described in a previous com- munication regarding globulicidal action of the animal organism ' led me to the view that the immune body combines with a particular group of the blood-cells and thus leads to their solution. This con- ception was based on the fact that a specific affinity exists between erythrocyte and the corresponding immune body, which affinity must be the same in the production as in the action of the immune body. According to the side-chain theory just this affinity is the driving force which on the one hand anchors the corresponding group of the erythrocyte to the preformed side-chains (such side-chains when thrust off constituting the immune body), and on the other, in hemolysis, anchors the immune body, and with it the complement, to the blood -cells. It must always be conceded to the opponents of this view that the evidence to prove such complicated processes as wOl develop in the cells after inoculations of blood into an animal body will not, perhaps, be absolutely conclusive. If one were willing to forego an explana- tion of the specificity, one could assume that the immunity reaction is based on an increase of the normal fimction of certain cells whose products are formed without requiring a certain group to fit into a corresponding one. It was therefore of great interest to be able to show experimentally that the group which in hemolysis combines with the inamune body actually gives rise to the production of the immune body. This demonstration was effected by injecting blood together with iiuio> tivated blood immune serum. If the development of the antibody is independent of the group ' Munch, med. Wochenachrift, 1899, Ndb. 13 and 14. J CONTRIBUTIONS TO THE STUDY OF IMMUNITY. 41 hich the immune body is attached, the immunity reaction will be exactly the same whether the injected blood is loaded with immune body or not. If, however, the production of the immune body is dependent entirety on the molecular group for which t!ie immune body possesses a specific affinity, no immune body will be developed when a sufficient amount of inactivated blood immune serum is added to the injected blood, since the group is already occupied by immune body and no longer offers the cells a point of attachment, The experiments completely confirm the latter assumption. When the blood loaded u-ith ivimune body was injected, no immune body vfluilever icaa developed in Ike injeeied animal: whereas in a con- trol rabbit, injected with exactly the same amount of cattle blood (30 cc), but without immune body, so much was produced that the serum eleven days after the injection was able to dissolve com- pletely eight times its volume of full blood provided sufficient com- plement was added. This fact, like many others, speaks against the idea that the lune bodies or the analogous antitoxins are not reaction producta of the organism but are derived by modification from the substances introduced, a view still maintained by certain high authorities. The phenomenon, however, is readily explained on the basis of the side- chain theory. Since the particular groups of the erythrocytes, which otherwise give rise to the immunity reaction, are already occupied by immune body, it is impossible for them to be bound by the side-chains, which are absolutely similar to the immune body. 3. According to the researches of Ehrhch and Morgenroth, the rhrocytee of sheep possess no affinity whatever for the complement normal goat serum. If instead of sheep blood-cells, one employs those of cattle and allows them to act on rabbit blood serum, exactly the same thing will be observed; the rabbit blood serum, centrifuged after prolonged contact with the bJood-cells, shows no diminution in the content of complement. //, however, other cells, e.g., ciliated epithelium }rom the trachea oj cattle, be mixed with rabbit serum, the reeult is directly opposite, the complement decreasing, and «ten under e circumstances disappearing entirely. In like manner the rabbit im may lose its complement through the action of other cells. the case of various mammals and birds, every one of the organs ited — liver, spleen, kidney, testis, lung, and brain — was able to ibstract more or less complement from the rabbit serum. Yeast and fission-fungi were also able to effect this. Especially remark- able, however, is the fact that the body cells of the same animal are able to produce tliia phenomenon. Exact quantitative examinations showed that there were dis- tinct differences. The spleen and kidney of a rat, for example, wae more strongly active than the same organs of a guinea-pig, while the liver tissue of the two species possessed equal activity; the spleen and kidney of the rat abstracted more complement from rabbit senim than did the same quantity of liver tissue, whereas in the guinea-pig the liver acted more strongly than the spleen, and the latter, again, more strongly than the kidney. Virulent cholera vibrios acted only one-quarter as strongly as the completely avtrulent "cholera Calcutta." (The number of active individuals could not, of course, be regarded.) Yeast cells were weakly active, anthrax bacilli strongly so. In the case of anthrax bacilli I tested the action of heat on this property to abstract complement from rabbit serum, and found that it is not destroyed by heating the bacilU for twenty minutes to 56*0., but that it is destroyed by heating them for only a short time to 98° C. But the property of the cells to abstract complement from rabbit serum is lost not only through the action of heat, but also when the particular cells prefious to their mixturt with rabbit serum have been allowed to remain in contact with another serum. For example, 1 grm. finely crushed kidney tissue of cattle is mixed with 2 cc. cattle serum, allowed to act at 37" C. for half an hour and then separated from the serum by centrifuge. If 2 cc. rabbit serum are now added to the sediment, and this is allowed to stand for half an hour at 37°, it will be found on testing with cattle blood immune body that there is no diminution of complement content; but such a diminution does occur when, with exactly the same procedure, 8 p. m. NaCI solution is used in place of the cattle serum. These phenomena are best explained by assuming that the ceHa in question, in contrast to the erythrocytes, possess groups which have a ver>- close chemical relation to those of the comiilement which reactivates the cattle blood immune body. The affinity of the cells may, in fact, be greater for the complement than for any immune body directed against other cells of the same animal species. For example, if we add ciliated epithelial cells from the trachea of cattle to an immune serum deri\'pd from a rabbit by treatment with cattle blood, we shall under favorable circumstances find that the immune body has been partially, but the complement completely, abstracted racted I J CONTRIBUTIONS TO THE STUDY OF IMMUNITY from the eenim. In this, therefore, the combining relations are just the opposite of those found by Ehrlich and Morgenroth to exist between blood-cells and their corresponding immune body. The tracheal epithelial cells must therefore possess com piemen tophile groups. The immune bodies, which according to the aide-chain theory are only the side-chains thrust off into the circulation, are similarly supplied with complementophile groups. These facts speak (or the correctness of the views of Ehrlich and Morgenroth, eapecially when we consider that a cell, corresponding to its many-sided func- tions, possesses not merely one kind of side-chain, but side-chains of the most highly developed form. Mammalian erythrocytes in contrast to the tissue cells seem not to possess complex side-chains," and this is readily understood when we consider that the red blood-cells of these animals, being without a nucleus and unable to maintain their nutrition independently are not complete analogues of the tissue cells; and further that their conditions of nutrition, corresponding to their simpler func- tions, must be less compUcated than those of the typical tissue cell. Among the hving constituents of the body, the red blood-cells con- stitute the simplest case and are therefore particularly adapted to the solution of many special problems in immunity, as can be seen from the course of the last experiments. The phenomenon, that body cells are able to abstract complement from the serum, furnishes us with a good explanation of the fact that immune sera are often so little active in an organism of a dif- ferent species. The immune body, which in stronger concentrations is not saturated with complement, even when the immune serum is perfectly fresh, can lose its complement entirely in the body of an animal of different species; it will therefore become active only when it finds a fitting complement in the new organism. Hence in serum therapy it is advisable, as Ehrlich has proposed, to employ for pur- s of immimization, animals closely related to man, and further^ femore to search for anthropostable complements. B, Phagocytosis and Globullcldal Immunltj'. In a previous communication ' lexpressed the view that the specific icrease of the globulicidal function of the organism, following the JDtroduction of chicken and pigeon blood, is due to the action of the 44 COLLECTED STTDIEa IN IMMUNITY. serum and not to the activity of the phagocytes. That the takit^ up of the blood-cells by the phagocytes in the epecifically treated guinea-pig is necessary for the solution of the blood-cells was ex- cluded by the fact that hEemolysis is also effected in the peritoneal cavity of the animals apart from the phagocytic cells. Furthermore, a transference by the phagocytes of the substances necessary for solution was not suggested because the exudate, rich in leucocytes, which was produced in specifically immunized guinea-pigs by in- jections of an aleuronat mixture, showed a much smaller content of both immune body and complement than the blood which waa poor in leucocytes. Metchnikoff has objected to these experiments.^ He states that aleuronat exudates contain principally microphages, whereas the blood is richer in macrophages, and that the latter alone are con- cerned in haimolysis. I have therefore tested the spleen (rich in macrophages) of normal rabbits and guinea-pigs with a cattle blood immune boily derived from rabbits in order to determine the amount of complement present. The experiments have demonstrated that the spleen also contains much less complement than the blood serum. For example, 1 grm, finely crushed spleen of an exsanguinated rabbit was mixed with 4 cc. of an 8 p. m. NaCl solution. This fluid, like similar mixtures derived from liver and kidney, when tested in the usual manner proved from eight to sixteen times weaker than the blood serum. Moreover, if the suspended organic particles were first washed with physiological salt solution, they yielded no com- plement whatever to the immune body. The spleen of a guinea-pig contained still less complement, although the serum of this same animal completely activated the cattle blood immune body derived from rabbits, and did so in even smaller quantity than the rabbit serum. We must therefore in conformity with the side-chain theory look to the blood serum as the chief source of complement. It is self-evident that the complement cannot originate in the blood plasma; it must, of course, be derived from some kind of cells. How- ever, that it is especially abundant in the phagocytes is not at all borne out by the above experiments. As for the immune body, Metchnikoff too believes this to circulate free in the blood plasma. According to his conception the macro- ■ Annalea de rinst. Pasteur I, No. 10. \ CONTRIBUTIONS TO THE STUDY OF IMMUNITY. phages yield this to the blood at the end of their intracellular diges- tion. Metchnikoff bases this view chiefly on his observations that the destruction of avian blood-cells in the peritoneal cavity of normal guinea-pigs is effected exclusively by the macrophages. This statement is in direct opposition to mine, according to which even in imtreated animals, the solution takes place free in the peri- toneal exudate independently of the phagocytes. I believe, however, that these apparently contrary results can well be harmonized. According to Metchnikoff the solution of goose blood-cella in the subcutaneous connective tissue of even non-immunized ardmab, 13 eftect€d almost exchisively extraccllularly. Hemolysis in this case must be due to a passage of complement and interbody from the blood into the subcutaneous tissues; this will naturally proceed more rapidly when, as a result of substances exciting inflammation, a stronger exudation ensues. It would be very curious if the same conditions for the passage of hieraolytic substances from the blood were not present in the peri- toneal cavity. We know, for example, that Pfeiffer's phenomenon is especially marked in the peritoneal cavity. As a matter of fact, shortly after an injection of avian blood-cella into the peritoneal cavity of normal guinea-pigs, one always observes free nuclei, even when the serum has been removed from the cells by centrifugation. Of this 1 convinced myself by repeated observations. If one employs blood-cells of low resistance (chicken-blood), and these in small doses, they will be degenerated and for the most part dissolved before they are taken up by the macrophages in any considerable number. When blood-cella of greater resistance are employed, and these in larger doses, the solution effected by the body juices will be com- jmratively slight and occupy more time. The taking up of these cells by the macrophages, which Metchnikoff in his splendid experi- .inents was able to follow mto the organs, will then come more to 'Ihe front. If therefore, a& a result of experiments in which I used sensitive ■cells in small doses, I underrated the significance of phago- losis, Metchnikoff, through the conditions in his experiments, fell ito the opposite error. The truth lies between these views; in the peritoneal cavity, according to the prevailing conditions, tuemol- ysis can be effected free in the peritoneal exudate or in the interior of the macrophage. In any case, phagocytosis is not essential for the development of J the immune body. The immunity reaction occurs even under con- ditions in which phfigocytosis does not at all enter; and if, accord- ing to the observations of Jletchnikoff, somewhat less immune body b produced after subcutaneous injections than after equal injection* peritoneally, this may be explained as follows: In consequence of the slower absorption from the subcutaneous tissues, fewer cells come into contact with the group of the erythrocytes which excites the immunity reaction before an excess of immune body is thrust oS by these cells into the blood. This immune body, of course, prevents any further combination of the group in question with other cells. To what extent the phagocytes are concerned in the production of immune bodies must be determined separately in each case. No definite conclusions can be drawn from the expcrimentii of Metchni- koff on guinea-pigs with goose blood-cells, for at no time did the organs of the specifically treated guinea-pigs show a stronger glob- ulicidal action than those of normal animals, although such an increase in hemolytic power was exhibited by the blood serum. But the observation has been made that even in normal animals the organs rich in macrophages are able, in contrast to other tissues, to dissolve goose blood-cells, and this observation is well adapted in this ease to support the assumption of a special significance of the phagocytes for this function. However, that organs rich in macrophages effect hreraolytic action is not necessarily the case. For example, the spleen of a guinea-pig (1 grm. finely crushed spleen suspended in 1 c.c of an 8 p. m. NnCl solution), in contrast to the blood serum of the same animal b not globulicidal for cattle blood. Considering the large number of immune bodies, it will surely often occur that the phagocytes are preeminently concerned in the production of the immune body, especially since these cells frequently come into intimate relations with the injected substances. On the other hand, it b extremely improbable that the phagocytes alone produce immune body. After all that haa been said we shall have to bring this production into relation with the general conditions of nutrition. The most varied celb, according to the kind of side- chajns they possess and the affinities thereby brought about, are probably able to produce immune body. Like the closely related antitoxic immunity reaction, the globu- licidal and bactericidal reactions rest on a chemical process the course of which b beat expl^ned on the basb of the side-chain theory. J By Dr. von DnNQERN, University of Freiburg, Gennony, A. Receptors' and the Fomiatlon of Antibodies. AccxjRDiNG to Ehrlich'a view ^ the antitoxins are formed in those organs whieli, according to their content of receptors, have bound the toxin. Roux and Borrel * in combating to this view, have jiointed out that rabbits die of tetanus following an intracerebral injection of very small doses of tetanus poison, and that therefore the brain of these animab contains no active antitoxin. Weigert ^ has shown that this phenomenon entirely supports Ehrlich'a theorj'. Since L the antitoxin of the central nervous system, so long as it has not B been thrust off into the blood, still functionates as receptor, it must ' anchor the tetanus poison to the nerve cells and is therefore not at all adapted to protect these against the action of the toxophore group. Furthermore, the fact that immunized animals behave similarly proves merely that in these animals, after immunization, the ganglion cells still possess receptors. According to the side- chain theory the antitoxins present in the blood act merely by sat- isfying the toxins which gain access to the blood and deflect these from the organs still possessing receptors and hence still sensitive. The observations of Roux and Borrel are therefore in entire har- nKiny with the views of Ehrlich. t' Reprint from Munch, med. Woehenschritt, No. 28, 1900. ' Ehrlich and Morgenroth designate those combining groups of the proto- laamal molecule to which a foreign group, when introduced, attaches ilaelf Rectbptors." See bIm) page 24. ■ Klinischea Jahrbuch, 1897, Vol. VI; Werthbemeseung dea Diphtheria Heil-eerum, Jena. Fischer, 1897. '.\nnales de I'lnstitut Pasteur, 1SS8. * ErgebnissQ der allgemein, Fathologie, etc. IV. Jahrgang, uber 1S97 47 48 COLLECTED STUDIES IN IMMLTJITY. Metchnikoff ' has pursued thi'i question as to the origin of the antitoxins further, fjince a positive conclusion did not seem pos- sible to him by the use of the bacterial poisons, he employed a 6i>ecifio cell poison, epennotaxin, which can be produced by treating guinea^ pigs with the testicle and epidiJymus of a rabbit. The use of this poison has the advantage thai the organs against which it is directed can be removed from the animal without scriom injurj-. Aa the injection of this poison into the body of male rabbits is followed by the production of an antiViody, it was merely necessary to repeat this procedure on castrated rabbits to decide the question whether the antispermotoxin is produced only by the sexual cells or also by other organs. The results showed that the sera of rabbits which had been inject«d with this spermotoxin would protect rabbit spermatozoa against the action of the spermotoxin no matter whether the rabbits from whom these sera were derived had been castrated or not. According to Mctchnikoff's view, thU is opposed to the sidd- chain theory, "since," as he says, " an antitoxin is produced with- out the presence of corresponding receptors in the organism." In this, however, Metchnikoffstarts with the assumption that the spermo- toxin is absolutely specific and that it act« exclusively on sperma- tozoa. He believes that the li!£molytic action which he has observed in the spermatozoa immime serum may be explained by assuming that with the injection of testis and epididymus red blood-cells were introduced, and that thrae produced a hEcmolysin entirely independ- ent of the spermotoxin. Further, he thinks that any relation of the spermotoxin to other cells is excluded by the fact that in the serum of guinea-pigs which have been treated with epermatozoa these cells suffer no greater change than they do in normal guinea-pig serum. Having made observations in the course of my investigations on epithelial immimization, which contradict these assumptions of 1 Metchnikoff, I feel compelled to explain my \iews in order to clear up the entire matter. As I have mentioned in a prc\-ious commiinication ' the ciliated epithelial immune serum is able, besides its specific action on ciliated epithelium, to dissolve the red blood-celLs of the same animal species. ' Annalea de I'lnstitul Pasteur, 1900, No. 1. 'Munch, mod. Wochenechritt. 1809, No. 38. J CONTRIBUTIONS TO THE STl'DY OF IMMUNITY. 49 This hiemolytic property can in no way, as Metchnikoff believes, be due to the introduction of erythrocytes with the injection of the epitrhehal celb into the body of the guinea-pig,^ which introduction would then lead to the formation of a specific hemolysin directed against the red blood-cells. This posaibility is at once excluded by the method of procedure in this experiment. For reasons of asepsis, the trachcEB employed were scrupulously cleansed with physiological salt solution and th\ia all traces of blood adhering to the surface were removed. The epithelium itself could not contain any erythro- cytes, for it was obtained by carefully scraping the surface layer, which contains no blood-vessels. Errors due to any admixture of blood, therefore, do not enter into ray experiments. Besides, such a strong haimolytic action as is manifested by the ciliated epithelial immune serum is never produced by the injection of such small amounts of blood. In my experiments this action was greater than that following the injection of 2 cc. of cattle blood. The strongest proof that the blood-diesolving property of the ciliated epithelial immune serum is independent of injected blood- cells is afforded by the fact that the hicmolytic immune body of this serum possesses greater affinity for the ciliated epithelium than that specifically derived by the injection of blood. There is no doubt, therefore, that pure ciliated epithelial immune serum possesses a hiemolytic action, and that, furthermore, the hEraolysin produced by epithelial cells is different from that pro- duced by bIood-«elk. Moxter^ made very similar observations on spermatozoa immune serum. He fotmd that the serum of a guinea-pig which had been treated with sheep spermatozoa dissolves the blood-cells of sheep; and he demonstrated that the immune body concerned in this hemolysis is completely boimd by the spermatozoa of sheep. An absolute specificity, so that, for example, the immune body [uced by means of ciliated epithelium is bound only by ciliated epithelium, that produced by means of spermatozoa bound only by spermatozoa, that directed against red blood-cells only by erythrocytes, without the existence of any affinities between the immune body and other cells of the same species, does not therefore obtain. ^prodi ^«pith ' Just an with giiinea-piea, it is T^Rsihle, by injecl.iiiK rahbil.s with tracheal Apittieliiiffl of catl.le. to produce a senim hipinolytic for cattle blood. ' Deutsche med, Worhensclir.. 1900. No. 1. I ^ IS pro COLLECTED STUDIES IN IMMUNITY. This, of course, is readily tindet3tood by means of the side-chain theory. One could not well assume that all the side-chains of & certain group of cells are enlirdy different from aU the side-chains of llie rest of the cells. It is much more probable that certain groups which serve general functions of nutrition are common to the majority, if not to all, of the cells of the same animal. When, therefore, after the injection of ciliated epithelial cells we see a hiEmolytic immune body develop, we may aesume that among the groups of the ciliated epithelial cell which effect the immunity, there are some which are identical with those of the red blood-cell or at least closely related to them chemically. If this view is correct wo should expect that, conversely, the immune body of an immune serum derived by treatment with blood, would be bound by ciliated epithelial cells of the same species. The facts corres^TOnd entirely with this assumption. According to my experiments, epithelial cells from the trachea of cattle are able par^ tially to bind the blood immune body derived by treating rabbits with cattle blood, The affinity of the ciliated epithelium for the blood immune body is, however, as already mentioned, less than that for the hiEmolytic ciliated epithelial immune body of the rabbit immune serum. With this a further fact of considerable importance becomes manifest. Although the ciliated epithelial cells are destroyed by the ciliated epithelial immune body (provided sufficient complement is present), it has thus far been impossible to demonstrate any injury of these cells resulting from the binding of the active blood immune body. The epithelial cells thus differ from the red blood-cells, which are destroyed even by the antiepilhelial serum. We shall not enter into an explanation of these phenomena, which point to a multiplicity of antibodies produced in response to cell material. It will suiBce to point out that there is a whole series of substances which are designated as blood poisons, because they attack especially the red blood-celk while they have little or no effect on other cells. The fact that the blood immune body when supplied with com- plement is bound by the ciliated epithelial cells of cattle without causing any apparent injury, proves, at least, that the phenomenon of toxic action in no way shows whether or not a toxin or toxin-con- taining substance has been bound by the cells. The appearance of toxic symptoms, to be sure, in the case of antitoxin-forming poisons, is proof that the poison has been boimd. An absence of toxic symp- J CONTRIBUTIONS TO THE STUDY OF IMMUNITY. 61 toms may not, however, at once be ascribed to an absence of aflSnity between cells and the poisonous substance. The formation of an antibody, according to the side-chain theory, follows only from the binding of the haptophore group which excites the immunity, to the corresponding side-chain, and hence is not directly dependent on the toxophore group. As to which cells will be able to produce an antibody depends, therefore, on the possession of a receptor for the haptophore group in question. A highly toxic action of the substance bound by the ceU is not at all essential, and, is in fact, often injurious, as has been emphasized especially by Knorr.^ This action, as Ehrlich^ has shown in his experiments on toxoids, is produced by a molecular group entirely distinct from the haptophore group and having no relation to the antitoxin. If this law applies even to the true toxins, we shall all the more have to assume that it applies where compound substances, such as hsemolysin, epitheliotoxin, or spermotoxin are concerned. In these the toxophore group is only loosely combined with the haptophore group; it is nothing but the complement, which, according to my researches,^ can be bound by all kinds of cells, even independently of the immune body, and can, under certain conditions of affinity^ be separated from the inmiune body. We see, therefore, that the assumption by Metchnikoff, that the spermotoxin is related exclusively to the spermatozoa, is incorrect. As against it I have here shown that a toxin obtained by immuniza- tion with epithelial cells is able to destroy the red blood-cells in the same manner as a true hsemolysin. In the following short communication I can bring forward an additional instance in which the development of a haemolytic immune body results although the co-action of the red blood-cells is com- pletely excluded. Even this demonstration proves that the assump- tion on which Metchinkoff based his objections to the side-chain theory is contrary to the facts. The phenomenon that even in castrated rabbits an antispehnotoxin is formed is therefore readily explained according to the side-chain theory by assuming that re- ' Munch, med. Wochenschr.. 1898, Nos. 11 and 12. *Klin. Jahrbuch, 1897, Vol. VI, and Deutsch. med. Wochenschr., 1898, No. 38. ' See page 41. 52 COLLECTED STUDIES IN IMMUNITY. ceptors for the immune body of the spermatozoa immune serum are present not only in the organs of generation but also in other cells of the rabbit. When, in addition, we come to consider the results of these last experiments, we find that the demonstration of Metchnikoff (that even in castrated animals, in response to treat- ment with spermotoxin, a body is developed which prevents the action of the spermotoxin) loses all value as proof for the origin of a si>ecific antispermotoxin. The active spermotoxin employed by Metchnikoff is of course not a simple poison; it consists, just Uke a hsemolysin, of the specific inunune body obtained by immunization and the complement present in all guinea-pig serum. Now it has been shown independently by Ehrlich ^ and Bordet ^ that when the complement is injected into foreign species it excites the production of an anticomplement which inhibits the action of an active immune body by taking away the complement, and that it does this without possessing any specific affinity to this immune body. It is therefore possible that the action of the antispermotoxin obtained by Metchnikoff is to be explained thus: The injected guinea-pig serum by virtue of the complement (Bordet's alexin) which it contains, causes the production of an anticomplement serum which then renders the complement of the spermotoxin (de- rived from guinea-pigs) innocuous. With this idea, Bordet has ex- amined an antihaemolysin, which is analogous to the antispermo- toxin, and has found that the action of the anticomplement is much more pronounced than that of the anti-immune body. The forma- tion of an anticomplement does not, of course, according to the side- chain theory, presuppose the presence of spermatozoa; for accord- ing to my experiments the complement may possess affinities for the most varied cells of the organism. Ehrlich 's theory, that the antitoxins are produced by those organs which possess chemical relations to the toxins, is therefore in no way affected by the observations of Metchnikoff. B. Milk Immune Serum. After it had been found that it is possible to produce a specific immune serum by injecting guinea-pigs with ciliated epithelium from * Croonian lecture, Royal Society, London, March 1900. *Annal. de PInstitut Pasteur. May 1900. CONTRIBUTIONS TO THE STUDY OF IMMTTNITY. 63 I the trachea of cattle it was but a step to employ epithelial secretions 1 for the same purpose. In conjunction with this it was of considerable theoretical interest to determine in thia very way whether the specific i properties of cells are preserved in their secretion products. I I have therefore employed milk for immunization and have first i treated guinea-jiigs and rabbits with cow milk. The cow milk immune serum thus obtained is able, so far aa I have been able to observe, to kill ciliated cells in the peritoneal ca^-ity of rabbits, though in a smaller measure than the specific cihated epithehal immune serum. The affinities of an immune serum are readily determined when the serum, like the ciUated epithelial immune serum for example, acts also on red blood-cells, for then this can be used as a reagent. Cow milk immune serum possesses the property to dissolve cattle blood in a not inconsiderable degree. This hfemolytic action, as in the case of the blood immune scrum and of the ciliated epithelial immime serum, is due not to any increased content of complement but to the presence of a specific immune body. Hence here also it was pos- sible to compare the affinities of this immune body (for the ciUated epithelium on the one hand and for the red blood-cells on the other) with the aiEnities of the specific blood immune body. The two immune sera obtauied by injecting rabbits with cow milk and with cattle blood were therefore inactivated, equal quan- tities of normal rabbit serum to serve as complement were added to them in excess, and the nuxture tested tor its hiemolytic propertio on cattle blood. The cow milk immune serum usually showed such a degree of action that one part of the immune serum saturated with I complement was able to dissolve completely 20 parts of the custom- ary 5% cattle blood nuxture. Corresponding to thLs, therefore, the much stronger ha^raolytic ssttle blood inunune serum was diluted with inactivated normal [Bbbit serum or with physiological salt solution until, with an excess f complement, the hiemolytic action of the two sera on cattle blood J exactly equal. When the two iramime bodies have in this way been made entirely iqual so far as the hemolytic property is concerned, it is possible to [actly compare their chemical affinities for a particular group of _Jell9. It is then easily demoastrated that the two ha^molytic immime bodies differ in respect to their chemical relatioas to other cells of the same species. Thus if equal quantities of ciliated epithelium are added to the 54 COLLECTED STUDIES L\ IMMLTJITV. two sera and the mixture centrifuged some time aft«r, it will be found that the milk immune body has been completely abutracled from the serum, but tlie blood immune body only partially so. Qli- ated epithelium, therefore, combines more strongly witli the milk immune body than with the blood immune body. On the other hand, the blood immune body possesses a greater affinity to the eiythrocytori than doea the milk immune body. TliuB if equal amounts of cattle blood are added to the two inactivated immune sera (amounts which would be completely dissolved if suf- ficient complement were present), it will be found after a certain time - that the blood immune body has been completely bound by the red blood-cells, whereas the milk immune body can still parUoIly be demonstrated in the serum. If one tests a number of different cow milk immune sera in this way, the results will show marked variation.-s. My experiments were . conducted on four different cow milk immune bodies which had been obtained by injecting rabbits with cow milk. Three of these showed considerably less affinity to the red blood-cells than did the specific blood immune body obtained by treatment with blood. The fourth, however, was bound by the red blood-cells in about the same degree as was the blood immune body. On the other hand, cases were observed in which the serum of rabbits after these had been injected with cow milk showed only a very slight harmolytic action, and thb only on the most sensitive of the blood-cells. All of these differences manifested themselves quite indej^end- ently of the cattle blood employed in the experiment and must there- fore be ascribed to differences in the immune sera themselves. Pos- sibly they are due to variations in the kind of receptors, sucli as were found in a marked d^ree in the experiments of Ehrlich and Morgenroth on isolysins.' The strong affinity of the hiemolytic milk immune body for tracheal epithelium, however, was present in all the cases examined and it did not differ materially from the chemical relationship between ciliated epitheliiun and its specific ciliated epithel immune body. Hence by treatment with cow milk we obtain a hsemolytic immune serum which differs from the blood immune serum, but cannot with certainty be differentiated from the ciliated epithel immune senim. 1 CONTRIBUTIONS TO THE STUDY ^F IMMUNITY. 55 The cow milk immune serum, owing to the character of its affinities, is to be classed with the epithel immune serum. The interesting fact to be deduced from this is that milk con- tains the same specific groups as the epithelial cells which produce it; and this agrees very well with histological observations accord- ing to which the protoplasm of the gland cells is itself used in the production of the milk. After having found it possible to produce a specific epithel immune serum by injections of cow milk, it seemed to me that immunization with human milk might prove useful in the suppression of carcinoma, especially mammaiy carcinoma. Thus far, however, the treatment of dogs and rabbits with human milk has not yielded an immune seriun hemolytic for human blood, one corresponding to the cow milk inmiune senmL 68 CJOLLECTED STUDIES IN IMMUNITY. The difference between these two views is considerable. According to our views the complement (=Bordet's alexin) possesses a direct affinity, due to chemical relationship, to the immune body, while according to Bordet such a relation is excluded. Since this question concerns our scientific understanding of hsemolysins and bacterioly- sins, and concerns also a basic difference affecting the practical appli- cation of the bacteriolysins, we shall have to study the subject more closely. I. Conoeming Alexins. Buchner, who by his thorough investigations on the bactericidal and globulicidal properties of normal sera laid the most important foundations of this subject, assumes that the serum contains cer- tain protective bodies, alexins, which act equally on bacteria, foreign blood-cells, etc. These alexins, which are essentially of the character of proteolytic enzymes,^ are of most imstable (labile) nature and lose their power by being heated to 55*^ C. Bordet also seems to assume the presence, in normal serum, of alexins in Buchner's sense. According to Buchner, the serum of a given species always con- tains the alexin as a single definite substance. Now in our second commimication we showed that the matter was much more conu plicated than this; that in the hasmolysins of the normal sera examined by us the action depends on the combination of two substances which correspond entirely to the two components of the hsemolysin obtained by immunization. Hence an " alexin " also consists of an interbody which withstands heating, and a complement which is generally thermolabile.^ The interbody is in every respect the complete analogue of the immune body, and the only difference between these is that in one case the side-chains of the protoplasm are thrust off in the course of normal vital processes, in the other •case this is due to an immunizing procedure. Since our second communication we have been able to confirm this view by meaas of a large number of separate cases. Of these we shall mention only a few which serve, above all, to support the immediate consequences of our view, namely, the multiplicity of the hcBtnolyains of normal serum. Goat serum dissolves the blood-cells of rabbits as well as those * Buchner, Mvinch. med. Wochenschrift, 1900, No. 9. 'Moxter (Centralblatt fiir Bacteriologie, Vol. 26) has demonstrated this 4dso for a normal bacteriolysin. STimiES ON HEMOLYSINS. of guinea-pigs. Heating the senim for half an hour to 55" C. causes this property to be lost, owing to the destruction of the com- plements. On the other hand, one frequently finds horse sera, by themselves unable to dissolve the erythrocytes of rabbits or guinea- pigs, which are able through their content of complement to complete the inactive interbody of the goat serum and make this a complete hemolysin. According to Buchner's views, only a single alexin is concerned in haemolysis. We therefore next studied the question whether the interbodies which act on the blood-cells of rabbits and guinea-pigs are identical. For this purpose we first determined the dose of inacliee goat serum which, on reactivation by the addition of sufficient horse serum, was able to dissolve a certain amount on rabbit or guinea-pig blood-cells. On the basb of these data this amount of rabbit blood in physiological salt solution was mixed with the required amount of inactive goat serum and after standing a short time at room temperature the mixture was centrifuged. The result was as follows: The clear fluid mixed with additional raiAU blood cella and the activating horse serum showed no trace of solvent property; the red blood-cells, originally separated by eentrifuging, liissolved completely under the influence of horse serum. In a parallel series of experiments the clear fluid was mixed with guinea- pig blood. In this, complete solulion ensued. From these experiments the conclusion follows that rabbit blood t'Oombines with an interbody present in goat serum, and does so. ■In fact, completely; whereas the interbody acting on guinea-pig blood mh not at all fixed by the rabbit blood. By means of this elective ■Absorption, therefore, it is jmsitively determined that normal goat Wtentm contains two interbodies, one acting on rabbit blood and Ike r on guinea-pig blood. The question at once arose whether these interbodies possess H single complement in common or whether there ia a special comple- ait for each. Only after considerable labor were we able to decide I question experimentally. We were finally able to determine t in the filtration of normal goat serum through Pukall filters, W^e fiiBt portion (6-10 cc.) possesses a markedly different solvent 1; power for rabbit and guinea-pig blood. We herewith reproduce I experiment of this kind. ).15 cc. of goat-serum previous to filtration was able to dissolve f 2 cc. of a 5% mixture of guinea-pig blood, while 0.2 cc, I was able to dissolve the same amoimt of rabbit blood. After COLLECTED STUDIES IN IMMUNITY. the serum was filtered, the filtrate showed the same solvent power ! for guinea-pig blood, whereas the solvent power for rabbit blood had almost entirely disappeared, for 0.8 cc. effected only a traw of solution and 0.23 cc. none at all. This loss of solvent power could be due only to an absorption, by the filter, of (1) the interbody j fitting the rabbit blood, or (2) the complement, or (3) both. Since, however, the solvent action of the filtrate on rabbit blood was restored by the addition of complement-containing horse serum, while the addition of interbody had no effect, it follows that the filtration had removed only the complement. Fr^m this fact, namely that a serum may be deprived of its complement for rabbit blood while the complement for guinea-pig blood remains, we must conclude that there are two differed complements corresponding to these tvxt interbodies. According to this, then, at least Jour different substances are concerned in the case in question, two different immune bodies and two complements fitting thereto. One pair of these acts on guinea-pig blood and the other on rabbit blood. According to Buchner only one singk substance, the alexin of goat serum, would be concerned. Further details of these experimenta will be published later. We should, however, like to observe that in the horse serum used for reactivating, it was possible to prove the existence of two complements. This proof, moreover, was effected in two ways, by means of filtration and by the production of anticomplements. The following observation will show that a still greater multi- pUcity of normal haemolysins can exist in the serum. In our second communication we have given a detailed description of an experi- ment in which a normal interbody of dog serum was caused to combine with guinpa-pig blood and then reactivated by means of guinea-pig serum, which served to supply the complement. In this experiment the interbody contained in 0.2 cc. dog serum was bound by a certain quantity of giunea-pig blood-cells. This Ls the amount of dog serum which, when active, just suffices to completely dissolve -the given quantity of blood. On repeating this experiment, but employing horse serum as complement, it was found impossible to reactivate the dose of interbody just sufficient for solution (0.2 cc). By systematic trials, in which multiples of the dose of interbody I previously used were employed, we finally determined that it required ' six tim® the amount, i.e., 1.2 cc, in order that the interbody would be completely reactivated by the horse serum. That is, the first- employed dose of the inactive dog serum, which contained just sufficient I employed ■ A STUDIES OK H.EMOLVSINS, 61 interbody to be completely activated when the complement of giiinea- pig serum was used, contained only one-sixth the amount of interbody which was completely activated when horse serum was used as complement. From this, however, it follows that all the interbody present in dog-serum and possessing specific relations to the guinea- pig blood-cells is iwt ol iJte same uniform nature. In our case one- sixth of the interbody acting on guinea-pig blood can be reactivated by horse serum, while fully five-sixths can be reactivated by the complement of guinea-pig serum. Therefore the goat serum con- tains two different inierbodies }or the same species of bhod-cells, and these can be positively separated by means of the difference in activa- tion. In our second communication, by showing the existence of a thermostabile and a thermolabile complement in the goat serum, we also proved that the complements of a given serum need not be of uniform nature. At that time we showed that the sera of two bucks treated with sheep blood-cells, as well as the sera of a number of normal goats, contained a complement which, in con- tra-st to the other complements of the same sera (for rabbit blood and guinea-pig blood), was not destroyed by heating to 56° C. Buchner finds it so hard to emancipate himself from his views that he seeks to explain our observations by assuming we made a gross error in the experiment. He supposes that the sheep serum still present in the o% mixture of sheep blood-cells, and which we disregarded, reactivated the inactive serum and led ua to mistake it for a resistant complement. We were well aware of this source of error and had therefore, even in the first communication, stated that the slight amounts of sheep serum present in the blood mixture caused no dis. turbances whatever. How, by the way, could it be explained that these disturbances occurred only in the serum of certain animals although the method of procedure was the same? Or, that digestion of the serum with HCl, which does not injure the immune body, pre- _ vented all solution whatever? After wliat iiaa been said, we shall have to assume that in gen- I et>CTT/ serum which acts heen neu- tralized to complete inacti^'ity. After centrifuging it was found that the blood-cells dissolved readily on the further addition of coitq>Ie- ment. Solution also occurs if a small amount of complement is excess is added to the exactly balancetl mi.tture of complement and anticomplement. These experiments indicate that the anticomple- ment acta by fUtiTig into (he haptophore group of the complement and side-tracking this group. We have also convinced ourselves that it is possible to produfe anticomplements not only with horse serum but also with other sera, such as the sera of goats, dogs, cattle, rabbits, and guinea-pigs, by injecting the serum into foreign species. In these experiments the choice of animals employed for purposes of immunization also plays an important rflle. For example, a rabbit treated with goat serum very readily yields an anticomplement, whereas when a dog was similarly injected no anticomplement (at least in the two casts examined by us) could be demonstrated. So far as we were able to determine, the protection afforded by the anticomplement extends to all the species of blood-cells on which the serum used for immuni- zation exerts its action. Since the sera in question, so far as lysin action is concerned, contain a plurality of complements, the anti- complementary senmi must contain a whole series of anticomple- ments which correspond to the different complements present lu the immunizing serum. Perhaps tliis jiolyvalence of the anticom- plementary serum accounts tor the phenomenon that certain anti- sera produced by means of a particular blood serum are able to inhibit the injurious action of many other kinds of blood serum. These facts indicate that this interchange of protection is due to the preseTice in the two sera of a certain number of common com- plements. In fact there seem to be cases in which certain species have the majority of their complements similar. Such a case in all probability is that of the goat and the sheep, as is evidenced by the fact that in the reactivating action goat scrum can be completely replacW by sheep serum and vice versa. This at least is true for I STUDIES ON HEMOLYSINS, all the cases observed by us. Still more convinring, however, is the fact that neither the injection of a sheep with goat serum nor of a goat with sheep serum results in the production of anticomplenients. All experiences indicate that the complements normally present in the eerura of a certain species of animal are nol able to excite the formation of anticoraplements in such an animal's ovm body. Per- haps this may be explained thus, that the relation between com- plement and complementophile group is extremely slight (as was shown by the binding experiments previously described by us) and that therefore one of the conditions necessary for the thrusting off — a per- manent and firm union with the receptor — is not in this case fulfilled. We reahze that we have been able here merely to point out some of the principles applying to this subject. Their closer analysis encounters extraordinary difficulties in consequence of one of the facts demonstrated by us, namely, the multiplicity of interbodies, complements, and anticoraplements. Thus far these difficulties have been overcome in only a few favorable instances, III. One ot Bordet's Objections CoDtroverted. Bordet, in his most recent work (loc. cit.) has described the follow- ing interesting experiment, by means of which he believes to prove that our views concerning the mechanism of hEBmolysis are incor- tect. As hsemolysin, Bordet employed the serum of guinea-pigs after these had been treated with rabbit blood. This then possessed a high degree of solvent power for rabbit blood. If this heemolysin is inactiviated by heating, it is possible to restore the h^emolytic action, as well by the addition of normal guinea-pig serum as by that of normal rabbit senmi. These two sera, therefore, contain complements (alexins) which make the reactivation possible. Bordet now sought to discover whether the "alexin" of rabbits is identical with that of guinea-pigs. For this purpose he treated rabbits with the serum of the inmiunized guinea-pigs and obtained an antiserum which, while it contained a small amount of anti-iromune body, contained considerable anticomplement. He then determined that this "antialexin" acted only against the "alexin" of the guinea- pig and not at all against that ot rabbits and some other animals. At the same time a certain degree of action against the complement of pigeon serum wa.? noted, so that this antiserum was not absolutely specific. From this Bordet concludes that his theory of sensitiza- must be correct, namely, that the variotLs alexins dcrivcfl from [ ss COLLECTED STUDIES TN IMMTNTTY. different species act direcUy injuriomly on the Rensitized blood-cell';. Against each of these alexins an antialexin exists which protects the t^ensitized blood-celLs against just this particular alexin. It cannot be denied that at first sight this experiment appears to speak strongly in favor of Bordet's theory. If one assumes, aa Bordet of course does, that in the immune aenim produced by hitn, one single immune body comes into play, then since this can be reac- tivated as well by rabbit serum as by guinea-pig serum, the com- plement contained in these two species of sera must, according to our theory, possess the same haptophore group. If thb were the case, however, the aame anticomplement should protect against holh complements, and this it does not do. We have therefore subjected Bordet's experiment to an exact reexamination and have been able to determine that an exhaustive quantitative analysis presents the experiment in an entirely different light, A hiimolytic serum was produced by treating guinea-pi^ with rabbit blood. A preliminary trial of this serum showed that when inactivated it could be reactivated in large amotmts as well by guinea-pig serum as by rabbit serum. Tlie an ti complement, derived from other rabbits by treatment witli norma! guinea-pig BCrum,' was able in the inactive state to completely inhibit the reacti- vation with guinea-pig serum, although the same anticomplement serum in its active state reactivated the inactive immune body. We next proceeded to examine these facte quantitatively and found that the simple solvent dose of the serum for 0.5 cc. of a 5% rabbit-blood mixture amounted to 0.075 cc. Then we tried von Dungem's experiment {loc, cit.) to increase this action, by adding to the native immune serum normal guinea-pig serum in amounts so small that they did not themselves exert any solvent action. We found that the full solvent dose had thus been decreased to 0.025 cc. This proved, as in von Dungem's case, that in the immunization a large excess of free immune body was present which could not nearly be satisfied by the amount of complement normally present. Now we could expect that this same increase in power would be effected by the addition of rubbit serum, but we Jound instewt that rahbU serum even in large amounts did not produce any increase whatever. According to Bordet's view such a deviation is al>soluteIy incom- prehensible, and this led us to pursue the case further. We first ' In contrast to lioriiet ne chose nnrnini guinea-pig serum for iintnuniiatioil .in order to avoid the disturbing action of an immune body, J ■ STUDIES ON H.HMOI.VSINS. 69 mactivated the immune serum and determined the minimal amoimt of the inactive serum which would cause complete solution in the presence of (1) normal rabbit serum, or (2) of guinea-pig serum. We found that it required 0.25 cc. of the inactive immune serum to effect complete solution of the given amount of rabbit blood when rabbit complement was employed, whereas only 0.025 cc. of the immune serum was required when guinea-pig complement was employed. This result, however, cannot be harmonized with Bordet's theory of sensitization. According to his view one would expect that a blood- cell which is sensitized by the presence of the immune body is subject equally to the action of variom alexins. In both cases the same amount of inamune body should then suffice to make the blood-cells sensitive to the alexins (complements). As a matter of fact, how- ever, it requires t^n times as much in the one case as in the other, if one desired to hold to Bordet's theory one might possibly say that it requires ten times as strong a sensitization nith the same immune body in order to make the cells sensitive lo the alexin of rabbit seriim. If this highly compUcated a.ssumption were correct, the relation as above determined, 1 : 10, should rcpre3cnt a constant ratio. Owing to a lack of animal material we were unable to study this question of constant ratio on the example selected by Bordet. However, in an analogous series of cases for which we had abundant material, we were able to pursue this question further. We made use of a goat which had been treated with sheep blood and whose serum therefore dissolved sheep blood-cells. The inac- tivated serum of this goat could be reactivated by two complements, that of normal goat serum and that of horse serum. The anticom- plement obtained by treating a goat with horse serum inhibited, even in small amounts, the action of the horse complement; whereas its action on the goat complement was so slight as to be practically negligible. The conditions here, therefore, are exactly the same as in the ease described by Bordet. In the beginning of the observations it was found that 1 cc. of a 57c niLxture of sheep-blood, mL\ed with normal horse serum to serve as complement, was completely dissolved on the addition of 0.35 ee. immune body (inactivated immune serum) ; whereas when normal goat serum was used as complement only 0.025 cc. of the immune body was required. This corresponds to a ratio of 14 : 1. On repeating the test a week later with serum freshly drawn from ^)£ immunized goat we found that the constituents which were I 70 COLLECTED STUDIES IN IMMUNITY. reactivated by horse serum were unchanged (0.35), but that it required considerably more immune body when goat serum was used for reactivation than it had before, namely, 0.1 cc. This corresponds to a ratio of 3.5 : 1 as compared to the former ratio of 14 : 1. This shows that a constant ratio does not as a matter of fact exist. We must rather assume, as we did for a normal hsemolytic serum, that two entirely independent immune bodies, A and B, are present in the immune seruni and that these differ in the ratio of their quan- tities and in the manner in which they are reactivated. The amount of immune body A contained in the immune serum has remained constant, while B after a short time has considerably decreased (to one quarter). This divergence would in fact indicate that the two immune bodies are formed independently of each other. We have thus demonstrated that in the phenomenon observed by Bordet not a single immune body, but two different ones, come into play, one of which is related to a complement found only in guinea-pig serum, while the other is related to a complement found in rabbit serum. Through this demonstration Borders objection loses all its force and his experiment becomes in fact a new argu- ment far our theory. The occurrence of different immune bodies in a hsemolytic serum obtained by immunizing with red blood-cells is not at all surprising in view of our experiments on isolysins described in our third com- munication. 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 inmiune bodies. All 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 immunized with blood-cells of species B a haemolytic serum will be produced which contains a great host of immune bodies. These immune bodies in their entirety are anchored by the blood-cells of species A. We are convinced that the duality found by us in the two cases examined is much below the actuality, and that thorough, though to be sure arduous, studies will succeed in discovering a multiplicity heretofore unexpected. For the present, however, this duality of the immune body should sufRce to controvert the objections made by Bordet from the unitarian standpoint. Vn. STUDIES ON ILEM0LYSINS.1 Fifth Communication. By Professor Dr. P. Ehrlich and Dr. J. Moboenroth. In the few years since its formulatioa the side-chain theory has exercised a marked influence on the direction of the investigations in immunity. The subject of toxins and antitoxins has to a certain extent been concluded, at least for the present. Several objections raised by Roux and BorreP in connection with their splendid work on cerebral tetanus, as well as those made by Metchni- koflf 2 and Marie,^ rested on a misconception of the theory, and the facts on which these are based serve rather as a complete confirma- tion of the theory.^ The attempt of Pohl* to place the doctrine of antitoxins purely on the basis of inorganic chemistry has been completely controverted by Bashford.^ Thus the facts proved themselves thoroughly in harmony with the theory, and the latter furthermore proved its inventive value in many directions. It was but natural that the side-chain theory originally formulated for the antitoxins, if it had any general biological significance at all, should also include the complicated phenomena of immunity which result from the introduction of bacteria or tissue-cells. Hence we began two years ago to investigate experimentally the applicability of the doctrines resulting from this theory to the specific haemolysins obtained by immunization, which had been discovered by Bordet a short time previously. These studies ^ Reprint from the Berliner klin. Wochenschrift, 1901, No. 10. ' Annales de Tlnstitut Pasteur, 1898. 'See Weigert, Lubarsch's Ergebnisse der Pathologie, 1897; also Levaditi Press mddlcale, 1900, No. 95. * Arch, intemat. de Pharmacodyn., 1900. •Arch, intemat. de Pharmacodyn. et Therapie, Vol. VIII, fasc. I and 11, 1901. 71 served to demonstrate the complete harmony of the theory with the facta on this subject. Furthermore aft«r overcoming considerable experimental difficulties we succeeded in demonstrating the same behavior for the hemolysins of normal aenim and thus brought ihsse also under the laws of tlie aide-chain theory. Reexaminations from various directions confirmed the correctness of our fundamentftl experiments and we may say that at present the majority of workere in thb field, partly as a result of their own experiments, have accepted our viewa and regard the side-chain theory as a justified hypolheoa which beat explains most of the phenomena thus far observed m the subject of immunity. Since this in part concerns processes in which the animal organism acts with all its highly complicated con- ditions, it is no wonder that now and then a fact has appeared in the course of the investigations which at firet seemed to be irrecon- cilable with the theory. The latter, however, is in no way injured thereby, for the solution of such apparent contradictions results in a deeper understanding of the subject and makes for progress. An instructive example of this was recently afforded in physical chemistry. As is well known, several at first inexplicable contra- dictions to van't Hoff's theory of solutions, resulting from certain deviations in osmotic tension, found their explanation in the theory of electrolytic dissociation of Arrhenius, and this theory served to again obtain general acceptance for the theory of solutions itself. We have therefore endeavored to analyze carefully the objections urged against our views by high authorities. The objection raised by Metchnikoff ' against the specific formation of the toxins was based on the fact that e\*en castrated rabbits j-idd an antispermotoxin. In a recent study ^ from the laboratorj' of Metchnikoff, this objection is withdrawn. It was found that in this antispermotoxin an anticomplement is principally concerned and not an anti-immune body, for it was produced even by treatment with normal aerum.^ It is therefore especially gratifying that Metch- nikoff also has recently accepted our view that the complement is anchored to the immune body by means of the latter's complement- ophile group. An important objection made by Bordet * based on some extremely ■ Annales de I'lostitut Pasteur. 1900, So. ' Ibid., No. 9. 'See von Dungern, page 47. 'Annales de I'lnstilut Paslcur, 1000, No. A STUDIES u.\ u.l;moly8I-\s. ^ interesting experiments, by which he believed to refute our theory of the meclianiam of hiemolysia, has been discussed Ijy us in our fourth communication ^ and controverted by means of extended quan- titative experiments. It is necessarj-, however, once more to thoroughly discuss the binding of immime body to the erythrocyte, for on this point the views seem not at all clear, because the purely chemical conception is denied ixy some authore or is regarded as unimixirtant. I. The Manner In which the Immune Body Combines with the ErythrocytfiB. In our first communication we had already shown that the ery- throcytes as such behave quite differently toward the two components which effect hseraolysis. The blood-cells abstract the immune body from its medium with great a-vidity, whereas they do not take up tlie slightest trace of complement. When loaded with immune body, however, they are able to anchor the complement also. From this we have concluded primarily that the immune body possesses two combining groups of different afTinity, of which the one combines with a corresponding group, the receptor of the blood-cell; the olhir com- bines with the comptemcnt. But according to our view these combi- nations are pure cliemical phenomena proceeding between immune body and blood-cells and between immune body and com|Diement. The function of the immune body can be elucidated by means of a chemical example, that for instance afforded by the beha\ior of diazobenzaldehyd. Through its diazo group this substance can unite with a series of bodies, especially with amines, phenols, keto- methylen groups, whilst the aldehyd group on its part can effect a of syntheses — e.g. with hydrazins, hydrocyanic acid, etc. It thus becomes easy by means of diazobenzaldehyd to effect a com- bination between substances which by themselves do not combine, as phenol and hydrocyanic acid. Such a combination includes bolk substances. In order to make the comparison still closer let us imagine that certain constituents of the living cell, say by means of an aro- matic group, are able to unite with the diazo combination. In this case it, follows that by means of the aldehyd group of the diazo- benzaldehyd a second highly toxic nucleus — e.g. that of hydrocyanic acid — can be joined to the combination in such fashion that the proto- ■mal raoleciile is now subjected to the action of the strongly ' See page 56. 74 COLLECTED STUDIES' IN IMMUNITY. acting nitril group. In this schematic example the diazo group which fits directly into the protoplasm would correspond to the haptophore group of the immune body which fits into the receptor of the blood- cells; the aldehyd remnant would correspond to the complemento- phile group of the immune body. The complement, which as we know possesses toxic properties, would then be compared to the hydro- cyanic acid.^ The facts described by us have been confirmed from various sides (v. Dimgem, Buchner, Bordet) by experiments on blood-cells. Bor- det 2 and also Nolf ^ showed that the stromata of the blood-cells, which represent the protoplasma, effect the anchoring of the inmiune body, while the haemoglobin, which is to be regarded as paraplasma, takes no part whatever in this binding. This fact corresponds entirdy to the views expressed by Ehrlich in an earlier study on blood-cell poisons.* Furthermore, it has been shown by von Dungem * that the power of the blood-cells to excite a specific hemolysin by immuniza- tion can be entirely inhibited by completely loading the receptors of the blood-cells with the immune body in question. These addi- tional facts were well fitted to still further support the chemical con- ception of these processes. Now, however, Bordet has described an experiment which he believes shows that the fixation of the immune body is not a chemical process in the strict sense, but that this phenomenon is to be classed rather with surface attraction and similar actions, and that it is completely analogous to staining processes. These views are also shared by Nolf ^ and Nicolle.*^ Bordet's experiment in the main is as follows: By treating a guinea-pig with rabbit blood a hjpmolytic serum is obtained specific for rabbit blood-cells. A certain amount of the serum dissolves an absolutely definite amount of rabbit blood-cells if all the cells are added to the serum at once. If, however, to the same amount of serum ^ One could designate substances which, like the immune bodies, are supplied with two different combining groups as ainhoceptors. This name would indicate the double binding function as well as the fact that they correspond to thrust- off receptors. ' Loc. cit. > Annal. de Tlnstitut Pasteur, 1900. * Charity Annalen, Vol. X. » See page 36. • Loc. cit. ' Revue g<»n^rale des Materies Colorantes, 1900, Nos. 43 and 44. STUDIES ON HEMOLYSINS. 75 only one-half the Amount of blood-cells is first added, sufficient time allowed lor these to completely dissolve and the second half of the biood-cella added, it will be foimd that these are no longer dissolved. It appears, therefore, as though the blood-cells were capable of com- bining with double the amount of immune body necessary for their solulion. In order to explain this result Bordet describes the follow- ing staining ex|)criment: If one dissolves methyl violet in water, it is possible, by means of a strip of filter-paper dipped into the solution, to abstract all the coloring-matter from the solution. The strip will assume a color of very definite intensity. If, howe\'er, the strip is divided into several smaller atrii« and these are dipped into the fluid oTie after Ike otlur, the first strip will assume a considerably deeper color, whereas the strips last introduced will be unable to abstract any color from the now colorless fluid. From this Bordet draws the following conclusion: "On peut admettre, par comparaison, que les premiers globules introtluits dans I'h^motoxine sont d6'^k susceptibles de perdre leur h^moglobine lorsqu'ils ne sont encore que " faiblements teints" par les princijwa actifs, mais qu'ull^rieurement ils peuvent absorber une dose beaucoup plus grande de cea substances, i?puiser ainsi le .s(;nira et empScher la destruction de nouveaux globules introduits dans la suite." Phenomena such as those here described have long manifested themselves in our experiments on the binding of the immune body by the erythrocytes although these experiments were of somewhat different form. But before we proceed to discuss these results and our conclusions, we should like to describe the facts observed by us. In order to determine the combining ability of the erj-throcytes for an immune body, especially when quantitatively accurate results are desired, it ia best to proceed as follows; The immune body L^hsemolysin heated to56^ C.} h added to the red blood-cella and, after Rft certain time, the mixture is centrifuged. The clear fluid so obtained "ia tested for free immune body by adding an excess of complement and allowing this mixtiue to act on the same quantity of fresh blood- cells. If one proceeds in thb marmer in a large series of cases, employ- ing varj-ing multiples of the solvent dose of immune body, it is possible - to determine accurately the combining power of the cells. The tiollowing experiment will very readily make tliis clear. Tlie immune body was present in the serum of a sheep which 1 been treated with dog blood. When tliis serum was inactivated 1 COLLECTED aTlIDIES IN IMMirNITY by heating to 56° C, it could be reactivated either with the com- plement of sheep serum or of goat serum. To begin, the exact quantity of immune body was determined which would just com- pletely dissolve 2 cc. of a 5% mixture of dog blood-cells when suf- ficient complement was present. This dose was found to be 0.15 re. To ft number of separate portions of blood mixture (each of 2 cc.) mul- tiples of this dose were then added, thus, 1, H, U, I}, 2, 24, 3 time- the solvent dose, and the mixtures kept at room temperature for an hour and frequently shaken. Since the complement was absent, haemolysis could not occm'. After centrifuging, the clear fluid, which had the appearance of water, was again mixed with the corre- aponding amount of blood (0,1 cc. of undiluted blood) and with complement.^ It was found that even the last trace of .the single solvent dose had disap[>eared from the fluid; whereas in the case where double the dose had been added, the fluid still contained just a solvent dose, i.e., it completely dissolved the freshly added blood- cells. In tiiis case, therefore, tlie blood-cells were able to combine with only a single dose of ike immurie boilij. This, however, is not at all the general rule, for by extendiny our experiments to other eases we found tliat there is a verj' Urge variability in this binding of the immune l»dy, and that frequently a larger or smaller multiple of the solvent dose is bound. The follow- ing case will illustrate the extreme in the other direction, in whidt aimost a hundred limes the solveTit dnse 0} immune body iras taken up by the hlood-ctills. A rabbit had been treated with goat blood, and its serum therefore contained an immune lK>dy fitting to goat blood. Nor- mal guinea-pig serum ser\-ed as complement and 0.2 cc, represented considerably more than .sufficient for 2 cc. of the goat blood mixture. When this amount of complement wa.s employed, the solvent doee of the immune body for 2 cc, of the blood mixture amounted to O.OOS cc. On allowing 0.48 cc. (sLxty times the solvent dose) to act on the blood-cells in the manner previously described, and then centrifuging, it was found that the clear fluid did not contain even a trace of immune body. When eighty times the dose was employed the clear fluid showed a very faint solvent action, corresponding to about i to 1 of a solvent dose. Not until one hundred times the dose ' Ab a counter test tlie blood-cells separated by ceatriruge were mixed with ealt solution and with the complement. Those BpecUneiiB in which just the solvent doae (0.15 cc.) of the immune body or more was present, disaolved completely. STUDIES ON H-EMULYSINS. (7 was employed did the centrifuged fluid contain a full solvent dose and effect complete solution. Hence out of one hundred solvent doses about ninety-nine had been bound by the blooil-t^elb, for only about one solvent dose of immune Iwdy remained in the fluid. By meana of parallel experiments we have found that one hour's contact of immune body with blood-cells results in the maximum amount of binding, for the experiments at 45° C. anrl room tem- perature yielded results exactly alike. Between the extremes repre- sented by these two experiments ft great variety of figures was obtained. The significance of these experiments offers no difficulties from the point of view of the side-chain theory. The facts are readily understood when we stop to consider the pecuLarities of the receptor apparatus of the blood-celk. As a result of our pre\nous experiments on the isolysins of goats we assume that a given blood-cell contains a large number of different types of receptors which in general fit to different immune bodies and hjemotoxina. Referring ttie reader to an exhaustive study by Ehrlich,' we shall content ourselves here [by remarking that certain kinds of receptors may be present in the id-cell in great excess. This excess cannot only be demonstrated. Kit, by means of the method just described, can be exactly measured. In ti rely analogous conditioas arise \mder other circumstances. : interesting fact discovered by Wassermann, that the central r\'ou3 system of various animals binds much more tetanus pobon I viiro than is necessary to fatally poison tlie animal, b probably [lie to such an excess of receptors for tetanas poison. From this point of view the experiments alxive mentioned are isily explained without departing from the side-chain theory. Thus, t us assiune that with a certain jjoison a it is necessary that x n-re- iptors are boimd in order that a blood-cell be completely dissolved, and let us further assume that the blood-cell jjosseses a much greater number, say 2x n-receptors. ^Vhen Bordet'a experiment ia now carried out, the conditions arising will be exactly those descrilred mhy Bordet, It Is at once apparent that the red blood-cell in this ; will combine with just twice Ihe amount of jioison necessary ^r its solution. If therefore double the solvent dose of immune »dy is added to a given amount of such blood-cells, the entire receptor ' Speeielle Patholagie und Thempie, edited by Notlinage!, Vol. VIIJ, seo- I. pages l(i3-lS4, 78 COLLECTED OTCDIES IN IMMUNITY. syste attacked. Such phenomena are exceedingly plentiful in chemiatrj-, and it may pay us to glance at some of them. Napthalin, as is well known, consists of two benzole nuclei joined together. When, now, a salt- forming group, hijdroxyl or amido group, is introtlueed into each of the two benzole nuclei, the heteronuclear substitution products, e.g., dioxynaphlhalin, amidonapkllwl, and naphtkylenediamine, or thdr sulfo acids, will be able to combine with either one or with two mole- cules of a diazo combination. When two molecules of dioxynajth- ihalin are mixed with Ueo molecules of diazobcfisol, the result is ex- clusively the mono-azo combination; when however two molecules of diaiobemol are added to one molecule of dioxynaphthalin, the result J is the diazo combination. If an additional molecule of dioxi/iuiph- I thalin is added to the finished diazo combination, this molecule will be unable to dissociate the latter, and the two substances, the dituo combination and the unchanged dioxi/naplUhalin, will exist side by side. This example, to which others, such as the esterification of dibasic acids, the methylation of anilin with iodomethyl, could easily be added, corresponds entirely to the relations between im- mune body and erythrocytes as described by Bordet. It may at once be admitted that where the binding of small multiples of the immune body is concemetl, it b very natural to think of a mechanical absorption due to the degree of concentration; j and that therefore the circumstances in Bordet'a case, in which the j binding was merely doubleil, justified the comparison with i^taining I processes. The cases examined by us, however, in which at one time I just the solvent dose of immune body, at another an extraordmarily j| large multiple of the dose was bound, weigh heavily against ttis j assumption. j Our decision, however, is especially determined by certain general I considerations. Thus, charcoal, the type of siu^ace-attractive agents 1 attracts thousands of substances of the most varied kind. A dye can j stain a large number of different substances, as is shown in every I stained microscopic preparation. In marked contrast to this is j the specificity of the numerous antibodies, which primarily ar« always directed against the exciting bacterial or other oeQ species. ) In the cases in which apparent deviations from this rulg TO^^ ► STUDIES ON H-EMOLYSms. noted, exsct investigation has shown ' that these are due to the pres- ence of one and the same receptor group in various elements. Thus we have shown that the isolysias produced by injecting goats with goat blood-ceUa act also on sheep blood-cells. We have further shown that these sheep blood-celly possess certain kinds of receptors which bind the goat lysin just as the receptors wliich are present in the goat blood-cells do. We produced the strongest proof for this community of receptors by means of crossed immunization, for we succeeded in producing a typical isolysin by injecting goats with sheep blood. Since all experiences, therefore, lead us to assume that each par- tjeidar complex produces just the specific antibody, and since this agrees exceedingly well with the assumption of a chemical union, it would be a distinct backward st*p to adopt so vague a conception as that of mechanical surface attraction. Were we to assume that the immune body enters the cell merely mechanically, it ^ould be necessary to drop the entire unity of tlie immunization phenomena which follows from the side-chain theory. It is probably quite generally conceded that the antitoxin acts on the toxin in a purely chemical manner. Hence so far as dissolved Bubstances developed by the immunity reaction are concenierl, the chemical conception applies. Why then should this chemical action suddenly cease when the substances instead of l>eing in solution are present within the cell, and a new principle be assumed for this case? This leads to the contradiction that in one case (when com- bining with the erythrocytes) the immune body is bound, specifically to be sure, but tnechanicfiUy, while in the other case (when anchored to an artificially jtroduced anti-immune body in solution) it is bound iBpecifically but chemically. These considerations, and they could readily be greatly extended, will suffice to show that the above-mentioned experiments are not at all capable of shaking the side-chain theory, for by it alone is a single uniform conception of the phenomena of immunity rendered possible. n. Concerning Complementoids. The complements, which effect the activation of the normal ^immune bodies and of those produced by immunization (amboceptors) do not possess great theoretical or practical importance in the study 80 OOLLECTED STUDIES IN WMrNITY. of immunity. They seem to play an important role in the uonnal processes of cell nutrition. As a result of experiments already described we must assume that in the blood serum of a particular animal species not merely a single complement exists but a laige number of different complements. It is understood, of course, that not all the complements occurring in a large number of species differ from one another. On the contrary it is to be regarded as certain that particular types find a wide distribution extending over several animal species. This explains why, for example, a hasmolytic or bacteriolytic immune body can be reactivated by the sera of different animal species. We have previously explained that a complement b to be con- ceived as possessing iwo characteristic groups, a haptophore group wliich fits into the complementophile group of the immune body, and a zymotosic group which is the actual carrier of the specific action. A complement therefore, to a certain extent, corresponds to a toxin, which possesses a haptophore and a toxophore group. Hence by the immunization of suitable animals it is easy to obtain anti- complements whose behavior corresponds exactly to that of anti- toxins. For example, if a goat or rabbit is injected with horse serum, an an tic om piemen t will be formed which is able to specifically inhibit the action of the complement contained in hotse serum. We have already shown ' that this is due to a deflection of the comple- ment. We ha\'e now tried to follow this analogy (between complements and toxins) further. We take it for granted that it is generally known that toxins, either through spontaneous changes or through the action of chemical agents, become modified into loToids, whose distinguishing character is that they no longer possess a toxophore group although the haptophore group remains. These toxoids, then, are relatively non-toxic substances which are nevertheless able to cause the formation of antitoxins in the animal body. Now vn know that the zj-motoxic group is extremely sensitive to the most varied influences; hence the attempt to study modifications of the complements analogous to the toxoids seemed to promise favorable results. Such modified complements would then be designated comjAemenioids. The first step was to see whether the well-known inactivation of a serum by heating to 56° C. completely destroyed ' See Fourth Communicaticiu, page £6. STUDIKS ON H.'EMOLYSlNS. 81 Xe complements or merely changed tiiem into inactive derivatives, jomplementoids,' In order to be certain of tlie destruction ot the zymotoxic group, fve heated the sera for fifty minutes to 60° C, a procedure, as shown t>y numerous subsequent examinations, which absolutely destroj-s ;very trace of complement action in the sera so treated. By treating animals with the sera thus prepared, it is actually I'erj' easy to obtain anticomplements. We injected rabbits, guinea- pigs, and dogs with inactive goat serum, and goats and numerous rabbits with inactive horse serum. A parallel series of animals ft-aa treated with active serum. The anti complement action of the senun from the animals treated with compIemeJitoids proved [ully as strong and often stronger than that of the control animals treated with active serum. By means of the procedure described in detail in our Fourth Comnmni cation it was readily shown that these were really anticomplements. The injection of the heated serum, therefore, possesses the same value as that of the unchanged serum.^ Since, however, accord- ing to otu- view it is the haptophore group which causes the immu- nity reaction, it follows that inaclivalion of (he complement has de- stroyed only the zymotoxic group, having the haptophore group intact. The important question now arises as to how the presence of complementoids influences the activation of the immune body; for whenever a serum is inactivated by heating a formation of com- plcmentoid ensues, and it is well known that such a mixture of immune body and complement is reacti\-atGd without any trouble by the addition of complement. It seems therefore as though the presence ot the complementoid does not hinder the union of inunune body and complement. On this point we have made special experiments by alternately ' At about tlio same time, exactly aimilar consi derations led Paul Miiller {CcntrnB'lalt f. Barteriolngie, Vol. 29, No. 5) to attempt the produgtion of antitomplement by the injection of Eerum which had been heated. In his ease, however (immunization with chicken blood), anti-interbody was prin- cipally developed, while anti complement could not positively be demon- etrateil. It is possible that this negative result indicates that not all tlie com- plements of the different animal species are able to undergo tliis metamorphosis into complementoid. ' We should like lo mention that in addition to this, in the case of the goat treated with inactive horse eerum, we observed the development ot a oowerful fcnilin. 82 COLLECTED STTOIES IN IMMUNTTY. inactivating and adding complement without finding that the con- stantly increasing amount of complementoid hindered the aiition of the complement. This phenomenon can be explained only by assuming that in Ihe change to complementoid, the haptophore groiiji of the complemenl suffers a diminution of its affinity for the comjJt- menlophile group of lite immune body. In the toxoids of diphtheria poisons the circumstances are some- what different, tor Ehrlich found that in the hemitoxin zone of the poison spectrum the afUnily suffers no change through the forma- tion of toxoid. On the other hand, M. Neisser and Wechsberg in another case, namely that of staphylotoxin, have been able to demon- strate a decrease in affinity occurring with the change into toxoid. This behavior is analogous to that of the compiementoids observed by us. Hence no general rules governing the affinities in toxoid and complementoid formation can be laid down: the circumstances must be investigated separately in each case. From what slight differences in the constitution of the moleciile enormous differences in affinity may arise is seen by studying certain organic acids. Thus, for example, a and fi resorcyiic acids differ from each other merely in the position of the two hydroxyl groups; the constants of their affinities, however, differ from each other by over a hundred times. We may therefore perhaps assume that in our special case it depends on the relative positions of the haptophore and hoxophore group and the corresjjonding relations thereby determined whether any change in one group can retroactively affect the other. ni. Concerning Autoantlcomplements. In the third communication, on isolysins, we pointed out that the organism possesses certain contrivances by means of which the immunity reaction, so easily produced by all kinds of cells, is prevented from acting against the organism's own elements and so give rise to autotoxins. Further investigations made by us have confirmed this view, so that one might be justified in speaking of a "horror auiotoxicus" of the organism. These contrivances are naturally of the highest importance for the existence of the indi- i vidual. During the individual's life, even under physiological though J especially under pathological conditions, the absorption of all material of its own body can and must occur very frequently. The formation of tissue autotoxins would therefore constitute a danger threatening the organism more frequently and much more severely than all STUDIES ON H.W10LYSINS. 83 US injuries. We believe that the study of these regulating nces is of the greateflt importance and according to our investigations either the disappearance of receptors or the presence of atitoantitoxins ia foremost among these contrivances. It will therefore be necessary to subject all the factors which are of importance in this r^pcct to a thorough analysis.' We shall now mention a few observations relating to the com- plements which seem to point to a regulatory contrivance a^ yet und escribed. Normal rabbit serum possesses a number of properties which are to be ascribed to the presence of complements. First to be mentioned is the property by means of which freshly derived labbit serum is able to dissolve guinea-pig blood-cells. This is due to the combined action of a complement and an inmiune body which is present in the serum in comparatively small amounts. Further- more, rabbit serum is regularly able to activate an immune Ijody derived by treating rabbits with ox blood. Now we noticed that rabbits which a week previously had been treated with goat senim (whether active or inactive is immaterial) had completely or almost completely lost these properf.ies, and that these changes persisted for weeks after the injection. Hence it fol- lows that owing to the injection of goat serum, complement nor- mally present had been made to disappear. It was therefore essen- tial that the cause of this remarkable phenomenon be determined. We could ne,\t show that frequently the serum of these rabbits in its native state, though more surely after heating to 56" C, ia able to prevent the above-described complementary action of normal rabbit serum. Hence in the above case normal complement lias evidently disappeared from the rabbit treated as described, and has been replaced by an anli-complemenl which we shall have to term an auloanlicomptemmt.^ ' MetalnikolTs interesting obsenation is only apparently a. contradiction of these r^ulatitig phenomena. He found that n typical avlmpermolaxm is developed in the blood of guinea-piga which have been treated with guinea-pig Epennatozoa, and that this ix able in vUto to kill the fpermatozoa of the unimal itself. But such an injurioua action ou the Hpermatoxoa does not take place, even in the slighMet degree, in the living animal, tiecause, as Metalnikofl'a reaearcheB show, only the immune body combines with the spemiafozoa, not the complement. In this ea«e, therefore, an autotoxin within our meaning. ttfuU destroys the crlln of iU avn l-ody. does not exiRt. ' Aocoiding to the investigations of T)r. M. Neither and Dr. Wechsberg still \ 84 CXJLLBCTED STUDIES IN IMMUNITV. It has previously been shown that such a rabbit serum is rich in antigoat complement. We observed an analogous phenomenon, whose nature may perhaps be identical with the above, in a rabbit which had been treated with ox blood (blood-cells and seniin) in order to produce a specific hfemolysin. Ten days after the injection of ox blood, the serum failed to show any solvent action whatever on ox blood, in direct contrast to numerous previous cases. At first we thought it possible that no inunune body had been formed in this case, tor even the addition of an excess of complement in the form of rabbit serum produced no solution. However, on centrifuging the ox blood-cells after treatment with this abnormal eerum, and mixing them with salt solution and complement, we found that oven slight doses of immune serum caused marked so- lution. The serum therefore contained plenty of immune body, and this had been anchored by the blood-cells. The presence of this immune body was obscured not only because the complement was absent, but because this had been replaced by an antJcom- piement which neutralized the complement subsequently added. Because of the anticomplcment which it contained, this rabt^ serum manifested a marked inhibitory action on the strongly hasn- olytie serum of another rabbit (one which had been treated with ox blood). But what happened in this case after injection of ox blood rarely occurs in such a conspicuous manner. Jlore frequently it is found that the scrum in its active state j.x>ssesaea an exceedingly slight solvent action, corresponding to a very small content of complement, and that after heating it manifests a distinct anticomplementary' action. This evidently leads to the extreme case above described, as is readily seen when the relations are expressed by means of a diagram. (See figure.) In .studying the question as to how these autoanticomplements are formed, we must constantly bear in mind that normal scrum abvays contains complements in excess. Now it is difficult to see what purpose would be served if at any time the normal comple- ments, so important in cell economy, were paralyzed by autoanticom- plements. We shall therefore have to assume that the normal in progress, this eenim also lacks the power to ai'tivatc certain harlmeidiU bodies. The animals at the same time seem to suffer a decrease in resisting power ftgainst certain infections, a fact which may perbapa sen-s hibit in the purest form the fuiiclioii of eertain 'UM. ^^^ STUDIES ON aSMOLYSINB If- II I i8-St. a =.2. K " '=^~'-p 3 « 5 l-iiH|r 1 g a. frl I si r? ri §=3 Sr3 86 COLLECTED STUDIES IN UnfUNITY. complements circulating in the serum do not cause the formation d autoanticomplement.s. Confirmation of this view is furnished by the fact that even in animal species possessing identical complemente it is impossible to produce anticomplements by means of serum injections. Thas, neither sheep when injected with goat serum, nor, conversely, goats* when injected with sheep serum 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 serum, althaugk related, is not identical with the complement of the rabbit. If we assume that a certaih goat complement possesses the same hajdophare group as does a certain rabbit complement, but that it differs in ihe rest of its constitution, then the assumption that identical complements do not form anticomplements will not apply. In this case, by means of the hapto]ihore 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 oflf 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 name places as the idiocomplements with the same haptophore group. Foremost among these places we may consider the complex receptors which possess two haptophore groups (amboceptors). In this case, contrar\' to what we asually observe, the thrusting^ff of an amboceptor v:oiM be effected through the anchoring of its complementophile group, and we sliould then have additional proof for our xiew that the com- plex receptors i)Ossess two binding groups. In any case it would seem to be of the greatest importance to gain an insight into the conditions governing the disappearance of the i(liocomi)lements. 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 ixwsesses but little significance pathologically. Of considerable importance for these occurrences under natural circumstances are the vital conditions governing the disappearance of complement through internal metabolic processes. The origin of the autoanti- complements as it has jast 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 ciu'ative 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 immimizing a rabbit with spermatozoa and noticed that in consequences of a purulent process which developed during the course of the immuni- sation, 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 immime bodies (amboceptors) which in bacteriol3rsis as well as in hsemol3rsis 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 will we gain an insight into the nature of "natural predisposition" and its changes, "increased resistance," "loss of resistance," etc. The Bteady progress of the investigations in immunity is rendered extremely difficult by the fact that in the inununizatioii with living cells and in the study of the immune sera thus obtained a large number of different substances which exist simultaneously is concerned. In our second communication we pointed out that the hfcmolyslns present in normal serum, 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 imable 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 nimiber of com- plements exLst together in blood serum, In like manner Bordet's absorption experimente indicate a plurality of the bacterial agglutinins and those of Malkoff a plurality of the normal hsemagglutinins. 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 aniiloxic antibodies occurring in nor- mal serum. In conformity to this, the reactive antibodies produced by injections of senim 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 antifermenta 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 ' Reprinted from the Berliner klin, Wochenschr., 1901, Nob. 21 and 22. 'Deutsche med. Wochenaohr., ISOO, No. 49. J ffrUDIES ON H-EMOLYSms. formed through immunization, corresponding to the multiplicity 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 commimication the occiurence of different immune bodies in a hiemolytic serum obtained by immunizbg with red blood-cells is not at all surprising. We have obtained a whole series of difJerent 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. All 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 immunized with blood-cells of sf>ecies 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^ has adopted the same view for the bacterioagglutinins. 3 a number of "components " {corresponding to our reccp- riors) ra 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 asaumijtion of a plurality of immune bodies. This view permits Durham to offer a sufficient and natural explanation of the var>'ing degree of action of typhoid agglutinins on typhiod Elli of different origin, and of the extension of the agglutinating jn of specific sera to related species of bacteria. It would be anc ■ tOTf Compare the thorough exposiiion by Ehrlich in VoL Vlll of Not [pecielle Pathologie und Therapie, Holder, ^'ie^na, 1901. Durham, Journ. of Experimental .Medicine. New York, Vol. V, No. 4, 1901. 00 COLLECTED STUDIES IN IMMUNITY. verj- 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 PEuralhtlc Conception ot the Cellular Immunft}' 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 will 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 ot 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 inmiune 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 arc 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 ia, in fact, conceivable that the occupation of n single group only produces a certain amouBt of injury to the cell without being able to cause its death. The danger to the life of the Ijacterial cells would increase in proportion to the number of partial injuries, which again would correspond to the increase in the Dumber of typos of receptors. It is possible that the potent bacl«ricidal 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, Ergcbnisse der Pathologie, 1897, page 138. I I STUDIES 0?J H.EMOLYSINS. stances, provided the receptors fitting these are present exclusively in vitally important organs, e.g., the central nervous system. This perhaps explains the circumstance that it is exceedingly difficult lo prodxice 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 unchanged tetanus poison ia 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 illness is avoided by keeping them cool (as we know from Courmont's beau- tiful observations) it is impossible to produce any tetanus antitoxin. Investigations bj' 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 will have found, particularly with the artificially produced cell poisons, that in some caaea it is extremely difficult if not impoa- fflble to effect the production of anli-immune bodies. Thus, Metchnikoff injected a scries of guinea-pigs with specific spermo- toxin, a substance which certainly finds receptors in the guinea-pig'a organism. Despite this, he found but two cases in which even a euggestion 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 impoB- sible in a number of animal species to produce antienzymes by the continued injection of certain enzymes. The explanation of these facts presents two possibilities: First, the receptors concerned in the particular case may be of peculiar COLLECTED STUDIES IN IMinjNITY. constitution in one respect, i.e. in being firmly bound 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; (n) a thrusting-off of the receptors ensues, and with this a formation of antibody; (b) in the case of sessile receptors, a hj-pertrophic process sets in comparable perhaps to a simple muscle hypertrophy, according to Weigert's conception. Second, it is concdvable, as Morgenroth ' has sliowTj 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 tliis species the haptophore groups of the cells will find a receptor apparatu-s 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 principie for the practical production of curative sera VA& follow, namely, titat in any single «ise one would 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^ ' CenlralbUtt fiir Bscteriologie, Vol. 26, 1899. STUDIES ON II.-EMOLYSINS. 93 taken the experimental study of the preliminary question whether mmune sera derived by treating two different animal spcrips with the same cells are identical so far as their antihodies are concerned, or whether they are partly or wholly different. Of these antibodies the most important are the bacteriolytic and hemolytic immune bodies. According to our conception, as is well known, these possess two haptophore groups, one, the complemcniophile group, and the other (which anchors itaelf to the receptors of the cells causing the immunity) which we can briefly designate the q/topkUe group. According to what has been said above it is this second group which possesses sjiecial significance in the question under discussion, and we may therefore formulate our problem as follows: To determine whether, in tlie immunizulion of difftrcnl animal species with cells of one kind, amboceptors (immune bodies) possessing different cylophile 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 bactcriolj'sina as well ss to hemolysins; 2, by neutralization with antiamboceptors (anti- immune bodies). The latter way, the more el^ant of the two, ia, howe\-er, I presumably applicable only to those immune bodies which are I directed against cells of the oi^nism. A henmohjtic or cytotoxic mune body, as is to be expected, alwaj-s finds points of attack [ in the organism of the corresponding animal species, for this is the first preref|uiaite for the possibility of an anti-immune body. As a matter of fact also, such anti-immune bodies have already I Tjeen observed. On the other hand, the immune bodies of bacteri- cidal sera, since their natural counter groups are found in the ' bacterial cells, will in all 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 directetl against the haeiericidal immune bodies will be produced. I ^H Wo selected immunization with ox blood-cells as being especially ^H Adapted for these experiments. Huch immunization had already ^Hbeen carried out by von Dungern on rabbits. The production of ^H immune bodies in high concentration succeeds particularly well in COLLECTED STUDIES IN IMMUNITY. this case, so that later investigators (Buchner, Rehns, Bulloch) have also employed this useful combination. In many cases, meet easily by means of intraperitoneal injections of the ox blood, a potent hiemolysin is produced of which 0.005-0.0005 cc. suffices to dissolve 1 cc. of the 5% ox-blood mixture. Since the production of the immune 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 obsejred that the serum of these rabbits which had been immunized with ox blood is able to dissolve not only the biood- oells of oxen, but also those of goals. The following table shows a comparison of the solvent action of several of these sera on the blood- cells of oxen and of goata. Guinea-pig senmi (0.1 or 0.15 cc.) was used as complement since rabbit serum itself, in the doses required, often exerted a hmmolytic action on the goat blood-cells. TABLE I. ■"-'-A%"l».-^'^ CompleW SolvBDl Doae (or 1 oc, of Ox Blood. Cooipteta SalYBDt DoH Tor 1 m o( Gout Blooti. CO. Ratio or tlH Solvent OoM 0.^1 1. 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 O.Ofl 1:1.5 1:1.7 1:1,8 1:3.3 1:3.6 1:3.6 1:5 1:9,8 1:10 1:17 ' xn-17-00 This table shows that the hiemolytic action of the immune body is always less on goat blood tlian on ox blood, and that the ratio of the solvent doses tor the two species of blood is not constant but varies within fairly wide limits, as can be seen from the last column. STUDIEB ON HEMOLYSINS. 95 Hub variable ratio indicates that the s<^vent 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 will be found that the serum has lost its solvent action for both species Blood-oell of a t and of a goat, showing epooi&s and oommou reoeptora of blood; for by means of the ox blood-celk, 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 hat its solvent pouxr far goat blood, wkiU thai 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 COLLECTED STUDIES IN IMMUNHY. the ox blood-cells, the Becond, ;-, 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-eella, by means of their a and /3 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 diffras, though of course the relations are reversed from those in that as can be seen by reference to Table II. Action of the InratTfJE Boot Goat Blood, CRucilv TABLE IL or THE Rahbit which had been N Goat Blood, and Ox Blood. alian with guinM-pig wrum. ) TBEAISD-Wa Number of tbe Rabtal Treated "^ITb'i^ cc. Compleu Solvent Dwe lot Goal 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 _ liao 1 " 4 " Xll 18-00 ' On employing the Bame serum on a dilfernd ox blood, 0.05 co, produced no solution at all, ajid O.I cc. merely a trace. This is evidently due to a caEual, individual lack of receptors in the ox. blood-cells in ([uestion, such as ah< itself BO frequently in goat blood when we studied isolysins. ! Because of these ratios we shall have to assume that the blood-cells in this case possess a second system of binding groups which is peculiar to them and represented in the above diagram by y. They possess these, of course, in addition to the receptors, ^ I which they have in common with the ox blood-cells. In accordance i with this, in the elective absorption test in this case, the goat blood- L cells will bind the entire lot of immune bodies; whereas when ox a >at blood- when ox J STUDIES ON HyKMOLYSINS. 97 •lood-cells are used, the group y will be left behind, for this possesses flinity only for the goat blood-cells. The following protocol shows the results of two series of experi- aents, which exhibits the effect of such reciprocal binding: To each 5 cc. of a 5% mixture of ox blood-cells or goat blood- ells (freed from serum by centrifuge) varying amounts of the immune lody of a rabbit which had been immunized with ox blood are dded. The amount of immune body is seen in the first column f the table; in the second and third columns the complete solvent OSes (for ox blood and for goat blood) contained in each specimen re given, as they were determined by tests made at the same time. Tie mixtures are made up to 6 cc. with physiological salt solution, :ept at 37® C. for IJ hours and then centrifuged. Two equal por- ions of each of the decanted fluids are then taken and again mixed rith corresponding amoimts of blood-cells. Finally guinea-pig erum is added to activate the mixtures. The haemolytic action rhich the decanted portions exerted on ox blood-cells and on goat Jood-cells is seen in the table. (See Table III.) The imion of the inmiune body with the ox blood-cells has resulted n a considerable abstraction of both portions of immune body. On he other hand, the imion with goat blood-cells , by which the action •f the fluid is considerably decreased for goat blood-cells, causes very ittle decrease in the solvent power for ox blood. In contrast to this experiment we here reproduce an analogous xperiment which shows a directly opposite behavior of the two factions of inmiune body of a rabbit inmiunized with goai blood. See Table IV.) Here the goat blood-cells bind both portions of the immune body, vhile after treatment with ox blood-cells the fraction adding on goai load is left almost iniact. Hence by means of this crossed immunization and reciprocal elec- ive absorption we succeed in demonstrating that in the case of the abbits treated respectively with goat blood and ox blood two arge fractions of immune bodies can be separated. Of these, one faction is common to both sera; the other is peculiar to each of hem. The main groups of receptors shown in the above illustra- ion and designated a and /? for ox blood, and ^ and y for goat blood, ,re thus to be differentiated. We have deemed it important to supplement this analysis by ixperiments on a second species of animal, and have therefore treated ^^^^^■^^■PIIV^^ 9S COLLECTED STITDIES TN IMMtTOTTr. a goat with ox blood. Naturally the serum of the goat so treated dissolves ox blood-cells. Besides this, however, it manifests the ability to dissolve the blood-cells of a few other goals, 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 T.\BLE III. BlNDINQ OF THB ImMCNE BoDT OP A RaUHIT TREATED UTITH Ox BlOOD, WITH J Ox Bloor-cells, and Ghat Blood-cellb. j Solvenl Power al the Decmnled Fliud.. Atnousl of At Number of Solv- (KS. L^e.] Therein. On BlnotJ, '■■'S.?SiS3.-"' '^IrS BJ<^, m ona. (i)On oiiau. (b)On OoBl Blood. Go.( Blood. 1 No. m. 1 0.001 iV 0 0 0 0 2 0.002 s 0 0 trace 0 3 0 003 A 0 0 veiy little 0 4 0,004 I 0 0 very little lo little 0 5 0.005 1 0 0 mod, to little 0 6 0.006 1 0 0 modera<« 0 7 0 007 1 0 0 0 8 0,008 1 0 0 aim' Hi comp 0 g 0.01 1 j 0 0 complele 0 10 0 012 2 0 0 faint trace n 0 016 2i 0 0 faint trace 12 0.02 3i faint trace vep- little verj- litlJe.top 13 0.024 4 U very Uttle little, top 14 0 032 51 It lit, W mod. little 10 very little little 15 0.048 8 21 little 16 0.06 10 3 dm' 8t com p. moderate 17 0.08 13* 4 complete fair 18 0.1 m 5 complete strong to al- moHt com p. litUetomod. J9 0,14 231 7 mod, toUttta 1 one o£ our goata, on the addition of complement, dissolved the usual amount of ox blood-eella. 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 amomifs, so that it reqiured 0.75 cc, serum (thirty times the above amount) to effect complete hemolysis of sensitive goat blood-cells. Hence in this case also the development of all such amboceptors as could find a point d STUDIES ON ILEMOLYSINS. attachmfflt (receptor) in the blood-cella of the individual goat its^ has been avoided, and the phenomenon which we have previously designated as a "horror aulotoxicm '"is again presented. TABLE IV. BnroiKoofTB« Ininna BoDT or A Rabbit Iiutonizbd wrra Goat Blood. ON Ox Blood and Goat Biooi>-CBLLa. Number of Solvect l>o«« Sulvent Po«er of Ii» D««Dlod Fluids AmouDlof ConHiDKl A. Aftaf BindiDgwili "■ ^^if-sa"" (Denvsd from TbereiB, O. Blood. . Ksbbit by Tn»lingwi[h Goal Bibod.) (o) For (blFor O^^S, (WFor {Q)For (b) For BWl. iiSSS. Gont Blood Ox Blood. G«I Blood. No. «. 1 0.038 A 1 0 (air to mod- erate 0 0 2 0.05 1 0 0 0 3 0062 iii 0 complete 0 0 4 0.074 2 0 0 0 5 O.l t 21 0 0 minimal ttsco 6 0,13 31 0 0 7 0.15 4 0 0 8 0.2 5 0 0 very little little 9 0 25 2 61 0 n 0 10 0.3 8 0 0 11 0.38 .1 10 0 0 lair 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 found 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 i^omplete in themselves — is this: By treating animalawith ox blood, two fractions of immune bodies are formed, of which one acts only on ox blood and the other also on goal blood ; whereas by treatment with goat blood the contrary though entirely analogous result ensues. These two frao- ■ We were also &ble to observe that the immune body of the rabbits which had been immunized with ox blood and goat blood acted also on sheep blood. Ooaer iaveetigatioo would probably show that this behavior is analogous to its action on goat blood. This corresponds entirely to our earlier obaervationa on the extensive similarity of the receptor apparatus of goat and sheep blood «s it was manifeatod particularly by the experiments on isolysins. 100 COLLECTED STUDIES IN IMMUNITY. lions do not correspond to two different single immune bodies, btd mcA fraction includes several, perfuips an entire host of immune bodies. The experiments also lead to conclusions of considerable impor- tance ID another direction, namely, as affecting our conception of cellular specificity and of the specificity of nacHon 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 Metchnikoff ' has recently expressed this view. We had already become acquainted with certain exceptions to this principle. The isolysin, for example, produced by injecting goata with goat blood, also dissolves sheep blood; and, in'ce versa, the immune body of goats which have been injected with sheep blood acta also as an isolysin. At that time we emphasized that these results are onij to be explained by assuming that certain tj'pes 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 cihated epithelium acts also on the blood- cells of the same species, and that conversely the hemolytic immune body produced by injection of blood-cells is partially bound 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 haptoj>hore groups cor- respond to the receptors of the exciting cclb. Hence sv£h an immune scrum 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 ceU "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 Woigert'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, crolin, and tetanolysiit , and gan- glion cells of the most divergent animals possess receptors for letano- ' Revue g«''nif-ra]e des ' See page 47. STUDIES ON B.F.MOLYSINS. 101 ' Bpasmin 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 apecificUt/ 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 anli-immune bodies.^ The highest concentration of inmaune 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- I tective power. That this was really an anli-immune body which Hinhibited the anchoring of the immune body to the red blood-cells, Bis seen by the following combining experiment. 0.5 cc. of the anli-immune body {inactive serum of a goat treated as just described) are mixed with varjHng amounts of the immune body (inactive serum) of a rabbit treated with ox blood. Thereupon I cc. of a 5% mixture of blood-cells is added to each specimen. These are then kept at 40T. for one hour and centrifuged. The various sediments are then mixed with salt solution and 0,15 cc. normal Kinea-pig serum. A parallel experiment (control test) b made i ' See EhrlitU'ri recent atudy, jia^e , 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. Dflgree of of IbgBed ths Conlnil T»t. io. 1 0.00125 " 2 0-0025 0-00375 " 4 0,003 " 5 0.0075 " 6 0.01 " 7 0,025 complete solution trace solution little solution almost complete solutioD completo tolution 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 competeJy dissolved on the addition of com|3lemenl.^ By this experiment the inhibiting substance is definitely characterized as an anii-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, i 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 liko to remark that in the course of numerous experiments hax-e now and then found normal goat sera containing slight amounts an anti-immune body acting on the immune body of rabbits treati^ with blood. This is to be brought into connection with the law (see also Neiaaer- •.) tliat the artificially produced antibodies frequently represent only on " normal functions. STUDIES ON HiEMOLYSma 103 TABLE VI. Experiment with 0.4 co. Anti-immune Body. Control Test with 0.4 co. Normal Cioat Serum. Amount of Immune Body. Solvent Action. Amount of Immune Body. cc. Solvent Action. • 0.0175 0.0145 0.012 0.01 0.0085 0.007 0.006 0.005 0.0044 0.00375 0.003 0.0025 0.002 0.0018 complete solution strong '* fairly strong solution moderate solution little solution it tt trace solution small trace solution tt tt ft tt tt tt minimal trace solution tt tt tt tt tt tt 0 0.001 0.00085 0.0007 0.0006 0.0005 complete solution almost complete solution strong solution tt tt moderate solution • 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 immimization 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) du^cted 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 wm in the Jorm of goat serum instead of guinea-pig serum. (See Table Via.) EiperitOBOt with 0.4 «. ADti-immUDe Body. Control Teal with 0.4 ce. Monii«l ^ SolVBUt Action. Amount ol .,,„,.„„. 0.051 0.042 0.029 0.02 0.017 0-014 complete solution ^ aJmoHt complete Eolution moderate solution trace aolution faint trace aolutioD 0 0.051 0,042 0.029 0.02 0 017 0.014 complete solution almost complet« solution moderikte solution very little solution trace solution 0 In this combination the anti-immune body exerts no action. Ifcticr we must here be dealing with a particuloT type of immune body which effects a combination with a comj^emeni 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 groui> 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 guinesrpig serum were used as complement. This ia well shown by Table VII. TABLE Vn. No. Complete Soii-enl Don of li^une Body when Coinpele Solvent Don GoBt Serum 0.6. R.lio of t « Two Do^. 2 3 4 0,005 0,0075 0,0075 0,0025 0.05 0.07.5 0.07.5 1:10 1:10 1:13 1:30 That this behavior is not due fo a smaller content of complement in the goat serum can readily be determined by suitable esperiments especially by increasing the dose of the latter. I STITDIES ON H-KMOLVSINS. 105 This can only be explained by assuming that only part of the total number of immune bodies find fitting complements m 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 aTiti-immune body derived by injecting goats with immune bodies of rabbits is not a simple uniform [einheUHch] substance, but is madt 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 probabilili/ con- tains a host of partial immuTie bodies, and we must assurne that, corresponding to this, the anti-immune bodies also possess a complex constitution. In the following diagram it is not sought to expresB 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 unmu- nized with ox blood contains only two different types of immune bodies and these, ftirthermore, in unequal amounts. Let the main irtion be represented by immune body type a, which is activated a particular complement present in the animal's own (rabbit's) Further, let the second portion, present in much less amount, J 106 COLLECTED STUDIES IN IMMUNITY. be represented by immune body type b, which b activated by a different complement also present in rabbit serum but found in goat serum aa well. Iiet the proportion of a: 6 be as 10: l;i.e., a quan- tity of immune serum containing one complete solvent dose of & 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 ia the case when goat serum, which contains complement only for b, serves for reactivation) used when immune body a is employed. The composition of thia immune serum can be represented by the formula 10a + 16. Diagram to show the tun types of imniuoe bodies preaent in the iiumuiw Berum ot a rabbit treated with ox blood. Each immune body symbol corre- sponds to ojK solvent dose for the amount of ox lilood employed in the eJi- periment. Immune body type a ia present in ten times the amount of type 6. The tomplementophile groups ot a and b differ; hence also the complement* differ. Tbs anti-immune body serum possesseB anti-immune bodies only 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} & large quanlily 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 b is present which is unaffected by the anti- immune body although this was able to neutralize ten solvent doses ■of a. One-tenth of the above amoimt of immune body, on the other STUDIES ON ILEMOLYSINS. hand, will be completely inhibited m ita action. For this contains one complete solvent dose of immune body a which is neutralized by the anti-immune body, and only one-k-nth of a solvent dose of 6 which, although not affected by the anti-immime body, is bo slight as not to cause any appreciable solution. Only when larger amounts of immune body are employed in which 6 becomes active docs solution occiy, and this becomes complete only when that quantity is reached which contains lOa+lii. Naturally, if the ratio is 1:20, a quantity will be required which can be represented by the formula 20a + 1ft. 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- immunt-body seruvi and the dose which cavtes complete solution a large number of intermediary stages exist in which the degree of solution graduaily 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 eans of the anti-immune body to demonstrate the difference of the I immune bodies produced by injections of ox blood into different species f Our first experiments were undertaken with the serum of goats I which had been immunized with ox blood. As will be seen by the I following figures, our anti-imraune body (derived by injections of an immune body obtained from rabbits) in this ca.se exerta 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 1 and 0.5 cc. normal active goat senim to activate the mixture. In I the control test 0.4 cc, inactive normal goat serum are used instead I of the anti-immune body. (See Table VIII,) That this serum differed markedly with respect to its content of I )ndi^■idual immime bodies was already shown by the fact that, in I contrast to the serum of immunized rabbits, it did not possess & I Wmolysin acting on all goat blood-cells in general, since such a hffimoly- i Bin would have exerted a most injurious action in the form of an auto- I lysin. As a matter of fact, the law already mentioned under the name 1 " horror autotoxicus " applied here also, and hence merely an isolysin lOS COLLECTED STUDIES IN IMMUNITY. was de\'eIop«i which acted only on goat blood -cells of a few individuals and which therefore possessed only a few individual special groupe 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 Been in Table IX. TABLE VIII. Etperirnant with Anti-immuiui Body. Owlrol T«t. 1 AmauDt Solvent Action. lS?y. ^1 Solvent Action. 0 051 0,043 0.035 0.029 0.02 0.017 0.014 complete Holution almost complete aoIutioD moderate solution trace solution doubtful 0 0.051 0.042 0.035 0.02fl 0.02 0.017 0.014 complete Bolutioo almost completely disaoU-ed almost dissolved moderate solutioii very little solution trace solution I In this experiment the method was exactly dmilar to tliat of ti previous ones. The blood was from goat No. Ill, 1 cc. of a J suspension being used. TABLE IX. Cnntrol Tut with 0.4 cc. NonnkI Inutiia ^^A. Solvent Action. Immi^B^y. Solvent ActloD. 1,5 1.0 0^61 0..51 0.42 0,35 complete slrong strong moderate little trace 0 1.5 1.0 0 88 0.61 0.51 0,42 0.35 complete strong strong moderate little trace 0 We see from this that by treating a goat mlh ox blood-cells, immune bodies hare been formed the main portion of which differs from those obtained by immunizing rabbits wilk ox blood or goal 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 L A STUDIES ON iL'EMOLYSINS. 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 wliicli leada to the formation of immune bodies of entirely different character. Our further experiments concerned themselves with the action exerted by our anti-immune body on immune bodies derived from rats, guinea-pigs, and dogs by treatment with ox blood. We found that the anti-immune body exerted a distinct protective action against all three sera, but that this was less strong than that against the immune 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-noutraliz- able types of immune bodies, in any case an amount far greater than I fe contained in the rabbit serum. In the guinea-pig the case is very I similar, the proportion of double the solvent doses being as 1:3, I The nearest approach to the ratio as found in the rabbit ia seen in l>the serum of a dog treated with ox blood. In tliis it required six rtimes the usual solvent dose to effect complete solution in the presence ■ of the anti-immune body.' All this leads to the conclusion that in the immime serum of Bthese three species the eytophile group of certain portifins is identical |*ith the eytophile group of certain immune bodies in the rabbit. ■ Certain particular groups of the ox blood-cells therefore must fit I equally into the receptors of these different animals. In view of I this fact, the entire alisence in the goat of that portion of immune I body which can be neutralized by the anti-immune body is of special I interest. As already stated, we are here dealing with an exception iwhich 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 tho I thefle Ihree species (guinen-pig, rat, and dog) differed H-fonJ blnod-crUs. It was found that while the 1 sod lats acted on goat blood, those of the dog did Vltiie dog, in contrast to rabbits, guinea-pigs, and rati ■ lor the groups (^ of the diagram, Fig. 1) bodies derived from their behavior toward bodies of guinea-pigs Thifl indicates that receplorB to the blood-celiB of oxen COLLECTED OTUDIES IN IMMUNiry. tophile groups come into play. With the means at present at oiir disposal it 13 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, quadriixploT). In any case it may be considered a fact that in the immune-body mbfture different kinds of complementophile groups come into play. ^^'ere 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 se^^'ice in case the organism were injected with foreign celb. It is much simpler and more natural to view these circumstances from our standpoint, namely, thai 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 hiemolysis. By filtering goat and horse sera through I'ukall filters we were able to demonstrate two complements. One of these, fitting ' In our fourth communicatioo we have clisfus.-ied analogous caeea in detail, subjecting them to thorough experimental inieBtigatlons. At that time, however, our sttidicn were directed only to the com piemen I ophite groups. In that case the serum of guineo-piga immunised with rabbit blood contained two immune hodies, of which one found ita complement In guinea-pig senun but not in rabbit aerum. These immune bodies were present in the propor- tion of I :I0. In another case mentioned at that time we obsen'ed consider- able chronological variations in the proportion of two immune bodies with different complenientopliile groups. J H Ot ( STUDIES ON H.liMOLYSINS. 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 ihermoslabilc complement in greater or smaller amounts in the serum of normal goats and calves. To again call attention to these experiments ia 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 altxin." It would be natural to conclude that there ia a plurality of com- jilemcnts from the manifold variations observed in the comple- tion of various inactive sera by nornml 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 acti\'ated 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 resta on the assumption that for a certain species ot blood a serum contains only a single interbody (or immune body). In our fourth communication we have already shown that this assimiption does not hold, even for the interbodies of normal sera. The assumption 0/ a plurality of complements in normal sera I'a aupported by the fact that by injections 0/ 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) untiaera are formed which act against (he complements of various other aera. In a number of different anioaals by injecting various sera we have succeeded in obtaining anti complements acting not only against the serum originally employed, but also against certain eomple- 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 anticoniplements, at least two different complements A 114 COLLECTED STUOrES 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 anticomplanenl 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. rrom Treated wilb ProlMlinn uuiul Rabbit + + + + + + + + + + + + + + + Horse seriun':;:;;;;::::;::;;:::' Rabbit + + + ■= strong protection; + + = fairly strong proleolion; + " very slight protection. With the assumption of a plurality of complements we are Ud 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 posai- bilily exists tliat certain of these complements are not constant, occurring in the blood only from lime 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. Aa 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 imrauni3ed with ox blood. How- ever, when thirty times the amount of immune body was employed complete solution ensued. Evidently the principal portion of the comjdements -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 HiEMOLYSINS. 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 inmiune 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 inmiune 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 but with the immune body from a goose immunized with ox blood. (See Table XII.) TABLE XII. Rea4stivBting Normal Sera. Guinea-pig serum, Rabbit serum Rat serum Goose serum Chicken serum. . . Goat serum Pigeon serum. . . . Horse serum Amount of the Rabbit Immune Body Sufficient to ESeot Complete Solution. 00. 0.0025 0.005 0.005 0.015 0.015 0.05 no "completion" no ''completion" Amount of the Goose Immune Body Sufficient to Effect Complete Solution. cc. 0.025 0.05 0.1 0.035 0.035 no "completion" 0.035 no "completion tf 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 T.V IMMtTNTTV. by horse serum but ia reactivated by gc»ose serum, we should Deces- sarily have to conclude that " the " complement of the goose is much more closely related to " the " complement of the rabbit than m 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 thia 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 pluralbtic 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 l>e completely similar, or, what is of primary impor- tance, their haptophore groups may be identical. It may very well be that the anan sera are alike in the gri-ater part of their partial complemerUs, and that therefore all three sera may in certain cases — e.g., witli the immune body of a goat immunized mlh ox blood — reactivate in like manner. But it la 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 fad 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, po.sscss 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 Ije understood why pigeon serum is unable to reactivate the immune body of the rabbit immimized 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. J STUDIES ON H.EM0LYS1NS. 117 I This brief analysis will show us that the complementopliile 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- piementophile groups and the provision of suitable complements cannot, of course, be neglected. In this connection Donitz (Klin. Jahrbiich, 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 hia 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, on 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 bacteriolvsm with a 'complement' which is stable in man {homoslabilc complement), or at least a bacterioly^in the immune body of which finds m human serum an appropriate 'com- plement.' The latt-er 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 sjiecies so distantly removed from man as the horse, goat, or dog. However this may be, tlie 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 would seem to indicate that our therapeutic efforts be directed primarily to excHing the greatest production of the oryunism'a 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. Miiller, 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 complemerUs orginoUy peculiar to the organism will be able to act when fitting complenientophile 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- meiits.^ The question raised by Donitz, relative to the provision of really plentiful sources of complement, has not thus far been solved. It still 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 amount of serum in itself causes severe injury to the animals experimented upon. Suck hdng the case, it seems that in the matter oj supplying com- plements, the method suggested by vs, namely, the employment of ' In hiB recent study (Zeitschrift fiir Hygiene, No, 37} Waaaermann also lays great stress on the increase of the individual's own complenienta. Wo 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 experimenta of i-on Dungern (Miinch. medizin. Woehenschrcft) concerning the binding of complement by certain cells ' Deutsche medizinische Wochenschrift, 1900, No. 18. J STUDIES ON ELEMOLYSINS. 119 mijced sera which contain a greai many different immune bodies would prove the most effective; for with the mvUijMdty of the immune bodies an increase of the different complementophUe 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. DC. CONCERNING THE MODE OF ACTION OF BACTERI- CIDAL SERA.* By Max Neisser, Member of the Institute, and Dr. Frederick WECRSBBito. 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, howe\'er, the lilera- tiire 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 Luffier and Abel ^ on their experiments with bacillus coH 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. PfeifTer,^ 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. IS, ' F. LOffler and R. Abel. Centrnlbi. (. Bact., 1896, Vol. 19, page 51. • R. Pfeifier, Zeitachr. f. Hygiene, 1895, Vol. 20, page 215. MODE OF ACTION OF BACTERICIDAL SERA. The same phenomenon was noticed by Leclainche and Morel ^ in their work on the bacillus of malignant cedema, and these authors had similar experiences with erysipelas of swine and with sj'mpto- matic anthrax. As a result of this they concluded that there was a "dosis optima Tieulralisans" of the immune serum. Since we encountered the same phenomenon in bactericidal Ust-tube experiments it seemed advisable to undertake a study of tliese 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 resuits 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 varj'ing amounts of immune senmi 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 0uid, usually 2.5 cc. Dilutions were made with 0.85% salt solution. Fiu'thermore three drops of bouillon were added to each tube, tor 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 ; serum employed was obtained from a rabbit by treatment ' I,«clsini?he and Morel, La Si^rotb^rapia da la septicemia gangreneuse, Annal. del'lDst. Pasteur, 1901, No. 1. •Munch, med. Wodieuachr,. 1900, No, 37. J 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. In.cti™ Im- Number o( erf Serum anunst lUbbit Berum Colonies DO tbo VibTIb MetchnikoS. u ComplemBil- Flste. S-'o 1-0 0.3 O) "5 ta* 0.5 0.3 0 3 Many thousands 0,1 0.3 Several hundred -1l 0 05 0.3 About 100 0.026 0.3 About 50 ■ OcE 0.01 0,3 0 h tj|x 0 005 0.3 0 ■ 0.0025 0.3 About 100 F -f' 0 001 0,3 1 0.0005 0,3 Control I 11 0,01 " III 1-0 0 '■ IV 0-3 " V ~ 1-0 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 eath plated on agar. This experiment shows that the inactive hnmune serum alone is innocuous to i-iitrio 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 tlie same way 0.005 cc. immune serum plus 0.3 cc. norma] 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 senim as before) the destruction of the germs is incomplete, while with still smaller amounts there is no destruction whatever. But the destrMUii'c effect also becomrs 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 immuneserum. That this action J 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 CuIturB. T^pf cc. of a one-day bouil- lon culture of vibrio Metchnikoff Amount of the Com- plementing Normal. Active Rabbit. serum. 00. 1.0 } ( Number of Colonies on a Plate on the Addition of Serum from a Rabbit Immuniaed against Vibrio Bftetohnikoff , the Serum having been Inactivated. 00 00 1.0 oc. 00 00 00 ioc. a few many thousands 00 A 00. 0 0 00 A 00. 0 0 00 Amount of Culture. Amount of Normal Active Rabbit- serum. 00. Number of Colonies on a Plate on the Addition of Inactive Normal Rabbit-serum. Ioc. ioc. Aoc. Aoc. j^ CC. of a one-day bouil- lon culture of vibrio Metchnikoff r 1.0 » 00 00 00 00 00 00 00 00 00 00 00 (( t€ ft Control I. T^ cc. bouillon culture + 2 cc. 0.85% salt sol. +3 drops of bouillon, planted as above, result oo . n. Sterility of the immune serum, 0. III. " tt t€ inactive normal rabbit-serum, 0. rV. " " " 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 Va cc. normal active rabbit serum kills the germs completely; while larger doses of the immune serum are less effective. The addi- tion of 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 foimd that small quantities of the latter increase 124 COLLECTED STUDIBS 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 heatmg it to 57° C. Normal active goat serum served as complement. (See Table 111.) 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 im- 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 in. Amoupt . 1 . 1 . • h BujActivf Culture. iDWlive Go*t Immime Serui Addition of lane- Stiam. Cost S« rum. - 1.0 CO. 0.1k. 1.0 m. O.Ieo. linCCOtn 1-0 0 about 50 0 0 0 one-day 0.5 0 many hundreds 0 0 0 0.2S 0 0 0 0 0.1 0 0 0.0.5 ttboiit 5( Nord- 0.025 hafen. ■~ « " «) CO Control I. tH "c bouillon culture + 3 cc. 0,S5% salt solution + 3 drops bouillon =00. " II. Sterility of the inactive immune serum, 0. " III. " " " " normal goat serum, 0. " IV. " " " 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 BACTERICroAL 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 Inactive Ooai Immune Serum directed against Vibrio Nordhafen. Culture. — 1.0 CO. 0.1 CC. 0.01 CO. tItj cc. of a one-day bouillon culture of vibrio Nord- hafen. • 1.0 0.5 0.25 0.1 0.05 0.025 0 0 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 0 0 a few about 100 many hui>- dred 00 00 Amount of Culture. Amount of the Comple- menting Active Nor- mal Guinea- 4 Number of Colonies on a plate on the Addition of Inactive Normal Ooat Serum. pig Serum, cc. 1.0 cc. 0.1 cc. 0.01 cc. -jii, CO. of a one- day bouillon culture of vibrio Nordhafen i \ 1.0 0.5 0.25 0.1 0.05 0.025 • 0 about 100 a few hundred 00 oo 00 00 0 0 a few a few hundred 00 00 00 0 0 a few several thous. oo 00 00 Control I. liji cc. bouillon culture + 2 cc. 0.85% salt solution + 3 drops bouillon. Result, oo. " II. Sterility of the goat immune seriun, 0. " III. " " '* normal goat seriun, 0. " rV. " " *' " guinea-pig serum, 0. Three drops of bouiUon 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 : ^ * 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 MODE OF ACTION OF BACTERICIDAL SERA. 129 bacteria, and a bacterium all of whose receptors are laden with b need not at all be injured in its vitality, liody b normally possesses a peculiar function, namely, to serve as a coupling member or link, and hence it poaaesses two groups (amboceptor). In this particular case one of these groups fits the receptor of the bacterium, the other possesses a peculiar relation to those normal ferment-like constitu- ents of sera which Ehrlich has termed complements. When there- fore to a normal serum which contains suitable complement we add equivalent amounts of immune serum, the condition pictured in A I will result. On adding the corresponding bacterium to this we get the condition shown in A II, in which all the bacterial receptors are occupied with immune bodies, or, more accurately, with immune bodies which on their part are loaded with bacteriolytic comple- ment c. In the case here presented we shall say that it requires the occupation of all the receptors with complemented interbodies to cause the death of the bacterium. If now to an equivalent mixture of complement and interbody we add an excess of interbody, it will be possible for only a part of the interbody to be loaded with complement, leaving a portion of the interbody uncomplemented. On adding the corresponding bacteria a number of conditions may result ; the affinity of the inter- body for the bacterial receptor may, as a result of the loading with complement, (1) remain unchanged, (2) it may thereby be increased, or (3) be diminished. In the figure, B II shows the condition of increased affinity. Of the six interbodies only those combine with the bacterium which have become laden with complement. In this case therefore the excess of interbodies will have no influence on the bactericidal effect. The condition b really the same as All, except that free interbody is also present. C II shows the condition of unchanged affinity. In this case, if we add the bacterium to the mixtm-e of complement and excess of interbody, all the receptors of the bacterium will, to be sure, be occupied by interbodies, but this will be entirely without any regard to the fact that these interbodies are or are not loaded with com- plement. It may therefore happen that only a few of the bacterial receptors will be occupied by complemented (i.e. active) interbodies, while the rest of the bacterial receptors are occupied by uncom- ]ilemented (hence inactive) interbodies. As already mentioned, hnn'ever, the vitality of such a bacterium is not necessarily destroyed. A COLLECTED STUDIES IN IMMUNITY. D II represents the last conceivable case. It is assumed that the " completion " of the interbody has resulted in a diminution of the latter's affinity for the bacterial receptor. In this case pri- marily only the uncomplemented interbodies will combine with the bacterial receptors, while the free fluid will contain complemented interbodies. In cases C II and D II, therefore, the excess of interbody is not without important results; for whereas in mixtures of equivalent amounts of complement and interbody all the interbodies are com- plemented and so made active, the excess of interbody will exert a deflecting action on the complements in case C II as well as in D II, thus diminishing the end results. The conditions shown in B II are apparently those which apply to the hfemolyains, for extensive investigations in this direction by Ehrlich and Morgenroth, concerning which we are permitted to report, have shown that deflection of complement by an excess of interbody is not observed in hemolysis. In this case only the complemented interbodies seem primarOy to be anchored by the receptors of the erythrocytes. In the baclericidal sera investigated by us, however, the deflec- tion of complement shown in C II and D II ia observed, though of course we are as yet unable to say which of the two possible modes is present in any particular case. The same explanation which we have given for the phenomenon observed in vitro must also be held to apply to the experiments on animals, at least so far as the phenomenon above described was observed. It is perfectly obvious that when appropriate affinities of the interbody exist and when there is a marked disproportion between complement and inter- body, a deflection of complement by the excess of interbody can occur in the animal body. The phenomenon of deflection as described may perhaps present further points for study. We know that inmiunization causes an increase only of the interbody and that therefore every immune serum presents a deficiency of complement in comparison fo inter- body. Hence it is conceivable in a highly immune animal, i.e. one in which through immunization a great increase of interbody has occurred, that after infection the phenomenon of complement deflec- tion through the excess of immune body could occur. That it actu- ally does occur we conclude from the following statement by R. Pfeiffer: MODE OF ACTION OF BACTERICIDAL SERA. 131 " It has frequently happened to me that highly immunized guinea- pigs died after an injection of moderate amounts of virus. On section there were then found in the peritoneum living vibrios, some- times even in considerable numbers. Notwithstanding this the heart blood of the cadaver when introduced into new guinea-pigs manifested the strongest power to dissolve vibrios." It is therefore conceivable that an individual can lose its natural resistance by producing too large an amount of interbody in pro- portion to the amount of its complement. Such an excess of inter- body then would act injuriously rather than helpfully. • This phenomenon is also of some theoretical significance. While it can readily be explained by means of the views of Ehrlich and Morgenroth, it appears, to us at least, to be absolutely irreconcilable with the theory of Bordet. This author, as is well known, regards Ehrlich's interbody as a substance capable of sensitizing the bac- teria whereby they are made vulnerable to the action of the solvent " alexin " (Ehrlich's complement). If this were the case it would be absolutely incomprehensible how an excess of sensitizing sub- stance could diminish the total effect; at the most such an excess could only increase the sensitizing action, not decrease it. Since, however, we have actually observed this decrease very frequently, we must regard this as a weighty objection to Bordet's theory. Equally incomprehensible from Bordet's standpoint is the fol- lowing observation. As has been shown above it is possible to over- come the action of an equivalent mixture of interbody and com- plement (the mixture itself being fatal) by adding a large excess of interbody to it. When, however, through the addition of more complement the equivalence of the mixture is again restored, the action returns. This action therefore depends not only on the abso- lute amoimts of complement and interbody, but also and essentially on the proportion in which these two substances are present. This is true at least in the sense that not very much more interbody should be present than is required. X. THE DEFLECTION OF COMPLEMENTS IN BACTERI- CIDAL TEST-TUBE EXPERIMENTS.' By Dr. A. Lipstein, AssiBtant in the Bact«riological Division. I^f a study published in 1901,^ Neiseer and Wechstwrg described a peculiar jihenomenon occurring in bactericidal test-tube experi- ments. This phenomenon conebted in the fact that the bacteria were not killed despite the presence of the appropriate bacterial amboceptor (immune body) and complements when a compara- tively large excess of amboceptor waa present. This fact, for which all other explanations failed, was explained by the authors on the basis of Elirlich and Morgenroth's views. They assumed that, with certain conditions of affinity, an excess of amboce]itor8 exerts a deflecting and at the same time a diluting action on the complement; aa a result the complement does not combine with the amboceptors anchored to the bacteria, but with the superfluous free amboceptors, while the amboceptors which are anchored to the cells remain without any complement. Now since only those complements exert a bac- tericidal action which are anchored to the bacteria by means of the amlwceptors, it follows that in this case there will be no bactericidal action. Naturally this phenomenon of deflection of complement does not occur with ever>' combination of amboceptor and comple- ment, but oiJy when certain conditions of affinity are present. Later I shall be able to show how the same amboceptor in excess exerts a deflecting action on one complement while it fails to do so on two other complements. I?ecause of the theoretical importance of this phenomenon and its explanation, a continuation of the experiments of Nei.sser and Wechsberg, taking special cognizance of the objectiona since made, seemed desirable. ' Reprinted from Centralblatt fiir Bact,, Vol. XXXI. No. 10, 1002. • See page 120 of this volume; also Wechsberg, Zeitschr, t. Hygiene, Vol. 39, 1902. BACTERICIDAL TEST-TUBE EXPEHLMENTS. 133 In the following experiments the method is the same as that of the above authors, to whose work I refer for these details. The phenomenon of the deflection of complement can be exhibited in two ways: First by emploj'ing aa a source of complement an active, in itself not bactericidal serum and showing that when decreasing amounts of inactive immune serum are added, only the medium amounts of the same exert a bactericidal effect, whereas both tlie larger and the smaller amounts are ineffective. The results shown in Table I will serve as an e.\ample of this. PiKB"n Number of Coloni™ on a PUile on the Addition »tive Goat Serum itBUut Vbrio Metehailiafl. Amaunl of Culture. i S S a 1 a PreviourLy Tnatnl with Dead II p P P tA. ec. of a one-day bouil- lon culture of vibrio Metch- nikoff pig serum plus 0.01 Metchnikoff 1.0 0,5 0.2. 0,1 [o,o,^ 10-20 0 0 0 0 0 0 10-20 0 10-20 0 100 0 Control I. tAi cc bouHloii culture -f 2 cc. 0.85% salt eolutioa-co. " II. 0,1 ee, active guinea-pig Benim -1-0,01 cc. inactive goat immune senimagaiDst vibrio Metchnikoff +17/1^100. bouillon culture—0. " IIL Sterility of all the sera-0. This experiment shows that by previously adding dead bacteria of the corresponding species and then using the centrifuged serum, it is possible to remove the property by means of which an immune serum when in excess can exert a complement-deflecting action. This absorption, however, did not succeed with three other species of bacteria. Hence we can conclude that the deflecting agent of the immune serum is a substance produced by immunization and 142 COLLECTED STUDIES IN IMMUNITY. related to the complement on the one hand (complement deflection I) and to the corresponding bacterium on the other (specific absorption) » It is therefore an amboceptor in Ehrlich's sense, and not an anti- alexin in that of Gruber. The results of my experiments may be simmiarized as follows: (1) By comparing two bactericidal unmune sera both possessing a strong agglutinating property, while, in certain combinations, only one manifested the phenomenon of deflection of complement, the objection was controverted that this deflection is due to the mechanical action of agglutination. (2) It was possible to show in several different ways that the deflection of complement is not caused by a constituent of normal serum. (3) It was directly proven that the deflecting agent of the immime serum is the specific amboceptor (immune body) produced by immunization. From this it follows that the amboceptor merely plays the role of a coupling element between bacteria and complement and that the property of "sensitizing" (Bordet) or of "preparing" (Gruber) cannot be ascribed to it. The latter assumptions seem to be irrecon- cilable with the phenomenon of deflection of complements described by Neisser and Wechsberg. XI. ACTIVE IMMUNITY AND OVERNEUTRALIZED DIPHTHERIA TOXINS.^ By Dr. Jules Rehns. Instead of following the classical method of immunizing against diphtheria, namely, by inoculating the toxin in gradually increasing doses, a number of workers have attempted to produce immunity by inoculating, either from the outset or during the course of the immunizing process, mixtures in which the toxin was partly, wholly^ or over neutralized. It will at once be realized that these methods, with which the names Babes, Pavlovsky, Arloing, Madsen, and Kretz are principally associated, possess an entirely different significance. Under the direction of Professor Ehrlich I have tried to see whether active immimity could be conferred upon a given normal organism by the injection of increasing doses of diphtheria toxin mixed with one or more times its equivalent of antitoxin. Rabbits weighing about 2000 grams were used and these were injected with mixtures composed of a toxin L and the Standard Serum of the Institute. The constants of this poison, determined according to the clas- sical methods devised by Ehrlich were as follows: (1) The amoimt of poison which just corresponded to an immu- nizing unit, i.e., the limit of no action whatever, Lo=0.3 cc. (2) The amount of poison which, mixed with one immimizing^ imit of serum, was just sufficient to kill the animal, the so-called Lt dose, Lt=0.45 cc. (3) The fatal dose for a rabbit weighing about 2000 grams, deatb occurring in four days: This was about 0.01. ' Reprinted from Compt. rend, de la Soc. de Biologie, 1901, page 141. 14a 144 COLLECTED STUDIES IN IMMUNITY Two rabbits were inoculated intravenously, one with 3 units aerum + 0.3 cc. toxin, B. mixture neutralized about three fold ; the other with 3 units serum + 0.45 cc, toxin, a mixture about doubly neutralized. The animals received daily increasing doses from the 5th t ISth of December, 1900, at the end of which time the total amount of toxin they had received was for the one 7.5 cc or 750 fatal doses, ^H for the other 4.27 cc. or 429 fatal doses. ^| The animals showed no change in health and lost no weight. In order to allow the excess of serum introduced time to be eliminated, four weeks were ailowetl to elapse before testing the serum for its antitoxic strength. A control rabbit treated with serum alone died accidentally, but, as will be seen from the results of the experiment, a control was superfluoui. Both rabbits were killed Jan. 24, 1901, and 1 cc. of the serum was mixed with one-quarter a Lt dose. The test animals died in twenty-four hours. By decreasing the quantity of toxin to one- eighth Lt dose, death occurred in forty eight-hours. From this we see that the serum of these animals certainly con- tains no more than one-eighth of an immunizing unit, an amount which at once eliminates any idea of a passive immunity. One must therefore conclude that the organism of a normal rabbit not sensilized through previous immunization is unable to break up the combination of diphtheria toxin with antitoxin. Not a trace of this toxin is tree at any moment, and the strongest doses of the mix- ture are destitute of any injurious effects. Twenty fatal doses, for instance, were given at the beginning. But we see further that these mixtures do not cause even the slightest production of antitoxin. We must therefore conclude, with Arloing, that the injection of ovemeutralized toxin is absolutely useless for purposes of immuni- zation. These results do not in the least resemble those of other authore who have used partially neutralized mixtures in which toxons and toxonoids are present in a free state. So far as immunizing power DIPHTHERIA TOXINS. 145 is concerned, Madsen has found that these substances, though abso- lutely free from pathogenic action, are entirely equal to pure toxin. In these, then, toxicity and immunizing power are entirely luiasso- ciated. These facts make Ehrlich's hypothesis very plausible, ac- cording to which the toxin molecule contains separate groups, " hapto- phore" and " toxophore." The combination of the former with corresponding groups in the receptive organs furnishes the condi- tions necessary and sufficient for the production of antitoxin by these organs. Translator's Note. — Park and Atkinson report quite difiPerent results in a similar set of experiments. By treating horses with toxin neutralized threefold (for guinea-pigs) , they produced a considerable amount of antitoxin. Even when the toxin was neutralized sixfold there was a slight production of antitoxin. See Proceedings of the New York Pathological Society, 1903. I Xn. IS IT POSSIBLE BY INJECTING AGGLUTINATED TYPHOID BACILLI TO CAUSE THE PRODUCTION OF AN AGGLUTININ?' lei^^^^ By Prof. M. Neisser, Memlier of the Institute, and Dr. R. Ldbowsei, fomii Assigtant in the Bacteriological Division. Especially important for Ehrlich's conception of the chemical union of toxin and antitoxin are the experiments in which immu- nization of animals was attempted with neutral, and therefore non- poisonous, toxin-antitoxin mixtures. Such experiments, inaugu- rated by Babea, were recently published by ICretz,^ among others. At first it appeared to this author that he could really immunize with such neutral mixtures, but exact reexamination convinced him of the contrary. Jules Rchn.^^ also was unable to obtain any results with neutralized toxin-antitoxin mixtures. All of these experi- ments showed that Ehrlich's conception, that of a chemical union of toxin and antitoxin, most readily sufficed to explain the facts. In immunization with celbilar material the circumstances are far more complex, von Pungem^ therefore first attempted to rule out the immunizing action of the injected cells (cn,'throcytes) by simultaneously injecting the corresponding immune serum obtained elsewhere. This mixture, therefore, was neutral, and caused no immunity reaction. Our colleague, Dr. Sachs, has continued these researches at the suggestion of Professor Shield, and will report thereon the following article. In direct contrast to v. Dungem's experiments are the results ■ Reprint from the Centralblatt f. Baeteriologie ParoGiteokunde und Infec- tions Krankheiten, Vol XXX, 1901, No 13. ^ ' R. Kretz, Ueber die Beziehungen von Toxin und Antitoxin, 2 Heilkunde, 1901, No. 4. * See pages 143 et seq. * See pogea 30 et seq. L AGGLUTINATED TYPHOID BACILLI. 147 I obtained by Jules Rehns ' by the injection of agglutinated typhoid bafiili. He found that it was inunaterial, so far as effect was con- cerned, whether he injected the typhoid bacilli agglutinated or not agglutinated. An entirely similar experiment has also been pub- lished by Nicolle and Tr^nell.^ Having previously and independently of Rehns busied ourselves with this question, and having seen that it is attended with con- siderable experimental difficulties, we again took up the problem on the publication of Reims' article, especially because of the theo- retical importance of the subject. Furthermore, our previous experi- ences had given ua the impression that Rehns' results were not gen- erally applicable. The technique of our experiments was as follows: The typhoid culture employed was an old laboratory culture es])eeially adapted to agglutination exjieriments. Of this, one-day agar cultures, sus- pended in physiological salt solution and killed by exposure for one hour to eO^-TO" C, were used for the injections. Tlic preparation of the agglutinated typhoid bacilli was moefc carefully attended to, it being deemed especially imjxirtant to fully satisfy the bacilli with the agglutinin. The agglutinin was a highly active typhoid agglutinin derived from a horse, and agglutinated even in dilutions of 1:50,000; only in the last experiments was a weaker serum used. The agglutinin was added to the bacteria in Buch amounts that about 500-1000 times the amount calculated to be necessary was used. In order to cfTect as firm a union as pos- sible between bacilli and agglutinin the latter was allowed to act on the bacilli for one hour at 42°-44'' C, during which time the tubes were shaken everj' ten minutes (at times with glass beads) in order to loosen the larger clumps and secure the penetration of the agglutinin to the central portions of the clumps. And in order to be on the Bafe side, we centrifuged the bacteria from the first mixture and repeated the saturating process in the same manner. After the Kcond saturation the mixture was again centrifuged, filled up with salt solution, again centrifuged, and then washed several limes. The various decantations were saved and tested for the presence mogIobin 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.hsmofi^obin even in the presence of substances abstracting mercury. We did not deny that the stromata could be digested by means of proteolytic ferment. Our objection was directed only to regarding the escape of hsemoglobin, an indi* cation of a digestion, or of digesting complements. I I I XV- A CONTRIBUTION TO THE STUDY OF THE POISON OF THE COMMON GARDEN SPIDER.> By Dr. Hans Sachs, AssistBiDt Bt the Institute. The studies in hsemolysis, constantly keeping pace with the develop- ment of the doctrine of immunity, have siiown-that besides the usiial blood poisons sharply defined chemically, there is another group of hsemolysina 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 ataphylolysin, tox- albumins of higher planta, such as crotin. Besides this there ia the endless series of htemolysina, 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-cdls are sat- sitive to these hcemolysins which are cajxibU of anchoring them. This fundamental law, which waa first recognized and clearly formulated by Ehrlich and Morgenroth^ has constantly been confirmed, espe- cially in the study of the serum hiemolysins artificially produced, -4s a result of this the mode of action of these poisoTis as well as of the toxins has been concriivd from the standpoint of tlie 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 receptore (side chains) which fit into the haptophore 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 poi.-^oas is of great significance for the study of the chemischeii FliysoloKie u. Fatbologie, Vol II, 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 hicmolysin which showed itself particuiariy well adapted to researches in this direction. The description of a complete experiment will give an idea of the method of obtaining and testing tliis poison. A garden spider weighing 1.4 graxns is rubbed up with 5 cc. toluol water containing 10% NaCl and the fluid kept iit the refrigerator for twentf-four hours. Then water ia added to make the total volume 25 cc. and the mixture filtered (or centrifuged). The ha;molytic experimentB are made in the uaual manaer with thia cloudy, brownish-yellow filtrate. Decreasing amounts of the poison solution are placed in a series of test-tubes, each of which is then filled up to 1.0 cc. with physiological (0.85%) salt solution. Elach tube now receives one drop of undiluted blood or I cc. of a 5% suspension of blood in physiological salt solution. The specimens are kept in the incubator at37°C. for two boura, and then in tbe 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 scriun and go exclude any possible disturbance from that source. The Aracknolysin, 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, arachnolj'sin is somewhat analogous to snake venom, while it differs therein from the hsemolysins of blood serum, in which, as is well known, actual hiemolysis Ls 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. ,ls can be seen from Ike table we are here dealing with a hiemolysin of extraordinary power, the action of which on the individual species of blood, however, is very variable. Thus a number of species of blood are destroyed even in dilution of 1 : 1(K)0 or 1 : 10000 (this refers to the original poison solution) ; others remain imaffected even by large amounts of poison. Next to rat blood, the most sensitive was rabbit blood, for 0.(XX)1 cc. of the original solution, i.e., 0.QQQ028 g. spider substance, sufficed to completely dissolve 0.05 cc. blood C=200,-000,000 blood-cells). A garden spider weighing 1.4 g. there- J THE POISON OF THE COMMON GARDEN SPIDER. 169 ArAehnolsrBin. Hsmolsrtio Aotion on the Blood of Rabbit. Rat. Mouse. Man. 00. 1/1000 1.0 0.75 0.5 0.35 0.25 0.15 1/10000 1.0 0.75 0.5 complete almost complete strong complete almost complete Strong complete moIytic 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-cetls. 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. Tliis confirraed their view of the complex nature of normal hemolysins. They were further able to activate inactive hiemolytic 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 182 COLLECTED STUDIES IN IMMUNITY. used. Tliis showed conclusively that the globuliddal yropcrty of normal serum is du£ to Ike co-action oj two bodies, one which wUkelands keaiing {thermostabk} and llie other which docs not {tluTmolabilc.) These views have been accepted by the majority of investiga- tors, and numerous obsen'ers, P. Miiller,' London,^ E. Neisser, and Doring' ha\'e constantly added new facts, the analysis of which in every instance demonstrates the complex nature of nor- mal hemolysins. 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 negati^'e result of part of their experiments, hold that Ehrlich and Morgenroth's conception of the nature of normal hemolysins 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 ciTcumslances. 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 IVof. 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 presenae of thermostabile bodies (his " Hilfskorper ") on the occurrence of hfemolysis, by reactivate 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 senun which was derived from the same species that furnished 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. Miiller, iJber Antiharaolyaine, Centralblatt fur Bacteriologie, Vol, 26, 1001. ' E. S. London, Contribution h. I'^tude dea b^molysines, Arch, des ScieoceB biolog. (Inst, imperial de mfid. exper. i St. Pet«rebourg), T. VIII. 1901. ' E. NeisBer u. Doring, Zur Kenntniss der haniolytischen EigenHchatten dea menschlichen Serums, Berl. klin, Wochen. 1901, No. 22. ' Buchner. Sind die Alcxine einfuche oder complexe Korper? fieil. klin. Wochenschr. 1901, No 33. ' M. Gniber. Zur Theorie der AntikOrper, II, tfber Bacteriolyse u. HceiuoIyaG, Huncb, nied. Wochenschr. 1901, 4S and 49. • Ehrlich, Vortrag im Verein fiir innera Medicin, Dec. 16, 1901. CONCERNING ALEXIN' ACTION. complement for any given amboceptor. In testing a series of com- binstions, 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, e\ea though often only after consider- able labors, this has always led to a certain realization of the com- plex nature of the hemolysin. Buehner was successfid in two of his cases in activating the combi- nation chosen by him; btood-ccUs A + inactive scrum (amboceptor) B +active serum {complement) A. (Guinea-pig blood and ox serum; goat blood and rabbit serum.) In three other eases, 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); gmnea-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 hiEmolysis 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 Bubsequently discuss in detail, have shown us that the temperature at which the senmi 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 hEemoIytic 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 amoimt of blood, as in all our experiments, waa 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 senim 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 ec. 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 htemolytic action of the decanted fluids is shown by Table I. TABLE I. Absorption op Sbeep Sebum by GtriNEA-Pio Blood at 0' C, ^^ Sulveot Power of tbe Dwcnted FluicU for A, Nuive GuinBi.-pi« Blood. B, Guinea-pijc Blood Previnurly TreoMd Sheep Serum. 1 0.7 2 0.6 3 0,5 4 0.4 5 0.35 6 0 moderate little trac« 0 complete almoEt complete fltrong 0 Buchner's second negative case deals with the combination sheep blood and rabbit semm. In the following experiment, entirely ana- logous to the preceding, the complete solvent dose of rabbit serum 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 mailer of fact, hemolysis 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 hsemolysis when it acts on blood-cells 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-ceU on the one CONCERNING ALEXIN ACTION. hand, and amboceptor and complement, on the other, may vary from case to case. Whereas in Case II the decanted fluids were TABLE II. Absobftion of the Rabbit Serum bt Sheep Blood at 0° C. Acided. Solvent Power of the Deeanied Fluidn iar A, Nmive Sheep Blood. B, Sheep Blood Babbit Serum. 1 0.6 2 0.45 3 0.35 4 0.25 5 0.2 6 0 trace 0 0 0 0 0 coin[)lete almost complete moderate 0 absolutely inactive against native blood (i,e., all the amboceptor had been bound at CC. 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 doae. This indicates that in this case the affinity of the amboceptor's eytophile group for the receptor of the cell is relatively slight at O^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 tlie 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 fu^t three tubes showed mod- erate, little, and trace of solution respectively. Both normal hiemolysma (Buchntr's negative Cases I and 11) therefore correspond in their main behavior. They amsist of two componenis (readily separable by the "cold method") which in their mutual relationt manifest a certain variation in (he behavior 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 dt^ serum), difficulties presented themselves which at firet ap- lob COLLECTED STUDIES IN IMMLTNITY. peared to be insurmountable. Despite numerous variations in the conditions of tlie experiment we did not succeed with approjjriate procedures in effecting a separation by means of the "cold method." The fluids decanted from the mixture of guinea-pig biood-eells and active dog scrum manifested tlie 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 j'et the)- showed slight differences so tjiat we did not feel justified in drawing any conclusion. However, we soon became convinced that a separation of two substances causing hicmolysis had nevertheless been effected by the absorption in the cold. We allowed the fluid decanted from the guinea-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 abie 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 only in traces. An experiment of this kind is shown in Table III. Ahbc TABLE m. RPTioN OF Doo Serum by Guinea-piq Blood at 0° C. HsmolyiiK of tho ^utEoB^t 37°. 8olv.nt Poirer ol Ibe Dwnnled Fluid» for ^ffii A.NMtivsGuine.- pig Blood. Tr«Kleerature. Be this as it may, these experiments certainly show that the power oj a dog serum (inactivated at a suitable temperature, e.g., 50° C.) la be activated by guinea-pig sentm is lessened by heaiing the dog serum to 55° C. and destroyed at 60° C. Table IV shows such an experiment: COMPLBTIOS (W TABLE IV. 1 GntNEA-pia Sbiipm) op Dog Sbrcm iNAcmvATED j DiFrBRBNT TEMPBRATtniBS. Degroe at Solution of the Guinm-pig Blood M™a with 0,1& «. Dob Serum and luutivsted by HbU bd Bour's SeBiicw to A.*.-, B, SS". C, 50°. 1 0,5 2 0,25 3 0.1 4 0 0 moderate little 0 complete strong little 0 4 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-cella 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 or Doo Sbrfm by Gcinka-piq Blood at 0" C. (0,075 cc dog Bcrutn just (>omplBte!y disBolvea 1 cc. 5% guinea-pig blood.) Sn vent Powir of tl« on ^&r A, Nilive "as™ Sonan liuunLvBled ruled with Dob CO. I. 60°, II. 55'. m. 00°. 1 0,15 2 0.1 3 0,075 complete little complete complete strong complete In this case, therefore, we have demonstrated a thermolabOity of the amboceptor' 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 hemolysins. After ha\'ing determined that certain amboceptors will only ' It is theretore not at all pemiiKsible to (Icfine the two components of the hH?molysin, as Gruber would do (Uiscuasion of Gruber'a AddreES, Wiener Klin, Wochensch, 1901, No. 50), only according to the temperatarr, and to say that at a. certain degree ot lieat the ambocept-or remainB intact while the complement does not. As long ago as their second communication Ehrlich and MorgeDRith 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 witlistand heating to 55° C, is absolutely impoa- sible. The itifluence of temperature on amboceptor and complement mriea from case to cage. Hence that these two factors act together in ha-molysis we know only from this, that (idd subatatires, in themeclfs not capable n] causing aohttion, when combined, effed htBmolyma; and that one of these suhstancea (the complemml) can nei{o in horse serum. This amboceptor, which fita giiineft-pig blood, and can no longer be d 190 COLLECTED STUDIES IN IMMUNITY The com l)i nation, sheep blood and rabbit serum (Buchner's second case) preaenW 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. 1 can therefore confirm the facts found by Buchner in this case, namely that sheep serum ia 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 hiemoysin because no( every aerum need con- tain a filling compkment jar 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 t« the peculiarity of the combining relations of the separate components, is entirely inapplicable in a number of cases. Gniber, the second author to come out against the conception of the complex nature of normal serum hemolysins, 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 hcemotysin. Ehrlieh and Morgenroth in their second communication on hi»- molysins have already analyzed the conditions for separating the interbody by means of absorption, emphasizing " thai Ike solution of the problem tiurefore is now possible only under either of the two above mentioned favorable conditions; (1) When Ike luv hnptopkore groups of Ike ijilcrliodi/ differ greatly in tlteir affinity; and (2) wken, by means of a combination whose discovery depends on chance, an acti- vating complcmtrnt is found." The limitations of the two methods applicable to an analysis of the complex nature of hipmolysine, 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 hEemolyains at all commensurate with the means at our disposal. The schematic application of only one nctivnted aUer healing lo 55° (', can be shown to exist ia aclitt horse eerum (whicli does not disHOlvc guinea-pig blood) by combining and completing it with guinea- pig serum. CONCERNING ALEXIN ACTION. 191 method can lead to the greatpst errors. In this respect a comparison of thie results obtained by Buchner and Gruber, is very instructive, for among their cases are two fombinations 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 Gniber's negative cases, which embrace the following com- binations: I, rabbit blood — dog scrum; II, rabbit blood — ox serum; III. gui ea-pig blood — ox serum, IV, rabbit blood — guinea-pig serum; I have syBtematically 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 strum, and sheep serum. II. For tlw amboceptor of ox scrum, acting on rabbit blood; in guinea-pig serum, rabb^U serum, and rat serum. III. For the amfioceptor 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 hieraolj-lic action or at least to act in a very much smaller degree, Gniber'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 purpo,se, we found only two sufficiently hsmolytically 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 of amboceptors. Buchner and Gmber 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 questioa of the complex nature of the normal serum hiemolysina. Against this we have in all these cases brought positive proof that the " alexin," coTiceivcd by Buchner to be a simple unit, always jrroduces its effects through the co-adion of two components, the existence of which ia demonstrable in different ways. We must therefore uphold Ehrhch and Moigenroth's view, thai normal and artificially produced hamo- 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 hajmolysin, and even a thorough analysis, therefore, need not necessarily achieve the desired result in every case. The method- adopted by Mullcr' for demonstrating the amboceptors in chicken serum, which is hjEmolytic for rabbit blood, is of interest in this connection. When the usual methods failed 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 liiemolysin, 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 hemolysins 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 ha-molytic behavior), for thus far no fitting complements have been found for this serum. In all other cases of hemolysis 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 CONCERNING ALEXIN ACTION. I'Jb to the co-action of two substances. Keiffer' 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 experimcnta in vitro. And according to the numerous investigations of M. Neisser and Wechsberg in this Institute, all of the bactfiriolysins of normal sera which they investigated, are of complex constitution. This is natural because in the cell-destrot/iiig properties of normal serum, as in t/ie development and increase oj these properties through immunization lite mechanism is exactly tite same in principle, although, owing to the tnvhiplicUy of the reodion products, the action of the latter appears more arnipUx. In my investigations of the ajtotoxic properties of normal serum J have included the widely distributed spermotoxic {unction. Accord- ing to the unanimous opinion of all authorities the speeiSc spermo- toxin produced by immunization consists of two substances. Thus far, however, this has not been demonstrated for normal spcrmo- 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 spennotozoa 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 pro|X)rtion of 3:1 or 3:2. In that case the guinea- pig spermatozoa were killed after 12-15 minutes, whereas, in the control teat with inactive rabbit serum or active guinea-pig serum alone, the spermatozoa showed lively movements even after !J-Ii hours, The proportion of amboceptor and complement employed by me is in direct contrast to that recommended for immune sera by MetchnikofT and his co-workers. The reason for this will be under- ' K. PfeiFFer, Weitere Mittheilungen iiber die Speiifischen Antikorper der Cholera, Zeitachr. f. Hygiene, XX, 1895. ' Moxter. Uber die Wirkiin^wi^iBe dpr barterienauflfisenden SuhBtarMn der tbieriachen Silfte. Centralbl. f. Bacieriol., XXVI. ISM. ' Metalnikoff, Etudes sur la Spermotoxine, Annales de I'lnBtitut Pasleur, 1000. 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 autospermotoxins. Subsequent Note. — In the meantime the French translation of a study by London, which had already been published in Russian, has appeared (Contribu- tion ik r^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 hsemoylsins 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 hsemolysis, 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 simple 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 amho- 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 I'Instit. Pasteur, May, 1901. 195 396 COLLECTEU STUDIES IN IMMUNITY. charact€r, in a recent work especially designed to refute the plural- istic view of the complementfi, haa 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 scrum specific for themselves were able to deprive a normal active serum of all ita complement activity. Bordet sensitized blood corpuscles with appropriate amboceptors, and then exjiosed them to the action of a freshly drawn normal serum. If now he waited for the occurrence of hiemolysis and then added sensitized cells, baet«ria, or blood coqjuscles of different species, they remained totally unchanged, although the serum that iiad been used as complement was capable in its original condition of destroy- ing these also. When fresh senmi was first brought into contact with sensitized bacteria, similar results were obtained. The blood corpuscles subsequently added did not then undergo hfemolysis. // such an action on otic of the sensitive substrata has imce taken place, the acUve sera, as a rule, arc deprived of all their co?nplcment 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 a.ssumes that a certain serum A, which is capable of complementing two different bodies B and C, one bac- tericidal and the other ha'niolytic, contains only a single comple- ment, Bordet's results would then most readily be explained by assuming that the two inmiune bodies are identical in their com- plementophile groups. In that case, of course, owing to the pre^-ioua exercise of the one function, the avaOable 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 t'ieti' is an artificial one, and does not correspond to the fads observed. For if it be a.ssumed that this particular serum A contains two different complements, both of which can be absorbed by the amiwceptors 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 Mo-genroth, p. 56. PLURALITY OF COMPLEMENTS OF THE SEIiUM. ly? piemen tophile groups. To one, therefore, conversant with this con- ception, Bordet's conclusion cannot appear otherwise than ioraed. 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 com pie men top hile group came into action, and not a plurality of groups. Despite these objections raised against liordet's evidence, and in spite of Ehrlich and llorgenroth's previous positive demonstration of the ]>lurality of the complements, it seemed advisable, owing to the irajjortance of the question, to enter once more upon a thorough investigation of the subject. We at first confined ourael\'es to the complements which effect the hemolytic 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 comptemertts is correct, it should be possible through appropriate experimental conditions to influence the serum in such a way that imly 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 assiimption 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 sliall designate them by the following numbers: Case I. Guinea-pig blood — inactive norma! goat serum. Case II. Rabbit blood — inactive normal goat serum. Case in. Rabbit blood — inactive serum of goats immunized with rabbit blood. Case IV, Ox blood — inactive serum of goats immunized with ox blood. i 198 COLLECTED STUDIES IN' IMMITNITY. Case V. Dog blood — inactive serum of goata immunized with dog blood. The various means by wluch we have succeeded in a eepanttiOQ 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 leas 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 ec. goat serum mLxed with 3 cc. of a 10% papain solution were placed in 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' demonstrated complete preservation of the complements for Case Til 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 op Goat Seihtm by Means op Papain. Solv. „t Power of the Gutti Semm. E«iii lei. Kiam nie II. E«m 1b 111. (<■) Dig«[«l. (6) NormiJ (a) Digeslo.; Kb) Noimid (a)DreoM«,j. Caw I 0,5 0-25 0,5 0 !5 (i 5 0.2S trace ooiuplele liioderule completo Case 11 1-0 complete 1.0 n complete ft. iraoe complete Caaelll complete complete complete complete Gomplete complete Case IV IJ.3 little 0 nil complete 0 ,■! little 0 07 (1 5 o.os Case V 0,5 O.OG trace .Im'l c'm'ie complete ' In all our experiments the a ■of a S*^ HU8pen,i[on, t of blood tued aa a reagent •kbx 1 ce. ] PLURALITY OF COMPLEMENTS OF THE SERUM. 199 When the papain was allowed to act longer, the resistant com- plement III was also affected, so that usually after one and one-half to two hours digestion, the goat serum was entirely deprived of all its complements. Treatment with alkali in place of papain digestion gave analogous results. We made use of soda and proceeded as follows: 10 cc. goat serum to w. ich 1 cc. 7% soda solution had been added, were kept in the incubator for one and one-quarter hours, and then neutral- ized with hydrochloric acid. The solvent power was compared with a goat serum which by the simultaneous addition of soda and hydro- chloric acid, had been brought to the same concentration of salt without having been subjected to the damaging influence of the soda.i (See Table II.) TABLE II. Destruction op the Goat Serum by Means op Soda. Solvent Power of the Goat Serum. (a) After Soda Treatment. (6) Normally. Case I '' II ** III '* IV " V 0.5 0 1.0 0 0.12 complete 0.5 0 0.3 0 0 . 1 complete 0.6 0.03 0.04 0.04 Hence, owing to the action of the soda the complements for Cases I, II, IV, and V have completely disappeared, whereas Complement III is 8tiU present, although its action is hut one-fourth of what it was. We have furthermore effected a separation of the complements by heating the goat serum to 49°-50° C. for half an hour. At this tempera- ture the solvent action of normal goat serum for rabbit and guinea- pig blood has been completely destroyed or almost so. On the other hand, the complement action for the artificially produced immune bodies is more or less preserved, as can be seen from Table III. The experiment shows that in this case complement IV is the most resistant, in contrast to its behavior with papain or soda. In the two latter cases, complement III had shown itself the most resist- ant. If we examine the table more closely we shall further see a * The resulting salt concentration, by the way, is so slight that the solvent power was in no way decreased thereby. COLLECTED STUDIES IN IMMUNITY. TABLE III. AN HnTiR's Hbatinr or the Goat Sbbttm to SulVBnt Power o( tba GcMit SeruB.. Uealcd. Normally. 0H^^^^8olv«„v Case I ■' 11 " III " IV '■ V 1-0 trace 1.0 ■■ 0,08 complete 0-035 " 0.75 0 , 1 complete 0,25 0,01 0,035 " 0.02 1 almost nothing 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 it<, III, IV, and V proceed vnde- prndentty 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 [einheitiich] com- plementa 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 ea.sily 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. ^^^ i PLLKALITY OF COMI'LEMENTS OF THE SERUM. 201 and diatom eartli, 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 haimolysis. It was just this phenomenon that led Bordet to his unitarian conception. \'et 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 complemcntophile 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 \', or tor both, without suffering any injury in the rest of its complement ' See p. 36 'The same results were obtained by Wilde (Bed. ktin. Wochenachr. 1901. No. 34] in absorption texts with anthrax, cholera, and typhoid bticteria^ but to conclude from this that the aleicin is a simple unit, as Wilde does, is not per- missible m view of our above statementB. • 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 Bhaken 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 tlie previous tables. TABLE IV. Bbibp Absorption op Goat Serum with Rabbit Blood. Solvent Power of the Goal Soruni. (a) After iba Abaorption. No^^ly. ■ Ca-w I " II " III ., IV 0.25 complete 0.5 " 0 04 0.3S Gomplola O.Z 0.25comfdete 0.5 0-04 0.08 oomptato 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 di\'ersity. It is of special interest that in this brief action the particular activating principle (complement 11) 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 • 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 h!pmolysin.s (Cases I and II) while the rest of the complements were in the main preser\-ed.^ An experi- ment of this kind is shown in Table V. ' In this also it is necessary first to determine the favorable condition* governing the experiment. Thus, in order to completely hinii ihe giiinea-p^ PLURALITY OF COMPLEMENTS OF THE SERUBt 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. Absorption qp the Goat Serum bt Guinba-piq Blood Smoic^TA. Solvent Power (a) Of the Deoanted Fluid. (6) Of the Normal Goat Serum. Case I " n " III " IV ti y 1.0 faint trace 1.0 *' 0.1 complete 0.15 complete 0.15 complete 0.15 comidete 0.25 ^' 0.1 complete 0.04 complete 0.15 complete - Hence after the absorption, the complements of the normal hemo- 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 hemolysin (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 ha?molysins, 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 TN IMMrNITY. therefore confirms the separation effected by means ot the stromata whereby the complements of the normal hiemolysins 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 acnmi. 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 tho 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 stnimata. By means of this, since the stromata plus the anchored complement is removed by centrifug^ng, Bordct's assumptions can be entirely excluded. Our absorption experiments therefore show that of the (wo possi- bUitifS, namely, of a complemcrU wilk seiwoi different zymoioxic groups, or of a pluralily of different complements, Ike latter assumption must be aaxpled. 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 VL C^pl.™ ntiiw Pomir of Oo.t S. rum after W (h) C") (.d) (O 1 The Aet[on AWjp^.™ ^'"■^iTh""' .^„ ""SO- Strommbi. Cue I n 0 0 + 0 0 0 0 + 0 0 " III -1- -t- i + + 0 0 + ^ 0 0 v» -1- + ' This objection, moreover, is entirely incomprehensible to us. Acoording .1 to our view, normal and artiScially produced h^molysina manifest their action ' by meons □[ the Eame meclianism; for when the normal anibooeptors ore re- placed by the hont of ftmbot'eptorw preBcnt in an immune serum, new compl»> mentophile groups come into action, and with these, of course, new partid complements. PLURAUTY OF COMPLEMENTS OF THE SERUM, 205 This shows us that the two complements I and II (normal hiemoly- is) cannot by these experiments be differentiated from each other, iat the olhir three complements, however, can absoluUhj be dislijiguisheH iiy llieir behavior, not only from one another but aho Irom the first group. ■£ence in the fi\-c different comlnnations tfie existence of at least four dtffercnt complemenis is positively demonstrated. And tliat the two I normal hiemolytic functions of goat serum are also effected by two different complements followis from a previous cxjjeriment of Erhlich and Morgenroth.' These authors showed by filtering a normal goat serum through Pukall filters, that the filtrate contained exactly the aame amount of complement for guinea-pig blood, whereas the com- iplement for rabbit blood was almost entirely absent. E. Neisser ^d 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 fire compleliona txamined, five different compleniaits of tlie gout scrum come into play.^ We have also examined the complementing properties of the :8era of other animal species, and have arrived at results which abso- lutely contradict the unitarian view of the complements. These igxperiments concern first the serum of rabbits. We shall proceed from the fact determined by 8chiitze and Scheller* under Wasser- mann's direction, that, following intravenou.'f 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 lass 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 ma numerous investigations we established the fact that the com- ' See page 5ft. ' E. Neisser and DOring, Berl, klin. Wocbenai^hr. 1901, No. 22. ' Through the courtesy of Dr. WendelstodC in Bonn, we leitm that thtLt investigator, by means of an int«resting method, hivs aluo succeeded in demon- strating a number of complements in goat Kerum. He immunized a goat with eeveral species of blood and vok then able by means of chemical and tbemuc influencea to separate the complement.s fitting the immune bodies produced, flee CentralblaH t. Bacteriologie, in which Ibis study is alwut fo appear * Schutze and Scheller. ExperimentelJe Beitruge '.ur Kennfiiisa der un normalen serum vorkonuneoden globuliciden Substomen, Zeitachrift f. Hygiene, Vol. 30.1901. i 2^6 COLLECTED STUDIES IN IMMUNITY. plement tor 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 goal serum which results from the injection may be seen from the following table; TABLE VII. Bqlvant Power of he Rabbit Serum. Betort the Injeclion. After tbe laiection. Goat blood — inactive normal rabbit serum Ok blood— inactive serum ot a rabbit im- munized with ox blood 0.05 1,0 no solution 0.25coEn|det« Similar results are obtained in the absorption of rabbit serum by means of goat blood in vilro, so that this experiment already justi- fies us in assuming two different complemenla 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 hsmolysin 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 beliavior, 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 /fe existence oj two different complements in rabbit serum; a proof which is strongly cor- roborated by the divergent bchatyior of the two complcmaits in the absorption with goat blood and pig blood respeciivclif. The difference between the two complements also manifests itaelf 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 hemolysin for goat blood is hardly affected, so tliat 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^ J 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 serum a half hour's heating to 49.5® and in the guinea-pig senmi 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 Vni. Half an Hour's Heating op Doq Serum to 49**.5 C. Blood-cell — Amboceptor Ck>mbmation. I. Rabbit blood — inactive dog serum II. Guinea-pig blood — inactive dog serum III. Sheep blood — inactive dog serum IV. Human blood — inactive se- rum of goats immunized with human blood V Ox blood — inactive senun of eoats immimized with ox blood VI. Ox blood — inactive senun of rabbits immunized with ox blood Solvent Power of the Dog Serum. (a) Heated. 0.5 0.6 0.5 0 0 0 0 . 5 moderate 0.35 complete 0 . 5 strong (b) NormaL 0.25 complete 0.1 0.08 0.15 0.06 0.045 Solvent Power Still Preserved. 0 0 0 less than i less than ^ TABLE IX. Half an Hour's Heating of the Guinea-pio Serum to 49® C. Blood-cell — Amboceptor Combination. Solvent Power of the Guinea-pig Serum. Solvent Power Still Preserved. (a) Heated to 40o. (6) Normal. I. Rabbit blood — inactive guinea-pi^ senim II. Ox blood — inactive guinea- pie serum 1.0 0 0.5 trace 0.008 complete 0.025 " 0.025 " 0.5 " 0 . 5 complete 0.5 0.008 " 0.025 " 0.006 " 0.25 0 almost 0 III. Ox blood — ^inactive serum of ^oats immimized with ox blood 1 IV. Ox blood — inactive serum of rabbits immunized with ox blood 1 V. Sheep blood — inactive se- rum of goats immunized with sheep blood VI. Dog blood — inactive serum of goats immunized with dog blood 1 Li i 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 lite other Itand, Jiarmonize best with the oasumptiGn of a number of different complements in the some scrum. 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 tliat in the Institut Pasteur a high authority (Metchnikoff ) ' 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- nikoff found that the exudates rich in macrophages acted Iiiemo- lytically, but were unable to effect bactfiriolyais. 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. wliich he terms mlcroci/tase, effects the bacteriolytic actions, the other, macroajtasc, is the carrier of the functions which destroy animal cells. He emphasizes that the demonstration of the duality of complementji does not affect the correctness of Bordet's experiments, and he says in explanation of Bordet's results: " II n'y a qu'i admettre que !es 616ments figurfe, uue fois qu'ils sont in^r^gnfo de fixateurs spficifiques, deviennent capables d'absorber non seulcment la cytase qui les dig6re, raais aussi une autre qui, sans les disaoudre, se fixe simplement sur eux." So far as this is concerned we should like again to emphasize that we al.so 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 mew. ' MetcbnikoS, L'tminimit^ (kns les malftdiew tnfectieuscs, page '20G, P&ris, 19(11. XIX. CONCERNING THE MECHANISM OF THE ACTION OF AMBOCEPTORS.' By Prof. Dr. P. Ehrlich and Dr. H. Sachs. I. Blocking of the Amboceptor by Complementolds. The complements which activate the amboceptors of btood 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 fiaptophore group, which coaibines with the complemento- phile group of the amboceptor, and the zymoioxic group, which represents the actual carrier of the complement's sjiecific 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 still able to excite the production of antibodies, and must therefore possess their haptophore group intact; in analogy with the toxoids, therefore, they are called compkmentoids. 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 htemolytic 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 coniplcmentoid), did not occur, e\-en 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 oj Ike compUmenl's hap- tophore group suffers a diminution. A similar assumption has been made by Myers ^ for the toxoids of cobra poison. ' lleprint from the Berl. klin. Wucliensjchr. IS02, No. 21. ' See page 79. • Myers, Cobra PoiBoaSi etc.. The Lancut, 1S98. ■JIO COLLECTED 8TDDIE3 IN IMMUNITY. It ia, 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 priori 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 oE 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 hemolytic properly 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 Euchner in hie 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 scrum 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 bsemolysis occurred. (See Table I.) Our first thought was that the amboceptor, despite the relatively long contact with the blood-cells (one hour), had perliaps not been bound by these. Such behavior, to be sm^, although conceivable and, as we shall see lat«r, 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, without first rcTnoving the fluid medium, no hemolysis took place. And we could see by the behavior of the fluid obtained by centrifuging the blood mLxture 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 tSl et seq. THE MECHANISM OF THE ACTION OF AMBOCEPTORS. 211 TABLE I. Inactive Dog Serum. cc. SolveDt Action on the Quinea-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 + Inactive Dog Serum + 0.5 cc. Guinea-pig Senmi Mixed Simultaneously. 1. 1.0 2. 0.5 3. 0.35 4. 0.25 5. 0.15 6. 0 0 complete almost complete 0 * The amount of blood used in our experiments is always 1 cc. of a 6% 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 complemeniophile groups of the dog serum*s amboceptor by the complemenioids 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 guineor 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 COLLECTED STITDIES IN IMMUNITY. Io.|tive^Dog pin Senun. nfUir Previously havinj (A) At 0°, (B) At37». 1. 1.0 2. O.S 3. 0,35 4. 0.25 5. 0.15 6. 0 0 0 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 complemeotophile 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 -Fcomplementoid). The tubes of series A were then centrituged 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 en.sued, the blood-cells of series B remained undis- solved, as can be seen from Table III. The subslance which caused the hlock'ivg oj the amboccpiers 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 TABLE III. Inactive Dog Serum. CO. Amount of Solution of Guinea-pig Blood on the Addition of 0.4 oo. Guinea-pig Serum. Series A. Series B. 1. 1.0 2. 0.5 3. 0.35 4. 0.25 5. 0.15 6. 0 complete atrong moderate (( 0 0 3. We know from the studies of v. Dungern^ and Erhlich and Sachs,^ 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. cc. 1. 2. 3. 4. 5. 6. 1.0 0.5 0.35 0.25 0.15 0 Amount of Solution of Quinea-pig Blood on the Addition of 0.4 oo. Quinea-pur Serum after Remaining at 37" C. for One Hour. Dog Serum Inactivated. (A) By Shaking with Yeaat.* complete almost complete strong 0 (B) •By Heating.- (a) Shaken with Yea«t.» complete If almost complete strong 0 (b) Employed Directly. 0 * 6 cc. serum are shaken with 0.2 grams yetfst. The complementoids had simply been removed by the yeast and the isolated amboceptors reacted in rurrmal 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 furnislieil by the results of the combining experiment when the molecular con- centration of the fluid medium was increased. As is well known the hemolytic 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 imder these circumstances the amboceptor is bound by the red blood-cells, whereas the complement la unable to take hold.^ 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 wore 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 haemolytic action even of targe amounts (1 cc.) of active dog serum. The result of the experiment corresponded exactly to our ex [jec tat ions. 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 aTialysis of this case furnishes the first proof by means of test- lube experimenis that completnenloids, the inactive modifications of the complements, actually exist in the inactive serum. To be sure, 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, Oljer Hemmang der Ilamolyso durch SnUe. Zcitsclir. f Hygiene, Vol. 39. 1902. 'These conditiona by the way, in our judgment, hai-e no connection with the osmotic conditiona of the cell membrane, as Markl bclie\ea. It seems to ua that tho 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 eicrt such an antireactive action is seen by the fact p()inted oiit by Knorr {MiJnc!i and Wochenseh. 1898. Nob. 11 and 12) that t«tanu8 antitoxin and toxin are absolutely prevented from combining by the addition of 10% NaCi. • In view of this new confirmation I should not want to deprive the reader of an exposition of the complementoid theory from tho standpoint oE an opponent THE MEt'HANlJiM O!'" THE ACTION OF AMBOCEPTOltS. 215 TABLE V. '°'&^- SedimentH ICenliirugfd.iifler Ibn tflxlures b>d bwu kBtit lo. (Joe Hour nt 37°) OQ Ihe Addition ol O.S ce. GuioM-rig Serum. ibe Mix I urea bavins been Previously Treiiled wilh 0.15«.307„(NH.l^O. 0.18 CO. 0.88% HtCl. 1. 1.0 2. 0.5 3. 0.35 4. 0.26 moderate little trace 0 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 Ihe Jomiation of compkmenloid. 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 fc.fc. Gesellschaft der Aerite in Wien, Wiener klin. Wochenachrifl, 1901, No. 51): "If tin animaJ ia injected with inactive serum of the same foreign species instead of active serum, it ia 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 thia time termed ^ymotoxic. As a reeult of the inactivation the zymotoxic group ia destroyed; the haptophore group remaina intact. Hence a continuance of the nsBimilation of comptementoid and the production of the anticomplement. So far, 90 good. Now. Iiuwever, we come to a questionable point. If tlie complemeat deprived of its Eymoto\ic group still posseHses ita haptophore group, it muat still bo able to satiafy and bind its ambopeptor. How then does it happen that an inactivated antiserum again becomes lytic on the addi- tion of suitable complement (active normal ECrum), a phenomenon which, according to Ehrlieb {despite Dr. Wechsberg). h due to the formation of lysin from amboceptor and complement. If the haptophore group of the amboceptor Itaii i^ready been bound by the remains of the old complement, the 'comple- mentoid,' it surely ia unable to bind cew complement. Hence by heating (inactivating) the serum the haptophore group of the complement cannot have remained unchanged; it must ha\'e completely lost its affinity for the amboceptoi. Now, gentlemen, I should like to know what ia left of the com- plement after this heating? The xymotoxic group is destroyed, the haptophore group BO changed that it is not recognizable. Nothing remains of the comple- ment except Ehriich's fervent wish that a little of it might bo left, because other- wise it would not harmonize with the thcoryl It ia thia wish that floats around urn under the name of com piemen to id." fur Gniberl I .-hjiU refrain from any personal remarks for which the ■ Thus 216 COLLECTED STUDIES IN IMML'NITV. plemeutoid still takes pl&ce. In this way we sought to fmd an approx- imate criterion for relative affinity of the eomplementoid. From the power to be reactivated possessed by the guinea-pig blood-cells previously treated at difTerent temperatures witli inactive dog serum it was seen that even at 3° C. a moderate binding of complementoids takes place, and thai complete blocking -pkenomena can already be obtained at 8° C, as is seen in the following experiment: TABLE VI. oiS^^i^. (A) 0° C. B Blood o» Ihe AddilioD of o'J^r."™. Th.H,lSn'..,... had been kept at tor oL Houj. CB) The Deeaoted Fluids from (A) TheUwn.ri/,?LMi.,„™ot Blood »Ed Dx Serum. W 1. 0.5 2. 0.35 3. 0.25 4. 0.15 5. 0.1 B. 0 faint trace 0 complete strong moderate 0 i completo BtroDK 0 CO„pl.„ almost complete Btroag 0 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 lo a very slight degree. Our attempts by means of horse serum to acti\-ate the guinea-pig blood-cells which had previously been treated at 0" C. with inactive ox serum and then centrifugcd, 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- bined with the complement, is of special significance for the method THE MECHANISM OF THE ACTION OF AMBOCEPTORS, 219 of analyzing hEEmolysins. 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 appiication of the second method employed to discover the com- plex nature of hEemolysins, 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 tor the union of amboceptor and cell, a condition which obtains here, cannot be fulfilled at low temperature, and if, on the other, it in ilaelf precludes any sepa- ration whatever? No wonder, therefore, that Gruberi 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 hsmolysm 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 hfemolysins to consist in this, that the amboceptors (substance sensibilatrice) sensitize the blood-ceUs 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 Buitable experiment was also made in this case and, as might have 'Gruber, Zur Theorio der Antikorper. Munch, med. WoobeiiKlir. 1901, No. 19. See also H. Sachs, 1. c. 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 Morgem'oth, we regard the amboceptor as a coupler possessing two haptophore groups. Owing to a mutual combination this traos- mita the action of the complement to the cell. In the case just described, it follows at once that the cytophiJe 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 niatt«r 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. Neieser and Wechsberg * 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 expert- mmlum crncis tor the correctness of the views formulated by Ehrlich and Morgenroth for the mechanism of hemolysin 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. SoBsEQiiBNT AnnrriOM. — According tt guinea-pig blootl-cells, which, because of can no longer be dissolved by guinea-pig inveatigations of Dr. Sachs, vith inactive dog serum, I, owing to blocking by com- ' See page 120 et acq. 'Grub«r, Protocoll der k.k. Gesellachaft der Aerzte in Wien, Wiener klin. Wocheoflchr. 1901. No. 50. * Metchnikoff, I'lmmunit^ 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 O^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 COMPLEMENTS BY MEANS OF A PARTIAL ANTICOMPLEMENT.' The question whether the serum of one and the same Bpecies 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 obser\'a- tiona of Ehrlieh and Jlorgenroth,^ of Wassermann,^ Wechsberg,* Wendelstadt/'' and by the recently published studies of Ehrlieh 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 Bufficient, but that we may caU attention to a method of demonstra- tion which, haa not heretofore been employed. Because of purely tfichnical 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, liowever, usually contain anticomplements cor- responding to the sum of all the complements originally injected,' and are, tlierefore, not adapted to the separation of complementa, ' Iteprinted from the Centralblalt f. Bftct. Originol \'ol. XXXI, No. 12, 1902. >See pages 11, 56, 86. 'Wassemiann, Zeitscbr. I. Hyg., Vol. XXXVD, 1901. * Wechsberg, Sitziing d. k. k. Gea. d. Aerate in Wien, Wiener klin. Wochen- •chr. 1901. No. 48. ' Wendelatadt. Oeotralblatt f. Bad. Part I, Vol. XXXI, No. 10. ' See page 195 et eeq. ' See page 63 et aeq. DIFFERENTIATING COMPLEMENTS. At least this had been the ease thus far, for a partial anticomplement, one acting only against a single complement, had not been obsen^ed. 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-inmiune 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 I 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 senim B, 0.2 cc. for each 1 cc. of the 5% suspension. On the addition of guinea-pig serum to these blood-cella, hffimolysis ensued very quickly in the thermostat; in both cases it required O.OOS cc. guinea-pig serum to fully dissolve 1 cc. of the suspension, while 0.0065 cc. caused incomplete solution and 0.002 ' In tho fallowing, for tbe Hake or simplicity, ne shul! speak only oF two coroplementa, whereas we wish iiere to remark that two groups of complemeDts are probably to be understood, each group made up of a host of angle comple- roents which it is impossible thus far to analyze. 224 COLLECTED STTJDIES IN IMMUNITY. only a, slight degree of solution. For the sake of clearness it waa 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 mbied each with 0.4 cc. of ascitic fluid inactivated at 56° C, and the mixtures kept at room temperature for halt an hour, after which the binding waa entirely completed.^ Thereupon the blood-cells loaded with amboceptor were added. The result of these experiments is shows in the following table: Case A (Ox Blood + Amboceptor). Ouiosu-pig Serum Alons. 0.008 complet« solution O.OOri.5 vcBlige 0.005 BtroDK 0.0055 considerable 0.003 0.0025 moderate "Btt 0.1 almost complc 0.08 0.065 ronwdemble 0.05 fairly littlo 0.035 very little 0.03 trace 0,025 " 0.03 0 ? Blood + Amboceptor). Guinea-pig Sanim Alooa. GuiDea-pif Sanim + 0.4 do. O.OOa complete solution 0.0OU5 almost complete 0.005 " 0.0035 strong O.OOS complete 0.0065 almost complete 0.005 " ^' 0 .0035 strong We see, therefore, that in case A the complement protects com- plelety against 2i times the complete solvent amount of complement, while the amount ot 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, therdore ' The union of complements and anticomplemeDts, aimlogous to the behavioi of certain toxins and antitoxins, is dependent on the time. Ileon: here ^ho this had to be considered and sufficient time aJloived for llie mixture to act DIFFERENTIATING CX)MPLEMENTS. 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 numerous other favorable cases which will 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 immunized animals; for in the latter there are also present innumerable primary, and (owing to internal regulative processes) secondary reactive products. ^ Erhlich and Morgenroth 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 ^his, that foreign complements combine with the complementophile group of certain cell receptors. According to this view the anticomplements are nothing else than thrust-off amboceptera whose com- plementophile groups possess a higher affinity than is usually the case. It is curious, therefore, that Gruber, nine months later (Sitzg. der k.k. 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 AMBOCEPTORS.! By Prof. Dr. P. Ehrlich and H. T. Marshall, 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 ^ 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 oui* 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 Borders 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. > See page 195. * See page 120. * See page 132. * See page 209. * Bordet, Annal. de Tlnstitut 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 nunAer 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; (b) haptophore group of the amboceptor; (c) domi- nant complement; (d) non-dominant complemeot. Comjdementophile groups of the amboceptor: (a) for the dominant com- plement; (^ for the non-dominant. The next question to be considered is whether it is necessary, in order to gel the specific lysin effect, for aU these complementa 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 complei- menta are termed "dominant complements," the rest are " non-dominant eompUmetiis." 328 COLLECTED STUDIES IN LMMUNITY. A case described by Ehrlich and Sachs makes this clear, and we shall therefore !>rielly reproduce it here; ' 'IVo amboceptors are concerned, namely, the normal ambocep- tor of goat serum for rabbit blood, and an amboceptor oblained by immunizing goats, which is anchored by ox blood-cells. We shall for the sake of simplicity designate these amboceptors as A and B, \aturally both these amboceptors are activated by goat serum, in which we shall have to assume at least two complements x and b. For immune body A, x is the dominant complement; for fi it is b. If in one of the two combinations, for example, in that of rabbit blood-cells loaded with immune body A, the senmi 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 b. We obser\'e 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 ca.se, tlierefore, amboceptor A's co m pie men tophi! e groups which combine with the complement must possess a higher affinity for the non-dominant complement b 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 comjAemetU does not combine until after the dominant complement has been bound. The demonstration of this relation succeeded only because in & certain human ascitic fluid an an ti complement was present which acted only against part of the complements of a scrum. The peculiar behavior of this anticompjement 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 Homenhat RimpliHed. The details of this experiment are found in Elirlich and Saolu, page 1.^i^ After BiDdinglhe Meuisof AmbD- After BiudLnmbe IVitQiilemenl by IV. Amount of Com- plement Vmi by Blood ^»Uii (Cms Menis of OA One of these cases deals with the combination guinea-pig bloods-chicken Berutn. From Ehrlich and Morgenrotb's earlier communications (see pages &S et aeii.l Gruber could liave seen that between animal Bpecles fo far removed aa chicken and guincB-pig the chances of complement ibillty are not ajj great aa they are between manimaUan species. If Gruber therefore employs as evi- dence such distantly related species lie must neceKsarily also ha^e uiied widely separated specieH when complementing the immune sera. We lia^e no doubt at all that by immimi^ing distantly related species (birds) with gninea-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 a -f inactive normal serum (am- boceptor) 6 -f complement a lead to haemolysis, in contradiction to their behavior as stated by Gruber. (See Table II.) TABLE II. Number. Species of Blood and of Complement. Amboceptor. 1 2 3 4 5 • 6 7 8 9 10 guinea-pig goat sheep guinea-pig rabbit guinea-pig dog calf rabbit sheep horse ox ft man 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 hy 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 ' Erhlich and Morgenroth, page 11 ; Neisser and D6ring, Berl. klin. Wochen- schr. 1901, No. 22; H. Buchner, Berl. klin. Wochenschr. 1901, No. 33; H. Sachs, page 181. 236 COLLEtTED STUDIES IN IMMU.MTY. by means of serum corresponding to the blood-cella employed has very recently been employed by Buchner * exclusively as a reaction for the presence of normal amboceptors. Although the principle advanced by Gniber as an invariable means of differentiation has failed, we are far from identifying normal and specific amboceptors. As already stated, we believe that in ttip 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 hscmolysia in a foreign serum entirely to the influence of the amboceptors, which he identilies with the agglutinins. He says that "while in themselves incapable of effecting hsemolysis, they put the red blood-cella into such a condition that they allow their haemoglobin to escape e^'en on relatively slight osmotic disturbances." Just these slight osmotic disturbances. according to Bauragarten, are caused by the foreign sera whose osmotic tension is changed by heating (inactivation). Hence Baum- garten regards the assumption of comi)lements as entirely unnecessary. In opposition to this we would like to call to mind the numerous combinations described by us (even Bordct has describetl such for the hemolysins obtained by immunization), in which the blood- cells dissolve in their own serum, i.e. in the ideal isotonic medium, it they have pre\'iously been treated with an inactive serum (ambo- ceptor) of a different species. Such cases clearly indicate that hae- 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 Varlabllftr ot the Complements. The plurality of the complements containeil in a senun has been proved by the most varied experiments. A separation ot the indi- vidual complements of the serum has been undertaken in various sera by means of chemical or thermic influences,^ by binding with ' Buchaer, Berl. klin. Wochenachr. 1901, No. 33. ' Baumgnrten, ibid., No. 5(1. ' Ehrlich and Morgenroth, see pages 11 et seq.; Ehrlich and Siichs, pages I et Mil,; Wendelstadt, CentralblatC f. Boct. 1902, Vol. 31, No. H. COMPLEMENTIBILITY OF THE AMBOCEPTORS. 237 blood-cells loaded with amboceptors,^ by filtration through porous filters,^ and by the action of a partial anticomplement.^ Eut 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 observ ed 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 hiemolytic effect by its.elf. 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 aIso observed marked chrono- logical variations in the senim 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. June 19. June 22. July 15. Amount of Serum. 2.0 1.5 0.5 2 1 0 5 1.0 0.5 2.0 0.6 0.3 Hflenfolyflis of Rabbit Blood (6% 1.0). very little trace 0 trace minimal 0 complete strong Guinea-pig Blood (5% 1.0). 0 0 0 complete little tt 0 0 0 ^ Ehrlich and Sachs, 1. c. ' Ehrlich and Morgenroth, page 56; £. Neisser and D6ring, Berl. klin. Wochenschr. 1901, No. 22. ' Marshall and Morgenroth, pages 222 et seq. 238 COLLECTED STUDIES IN IMMUNITY. Hence within three days the seriim of the horse has become strongly hemolytic for guinea-pig blood without altering its hremo- lytic property for rabbit blood, whereas within a further three weeks its proi^erties 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 coasiderable amount of amboceptor for guinea- pig blood. This ambo^epfor was characterized by a particularly high degree of thermolabilily, 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 sennn that this amboceptor becomes manifest. The cause of this varying hsemolytic property of the horse scrum, which is in contrast to the extraordinarily constant amount of normal hsemolysin 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 propertj', or nearly BO, 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 ia employed pureij' as a source of complement. We ha^'e frequently used horse serum as complement in the following combinations: Number. Blood. Ap.b™p..r. 1 3 5 6 7 S guinea-pig rabbit guinea-pig slieep goat serum aog serum ^og^rX* ueruni of a goat immunized \v[(h fiheep blood Of all these cases only the complement for 6 and for S was present in considerable amounts. So tar 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 AMBOC^EPTORS. ail of the completions of normal amboceptors succeeded, we found in Franlifurt 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 imnmnizing a goat with sheep blood. ^ 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 dbcovered just in the completion of normal amlmceptors, 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 obser\-a- 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 inacti^'e sera by means of several other active sera. For the evaluation of baclericidal sera in anhnal experiments we believe it to be especially important to consider cases of thii 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 themseh'es in the fact that now and then an animal dies of the infection even though in the zone of sufficient immune senim 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 dbtmbing. ' la respect to its complements horae serum occupiea a special place among most other sera used in the laboratory. Thus, for example, we were rarely Buccesaful in complementing the amboceptor of a rabbit immunized with ox blood; ' we never found a complemeuC in hoise Eera for the amboceptors of geese or goats immunized with ox blood. That the locality plays a certain role in these phenomena follows from our obBervationa thai here, in contrast to the slatemonta of so reliable an observer as P. Midler in Grnz, rabbit blood is not dissolved by duck Berum to any appreciuble extent. 'Another abnormal phenomenon nliicli is oft«n observed in this connec- tion, the disturbing action of large amounts of the immune senini, is explained by the peculiar deSection of complements by an excess of amboceptor, as has been determined by M. Neisser and Wechsberg (see pages 120 et scq). 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, Metchnikoff, Wassermann, Schiitze and Scheller). XXIII. THE PRODTTCnON OF ILEMOLYTIC AMBOCEP- TORS BY MEANS OF SERUM INJECTIONS.i A Ckintribution to Our Knowledge of Receptors. By J. MoROENROTH, 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 hemolysins 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 hemolytic 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 receiptors. 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 haemoglobin; 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 Sagella and cilia. The specific binding of the am- boceptors ia therefore not dependent on a coarser or finer morpho- logical structure: i( can occur wherever the spedficaUy related receptors are present. For the doctrine of immunity these views constitute a new and really 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 aa to the nature of the cell's receptors. Every ■product of immunizalion is specific for those receptors by which it was cailed forth, iirespeetii'e of where tlie receptors may 6e.' When injected into an animal the receptor produces antibodies, and these again, when they meet the receptor under suitable conditions, are bound 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 oblaioB Jutmolytic amboceptors by injectioDs of ciliated epithelial debris, such aa is contained ia goat milk. v. Dungem ' has very properly pjointed out thiH fact in emphasising the community of the receptors. The same holds tnie for the h«DmoI}^ic am- boceptors obtained by Moxter ' by injections of spermatozoa. Sever&l 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 aa 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-cells in Schiussbetrachtungen, Vol. VIII, of Nothnagels spczielle ■pathol. und Therspie, Vienna, 1901. 'v. Dungem. Miinch. med. Wochenschr, 1899, No. 38. 'Moxler, Deutsche med. Wochenschr. 1900, No. I. ' Ehrlich and Moi^nroth, page 88. ' It has already been shown that as a result of injection of amboceptors into e animals a considerable number of cell receptors are thrust off, whidi PRODUCTION OF H.^MOLYTIC AHBOCEFTORS. 243 The extraordinary multiplicity of such dissolved substances in blood serum has already been pointed out by Ehrlich.^ "The chief tools of the internal metabolism arc the receptors of the first, second, and third order. They are constantly being used up and produced anew, and can readOy 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 surjDrising 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 tj-sins, 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 wa}'. 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 hiemolysin directed against chicken erj'throcytes as a result of injections of chicken serum into guinea-pig serum, and Tschiatovitseh * has observed the formation of a htemolysin (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 er>-throcytes. 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 Ehrlieh and Morgenroth had shown to be due to an anti-immune body.' I am led to publish these experiments now then fimctionaie as i 23andS8. ' Ehrlieh, Sclilussbetrachtungen, 1. e. • V. Diingem, Miinch. med. Wochenschr. 1891 ' TschiBto^-ilsch, Annal. Inst. Pasteur, 1899. bodies. See Ehrlicli and Morgenroth, pages 'The increase in ha^molytit the iniectioD of chichen blood-pla I90I)> reatH apparently only on ar fflent of new amboceptors. ' See pages 88 ot seq. I of rabbit scrum for chicken blood after described by Nolf (Anna!. Inst. Pasteur, of complement, uot on the develop- 244 COLLECTED STUDIES IN IMMUNITY. because of a rattier important contradiction which exists between tht'in and certain exiJcrinienta recently published by Schattenfroh.' This author found tlmt one can produce htmutlytic immune bodies for goat blood by injecting rabblta with goai 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 antiamboccptor action of the serum prevented this, and |K)inted to the presence in this of the dissoh-ed receptors. .\8 a matter of fact, therefore, the "interesting contradiction" described by Schattenfroh aa existing between the action of the urine and the senmi does not obtain; for it is possible by injecting rabbits with goat serum completely deprived of blood-cells to produce specific amlK)ceptors. 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, readily and surely demonstrated if one attends to certain fine details. As a rule a hscmolytic serum obtained by specific immunization will, when fresh, dissolve the corresponding blood-cells; for, as v. Dungem has shown, in immunization with blood-cells the comple- menta 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 scrum 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 a nticom piemen ts directed against the complements of their own senma. These a nti complements are therefore to be regarded aa auto-anticomplements. They not only suffice to neutrahze the complements present in the senmi, but are al)le to bind complement eubaetiucntly added. Thus the amboceptor of a rabbit mixed with goat serum is completely o!>scured; for if the immune serum ia employed frcah, the fitting complements enabling it to act are lacking, while if the serum is inactivated and one seeks ' Miinch. med. Wochenschr. 1901, No. 31. ' See pages 71 et seq. PRODUCTION OF ILEMOL'iTIC AMBOCEPTORS. 245 to activate it by the addition of normal rabbit serum, the comjije- ments of the latter will be made inert by the auto-anticomplenient preaent. 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-eells are mixod with the serum of the immunized rabbits and the mixture allowed to stand until the amboceptors present have lieen bound by the blood-eells. The latter are then separated by centrifuge, the supernatant fluid which contains the cause of the trouble, the auto-antiromplement, being removed. If the blood-cells are now mixed witli fresh normal rabbit serum, the hemolysis which ensues in the incubator will show the presence of the anchored amboceptor. iShould this method, which guards against all errore, 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 haa been found that normal rabbit serum possesses a con- siderable though variable hemolytic action for goat blood (see Table I). Tlie 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 ver}- little, even though considerable comple- ment ia present. (See Table I.) l"he 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 3S° 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- COLLECTED STUDIES IS iMMUKITY. TABLE 1. F Goat Bix)od (t cc. 5%) bt Fresh Serum or Nobmal RABsm. K2 .. 11. ■■■ IV. V. V. VII. 0.05 Sa ""ihtta" liul. ve?y mc£ compleu fair 0 0,25 0.O75 OOffipteU. compbt, itrong ^ eotnplete ™mpk«< »f!one oom Plate Blroog 0,05 0.025 ■• \ c.1o%C ( 2 = ■■ = Sf 1 0 0 '-"s"" 0 flint trace 0 "v" -£?• J of O.S. do not by tbEo» vation was effected by the addition of normal guinea-pig Benun. The ha^molytic action of the immune sera is seen in Table II. Rabbits were treated nitb goat serum whieh had been ca.refull}' freed from aU blood-cells by continued ceatrifuging. Usually the serum was inactivated by heating It to 55° C. for liolf an hour, then it waa injected intra peritoneally. As a rule the animals received two to three injeetions of increasing doses of serum, in oil about 35-90 cc. More frequent injeetions caused no greater formatioD of amboceptorR, a behavior which corresponds to that seen with the injection of ox blood or gout blood. These experiinenls show that specific amboceptors were developed in all the rabbita treated with goat serum. Quantitatively this was subject to individual fluctuations just as is seen following the injec- tion of blood-ceils; in some cases the development was quite con- siderable. Most of the sera were examined itesh for their action on ox blood, and invariably showed themselves without action even in doses of 0.5 cc' The addition of largo amounts of normal rabbit ' The method here emjdoyed to disclose amboceptors whose presence is masked can often be used with Bucees.s. Dr. Marsliall and I shall shortly report an analogous case dealing with the amhoeeptort at a pathological exudate. PRODUCTION OF HiEMOLYTIC 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 Haemolysis. 0.05 cc. 0.05 " Serum rabbit I n tt tt ni. 0.25 tt B. Blood+ahboceftor+0.1-0.2 Guinea-piq Serum as Complement. Serum rabbit IV 0.1 cc. V 0.05 " VI 0.05 " VII 0.028 " Vin 0.013 " IX more than 0.25 X 0.05 XI less than 0.05 tt tt tt tt tt tt tt I < tt tt serum does not suflSce 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 0.26 '* 0.15" 0.1 tt 0 trace tt very little 0.075 cc very little 0.05 " " 0.025 " trace tt The abnormal course of this slight haemolysis 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 24S COLLECTED STUDFES IN LMMUNITY. each specimen, which was then kept on a water-bath at 38° C. for une hour, after which the mixtures were centrifuged. The blood- cell SEsdiment was again susi>ended 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. +0,3 Anliamboceplor. + 0,.'i Normal Inactive UoBl Serum. 0.25 0.15 0-1 0.075 0.05 0,025 complete solution strong little very little 0 0 complete Kolution strong 0.2 complete solution complete mlution 0,15 fltrong 0.1 0.075 0-06 0 0.05 0 moderate 0,025 0 little 0.012 0 0.009 0 0 TTie antiamboceptor b thus seen to ofTer exactly the same pro- tection agaiiLst the amboceptors obtained as a result of goat-blood injections and those resulting from goal-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 HiEMOLYTIC 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 fimction. 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 imsuita- ble process. In fact the free receptors evidently do not generally possess the character of antiautolysins, as Besredka ^ believes, for by injecting a rabbit with ox serum it was impossible to obtain any haemolytic 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." Subsequent Note, — In a recently published study (Miinch. med. Wochen- schr. 1902, No. 32) P. Th. Miiller reports on the production of hflBmolytic amboceptors by treating pigeons with guinea-pig serum, and he accepts the views here developed. ^ Besredka, Annal. de ITnstitut Pasteur, Oct. 1901. ' London, Arch, des Sciences biologiques, St. Peterabuig. 232 COLLECTED STUDIES IN IMMUNITY. The 6gures in Table 1 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 anumrHs of amboceptor smaller doses of complemenl suffice for complete biatwlysis. The relation is not exactly the same in the se])arate 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 — , 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 casea reproduced in Table II, in which the combination was ox blood-}- the amboceptor ot specifically immuoized rabbitsH-guinea-pig serum or rabbit serum as complement. Ambowplor. .h^-frrn?,if CompUiBcnt SufGcwnt for the ^oimU ot 0.002 IX 0.035 1 o.ooe 2iX 0,015 2l 0.01 SX 0.01 3:5 0.05 25 X 0.008 1 4.4 0.1 MX 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 II— ConHnued. B. The Same, but Rabbit 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 • 0.005 IX 0.5 1 0.01 2X 0.17 1 2.9 0.05 10 X 0.12 1 4.2 0.1 20X 0.14 1 3.6 0.2 40X 0.14 1 3.6 0.4 SOX I 0.15 I. 1 3.3 0.005 IX 0.6 1 0.01 2X 0.17 1 2.5 0.05 10 X 0.12 1 5 0.1 20 X 0.14 1 4.3 0.2 40X 0.14 1 4.3 0.4 SOX 0.15 1 4 II I. 0.005 IX 0.75 1 0.0075 lix 0.6 1 1.25 0.015 3X 0.14 1 5.3 0.03 6X 0.17 1 4.4 0.06 12X 0.14 1 5.3 0.12 24 X 0.12 1 6.3 254 COLLECTED STaDIES IN IMMUNITY. Here we see that the emplojinent even of \'ery high multiples of the amtoceptor effects a reduction in the amount of complement required of one-third to one-aixth 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 m which different kinds of complement are added, namely, sheep serum and horse serum. These cases constituts 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 whitk the decrease of complement required is but slightly or not at all affected by an increase in the anwunt of amboceptor. We see therefore that with 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 affinity. 3. TTie plurality of the amboceptors. So far as the first point is concerned we know that the amount of receptors of the red blood-cells may exhibit great differences in any individual case.^ 'Weuse the term " umliocpptor unit" to sjiecify that amount of nmboceptor which on the adiiilion of the optimal amount of complement just HUfiieesfor com- plete hiEmolyBiH. In the same sense R. Pteiffer unea the t«nn "immunity unit" when speaking of bactericidal sera. Corresponding to the smboceptor unit the "receptor unit" is that amount of receptor which binds the amboceptor 'See Ehrlich, Schlusabetrachtungen in Nothnagels spec. P.ithologie und Therapie, Vol.MII, Vienna, Holder. 1901; and Thrlichnnd Morgcnrolh. page 71. AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 255. TABLE III. A. 1 cc. 6% Sheep Blood + Amboceptor of Goats Treated with Sheep- Blood + Sheep Serum as Complement. B. The Same , but with Horse Serum as Complement. Amount of the Amboceptor. Proportion of the Amount of Amboceptor. Amount of Complement which Huffioes for Complete Solution. Proportion of the Amounte of Complement. A. 0.1 1 X 0.15 1 0.25 2.6X 0.035 1 4.3 0.5 5 X 0.05 1 3 0.75 7.5X 0.05-0.036 3^4.3 B. O.l IX 0.5 almost 1 0.2 2X [complete 0.1 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. Keififer) 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. 6% Ox Blood + Amboceptor op Goats Treated with Ox Bloods- 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 SufiSoes for Complete Solution. Proportion of the Amounts of Complement. 0.1 0.2 0.4 0.8 IX 2X 4X 8X 0.01 0.01 0.01 0.01 1 1 1 1 B. 0.1 IX •0.15 1 0.2 2X 0.15 1 0.4 4X 0.15 1 0.8 8X 0.15 1 c. 0.1 0.2 0.4 IX 2X 4X 0.1 0.1 0.1 1 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 cytophUe 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. AMBOCEPTOK, COMPLEMENT. AKD ANTICOMPLEMENT. 257 the requireti equi\'nlence will already be reached with the amount of complement just sufficient for solution, and ^n 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 ancliored amboceptor for the complement is only ver\' 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 ts in excess, the more of the completed combinalion will remain intact. Hence if there are very few receptor units in the blood-cells, it will be necessary to add ^'ery 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 arc present, less complement will suffice. The tables here given present numerous considerations which show that little amboceptor + muck complement and mvck amboceptor + little complement lead to the formation of the same- amount of complement-amboceptor combination (hemolysin unit) anchored by the receptors. A most conspicuous rfile, however, is played by the fact that the immune serum is not a simple substance, hut is made up of partial ambo- ceptors to which various dominant complements of Ike sera correspond. Of especial importance in this respect are partial amboceptors present in immune serum in small amounts (and which tlicrefore can only come into action when high multiples of the immune scrum are employed), but which, for their completion, find a partial complement which is particularly -plentijiil in the completing serum. Such a partial amboceptor present in these small amounts (such, for example, as haa been demonstrated in the senim of rabbits treated with ox blood) constitutes one of the main explanations for the phenomena above described. From these considerations wo see that the various phenomena which we obser\'e 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 COLLECTED STUDIES IN IMMUNITY. n. Amonnt ol Amboceptor and Antlcomplement Required. The following observations deal with the quantitative relations existing between the amount of amboceptor and that of the anticom- plemcnt required to prevent hemolysis, In a number of cases we determined the amount of anlicomplement which just suffices to prevent the solution of red blood-cells loaded with varying amounts of amboceptor, when that amount of complement was present which always just sufficed for complete solution. The majority of our experiments again refer to the solution of Bheep blood by an immune serum (deri\'ed from a goat) whose ambo- ceptor ifl complemented by guinea-pig serum. This, it will be re- calletl, is the case in which with large amounts of amboceptor the complement required decreases considerably. For the antlcomple- 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 communication, 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 antieomplement required in each instance to effect neutrali- zation was determined, whereupon complement and antieomplement 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 hemolysis, the pecuhar behavior is obscrv-ed that with small amounts of amboceptor 0.015 cc. antieomplement scrum neutralize the complement of 0.05 in guinea- pig serum, whereas with large amounts of amboceptor 0.35 cc, anti- complement senun are required to neutralize 0.006 guinea-pig serum. If we calculate the amount of complementing serum neutralized in both cases by 1 cc. antieomplement serum, we find that in one case it is 3.3 CO., in the other 0.017 ce. Hence when large nmonnta of ambo- ceptor are employed the antieomplement acts 195 times weaker. The required amount of antieomplement 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 b 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 anticomplemeni required is far from being 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. 0.3 0.06 0.01 0.005 Amount of the Complement Sufficient for Complete Solution. 0.005 0.005 0.01 0.035 B. Amount of a b e 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 0 0 0 0.25 faint trace 0 0 0 0.15 trace 0 0 0 0.1 II 0 0 0 0.075 moderate faint trace 0 0 0.05 complete trace faint trace 0 0.035 moderate little 0 0.025 II complete II 0 0.015 II . *' complete 0 0.01 II tt li faint trace 0 II 11 tt 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. !C0 COLLECTED STUDIES IN IMMUNITY. TABLE VI. Oi Blood +Ambocf.ptoi( of an Ox-blood Rabbit+Gitinba-ph) Seruu. Amtwcepior. ESeot CompleM Bolutioa. 0,2 O.OlM 0.05 0,075 AnlicoDiplB. ^^^X-0%, "SSSS-oT 0 75 0.5 0.35 0.25 0.15 0.1 0.075 0 05 0.035 0.025 0 015 0.01 0 strong almost complete 0 0 0 0 0 0 trace little strong almost complet* The study of the phenomena of uiimuniKatinn has taught us that nothing is so liable to error a-s premature generalization. Hence wc 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, imih this difference, however, that in this case the anti- complement was not the same, since it was derived from a rtibbit 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 oa is made necessary by the smaller amount of amboce]>tor, requires ten times as much anticomplement as it does with one-tenth the quantity of complement when lai^r amounts of amboceptor arc used. AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 261 TABLE Vll. Amounl of Compleui Solution. Aaiuuiit ot Comi.le- ™ptoU. T«'t. Amount of AnHeom- jjltmeat Inquired for Complete Neu- 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 l>etween complement and amount of anticompleraent required with different quantities of amboceptor, In other cases there b a wide divergence. How are these phenomena to be explained? To begin, let us assimie for the sake of simplicity that comple- ment and an ti complement are of simple constitution. In that case, it, aa all our experiments show, the affinity of complement is much greater for anticomplemcnt than for amboceptor, the neulralization of complement and anticomplement should follow etoichiomelric laws. As a matter of fact this is what we found in the last case (1 able VII). In the first two eases, however, the results diverge so widely from this, and are moreover so far beyond the limile 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 pluTulily oj the coviple- menls and anticompiemerUs. 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 tbe 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 {Ai and Bt), namely, an identity in the haptophore group. FoDowing the terminology of the theory of numlwrs in which "friendly ntimliers" {numeri amicahilcH) are fpoken of. one could designnte complements of difTerent species which correspond in their haptophore groupg, &s "friendly complements." 4 262 (X)LLECTED STUDIES IN IMMUNITY. Now if one injects any serum containing two dififerent 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 anticoraplement will be directed for the greater part against A in the one case, and against B in the other. It is therefore readily understood that with isogenic sera the yield of anticomplements can correspond fairly well to the nuxture 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 fitt^ fjo 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 -f Amboceptor of a Human-blood Rabbit + Rabbit Serum 4- Anticomplement from the Goat Treated with Rabbit Serum. Amount of Amboceptor. Amount of Ck>mplement Neoeasary for Complete Solution. 0.2 0.2 0.05 0.05 0.05 0.075 Anticomplemen t . Amboceptor, 0.2. Amboceptor, 0.1. Amboceptor, 0.05. Complement, 0.05 Complement, 0.05. Complement, 0.1. 0.1 0 0 0 0.075 0 0 0 0.05 trace 0 0 0 . 035 < ( 0 0 0.025 little trace 0 0.015 moderate < < trace 0.01 almost complete little moderate 0 complete complete 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, CUMPLEME.NT, AJJD ANTI COMPLEMENT. 263 A priori, it i3, of course, conceivable that in the rabbit the complements Ai and Bi exist exactly in the same projxirtion 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 alloiogenic anticomplement will result in a serum in which the proportion of the two anticoraplements 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 Lwgenic anticomplement serum produced by guinea-pig serum {presupposing that its constitution is uniform) will neutralize guinea-pig serum in euch 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 -4 Is completely neutralized, but that a larger or smaller excess of com- plement B is still 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 amboceptor comes into action which ia present in the immune serum in relatively small quantity. This partial aralxjceptor is complemented by complement B contained in guinea-pig aenim, whereas the preix>nderating amboceptor is sensitized by comple- ment A. Complement B finds a plentiful amount of anticomple- ment in the isogenic immune serum, liut not in the alloiogenic serum. In the latter case, therefore, disproportionately much serum contain- ing B anticomplement wOl 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 l.iy 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 cndeavorerl to establish this point of view on a wider experimental basis. With this end in view we first used small amounts of amboeejitor, adding \'ariou3 multiples of the complementing dose •i04 COLLECTED STUDIES IN IMMUNITY. of serum and then determining the amount of anticomplement required in each ease. In one of the experiments we made a parallel test with a Uirge excess of amboceptors. The results showed that under 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% Shbbp Blood + Amboceptor of Goats Iumumzed with Sheep Blood + Giiinba-piu Sescm ^ The serum of a goat treated with rabbit serum, i Neceuary lor Con A. LiUU Amboceptor ( — Amboceptvr Unit). I 0.22 B. Much Amboceptor (=25 Amboceptor Units), 0.125 I 0.006 I 0.24 0.125 0-012 0.42 0.125 0.024 0 8 1 CC, 5% Ox Blood + .^MBocEPTon of a Goat Immunized wrra Ox Blood+ Rabbit Sehum ab Coupleuent. The Mrum of a goat treated nilh rabbit seruin as anticompletneiit. Afflount of ..fe"^.. 0.16* 0.15 0,15 0.2 0.1 0 05 0.1 0,05 0 025 Here, then, we are dealing with the same phenomenon which in the domain of antitoxin immunity we know as the muUiplicoHon 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, quadrupling, etc., of the AMBOCEPTOR. COMPLEMENT, AND ANTICOMPLEMENT. 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 amboceptor 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 amboccptora varies. The relation just described indicates that we are here dealing mUk a chemical process jollcrwing stoichiometric laws. We should like to menlion further tbat this peculiar behavior observed by US is of Bome importanc* in refuting an objection made by Gniber (I. c.) against Wechsberg, Ah is well known, Gruber believed he had shown that in the bactericidal eera anticomplementa were present produced by the immuniza- tion. This he held (o be very important, tiince according to his view it showed tbat the deflection of complements by exceos of amboceptors, whieh had been described by Neisser and Wechsberg, was incorrect. This is not the place to enter into the great improbability of Gru be r's deductions, for this has already been well pointed out by Wechsberg, by Lipstein,' and by I,evaditi.' Wechs- berg * repeated Gruber's experiments, but was unable to confirm Ids results. Sacha also wiis unable to do this. Gruber has now objected to Wechsberg'a work on the score of a gross error, saying that Wechsberg worked with weakly sensitized blood-^^Us, whereas he had used strongly sensitized blood-cells. Wechsberg had therefore used considerably more complement than he. and had in connequence required much more anticomplement for neutralization, so tbat the presence of sniall quantities of anticomplement could easily have escaped Wechsberg. From what has been said abovt. kovrever, Jutt the contrary occurs; uitk itUaio- ffenie sera larger amounls of anticomjiUment arc used. That the anticomple- ment which would be produced artificially by injections of bacteria (even if that be r^arded as conceivable) would eminently be alloiogenic need not further be emphasized. It is shown by Table VIII tliat the conditions which Gruber assumed to exiat do not obtain, even with an isogenic anticomplement, in Gruber's case (human blood + human-blood rabbit +rabbit scrumV It is unnecessary to enter further into Gruber's objections, for Wechsberg ' has succeeded through the demonstration of complementophile amboceptoids in finding the source of the differences. These amboceptoids have tneantime been found independently by E. Neisser and Friedemann ' and by P. Th. Miiller,' It is immaterial in judging of this phenomenon whether in the anticomple- ment^ary sera used by Gniber the diverting amboceptoids developed as a result of long standing or under the inSuence 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. 1002. No. 25. 'Wechsberg, Wiener klin. Wochenschr, 1902, Nos. 13 and 28. *Ibid, ■ Neisser and FViedemann, Berl, klin. Wochenschr. 1902. No. 29. • P. Th. Miiller, Miinch. med. Wochenschr. 1902, No. 32. 266 COLLECTED STUDIES IN IMMUNITY. by him as an objection conBtitutes a new and telling demonstration of the correctness of the amboceptor theory. Thus we see that the antieomplement experiments give us a further insight into the mechanism of hemolysin action. This in its turn shows that the simple unitarian 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 HiEMOLYTIC PROPERTIES OF ORGAN EXTRACTS.* By Dr. S. Kobschun, of Charkow, and Dr. J. Morgenroth, Member of the Institute. The first observations concerning the hsemolytic properties of organ extracts were published, so far as we are aware, by Metchni- kofr.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 haemolytic 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 eases the spleen. Of the other organs the pancreas showed a marked, and the salivary glands a weak haemolytic action; the bone marrow, liver, kidney, brain and spinal cord, ovaries, testicles, and adrenals were inert. Metchnikoff found the haemolytic 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 PInstit. Pasteiu:, Oct. 1899; see further references in Metchnikoff, rimmunit^, Paris, 1901. ' Metchnikoff and his pupils use the term "cytase" for our complements as well as for the complex cytotoxins (ha?molysins, 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 8TCTD1ES IN IMMUNITY. by its behavior toward heat, completely losing ita action on being heai«d to 56° C. for three-quarters of an hour. Observations in this same direction have been made by Shibayama * and Klein,^ and a comprehensive study by Tarassevitach ^ has recently appeared from MetchniliofT's laboratory. Shibayama, working in Kitjisoto's lalwratory, studied the action of extracts of guinea-pig organs on dog blood and obtained hiemolysia 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 htemolytic substances of the organs and the specific hsemolysins which appear in the scrum 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 htemolytic action of the extract of pancreas; in a few cases the ex- tract of kidney and of intestinal mucosa also dissolved the red blood- ed Ls. Metchnikoff's experiments were continued in his laboratory by Tarassevitsch, who studied principally the organs of guinear-pigs, rabbits, and dogs. Corresponding to Metchnikoff's first experiments, he tested the hemolytic 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 hiemolj'tic. Besides this pancreas extract and in many cases salivary gland extract were hcemolytic. In general the hemolytic action of the organ extracts of rabbits is weaker than that from the organs of guinea-pigs. Omentum, spleen, and mesenteric glands frequently were ha^molytic; the salivary glands acted feebly; bone marrow, liver, and thymus were not hiemolytic. According to Taraasri-Hsck, therefore, only Ike macro-phagic organs arid the digestive glands possess a heriments. (See Table I.) clumps in the organ extract.^ which before had been free from visible particles. These clumps tould be (separated by centrifuge, and cTdiibiled a hiemolytio action when suspended in salt solution. THE affiMOLYTIC PROPERTIES OF ORGAN EXTRACTS. 271 TABLE I. Emulsion of Mouse Intestinb (10%). 1 CO. 5% Ox Blood. 1 CO. 5% Guinea- pis Blood. 1 eo. 5% Mouse Blood. 1.0 0.76 0.6 0.35 0.25 0.2 0.15 complete n almost complete trace 0 0 0 complete complete it tt trAce 0 0 Emulsion of Beef Pancreas (10%). 1 cc. 5% Rabbit Blood. 1 ec. 5% Guinea- pig Blood. 1 oc. 5% Ox Blood. 0.6 0.35 0.25 0.15 complete 11 strong 0(?) complete 0 0 0 complete 0 0 0 These experiments show that the susceptibility of the body's own blood may be very great, even as great as that of a foreign species of blood. Whether aU these extracts dissolve the blood of the own individual we have not determined; we regard it as probable, however, since positive results were obtained in all experiments which we made in this direction, especially with extracts of mouse intestine and of guinea-pig stomach. These experiments (especially those with the extract of guinea- pig spleen, which Shibayama too found to be active only for dog blood) show that we are not here dealing vnth hasmolytic poisons of a general kind (such as saponin, the gallic acid salts, and certain alka- loids, like solanin, which dissolve all blood-cells regardless of species), but that these hsemolytic poisons possess a certain specificity which is of special biologic interest. The property of organ extracts to dissolve the blood-cells from the same individual is of great significance because neither when normal nor after immimizing procedures does the blood-serum of these animals ever contain substances which damage the blood-cells of the animal itself (autohsemolysins). Tarassevitsch himself noticed the great dif- ference existing, on the one hand, between the absence of a marked haemolytic action of guinea-pig serum on foreign species of blood and the strong hsemolytic action of the extracts of certain guineorpig 272 COLLECTED STUDIES IN IMMUNITY. organs, on the other. He believes to explain this by assuming a difference in tlie macrocytaae extracted from the organs and that present in the senmi. In any case this constitutes a serious dilemma for Tarassevitsch; for either there are several "macrocytases" as opposed to the unitarian view ot Metchnilcoff or the macrocytase of eenim is identical with llmt of the organ extracts. In liew of this entirely different behavior, however, the latter does not appear acceptable to Tarasse\it3ch. Our first question was an entirely different one, for In all the cases of haemolysis and bacteriolysis sufficiently examined we had never met with a simple ale\in in the sense of Buchner and Metchnikoff, but invariably found a coaction of amboceptor and complement. In view of this our investigations had, above all, to determine whether the hfemolytic organ extracts could be shown to be characterized by complement and amboceptor. These first doubts, namely, whether these substances corresponded to what we concei^'C as the comjilex hemolysins of blood-serum, led us to study the hiemolytic organs in respect to those main character- istics which we have come to know in our study of the complex hie- molysins. These are: 1. The behavior toward thermic influences. 2. The behavior when bound to the red blood-cells at low tempera- tures. 3. The power of producing antibodies by immunization. We shall begin by describing a number of typical experiments which show the behavior of the organ extracts toward higher temperature. Let us glance first at the cxiwrimcnta dealing with the effect of organ extracts on goose blood-cells, for this is the blood species which has been mainly used by Metchnikoff and Tarassevitsch. (See Table II.) These exj^riraents clearly show that in most of the cases the hiemolytic action of organ extracts on goose blood-celts is not at all or but slightly affected by a three-hour heating, to 62" C. and that heating to 100° C. for one hour and even for three hours does not produce any further damage. Onlj' the hteraolytic effect of extract of mouse intestine is reduced to about one-half by the heating to 62° C; heating to 100° C. for three hours causes but little additional damage. But that this cannot be a true destruction of part of the htemolysin will be discussed lat«r. We wbh next to pre.aent additional experiments dealing with the behavior of heated organ emulsions on guinea-pig blood. (See Table III.) Nor is this result changed if stronger agents, such ns alkalies or acids, are employed at high temperatures, (See Table IV.) THE HiEMOLYTIC PROPERTIES OF ORGAN EXTRACTS. 273 TABLE II. A. Action of Heated Organ Extracts on Goose Blood-cells (1 cc. 5%). I. Extract of Dog Spleen (10%). Not Heated. 8 Hra. (62«»). 0.2 0.15 0.1 complete solution it ). 0.25 0.15 0.1 0.075 complete trace 0 complete 0 0 complete it faint trace 0 II. Extract of Ox Pancreas (10%). Not Heated. 1 Hr. (62»). 0.35 0.25 0.15 complete strong complete strong III. Extract of Ox Pancreas (20%). Not Heated. 1 Hr. W). U Hrs.dOO*). 0.15 0.1 0.075 0.05 complete << trace 0 complete n trace faint trace • complete « i trace 0 IV. Extract of Guinea-pig Stomach (10%). Not Heated. 3 Hre. (65«). 0.25 0.2 0.15 complete strong complete strong TABLE IV. Extract op Ox Pancreas (10%). 0.35 0.25 0.15 0.1 Not Treated. complete tt faint trace 0 Containing 1/50 n. HCl Heated to 60<> for 30 Min. and Neutralised complete almost complete 0 0 Containing 1 /50 n NaOH Heated to 60'' for 30 Min. and Neutralised. complete almost complete 0 0 THE HiEMOLYTIC PROPERTIES OF ORGAN EXTRACTS. 276 All these experiments show that the organ extracts will bear heating to 62-68® C. for hours, and even 100*^ for several hours, with- out suffering any change in their hsemolytic properties worth men- tioning. In these experiments, in fact, we have been unable thus far to find any limit for the thermostability of the organ extracts. We are therefore dealing with substances which withstand boiling (coctostabile), and this fact in itself is suflScient to disprove the assump- tion that they are "cytases." The next question, of course, is how such a fundamental divergence between our results and those from Metchnikoff's highly esteemed laboratory can be explained. We think we have discovered the cause of this difference. It is as follows: In the above experiments it is of the greatest importance to shake the fluid previous to testing its haemolytic property; in that way the more or less plentiful precipitate formed on heating is again uniformly distributed throughout the fluid. Only the coagulum produced by heating possesses a hsemolytic action. According to our experience, if a precipitate has been produced through heating, the clear fluid which is separated from this no longer possesses any luemolysin what- ever. If the precipitate is separated by centrifuge the clear fluid will be found inert; on suspending the sediment in the requisite quantity of physiological salt solution a new emulsion is obtained which has preserved the hsemolytic property. This is shown in the following table.i According to these experiments it would seem very probable that the contradictory results obtained by us on the one hand and by Metchnikoff and Tarassevitsch on the other are due to insufficient regard being paid by the latter to the precipitates formed in the organ extracts on heating. If we assume that the hsemolytic, coctostable substance is present ^ The coagula formed on heating may be so plentiful that they render an exact observation of hsemolysis exceedingly difficult. It is frequently seen that haemolysis by means of heated organ extracts which are filled with coagula proceeds very slowly; apparently the precipitates offer considerable resistance to the escape of the hsemolytic substance. Naturally, this constitutes a source of error, since with low temperature and too short a time for observation the hsemolytic action is underrated. This may also explain the occasional weaken- ing of heated organ extracts, to which we have already referred ; in that case the weakening would not be due to a partial destruction of the hsemolytic substance. 276 COLLECTED STUDIES IN IMMUNITY. TABLE V. I. Extract op Doa Lymph Glands (10%). Guinea-pig blood (1 cc. 5%). 2.0 1.5 1.0 0.75 0.5 0.25 0.15 Fresh. complete tt tt strong 1 Hr. (62»). (No Coagulum.) complete tt tt strong very little 1 Hr. (lOO*). Slight Precipitate, Oentrifuoea, and Suspended in Salt Solution. complete tt tt complete 1 Hr. (lOO**). The Clear nuid obtained by Gentrifug^ic. 0 0 0 0 II. Extract op Doo Pancreas (20%). Guinea-pig blood (1 cc. 5%). Fresh. 1 Hour (62«). (No 0>agiilum.) 1 Hr. (lOO"). Slight Precipitate. Centrifuged, and Suspended in Salt Solution. 1 Hr. (100»). The (3ear Fluid obtained by Cbntrifuging. 2.0 1.5 * 1.0 ,v 0.75 ^ 0.5 0.25 0.15 complete i t tt little . tt complete tt little 0 0 complete tt moderate 0 0 0 0 0 ;t III. Extract of Dog Intestine (10%). r Gc^pse blood (1 cc. 5%). 1 Hr. (100°). Precipitate again Uniformly Distributed, 1 Hr. (100°). Precipitate after Centri- fuginff. Suspended in Salt Solution. IHr. (100«). Ontrifuged Fluid still somewhat (3oudy. 1.5 1.0 0.75 0.5 0.35 0.25 complete. tt •j^ it *' It almost complete complete tt tt almost complete 0 b'ttle trace 0 0 0 0 0.2 — — 0 0.15 ^^^w 0 THE HEMOLYTIC PROPERTIES OF ORGAN EXTRACTS. 277 TABLE V—Continwd. IV. EmtAcT OF Mouse iKTEsriNii (10%). Goose blood (1 ec.5%). 3h™. HOO"). 3 H (100°) CleaiC^nTrif^ Fluid. Pwipiutc BKAln Uniformly Dialnbuled. '"rai-aSs" 1,0 complete _ 0 0.75 cot,,plet« 0.5 0 0.35 strong 0 0.25 moderate 0.2 0 15 little faint trace 0 1 very little minimiU ~ in the organ extracts in dissolved form we find it difficult to under- stand tfie fact that it is abstracted from the fluid by means of the coagulum formed on heating. To be sure, one could think of an absorption by the coagulum. The complete abstraction by means of heating is, however, readily understood if the hjemolytic substance is present, not in solution, but in a state of finest suspension; for it is a matter of common experience that substances finely suspended in a fluid are carried down with a precipitate produced in the fluid. Tlie technitjue of clearing cloudy fluids rests to a large extent on such precipitations. We have not yet been able to decide definitely whether the hae- molytic substance is present in the fluid in dissolved form or in veiy fine suspension; we incline strongly to the latter view. We base this (1) on numerous experiences which show that by filtering tlie organ extracts through porous filtering candles the fluid obtained is entirely inert; (2) on the behavior of the hsemolytic substance when treated with alcohol. One part of a 1% extract of ox pancreas is mixed with ten parts 96% alcohol, and after a time the fluid is filtered off from the flaky precipitate which has formed. The entirely clear filtrate is distilled in vacuo and the portion left behind mixed with physiological salt solution. A coarsely flocculent suspension is thus obtained which possesses strong hiemolytic action, about one^half to one-third of the original strength. If this mixture is now filtered, the clear filtrate is found to be absolutely inert, whereas the flakes washed from the filter exhibit almost the full hemolytic effect. The following experi- ment will serve as an example. 278 COLLECTED STUDIES IN IMMUNITY. TABLE VI. Guinea-pig Blood (1 cc. 5%), Extract of Ox Pancreas (10%). Portion Left from the Alcoholic Distillate Suspended in 0.85% Salt Solu- tion. Total Fluid. Clear Filtrate Suspenaioii of the Flakes. 1.0 0.5 0.35 0.25 0.15 0.1 complete complete moderate 0 0 0 0 0 0 complete << l( (< strong trace We are therefore evidently dealing with a substance which in the above treatment is dissolved in the alcoholic fluid but which is soluble to only a very slight degree in salt solution. Naturally a certain degree of solubility is always one of the con- ditions of the hemolytic action observed, but this need only be a minimal one. The blood-cells can anchor the amount of hemolytic substance in solution at any given time and so render the fluid capable of taking up small amounts of the substance anew. This conception of a relative insolubility of the substance is readily reconciled with the hsemolytic action. The process which takes place reminds one of that occurring with certain dyes, which, although not given oflf to the water from the dyed fibre, are nevertheless able by means of the water}' medium to go from the dyed to undyed fibres. The coctostability of the haemolytic substances of organ extracts, their adherence to solid particles, their solubility in alcohol — ^all these; in our opinion, show that these substances cannot be classed as iden- tical either with the "cytases^' of Metchnikoff or with our complex haemolysins. Nevertheless we have still further examined these substances for properties which characterize the hemolysins. In one case, therefore, we studied the action of our organ emulsion on blood-cells at 0° C. in order to determine the possiblity of separating a possible amboceptor and complement. To each 1 cc. of a 5% suspension of guinea-pig blood which had been thoroughly cooled on ice, var\'ing amounts of cooled extract of ox pancreas were added and the mixture kept at 0° for two hours and frequently shaken. In, this case slight solution occurred only with large quantities of the extract. Then the mixtures were cen- trifuged, the sediment resuspended in vsalt solution (1.5 cc.),and the THE HiEMOLYTIC PROPERTIES OF ORGAN EXTRACTS. 279 decanted fluid mixed with 0.05 cc. of guinea-pig blood freed from serum. (See Table VII.) TABLE VII. GuiNBA-PiQ Blood (1 cc. 5%). Pancreas Extract. Solution at the End of Two Hours at 0°. Haemolysis with the Decanted Fluid. Hemolysis of Sediment. Control, Absolute Action in Warmth. 1.0 0.5 0.35 0.25 0.15 little 0 0 0 0 complete 0 0 0 0 complete <« almost complete strong complete it tt strong We see, therefore, that at 0° the single solvent dose has been com- pletely anchored by the blood-cells and that after centrifuging this leads to complete solution at higher temperatures; double the solvent dose is still completely anchored by the blood-cells. This condition of affairs does not at all correspond to the behavior of the complex hsemolvsis of serum. It still remained to study another fundamental characteristic, namely, the formation of antibodies. We made peritoneal injections into rabbits, using for this purpose a strongly active extract of ox pancreas that had been sterilized by heating to 60® C. for one hour. The precipitate which developed being regarded as the true active constituent, the mixtures were thoroughly shaken and the whole injected. Two rabbits received 20 cc, 45 cc, and 60 cc. of the extract at suitable intervals and were bled ten days after the last injection. The antihsBmolytic action of the serum against the extract was found to be exactly the same as that of normal rab- bits. (See Table VIII.) As can be seen from this experiment (the result of which is con- firmed by a number of similar experiments with the serum of other rabbits and of a goat treated in like manner) it has not been possible to produce antibodies by^injections of pancreas extract. The experiment, moreover, shows that normal rabbit serum already possesses a marked inhibiting action on the haemolysis through organ extracts.^ We have been able to demonstrate this on all the species .. ._ — ' * This action of the scrum must always be taken into account in the ex- periments, and the blood-cells first washed. 280 CX)LLECTED STLXllIS IN IMMUNITY. TABLE Vlll. 1 cc. GuiNBA-Pia Blood +0.5 Extract of Ox Pancreas -Twicb THE Solvent Dose. 1 Of Rabbits + Serum. Immuniaed with 2. Of Normal Pancrean Extract Rabbits iQaotive. cc. Inactive. 1 0.26 0 0 2 0.2 0 0 3 0.15 0 0 4 0.1 complete almost complete 5 0.075 1 1 complete 6 0.05 it r$ 7 0.015 t < i< 1. of sera investigated by us; it is especially marked in ox serum, be seen by the following examples. (See Table IX.) as can TABLE IX. 1 cc. 5% Guinea-pig Blood +0.5 cc. Extract of Ox Pancreas. cc. + Inactive Rabbit Serum. + Inactive Go«t Serum. 1.0 0.5 0.25 0.1 0.05 0.025 0 0 0 0 almost complete complete < ( 0 0 0 0 strong complete < t Guinea-pig Blood, 1 cc. 5% + Extract of Ox Pancreas, 1 cc. ( » 4 times the solvent dose) + Inactive Ox Serum (J hour at 56'' C). 0.05 0.025 0.01 0 0 0 strong complete That these antihaemolytic actions of normal sera are not due to antibodies in the proper sense is shown by the fact that this protective action withstands the action of high temperature, even 100® C. This is shown by the following table. THE H.fiMOLYnC PROPERTIES OF ORGAN EXTRACTS. 281 TABLE X. Extract of 1 oe. 6% Guinea-pis Blood +0.2 oc. (1 oo. -i) Goat Serum. Pancreas. Goat Serum was Heated for 1 Hour Without Serum. cc. at 70*. at 100». 1 1.0 complete complete complete 2 0.75 trace faint trace 3 0.5 0 0 Schreiber, Berlin thier&rztl. Wochenschr. 1902, No. 19. > ROmer, v. Graefe's Archiv f . Ophthalmologie, Vol. 54, 1902. XXVn. THE MODE OF ACTION OF COBRA VENOM.i By Preston Kyes, A.M., M.D., Associate in Anatomy, University of Chicago, Fellow of the Rockefeller Institute for Medical Research. I. ConceminK the Amboceptors of Cobra Poison. The most important contributions in recent years to our knowl- edge of the action of animal poisons are the recently published in- vestigations of Flexner and Noguchi^ on haemolysis by means of snake venom. These authors have found that although red blood- cells whose serum has been completely removed by washing with salt solution are agglutinated by snake venom, they are not dissolved. If, however, serum is added to the washed blood-cells, or if un- washed blood is used, haemolysis ensues. From this Flexner and Noguchi conclude that the hsemolytic action of the snake venom is due to two factors. One of the components is contained in the snake venom itself, and is said to bear heating to about 90® C. very well. The other component is a constituent of the serum; to a certain extent this activates the poison which in itself has no action. Flexner and Noguchi therefore arrive at the conclusion that snake venom is made up of a number of substances^ acting after the munner of amboceptors J which are activated by certain complements of the serum. The great significance of this interesting fact is at once evident. While formerly snake venom was regarded as a simple poison acting after the manner of toxins, this shows that the haemolytic action of snake venom is somewhat more complex, being identical with the mechanism of the haemolysins of blood senmi, as this has been conceived by Ehrlich and Morgenroth. For this reason Flexner and Noguchi's discovery was hailed with especial delight here in the Frankfurt Institute. * Reprint from the Berl. klin. Wochenschr. 1902, Nos. 38 and 39. ' S. Flexner and H. Noguchi, Snake Venom in relation to Hsemolysis, Bacteri- olysis, and Toxicity. Joum. of Exper. Medicine, Vol. VI, No. 3, 1902. 291 COLtECTED STUDIES IN IMMUNm'. In view of the exceeding importance of these questions it seenaed advisable to proceed from these new facts and attempt to penetrate more deeply into the mechanism of the snake venom's action. We had at our disposal two specimens of dried cobra poison the hemolytic strength of which had proved to be almost identical and for which we are indebted to Dr. Lamb and Prof. Calmette. A one per cent solution of the dried cobra poison in 0.85% salt solution served as our standard poison. This solution when kept on ice was preserved unchanged for several days. The experiments were made with the following animal species; man, ox, horse, goat, sheep, dog, rabbit and guinea-pig. Guided by Flexner and Noguchi's observations, we at first used only blood- cells which had been freed from serum. This was accomplished by making a 2i% suspension of the colls in 0.85% salt solution, centrifuging, decanting the fluid, and then adding anew the same amount of salt solution. This was always done twice and then & 5% suspension was made. All the tubes of a given series contained 1 cc. of a 5% blood suspension and they were all made up to the same volume (2 to 2.5 cc.) by the addition of salt solution. The specimens were kept in the incubator at 37° C. for two hours, and then placed on ice at 6° to 8° C. until the following morning. According to our experience there are two kinds of blood-cells so far as their behavior toward cobra venom is concerned: (1) Those that in themselves are dissolved by the cobra venom. (2) Those that are affected by the cobra venom only after the addition of other substances (complements, etc.). The following table will show the behavior of washed red blood- cells of various species toward cobra venom: TABLE I. AiDHuni pi«. ' Do,. ICu>. Rabbit. Horn. Ox. Bbnap. .... Ik 0 OOM o.oooi littin 0 aomt moderate Jm™"'om'lB>e fain^™ ' 0 j THE MODE OF ACTION OF COBRA VENOM. 293 From this, two groups of blood-cells can at once be recognized, namely, blood-cells like those of guinea-pig, dog, rabbit, man, and horse, which are dissolved by the cobra venom, and blood-cells which are not affected under these circumstances even with large amounts of the poison. The sensitive blood-cells do not all possess the same vulnerabiUty, but manifest considerable variations, depending on the species to which they belong. This is the case with all hsemolytic poisons. Naturally besides this there are certain individual fluctua- tions in vulnerablilty. The blood-cells of the dog and the guinea- pig are the most sensitive since as a rule 0.25 cc. of a 1 : 10,000 dilu- tion of the poison still produces complete solution. The blood-cells of the horse proved least sensitive, for here it required 1.0 cc. of a 1:1000 dilution of the poison to produce solution. The difference in vulnerability is therefore one of forty times. In view of Flexner and Noguchi's experiments by which the amboceptor character of the hsemolytic portion of snake venoms was demonstrated, it seemed advisable to undertake activating experiments in those cases in which the cobra venom did not effect spontaneous solution. It was actually very easy to produce solution by the addition of foreign sera. We shall shortly show that when the observations of Calmette ^ are taken into accoimt these activities are not all due to complements. According to our conception only such substances are complements which in general are inactivated at a temperature between 52° and 60®, in some cases even somewhat higher. This corresponds to the greater or less degree of lability of the complements thus far known. In our experiments such pure complementings were met with in the following combinations: Horse blood ox serum Ox blood guinea-pig serum Sheep blood guinea-pig serum Rabbit blood guinea-pig serum Table II shows such an activation of the cobra venom. It also shows that the serum employed lost its complementing property by half an hour's heating to 56®. * A. Calmette, Sur Taction h^molytique du venin de cobra. Comptes rend. de TAcad^mie des Sciences, T. 134, No. 24, 1902. COLLECTED STLTJIES IN IMMUNITY. TABLE II. Ice. 5%8h«pBlood + Amount of the uiMtpjg rum 0,02 M. 1% Cob™ Poison + Guilwa-pig Sonun. CO. I. Nomutl. Hwted H»1J an Hour 0.5 0,25 0.1 0,05 0,025 0-01 little trace 0 I tompleto strong little trace faint trace 0 0 0 0 0 0 0 From these experiments it can be seen that in the cases described the cobra venom haa the character of an amboceptor and that the amboceptors are activated by serum complements which possess the ordinary degree of thermolability, We ha^'e thought it necessary to determine the mode of action of both substances according to the method used in previous studies on hemolysis. Hence we next studied the behavior of sheep blood- ceils toward the isolated cobra venom and toward the complement. So far as the behavior toward the poison alone is concerned it can be shown tliat this poison is bound by the sheep blood-cells although these are not by themselves dissolved by cobra venom. This confirms the statement of Flexner and Noguchi. According to our experience, however, the blood-cells possess relatively feeble binding powers, especially in dilute solutions of the poison.' On the other hand the complement alone is not at all bound by the blood-cells. This is borne out by the fact that at 0°C. sheep blood-cells are not dissolved by cobra venom +guinea-pig serum; while at 8°C. only a trace of solution occurs. If a separation experiment is made, so that ambo- ceptor and complement are allowed to act on blood -cells at CC, ' The staleiiieata of Deeroly and Rousae (Areliiv. inlsmat, de pharma- codyuainie ct do th^rapic. Vol. VI, 1899) are in entire accord with the slight binding powera of red blood-cells for snake poison. They find that in the animal body also snake poison is bound very much more slowly than diphtheria or totanua poison. Rabbits which had been intravenously injected with a fatal dose of snake venom could be saved even after ten minutes by bleeding and trana- fusing fresh blood, whereas with diphtheria or tetanus poison, even though the some treatment was done immediately, the fatal ending could not be averted. THE MODE OF ACTIOX OF COBRA VENOM. 295 after which the blood cells and fluid are separated by centrifuge, it will be found that the blood-cells have taken up a certain portion of the amboceptors, but none of the complement. These experiments would seem to prove the amboceptor character of the cobra poison, at least for the above cases, entirely according to the ideas of Flexner and Noguchi. n. Concerning Endocomplements/ We shall now analyze the phenomena which we observe with those blood-cells which, like guinea-pig blood-cells, are directly dis- solved by cobra poison. This solution could be explained by assuming that cobra poison, besides the amboceptors, contains true toxins which are analogous to the diphtheria toxin and exert a toxic action, i.e., effect haemolysis, without the inter\'ention of a complement. In that case, however, one would be compelled to assiune further that only part of the species of blood-cells react to this poison. The incorrectness of this conception is readily demonstrated. The observation was made by earlier investigators (Stephens and Myers ^) that red blood-cells which are soluble in weak solutions of poison may be insoluble in stronger solutions; and the same observ^a- tion was made by us on rabbit blood. This phenomenon is entirely irreconcilable with the assumption of a preformed poison, for, ceteris jxiribus, the action of this should increase with the dose. This inhibi- tion in consequence of large doses of poison cannot be harmonized with the toxin theory On the contrary it indicates that we are here dealing with a phe- nomenon whose significance was first point Journal of Pathology and Bacteriology, Vol. V, 1898. * Munch* med. Wochenschr. 1901, No. 18. See also page 120. 296 COLLECTED STUDIES IN IMMUNITY. of very strong snake poison, they will not be dissolved. The mixture is 1WW centrijiiged and the sediment washed vAth salt solution. No solution takes place; as soon as fitting comfiemeni is added, however, solution ensues very promptly. This shows that by the treatment with the poison the complement contained in the red blood-cells has been abstracted. The following diagram will make this clear. I. Blood-cetl with receptor r ond endocomplement e. II. Blood-cell after treatment with a large amount of cobra poison. Tb© cobra amboceptor c has been anchored by the blood-cell receptor. The endocomplemcnt has been abstracted from the cells by the large excess of free amboceptor, HI. Blood-cell of stage II after the addition of complement or endocomplement«. The added endocomplement has combined with the cobra amboceptor e and can now effect solution. The following experiment may serve as an illustration. (See Table III.) TABLE m. • |p*.vsn.%"s'"'«Vu'^™f^'?:^r^"'"''^ "saa" N.C?lk™. b O.IS PC. Guinea- 0,8 ce. Guimm- piK Endoeom- pi^ E^dDC^^B- Solution effected 0 complete complete 0 The correctness of this view can readily be shown in anotlier way If the blood-cells actually do contain an endocomplement, it must be possible to demonstrate this by dissolving the blood-cells in water and finding that these dissolved celb are capable of acting a8 complement to cobra poison for such blood-cells as are incapable of being dissoh'cd by cobra poison alone. THE MODE OF ACTION OF COBRA VENOM. 297 As a matter of fact we have succeeded in a large number of casea in causing the solution of such cells by the addition of laky soliUions. of endocomplement.^ The amoimt of endocomplement contained in blood-cells varies; that of hiunan and guinea-pig blood appears to be the highest and also fairly constant. The following table shows the combinations in which, according to our experiments, cobra poison causes solution (+) of blood-cells, which are not dissolved by cobra poison alone (see Table IV). Endooomplexnent of Rabbit. . . Man Dog Guinea-pig Goat Ox Sheep TABLE IV • SpeoioB of Blood. Ox. Goa Sh«ep. + + -f + + -f + + + + + + + — — _ J " " It is in place here to mention another fact. The deflection of the endocomplement by large quantities of poison described in the case of blood-cells vulnerable to cobra poison succeeds equally well if the experiment is made with blood-cells insensitive to cobra poison alone (ox blood) and if dissolved endocomplements (guinea-pig) are used for activation. There is no dovbt therefore thai the blood-cells: themselves contain complement4ike substances, endocomplements. So far as the behavior of these endocomplements toward thermic influences is concerned, they are seen to be somewhat more resistant in general than are the complements contained in the serum, for it requires half an hour's heating to 62® C. to inactivate them (see Table V). In the light of our present knowledge, however, we probably cannot deny the complement character of these substances merely * As a rule these endocomplement solutions were prepared by twice washing and centrif uging a certain quantity of f uU blood, and then filling the sediment up to a certain voliune. Either the original volume or a greater or less dilu- tion was made up depending on circumstances. They were then salted to contain 0.85% NaCl. We have designated these dilutions as }, J, -Ar» etc., endo- complement. ' Even in these cases we noticed an activation with certain specimens of blood. 298 COLLECTED STUDIES IN IMMtTNITl*. because of this thermostability, especially since we know that Ehrlich and Moi^cnroth i have described a partial complement in goat Eerum which was much more thermostable. According to some unpublished studies by Shiga such thermostable complements seem to take part in the bacteriolysis of anthrax bacilli by rabbit serum. The active group of coagulins and agglutinins, which, according to Ehrlich, is analogous to the zymotoxic group of complements, is still more thermostable,^ for inactivation takes place only between 70 aod 75° C. From all this it follows that we must assume the blood-cells which are sensitive to the above poison, to be supphed bolk leilh reixptors and comjAcmenls. Through the inter\-ention of the amboceptors added, the discoplasma enters into that intimate combination with the complement which is necessary in order that the latter may act. We should like to add a few explanatory remarks to these state- ments, and shall begin with the conception of complements as endocom- plemenis. One could, for example, assume that the endocoraple- ments are derived not from blood-cells themselves but from the serum Btill adherent to these. Howe\'cr, we Ijelieve that the repeated wash- ing and centrifuging has completely freed the red blood-cella from serum. Guinea-pig blood-cells were washed and centrifuged eight times, yet even after that the dissolved blood-celb manifested tlie complement action. This excludes the possibility of the action being due to adherent senim. Anotlier thing which speaks against this is the fact, now and then obser\'ed by us (mostly, to be sure, merely indicated) that the last decantations activated more strongly even than the first. If the washmg removed adherent serum constituents, the first washing should contain more than the later ones. As a matter of fact just the reverse was found to be the case; which indicates that we are dcalmg with an extraction phenomenon. In one case we even succeeded in entirely rcmo\'ing the cndo- complement by means of salt solution. Tliis was a suspension (5% in 0.85% salt solution) of rabbit blood, which is dissolved by cobra poison. Tliis susjiension was kept in a refrigerator for twenty-four hours and then centrifuged, when it was found that the scdimented blood-cells suspended in fresh salt solution were no ' See pages 11 et seq. 'See Bail. Arrhiv fiir Hygiene, Vol. XLII, 1902; hIho Eisenberg and Volk, Zeitachr. t. Hygieoe, Vol. XXXIV. 1902. THE MODE OF ACTION OF COBRA VENOM. 299 TABLE V. In all Cases 0.02 cc. 1% Cobra Poison. A. Amount of the 1 00. 5% Ox Blood + Guinea-pic Blood Endooomplement (1/20). Endooom- plement (1/20). 00. o Normal. 6 Heated to 62*" for i Hour. 1.0 0.75 0.5 0.25 0.1 complete trace 0 OOOOO B. 1 CO. 5% Goat Blood + Guinea-pic Blood Endooomplement (1/10). Endooom- plement o Normal. ^ Heated Half an Hour to (1/10). 00. b 66«C. e 60«C. d 62«C. 1.0 0.5 0.25 0.1 complete moderate little faint trace strong little trace 0 trace 0 0 0 0 0 0 c. 1 00. 5% Sheep Blood + Guinea-pic Endooomplement (1/10). Endooom- plement o Normal. Heated Half Hour to (1/10). 00. 6 66«»C. c 60* C. d 62'»C. 1.0 0.5 0.25 0.1 almost complete little trace 0 moderate trace 0 trace 0 0 0 0 0 0 0 longer dissolved by the cobra poison, or were only very slightly dissolved. If our view was correct, the endocomplements would now be foimd in the decanted fluid. This proved to be the case, for the addition of suitable amounts of this fluid sufliced to cause solution of the blood-cells which were insoluble in cobra poison alone. We 300 COLLECTED STUDIES IN IMMUNITV. were unable to obtain a like result in two similar cases. Evidently slight variations in the experiment and jjossibly also minute changes, and impurities leading perhaps to certain ion actions, play a role which it is difficult to control. We were not interested enough to follow up tliese relations; but we believe that had we done so we could have made the conditions more favorable for washing out the endocomple- ments. We merely mention this because I'lexner and Noguchi state that in their experiments after repeated washings of the blood-cells all of these were found insoluble in cobra poison alone, These authors did most of their work with snake poisons differ- ent from ours {Crolalus adamanleus, Ancistrodon conlortn'^, etc.). How far this fact is responsible for the divergence cannot here be decided, nor whether the escape of the endocomplenienta was favor«d by other conditions in the experiments.' That the endocomplements cannot be derived from the serum is also shown by the obser\-ation frequently made by us that the senan of several species of blood, whose blood-cells exhibit a plentiful supply of endocomplement, does not possess the slightest activating power, but that, on the contrary (as in the case of rabbit eerum) , it sometimes hinders hicmolysis of the homologous blood-cells by snake poison. .So far as tlie condition is concerned in which the endocomple- ments exist, we must assume, in those cases in which the blood- cells are directly soluble, that the endocomplement is contained free in the blood-cells. In those blood-cells, which are primarily infloluble, it will cither be absent or be present in a latent form. We believe the endocomplements are absent in the goat, for in no ease were the dissolved goat blood-cells able to activate cobra venom for goat blood. Ou the other hand, ox blood is sensitized for cobra venom by dissolved ox blood-ccUa, so that we shall have to assume that ox blood does not contain endocomplements in available form and that these endocomplements are changed into an active form when the cells are dissolved. We shall reserve for subsequent study the question as to whether the endocomplements are of simple constitution or complex. Attention is called to the fact that the existence of endocom- plements furnishes another objection to Bordet's view that the iliall merely say that Daboia poiaon, wliich through Lamb's pretty '- has been shown to differ from cobra poison, does not dissolve rabbit THE MODE OF ACTION OF COBRA VENOM. 301 amboceptor is only a key which makes possible the entrance of the complement into cell. For in these cases the complements which are able to destroy the blood-cell are already present within the same before the amboceptor is anchored, and yet the blood-cell is in no way injured. The injury takes place only when a particular organic relation has been effected between complement and protoplasm by means of the amboceptor. Finally, the demonstration that the red blood-cells contain com- plementing substances is exceedingly important in other directions. The French school in particular was inclined to refer the source of the complements exclusively to the leucocytes. We now see that the red blood-cells, heretofore considered merely as concerned with the oxygen exchange, are also carriers of special complement-like substances. This confirms the view expressed by Ehrlich ^ in his "Schlussbetrachtungen," namely, that "the jed blood-discs also exercise other functions hitherto overlooked." "The red blood-cells serve as storage centres in the sense that they temporarily take up into themselves substances characterized by the presence of hapto- phore groups and derived from the internal metabolism or from the food." in. Cobra Venom and Lecithin. Having demonstrated that the amboceptor of snake poison can be complemented by easily destructible complements which may be present either in the serum or in the red blood-cells, we go on to a series of other phenomena in which activation is effected by more stable substances which are in no way related to the complements. Calmette,^ in following up the work of Flexner and Noguchi, found that certain normal sera when heated to 62® C. became much better able, in conjunction with cobra poison, to cause haemolysis of the washed blood-ceUs. In fact it was found that fresh sera, added in large excess, can retard or even inhibit haemolysis, while these same sera when heated cause immediate solution of the blood-cells in the presence of cobra poison. From this Calmette concludes that such a blood serum must contain a natural antihaemolysin which can pro- tect the red blood-cells up to a certain point against solution by the ' In NothnagePs Specielle Pathologie iind Therapie, Vol. 8. Vienna, 1901. M.C. 302 COLLECTED STUDIES IN IMMUNITY'. snake venom. This antihsemolyain is thermolabile, being destroyed by temperatures over 56° C. The other (the activating) constituent of the serum on the contrary is thermostable, since it does not lose its activity even by heating to 80° C, Calmette therefore assumes that the alexin (our complement) takes no pari in the acHvaium, but that a particularly thermostable "substance sens^rUatrice " is con- tained in the serutn besides the thermolabile antihtBtnotysin. By the term "substance sensibilatrice, " as used in French terminologj', la meant the lx)dy which we term "amboeeptor." The amboceptor capable of being anchored is supposed to render the red blood-cells sensitive to the attack of the alexin (complement). It is hard to see just how Caimette conceives this entire process. As we already know from the researches of Flexner and Noguchi snake \-enom is capable of being anchored, and from all of its properties is therefore surely a substance sensibtlatrice (amboceptor). If then the substance supposed by Calmette were also a sensitizer, we should have before us something absolutely unique, namely, the combined action of two sensitizers. Unfortunately Calmette has undertaken no combining experiments and therefore has furnished no proof for his view. Our own experiments, however, speak against this assumption. In our opinion the main reasons which led Calmette to conclude that complements play no role in the hEemolysis by means of cobra venom are : 1. That he overlooked the endocoraplements. 2. That he employed too schematic a manner of activation, namely, usually only at 62" C. We have convinced ourselves that in suitable cases (see Table VII, case IV) a blood serum, e.g. ox serum, when fresh, dissolves the red blood-cella. If this is inactivated by heating to 56° C, the action will be found to be completely inhiljited, or almost so. This same serum, however, when heated to 65° C. or higher is again able to effect hiemolysis. The serum heated in this manner possesses a stronger soh-ent power than the fresh serum, for even fractional parts of the complete solvent dose of fresh serum suffice to cause full solution (see Table VI). This experiment was repeated many times and pro\'ee that in this case two entirely differeni kinds of actimtions occur, namely, 1, Activation by means of complements. 2. Activation by means of substances which become riuiniffst only through heating. THE MODE OF ACTION OF COBRA VENOM. TABLE VT. 1 cc. 5% Horse Blood + Ox Serum. 303 I. Ox Serum Alone. II. 0.02 oc. 1% Cobra Poiaoo + Ox Serum, 1/10 Dilution. Amount of the Ox Serum (1/10). o Normal. Heated for One Half-bour to oc. b 66«»C. e 66*»C. 0.5 0.35 0.15 0.15 0.1 faint trace 0 0 0 0 complete almost complete strong little trace faint trace 0 0 0 complete little trace It seemed to us that it was of the highest importance to gain a further insight into these thermostable activating substances. To begin, we found that the substance is far more stable than Calmette assumed, for activation is effected even by sera which have been cooked for hours. Thereupon we investigated a number of sera in respect to their activating power and obtained results that were little less than confusing. We foimd sera which activated not only in the fresh state but also after heating to 56® C. and 100® C. (No. I of Table VII). Other sera did not activate either when fresh or after heating to 56® C; they did activate, however, when they were heated to 65® and 100® C. (No. II of Table VII). As a rule in these cases the serum heated to 100® C. proved more powerful than that heated to 65® C. A third class of sera was found which did not activate when fresh, but activated when heated to 56® C. or higher (No. Ill of Table VII). Finally, there is the type already mentioned, namely, a serum which activates when fresh, is made inactive by heating to 56® C. and again made active by heating to 65® (No. IV of Table VII). We have also observed sera which activate ordy when fresh and do not again acquire this property when heated to a greater or less degree (No. V of Table VIII). We see therefore that we are dealing with five different combinations,^ as is shown in Table VII. ' Naturally in the case of such bloods as rabbit blood, which are dissolved by cobra poison alone, only such amounts of poison have been used which by themselves are not active, but which cause haemolysis when they are combined with suitable reinforcing agents (complements, etc.). COLLECTED STLTIIES IN IMMUNITT.'. TABLE VIL Activ.t ns Powr if the Senim. CambiniiliDns. • HmUidto S«nun. Blood-eaU. NormiLl. Sfl-C. 6B° or lOO- C. horse 03C horee + + + horeo rabbbit horse goal* n 0 0 + a Keep rabbit sheep. ni 0 + + sheep Buinea-pig « + 0 + guiriea-pig -1- gumea-pig * Only slight nolution. Tliese contradictory results are not to be harmonized with Cal- metto's conception of a definite antibody which is destroyed at 56° C. One would have to assume that this normal antihaBmolysin were lacking in horse serum, for as a rule this does not become more strongly hffimolytic by heating to 56° C. On the other hand in the case of a serum like No, II, which has no activating properties even when heated to 56° C, it would be necessary to believe that the activator is entirely absent. The conditions are still more complicated by the fact that one and the same serum can behave differently toward various species of blood. Thus a horse scrum heated to 100° will activate cobra venom for ox blood in high dilutions (0.02 complete), whereas even in large amoimta it dissolves goat blood only in com- paratively slight degree (0.35 cc. moderate solution). In this case, then, the activator present is in the main one for ox blood, not for goat blood. Believing that an insight into the nature of this maze of facts could be gained only by a thorough chemical analysis, we sought to isolate the thermostable activating substance. First we succeeded in proving that when serum is precipitated with 8 to 10 volumes of alcohol, the activating substance passes into the alcohol, while the inhibiting substance is contained in the precipititte. For if the THE MODE OF ACTION OF COBRA VENOM. 305 alcoholic extract is evaporated in vacuo and the residue dissolved in an amount of 0.85% salt solution equal to the original amount of serum, a strong activating fluid is obtained. An alcoholic extract of horse serum, when treated in this way, in contrast to the native horse senun heated to 100*^ C, dissolves goat blood to a high degree (0.1 cc. dissolves completely). The alcohol precipitate must there- fore have contained a substance which inhibits the action of the activator, and we were actually able to demonstrate the existence of this inhibiting substance. If the precipitate is dissolved in salt water, a fluid is obtained which inhibits the haemolysis of goat blood by cobra venom and the activator derived from the alcoholic extract of horse serum. In larger, though unequal, doses it protects ox blood against solution by cobra venom and the activator. Before studying the nature of the inhibition effected by the albuminous precipitate we shall try to discover the nature of the activator. As already said, the residue obtained on evaporating the alcoholic extract was dissolved in salt water. On shaking this solution with ether, we found that the ether had taken up all of the activating substance. This proved that the activator is a substance soluble both in alcohol and ether, and one which has a wide distribution in the sera of animals. Constit- uents of the blood serum which are soluble in ether have long been known to us. Those mainly to be considered are cholesterin, lecithin, fats and fatty acids. After several negative trials with cholesterin we found that lecithin possesses the properties of the activator, since all blood-cells are rapidly dissolved when cobra venom and lecithin are allowed to act on them simultaneously. Not only blood-cells which are insoluble in cobra venom alone, such as goat blood-cells, but also those which are deprived of endocomplements when treated with strong solutions of poison (see § II, Endocomplements) are promptly dissolved by the lecithin. Our solution of lecithin ^ was made in the * The lecithin employed by us was derived from yolk of egg and obtained from E. Merck, Darmstadt. It was a neutral mass of salve-like consistency, which was entirely precipitated from its ethereal solution by aceton (Altmann- Henriquez). Even when thus purified it manifested the activating power unchanged. We reserve for further study our experiments with the pure lecithin prepared after the method of P. Bergell (Ber. der deutsch. chem. Gesellschaft, Jahrg. 33, 1900, page 2584) and with the homologues of this body. A specimen of lecithin obtained from J. D. Riedel, Berlin, corresponded exactly in its activity to Merck's lecithin. Cerebrin and Protagon, obtained through the courtesy of Prof. Kossel of Heidelberg, possess no activating power. 306 COLLECTED STUDIES IN IMMUNITY. purest methyl alcohol, for, as we know trora special experiments, this does not injure the red blood-cells even in concentration up to 9 or 10%. A 1% stock solution was diluted with 0.85% salt solution, and it was found that 0,0025 cc. to 0.0035 cc. of the 1% solution (i.e. 0.000025 g. lecithin) were sufficient to completely dissolve 1 t-c. 5% ox or goat blood on the addition of suitable amounts of cobra poison, (See Table VIII). TABLE VIII. i^tL^ 0.002 PC. 1% Cobts PoiKiD. Oi Blood. Go.t Blood. 0.005 0.0036 0.0025 0.0015 0.001 0.00075 comple.. almoat complete little 0 moderate trace 0 0 In what way now are we to picture the action of this lecithin? We know that lecithin is able to combine with albimiinous bodies, sugars (Henriquez and Bing), etc. A threefold question had to be decided. First, whether cobra venom unites with lecithin after the fashion of an amboceptor; second, whether perhaps the snake venom had made the blood-cells sensitive to lecithin; or third, whether the reverse holds true. A preliminary test was made to see whether lecithin and snake poison coml.iine with one another. The method of making this t^eriment is relatively simple. Lecithin can easily he shaken out of its solution in salt water by means of ether. As the following experiment will show, lecithin passes into the ether in great abundance, but not completely. This behavior corresponds to a general phe- nomenon which is the expression of the "loi de partage." If, how- ever, to the same amount of lecithin a suitable quantity of snake venom is added, it is found that but very little passes into the ether on shaking the ether with the mixture. Two portions each of 10 cc, were thus shaken out with ether: A. containing 2 cc. of a certain lecithin solution: B, containing besides this 1 cc. of a 1% solution of cobra venom. Previous to this both solutions were kept at 37° C. for half an hour. The ethereal extract was evaporated and the residue taken up in 10 cc. 0.85% salt solution The action, on ox THE MODE OF ACTION OF COBRA VENOM. 307 blood -f cobra venom, of the ethereal extract residues on the one hand, and of the solutions which had been shaken out on the other, is shown in Table IX. TABLE IX. Complete solvent dose of lecithin (stock solution) with 0.1 cc. of 0.1% cobra venom » 0.005 cc. (corresponding to 0.p25 cc. of the shaken-out solution). 1 cc. 5% Ox Blood +0.1 cc. 0 1% Cobra Poison. Amount of A or of B. A. Lecithin Only. B. Lecithin + Cobra Poison. cc. I. Ether Extract. II. Aqueous Portion. I. Ether Extract. II. Aqueous Portion. 1.0 0.5 • 0.25 0.1 0.05 0.025 0.015 complete solution ( < ( ( ft i ( it t i trace 0 complete solution i ( < < K it 0 complete solution moderate 0 complete solution 0 It can be seen from the table that on the addition of snake poison to the same lecithin solution only -^ part of that amount of lecithin passed into the ether which passes into ether when a pure lecithin solution is shaken out. The cobra venom had there- fore bound the lecithin. The next question to determine was how the red blood-cells behaved toward cobra venom and lecithin alone and toward mixtures of these substances. In order to retard the course of the reactions as much as possible and to secure a better view of the processes we sought to create such retarding conditions by making the test with dilute solutions and at 0° C. This necessitated a preliminary quantitative determination of the effect of each factor separately. Corresponding to the slight affinity of the cobra amboceptor for the red blood-cells, it was found that with suitable conditions (2 hours at 0° in dilute solutions of the poison) the amboceptor is not anchored; neither is lecithin by itself bound by the blood-cells. On the other hand blood-cells to which cobra venom -f lecithin were added in suitable quantities were rapidly dissolved even at 0^ C. Both com- ponents must therefore have been bound. The following table (Tabb X) illustrates this behavior. 308 COLLECTED STUDIES IN IMMUNITY. TABLE X. Complete solvent dose of cobra venom (0.1%) in the presence of 0.01 cc. lecithin « 0.005 cc. Complete solvent dose of lecithin in the presence of 0.1 cobra venom (0.1%) -0.005 cc. A Amount of Gobra Venom 1 cc. 5% Ox Blood + Decreasing Amounts of 0>bra Venom Kept Two Hours at 0^, then Centrifused and Washed. Thereupon 0.01 Lecithin Solution Added to Added (0.1%). cc. I. The Sediments. II. To the Decanted Fluid which had been Added to Native Ox Blood. 0.01 0.05 0.025 0.01 0.005 0.0025 faint trace solution 0 0 0 0 0 complete solution tt tt It tt tt tt almost complete 0 B. Amoimt of the Lecithin 1 cc. 5% Ox Blood + Decreasting Amounts of Lecithin Kept at 0^ C. for Two Hours, then Ontrifuged and Washed. Thereupon 0.1 cc. Cobra Venom (0.01%) Added to Solution Added. cc. I. The Sediments. II. To the Decanted Fluid which had been Added to Native Ox Blood. 0.075 0.05 0.025 0.01 0.0075 0.005 trace solution complete solution tt tt tt tt tt tt tt tt 0 c. 1 cc. 5% Ox Blood + 0.025 Lecithin Solution + Decreasing Amounts of Cobra Venom, Two Hours at 0** C. Amount of Ck>bra Venom Added (0.1%). cc. I. Degree of Solution Effected. II. Specimens not Dissolved are Centrifuged, the Sediments Washed. a Sediments Suspended in Salt Solution. (+0.01 cc. Lecithin). 6 Decanted Fluids Poured over Native Ox Blood. 0.1 0.05 0.025 0.01 0.005 0.0025 0.001 0 complete tt tt tt faint trace 0 0 0 0 0 0 0 complete moderate 0 0 THE MODE OF ACTION OF COBRA VENOM. 309 These results can be explained only by assuming that lecithin and cobra amboceptor have combined to form what may be termed the "lecithin" of cobra poison, and that the ajBSnity of the cobra amboceptors cytophile group is thereby increased. According to this the union with the lecithin causes the cobra poison to Le more rapidly anchored than the cobra amboceptor alone. The increase of the cytophile groups affinity through the occupation of another group is perfectly conceivable chemically. An analogy frequently met with is the fact that the anchoring of the haemolytic serum amboceptors by the blood-cells usuaUy causes an increase in the affinity of the complementophile group. Ehrlich and Sachs ^ have shown that the occupation of the complementophile group of serum amboceptors can cause an increase of the cytophile group's afiinity, such as is presented in this case. We therefore assume that the lecithin acts as a kind of comple- ment since it is anchored by certain definite groupings of the poison molecule. In this way a poisonous double combination is formed of which perhaps the cholin residue constitutes the toxophore group. There is another fact which supports the view here presented, namely, that the lecithin amboceptors effect solution of the red blood- cells even at 0° C, whereas the thermolabile complements of blood serum are anchored only at higher temperatures. Corresponding to the views formulated by Ehrlich and Marshall ^ for the amboceptors (polyceptors) of blood serum, we must therefore assume that the snake venom amboceptor in addition to its cytophile group possesses at least two haptophore groups, of which one as usual is able to bind complements, the other to bind lecithin. Each of these combina- tions by itself is dominant^ i.e., sufficient to effect solution of the blood- cells. It is very probable that occupation of both groups increases the solvent effect. The following experiment furnishes additional proof that the phenomena observed cannot be regarded in the light of a sensi- tization. The amount of lecithin required for complete haemoly- sis is determined in two parallel series, one on the addition of small amounts of cobra venom, the other with large amounts. It is found that far more lecithin is required for complete solution when there is a large excess of cobra venom. (See Table XI.) * See pages 209 et seq. > See pages 226 et seq. COLLECTED STUDIES IN IMMUNITY. TABLE XI. Lwiihi^Solu- lee. 6%0xBlood+ ] 0.4 ce, 6% Cobr. O.loc p,l%D.br« 0,05 0.035 0.025 0,015 O.OI 0-0075 0,005 0.0035 coinplel« solution moderate tittle faint trace 0 0 0 0 complete solution raoderalB 0 Now if the cobra ^■enom sensitized the biood-cells for the lecithin, leas lecithin would be required for solution the more cobra venom were added. As a matter of fact the reverse is the case. When we used a large excess of poison, five times as much lecithin was re- quired for complete solution as when smaller doaea were used. This ia readily explained by assuming that a large excess of amboceptor causes a deflection of the lecithin, a phenomenon which we have already met with in the endocomplements. The phenomena observed by us also serve to explain most easily the inhibiting action exert«i by certain sera. As is well known, lecithin is able to combine with albuminous bodies, sugars, etc. If this union is so firm that it is not disrupted by the affinity of the cobra amboceptor, it will be impossible for the lecithin to come into action. This is the case, for example, with ox serum, which when fresh does not exert a trace of activation on goat blood, and yet the OS serum contains sufficient lecithin, as we know by examining \\s alcoholic extract. Ox serum is even able to prevent hjemolysis on the addition of free lecithin, the reason being evidently because it contains an excess of inhibiting substances. On heating the serum these substances lose their action to a greater or less extent, so that the serum is able when mixed with cobra venom to effect htemolysis. As already mentioned, however, the hiemolytic action is usually considerably stronger when the sera are heated to 100° C. instead of only to 65° C. Perhaps this is due to substances possessing different degrees of thermolability. In other cases only a very slight difference ia to be observed THE MODE OF ACTION OF COBRA VENOM. 311 between the activating power of fresh and of heated serum. In this case evidently the fresh serum ahready contains free, i.e. active, lecithin, and the inhibiting substance is affected but to a slight degree by the heating. In view of all this it is certainly incorrect to speak, as Calmette^ does, of a definite thermolabile antibody which is destroyed at 56® C. It is natural to attempt a quantitative estimation of the cobra amboceptor by means of the binding of lecithin; also to think of the possibility of isolating the cobra amboceptors as lecithids. Ex- periments in this direction are now under way. The results of the experiments here given furnish a further in- sight into the nature and mode of action of the amboceptors. The demonstration of endocomplementSj as well as the significant fact that a definite chemical and crystalline substance, lecithin, can in a certain sense play the rdle of complement, would appear to be especially im- portant for the development of our knowledge concerning poisons. ^ One might assume that the hemolysis by cobra venom alone, ascribed in § II to the action of the endocomplements, was caused by the lecithin contained in the red blood-cells. This assumption, however, is at once excluded by the fact that the endocomplement solutions are inactivated by heating to 62^ C, showing that their action has nothing to do with that of the lecithin. K\ail. FURTHER STUDIES ON THE DYSENTERY BACILLUS.! By Dr. K. Shioa. When I discovered the dysentery bacillus in 1897 I found that althougii this organism apparently remains localized in the intestine and does not pass into the circulation, it nevertheless gives rise to the development of specific antibodies in the serum. This fact, made use of after the manner of the Gruber-Widal reaction, furnished me with an important aid in the diagnosis of the dysentery bacillus- In the course of the following years the facts which I obsen-ed in connection with epidemic dysentery have been confirmed in ^'a^iou3 parts of the world,^ especially since Kruse succeeded so well in his studies on this disease in Germany. To-day Uiere is no longer any doubt concerning the identity of the bacillus isolated by Kruse with mine, even though there is atill a slight divergence concerning certain morphological details. All of the important character- istics of the bacilli discovered by me, as well as their agglutinat tion by serum of the patients, have been confirmed by Kruse. Iha- certain slight differences in growth may occur is not at all uncommon in other bacteria, even in cholera. The question as to the presence of motility is especially hard to answer. At first I stated that my bacilli were motile; Kruse found them immotile. It is well known that it is not always e^sy to decide whether a bacillus is motile or not, and Kruse himself says concerning motility as a characteristic of the roli group (Flugge, Vol. II, page 361) that "one must be very careful In deciding this point, for the rao\-ement8 often last but a short time and are not present under all conditions of life (nutrient medium, ' Reprint from the Zeitsch. f. Hyg. und IntectionB-Krankheiten, Vo\. 41 . 1902. ' Compare also the siiidy published since this, entitled "Unterauchungen uberdie Ruhr," Berlin. 1902. FURTHER STUDIES ON THE DYSENTERY BACILLUS. 313 temperature, etc.)." In this connection I would call to mind the bacillus of erysipelas of swine, whose inmiotility is still questioned by many observers. I have always described the motility of my cultures as feeble, though I found it strange that I was unable at first to demonstrate flagella by staining methods. Later on, how- ever, I succeeded in finding two terminal flagella in one preparation, and thought that this question might now be regarded as closed. To what extent this was an error I should not yet like to say, and for the present I should also not like to regard the observations of Vedder and Duval,^ who found peritrichal flagella, as a confirma- tion of my findings. In 1898 I immunized horses with dysentery bacilli and obtained a high-grade serum with which in 1898-1900 almost three hundred people have been treated. It therefore seemed advisable to study this dysentery serum from the standpoint of the modem theory of inmiunity. At the same time I was anxious by means of serum diagnosis to again prove the identity of Kruse's bacillus with mine. The cultures employed were the following: One of my original cultures, one from Prof. Flexner, one culture of the Kruse bacillus from the Frankfurt Institute, and a Kruse bacillus from Dr. Conradi, Berlin. I may at once say that in all the various bactericidal experi- ments these cultures behaved exactly alike, and I shaU therefore in the following speak of the dysentery bacillus as such. When I come to speak of the agglutination I shall make mention of certain variations of Flexner's bacillus from mine and Kruse's. To begin, the bactericidal action of normal active sera was tested on the dysentery bacillus. The method employed corresponded exactly to that described by M. Neisser and Wechsberg, to whose paper I shall therefore refer .^ The amoimt of culture planted was always 1/500 mg. of a one- day agar culture, and in the dilution employed this was contained in 1.0 cc. salt solution. The total amount in each tube was always 2.0 cc, to which quantity three drops of bouillon were then added. The serum was allowed to act for three hours at 37® C, after which time six drops were made into agar plates. In judging the plates we did not make use of accurate counting, but always employed the * The Etiology of Acute Dysentery in the United States. Journal of Experi- mental Medicine, 1902, Vol. VI., No. 2. ' See pages 120 et seq. 314 COLLECTED STUDIES IN IMMUNITY. method of Neisser and Wechsberg, namely, approximate estimation, because only large results were regarded as conclusive. Frequently after the six drops had been taken from tlie tube, the residue was again placed into the incubator. In this way one often obtains valuable confirmation of the agar plates by noting whether or not there is a growth in the tubes. The strongest bactericidal power is possessed by goat and sheep .sera, but this is but slight in comparison to their action on many other species of bacteria, 0.3 cc. of these sera almost completely killed the bacteria under the conditions mentioned. Other sera are weaker, such as ox, horse, human, dog, guinea-pig, and rabbit serum, A reactivation of normal inactive sera succeeded only in the follow- ing combination; normal inactive goat serum could be completely reacti\'ated by normal active horse serum in an amount which by itself did not kill the bacteria. These experiments showed that only a few sera could be used for reactivation (e.g. horse serum) apparently because the other sera did not contain any considerable excess of free dominant complement, or contained none at all. Tliis was entirely confirmed by the complementing experiments which were made with a high-grade immune serum. The immune serum used was obtained from a horse which I myself had begun to immunize and which had been further immunized in the meantime. The serum was sent to me from Japan with the addition of 0,5% carbolic. In the small amounts in which the serum was used, this addition in no way disturbed the bactericidal experiments, as was shown by control .tests. The first experiments undertaken with the completion by means of active horse serum resulted negatively in so far as any destructive action was concerned. ITiis was soon found to be due to the phenomenon of complement deflection described by Neisser and Wechsberg; for when smaller and still smaller doses of the immune serum were employed the destructive action became more and more marked. Table I, in which column A gives the result of the plate tests, and B that of the test-tube experiment made at the same time, shows the destructive action as well as the phenomenon of comple- ment deflection. From this it is seen that even 0.0025 and 0.0005 cc. still have a -distinct bactericidal action. This result was obtained a great many times, with various strains, in almost the same manner. Besides the horse serum only one other serum could be used FURTHER STUDIES ON THE DYSENTERY BACILLUS. 315 for complementing the immune serum, namely, active human serum. Table II shows an experiment with this serum. TABLE I, Inactive Active A. B. Dysentery Horse Serum. Dysentery Culture. No. of Colonies Growth in the Serum, co. cc. on the Plate. Tubes. 0.01 0.3 1.0 CC. (1/500 mg.) 00 + 0.0075 0.3 00 + 0.005 0.3 00 + 0.0025 0.3 almost 0 0.001 0.3 0 — 0.00075 0.3 almost 0 .i~ 0.0005 0.3 about 50 — 0.00025 0.3 " 100 + 0.0001 0.3 " 1000 + 0.000075 0.3 several thous. + 0.00005 0.3 00 -f- f — 0.3 Control Qi 0.3 1.0 cc. (1/500 mg.) several thous. ** 00 — 0 — 0 TABLE II. 4- Inactive Dysen- tery Serum, cc 0.01 0.003 0.001 0.0003 0.0001 0.00003 0.00001 Active Human Serum, cc. 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Dysentery Culture. 1.0 CC. (1/500 mg.) No. of Colonies on the Plate. 00 X X few 0 about 100 1000 n Control 0.1 0.3 0.3 1.0 CC. (1/500 mg.) tt 00 00 0 0 Up to the present time I have tested the serum of six individuals and found it active in five cases (four times in placental serum and once adult serum) ; only once, in the case of a nephritis patient, was the fresh serum ineffective for complementing. It may be mentioned that one of these sera was my own, and this was considerably stronger than the rest. Whether this property has any connection with an active immunization which I underwent some four years previously 1 shall leave undecided. 316 COLLECTED STUDIES IN IMMUNITY. I believe this demonstrates that the horse immune serum em- ployed by me for therapeutic purposes, meets the requirements which are nowadays to be demanded of a bactericidal immune scrum, namely, (1) that it be high grade, and (2) that it find a fitting com- plement in normal human serum. This is the first serum employed in human therapy which fulfils the conditions laid down by Ehrlich in hisCroonian Lecture, 1900. The excellent curative results obtained by me in Japan ^ furnish abundant confirmation of the correctness of Ehrlich 's views. As already mentioned, the phenomenon of deflection of comple- ment could be demonstrated very prettily with the complement of this active horse serum. Since this deflection is primarily dependent on the amount of immune botly present, it may perhaps be possible to employ the degree of deflection as a measure of the titer of a serum. Some experiments in this direction which I have undertaken at the suggestion of Prof. M. Neisser have not yet been concluded. I have already stated that the other active sera {e.g. goat serum, etc.) could not be used for complementing the dysenterj' immune serum, altJiough in themselves they were bactericidal. But for this immune scrum the phenomenon of complement deflection can be demonstrated very nicely with these sera also. (See Table III.) TABLE III. Dys^tary Immune Active GoU Serum. Dv«,..C^,_. pu™!" on. 0-1 0.3 1/500 mg. :^ 0-03 0-3 O-Ol 0-3 0-003 0 3 0 0-001 0-3 0 1—0-3 — 0 Perhaps also this method of testing ia available for determining the grade of bactericidal sera. Furthermore by means of an absorption teat analogous to tlie experiments of A. Lipslein* I have convinced myself that the deflec- ' DeutBcho mcU. Woi;lienapiirifl, 1901. Nob, 43—15- •See pagea 132 et acq. FURTHER STUDIES ON THE DYSENTERY BACILLUS. 317 tion of complement described is actually due to an excess of im- mune body and not, for example, to the presence of an anticomple- ment. Prof. Neisser and I thought that this phenomenon of comple- ment deflection cduld be utilized in another direction. Ehrlich and his pupils, it will be remembered, have demonstrated the existence of a plurality of complements. In view of this it was conceivable that, following a large addition of inactive immune serum to a normal serum bactericidal per se, only that complement would be deflected which is able to complement the inunime serum, while the remaining complements were left unafifected. From this it would follow that the normal active serum in question would in the main have lost only this one bactericidal action, while it still retained almost all the others. One would thus have a serum which had lost a bactericidal action chiefly for that bacterium whose immune body has been added in excess; that is to say, a truly specific nutrient medium. Proceeding from these considerations we first infected a normal stool with a small quantity of dysentery bacilli. To small amounts of this infected stool 2.0 cc. normal active goat serum and 0.2 cc. inactive immune serum were added and the mixture kept in the thermostat. At the end of three hours six drops of this mixture were added to a second tube containing 2.0 cc. normal active goat serum and 0.2 inactive inmiune serum. Agar plates were made (1) from the original infected stool; (2) from the first tube; (3) and from the second tube after it also had been kept at 37® C. for three hours. A great many tests showed that a specific enriching in dysentery hacUli takes place, so that when the first plate shows only a few scattered colonies of dysen- tery bacilli, Plates II and III show numerous colonies. In one case we even succeeded in finding dysentery bacilli in Plates II and III, although none had been found on Plate I. It may be mentioned that we used the agar medium recommended by v. Drigalski and Conradi ^ for the diagnosis of typhoid bacilli, and found it of great advantage. The method just described for enriching cultures may perhaps be extended and perfected. » Zeitschrift fur Hygiene, Vol. XXXIX. 31S COLLECTED STUDIES IN IMMUNITY. ^H Proaggiulinoid. As a result of the brilliant investigations of Bail ' on the oae hand and of Eisenberg and \'olk^ on the other, two new phenomena have been described as occurring in the agglutination reaction, phenomena which are of great imiiortance in the study of agglutinins. Bail first showed that typhoid bacilli which had been added to an inac- tivated (by heat) agglutinin and then centrifuged could not longer be agglutinated by the addition of active agglutinin. The study of Eisenberg and Volk described an irregularity occurring in a series of agglutinations which manifested itself in this, that the tubes con- taining the largest amount of agglutinin showed only feeble agglutina- tion or none at all, while the tubes containing less agglutinin showed strong agglutination.^ Bail was of the opinion that the phenomenon observed by him was due to the interaction of two components (cor- responding to aralwceptor and complement), and he supi>orted this with several reactivating experiments. Eisenberg and Volk explained the irregular course of the agglutination by the presence of agglutlnoids, a view in which I fully agree. Following Ehrlich's nomenclature I should, however, like to term these agglutlnoids* proaggluiinouls, for we are dealing with the action of substances which arise from the agglutinins as a result of external influences. Furthermore the proagglutinoids possess a higher affinity for the bacilli than the unchanged agglutinin, and they have lost that group which is the real carrier of the agglutinating action, while the other group, which effects the combination with the bacteria, is left intact. 'Archiv. f. Hygiene, 1902, Vol. XLIII. ' ZeitBchr. f. Hygiene, 1902. Vol. XL. ' This paradoxical phenomenon is mentioned by Aaakawa in a report from the Inatitute for Infectious Diseases, Tokio (Sept., 1901). and is lertned by him a "rBverseiy behaving phenomenon" ("ein umgekehrt sich verhaltendes Phanoraen"). ' Since the conclusion of these experiments two new studies have appeared on precipitoida. R. Kraus (Centralhlatt ffir Bakt«riologie 1902. Vol. XXXII. No. 1), V. Pirquet and Eisenberg (Extnul d. Bull. d. I'Acad^ie des sciences de Cracovie, also Centralblatt f. Bakteriologie. 1902. Vol. XXXI, No. 15); also Wiener (Klin. Wochenschr. 1901, trber Precipitoide). The BUlhors arrive at the same resultB as have been described for agglutination . Their experiments for demonetrating these precipitoida are similar to mine for the proaggh it i no ids . FURTHER STUDIES ON THE DYSENTERY BACILLUS. 31» From the large number of experiments which I have made with dysentery and typhoid bacilli I have selected only those which may serve to demonstrate my point. Using the dysentery immune serum described above I found it easy to demonstrate the Eisenberg-Volk phenomenon both with my original dysentery culture and with a Kruse culture. The method was as follows: An agar culture was suspended in 10 cc. of an 0.85% salt solution. At first this was used in the living state; later on, after it had been found that there is no difference in the action of living and dead culture, the culture was used with the addition of 0.02 cc formalin (40%). One cubic centi- meter of this suspension was put into each tube, and decreasing amounts of the immune serum (^/lo, 2/20, V4o> etc., usually up to V6120) added, the total volume in each of the tubes being 2 cc. The tubes were then kept in the thermostat at 37° C. and inspected at the end of 2, 5, and 24 hours, both with the naked eye and with a magnify ing-glass. The results were noted as follows: — no agglutination; ± trace agglutination; + microscopically distinct but feeble; + + very distinct; + + + entirely clear fluid with an agglutinated sediment. TABLE IV. 1 Dilution of the Dysentery Serum. Two Hours. Five Hours. Twenty-four Hours. 1:10 — — d; 1:20 — ± + + 1:40 ± + + + -f- 1:80 + + + + -f--f- 1:160 ± + + -f- + 1:320 — + + + + 1:640 — ± + + 1:1280 — — ± 1:2560 — — 1 : 5120 ~" ^>M "-" The objection was made that the agglutination was hindered in the low dilutions by the large amount of serum present in the tubes. This was met by a corresponding addition of normal serum, and of other fluids (gelatine, mucilage, etc.) to the other dilutions. In the old dysentery serum the question as to the development of the 320 COLLECTED STUDIES IN IMMUNI-n'. proagglutinoid from the agglutinin could only be answered by showing that the amount of proagglutinoid already present in this serum could be increased by heating, by continued exposure to light, or by the addition of chloroform. See Table V. TABLE V. nunHB DilutloD of the Light for 17 Daya. 2Hr,. ah™. 24 H™ 2H™. SHm. 2* HfN. 2Hra. SHn. 2*H™. 1:10 _ _ _ _ _ _ _ _ _ 1:20 1:40 + 1:80 ± + + + + ± + + + + + ± + + + + 1:320 + + J. +. 4 + ± 1:640 ± ± + + ± ± 1:1280 1:2560 1:5120 - - ~ - ~ - - - - The development of the proagglutinoid from the agglutinin was still more distinct in a fresh typhoid immune serum (goat). This serum, which had shown no zone of proagglutinoid, showed a dis- tinct zone after being heated twice to 60° for four hours. Bj' tliis experiment the higher affinity of the proagglutinoid is already demonstrated. It can, however, l)e confirmed by other experiments. By shakmg the dysenterj' serum with chloroform, it was possible to effect almost a complete transformation of agglutinin into proagglutinoid so that the serum hardly agglutinated in any dilution. When to a dose of the unchanged dysonterj' sermn, suffi- cient by itself to effect agglutination, I added decreasing amounts of the serum treatod with (chloroform, no agglutination was obtained in the dilutions up to 1:160. {Control tests with chloroformed normal serum were invariably made.) The same result could be obtained with dysentery serum that had been heated. Dysentery serum heated for 3 hours to 65° C. was able b dilutions of I ; 10 to 1:320 to prevent agglutination by such a dose of the unchanged dysentery serum which by itself would have sufficed fo agglutinate l":160. '(See Tabic \1.) Finally it remained to prove that the proagglutinoid had really been anchored by the bacteria, i.e., that the agglutincbic group of the bacilli had been blocked. This was readily accomplished by FURTHER STUDIES ON THE DYSENTERY BACILLUS. 321 centrifuging, washing the bacilli from those tubes in which no agglu- tination had occurred, and adding to them a dose of agglutinin which by itself would suffice for agglutination. The result was that these bacilli always showed themselves to be no longer agglutinable. see (Table VII.) TABLE VI. Dysentery Dsrsentery Serum Suspension Serum Diluted, Heated to 65<* C. for of Dysentery 2 Hours. 5 Hours. 1:8. Three Hours. Cultures. 0.1 cc. 1:10 (1.0 CC.) 1.00 CC. __ ^ 20 — — 40 — — :80 — — :160 » — ;320 — — :640 — ± :1280 — -f- :2560 " — -f-f- :5120 " — + + Control 0.1 cc. Salt solution 1.0 CC. 1.0 CC. + -f- + One other point may T^e mentioned. In the experiments thuijk^ far described the quantity of bacteria was the same in all the tubes. (See above.) However, if the amount was greatly increased, other phenomena were observed. Table VIII shows that the zone of proagglutinoid disappears entirely if a sufficiently large quantity of bacteria are employed. The explanation of this phenomenon is not difficult if we bear in mind the experiments of M. Neisser and Lubowsky i on the one hand and those of Eisenberg and Volk on the other. The experiments of the latter show without doubt that typhoid bacilli, for example, are able to anchor a far greater quantity of agglutinin than is required for their agglutination. One may therefore assume that the dysentery bacillus also possesses a large nimiber of receptors which are able to unite with, i.e. anchor, the proagglutinoid. The occupation of only a few of these many receptors by the active agglutinin is apparently sufficient, however, to agglutinate the dysentery bacillus. Hence if we add comparatively few dysentery bacilli to a serum which contains much proagglutinoid and little agglutinin, a large number of receptors of the bacilli will be occupied by proagglutinoid. If, on the contrary. ' See pages 146 et seq. COLLECTED STUDIES LN IMMUNITY. TABLE VII. Dilution 2*Bn. To tbe llenidue aHn. 5Hts. Hem&rkit. 1;10 1:20 1:40 1:80 1:180 1:320 1:840 1:1280 1:2560 + + + + + + + + + + + + + 1 s 1 2.0_0C. Control 2.0 cc. -f-dyBentery blciUi + + + + + + + + + Not tested the second time because of the primary agglutina- tion SHouit. 24Houn. The Dyeeniery Senira aHoutm. SHourm. _ _ 1 a.oco. _ _ 1:20 1:40 =5 1:80 1:160 + + ± + + + 1:640 + + + + + + + + Control 2.0 cc. + dysentery bacilli -(- + + + + + + + + aHouni. 5 Hour.. MHoun. 2Hour«. 6Houn. 34Boura. 1:10 _ _ ± _ + + + + + 1:20 ± + + + + 1:40 ± + + + + + + + + + 1:80 ± + + + + + + + + + + 1:160 + + + + + + + 1:320 ± + + + + + + + 1:640 ± + ± + 1:1260 1:2560 1:5120 ~ " " ~ ~ ~ FURTHER STUDIES ON THE DYSENTERY BACILLUS. 323 we add a large quantity of bacteria to the same amount of serum, the proagglutinoid will not suflBce to occupy all the receptors and some agglutinin will be enabled to combine with the bacteria. This, how- ever, results in agglutination. As ah-eady mentioned, my original culture proved entirely identical with the Knise culture so far as the zone of proagglutinoid was con- cerned. The Flexner culture, on the contrary, behaved differently, for, although it was agglutinated in the same degree by the immune serum, the zone of proagglutinoid was entirely absent. This is well shown in the following table. TABLE IX. Dilution of the Dyaentery Serum. 2 Hours. 5 Hours. 24 Hours. 1:10 + + + + + + + + + 1:20 + + + + + + + + 1:40 + + + + + + + + 1:80 + + + + + + + 1:160 ± + + + + 1:320 — + + 1:640 — + + 1:1280 — ± + 1:2560 — — ^ 1:5120 ^ ^" ^ Absorption tests, which were then made, showed that the Eruse bacillus when added to my immune serum completely abstracted the agglutinin and proagglutinoid for this strain, while the agglutinin for the Flexner strain was abstracted to only a slight degree. Conversely, when the Flexner bacillus was added to my immune serum and the mixture centrifuged it was found that the agglutinin for Flexner's bacilli had been completely absorbed, but only a small part of the agglutinin and proagglutinoid for the Kruse strain. We shall therefore have to assume that my original strain corre- sponds completely to the Kruse strain so far as the receptor apparatus is concerned, while both these strains possess certain receptors identical with those of Flexner's strain, and others which differ from them. We may furthermore assume that the serum with which these experi- ments were made was obtained by immimizing not only with my original strain, but that in the course of years various other strains had been used for immunization. In this way agglutinins of various kinds were developed, and these, of course, also fitted strains with 324 COLLECTED STUDIES IN IMMUNITY. a somewhat different receptor apparatus. It may be remarked that the receptor apparatus of the bacteria need not i>crmanently remam the same qualitatively and quantitatively^ as is well shown by Bome experiments of mine in whieh I succeeded in producing a change in these properties by means of cultivation. Thus after having grown Kruse's bacilli on sterile milk' ten consecutive times (always trans- planting on the second day), and finally transplanted it to agar, it was found that this milk strain no longer showed the zone of the proagglutinoid reaction. On making mutual absorption tests it was seen that the organism was no longer like the original Kruae strain but entirely hke that of Flexner. That is to say, this cultivation on milk had effected a gradual change in the Knise strain which manifested itself in the changed proagglutinoid zone of the absorption power. (See Table X.) It remains for further experiments in this direction to see whether I shall succeed in cultivating the Milk-Kruse strain back to the original Kruse strain, or in changing the Flexner strain into the Kruse strain. Thus far the Flexner strain, as well aa the Flexner strain altered by cultivation, have preser\'ed their properties for months. 1. In the bactericidal tests, as well as in agglutination reactions, my original dysentery strain from Japan proved entirely identical with the t^vo Kruse cultures. Since these are the most refined methods at present at our disposal, there can be no doubt as to the identity of my original cultures of 1897 with Kruse's bacillus of 1900. 2. The dysentery immime serum derived from a horse and employed by me for therapeutic puqioses in 1S98-1900 is of \-etT high grade and 'This rnetliod nf cultivation waa really made hecauso of the b Celli ("Zur .\etiiilogie der Dysenterie, v. Leydena FcBtschrift") that my barillua would also coagulate milk like the bacillus found by bim, if it was transpluited 8-10 times on aikaJiue milk. The result of my experiment was absolutely different, tor neither my original strain, nor the strain of Kruse, nor that of D'lexner coagululed milk when the cultures were grown on milk ten roosecutive times, provided care was lakcn to protect the milk from contamination. 1 liad already tested Cclli's bacillus in Japan and found that it produced a considerabla amoimt of gas and coagulated milk, whereas my bacillus docs not do this. In view ct this and of the further fact that Celli's bacillus does not aggliiiinal« with the immune serum produced by means of my bacillus, I conclude tliat these two organisms are entirely distinct from one another — a view whicli I have already expressed in a previo FURTHER STUDIES ON THE DYSENTERY BACILLUS. 326 is the first of such sera whose complementibility by human serum has been proved. TABLE X. Dilution of the Agglu- tinating Normal Culture. First Generation of Milk Culture. Fourth Generation of Milk C^ture. Serum. 2Hr8. 5Hra. 24Hr8. 2Hr8. 5Hr8. 24Hra. 2Hr8. SHrs. 24Hr8. 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 ± ± ± + + + + + ± ± + + + + + + + + + + + + + + + + ± ± + + ± ± ± + + + + + + + + + + + + ± + + + + + 1 1 ff + + + + + + + + + + + + ± ± + + + db ± + + + + + + + + + + + + + ± + + + + + + + + + + + + + + + + + + + + Dilution of the A^lu- tinatmg Sixth Generation of Milk Culture. Eighth Generation of Milk Culture. Tenth Generation of Milk Culture. Serum. 2Hr8. 5Hr8. 24Hr8. 2HrB. 5Hr8. 24Hr8. 2Hr8. SHrs. 24Hr8. 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 + + + + + + + + ± + + + + + + + + + + + + + + + + db + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ± + + + + + + + + + + + + + + + ± ++++++ 1 1 1 + + + + + + + ++++++ + + + + + + + + + db + + + + + 1 1 1 H- + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 3. The deflection of complement of Neisser-Wechsberg could very readily be demonstrated with this serum and pointed the way for a new method of specifically enriching bacterial cultures in mixtures. 4. The change of the agglutinin into a proagglutinoid succeeded both in dysentery serum and typhoid serum. 5. Various strains may possess a somewhat different receptor apparatus. By means of continued culture on milk a certain change in the behavior of the receptor apparatus of dysentery bacilli could be effected. In conclusion, I wish to express my thanks to Prof. Ehrlich and Prof. M. Neisser for aiding me in this study. XXIX. METHODS OF STUDYING HEMOLYSINS. By Dr. J. Moi I, Member of Ibe Institute. The object of the following article is to give a brief outline of the principles governing the technique of htemolytic experiments. It may be taken for granted that the methods employed in the experiments already described will be applicable to many problems of hjemolysis Btill to be studied and to many questions concerning bacteriolyeins and cytotoxins. In view of this a systematic treatise on methods will prove of considerable value, especially to one who uses these methods only occasionally. In those cases where a particular technique has been sufficiently described in the prevouia papers. I have contented myself with merely giving the reference to this paper, Aside, however, from this practical object, a general survey of the subject is to be given which will show how a sj'stem of technique, intelligently built up on a comprehensive theory, Iiag made it possible to push our analylieal inquiries into a department of science which formerly constituted a sealed book to the ordinary methods of chem- istry. Disregard of these newer methods has invariably led to obscu- rity and error, as we iiave been able to show on several occasions*; and in the future, even if refined chanical methods can successfully be introduced into this domain, the general method of analysis here outlined will always form the basis of this study. According to our experience the study of hsemolysins will be much simplified by atten- tion to a number of technical details which are described in this article. I. Collecting and Pre§erTlng the Blood and Blood Serum. We shall begin with some remarks on the collection and preserva- tion of the blood and serum required for these experiments. Aa a general rule for hjpmolytic experiments it is not necessary tuple, paffas 181 et scq.; 2-tl e it seij., etc. METHODS OF STUDYING HEMOLYSINS. 327 to observe aseptic precautions; usually all that is required is to collect the blood in dry sterile vessels, avoiding contamination with dirt, etc. Hence the troublesome method of collecting blood from the carotid of the animals will only then be undertaken if for some reason asepsis is necessary or a large yield of blood is required. In the latter case the yield of blood can be considerably increased toward the end of exsanguination by rythmic compression of the cardiac region. With goats, sheep, etc., the blood can easily be obtained without any previous dissection by means of a suitable canula thrust through the skin directly into the jugular vein which has been distended by compression on the cardiac side. This is the method commonly employed in obtaining the therapeutic sera from horses. In this way small amounts of blood can be drawn from the animals a great many times. Smaller animals, such as dogs, rabbits, guinea-pigs, and rats, are most readily bled by ansesthetizing them, dis- secting off the skin of the thigh and then with one stroke cutting both the femoral artery and vein. From rabbits small amounts of blood are easily obtained by incising the ear with a scissors or by means of a hypodermic needle introduced into the marginal ear vein. Small amounts of blood can be obtained from birds from the large wing vein; in the case of geese and ducks the web of the foot can be incised. For purposes of obtaining serum the blood is collected in cyhndrical vessels and allowed to coagulate spontaneously. It is kept in the refrigerator until the serum has separated. Several hours after collecting the blood, it is well to loosen the clot from the sides of the tube by means of a glass rod or spatula, for if this is not done the serum may not separate. Small amoimts of blood are best allowed to clot in cylindrical glasses or tubes placed slantingly. After clotting has occurred the vessel is placed upright. The serum which separates will then flow to the bottom and can be poured off the next day. If the serum is clouded with blood-cells, these are to be removed as soon as possible.^ When the serum is poured off the first time the vessel containing * An excellent centrifuge with a capacity up to 200 cc., but which can also be had for larger quantities, is that made by Runne, the mechanic in Heidelberg University. This machine is made either for water or electric power, and runs exceedingly smoothly. For centrifuging smaller quantities of fluid, and espe- cially for sedimenting blood-ceUs from diliUe blood suspensions, the hand cen- trifuge designed by Steenbeck-Litten, and made by F. and M. Lautenschlager in Berlin, is excellent. 32S COLLECTED STUDIES IN IMMUNITY. the clot can be kept on ice for 24 hours longer. In that way a further yield la obtained. In order to obtain serum immediately the blood is defibrinated by whipping it with a stick of wood or by shaking it in a bottle containing some glass beads, or still better a little mara of dry- sterilized iron turnings. After the blood is defibrinated it is centri- fuged and the serum carefully separated by means of a pipette. It is well to fasten a long rubber tube to the upper end of the pipette and have an assistant suck while one watches the point of the pipette. So far as concerns preservation of the serum it may be said that our present experiences are not yet sufficient to permit us to formulate safe rules having general applicability. It is not only necessary to prevent putrefaction, but also to presen-e intact a large number of most unstable substances, the conditions necessarj' for whose existence are, in part, evidently very narrowly limited. Hence for the present it may be put down as a rule that in all important primary determina- tions only very fresh scrum should be emijjoyed. This applies above all to the study of the complements. Negative results with sera which have been kept several days and which have been exjxised to any kind of thermic or chemic influence, are particularly unreliable. Hence it is necessary that those properties of a serum which one purposes to study should be examined before the serum is presented, so that secondary changes can then be controlled at any time. The easiest substances to presen-e are the antitoxins, anticomple- ments, antiamboceptors and the majority of artificially-produced amboceptors. By the addition of carbonic acid, Pfeiffer ' has succeeded in keeping a cholera immune serum deri\'ed from a goat for five years without decrease in strength. We have preserved hemolytic ambo- ceptors for a long time without any addition, by keeping the sera in an ice-chest at 8° C. The development of bacteria is usually prevented by heating the serum in the test-tubes stoppered with cotton plugs to 67" for half an hour. In this way the serum is both inactivated and sterilized, t^o far as our experience goes the anticomplementB and antiamboceptors can be preserved in the refrigerator like the amboceptors. Drying the serum over sulphuric acid or over anhy- drous phosphoric acid in vacuum can also be used for these substances. Of all the substances here concerned the complements are by far the most labile; whenever possible, therefore, fresh serum is used ' See Mertens, Deutsche nied, Woe)ienBph. 1901, No. 24. METHODS OF STUDYING H.EMOLYSINS. 329 for activation. Mo8t of the compIemeDta will keep unchanged for a number of days provided the serum ia kept on ice. But this does not preclude unpleasant aurpriees, diminutions in the complementing power often occurring to a high degree without any assignable cause. According to our exjierience the complements of guinea-pig serum and goat serum are relatively stable. Tlie least reliable in this respect is horse serum, whose complementing powers are often partially or completely destroyed within twenty-four hours. The complements also suffer when the serum is dried : at least that has been the case in our rather limited experience. The best method of preserving the complements for a long time, and the one almost always reliable in all cases, consists in freezing the serum at —10° to — 15°C. This method has been employed in the Institute for a long time. The serum is bottled in little vials, which are then kept in a freezing apparatus or in a well-insulated freezing mixture of ice and salt, each vial being thawed out as needed. This procedure is at present the only one which is of general appli- cability and which presBn-es the various constituents of the serum for a long time. The blood used for the hiemolytic tests is defibrinated by one of the methods above mentioned. In special cases, instead of defibrina- ting, one can prevent coagulation by precipitating the lime salts. This is done by allowing the blood to flow into salt solution to which citrate of soda has been added, as was recommended by Ehrlich.' For the majority of experiments the blood ia diluted with physiological salt solution. If for any reason one wishes to remove the serum, the blood is separated by centrifuge and the suspending fluid renewed several times. As a rule blood which has been kept on ice for two days can still be used. It should also be mentioned that a suitable salt solution should be employed for each species of blood. For the blood-cells of most mammals a feebly hypertonic solution of NaCl 0.85% is best adapted. In 0.85% salt solution dog and horse blood frequently shows a slight amount of spontaneous haemolysis which can often be prevented by using a somewhat higher concentration (0.95%) of the salt. As a rule strongly hypertonic solutions of salt are to be avoided because the increased contents of salt markedly inhibits htemolysis.^ ' Ehrtioh, FortschriltB der Hedi^in, 1897, No. 2. ■ S. Markl, Zeitsch. f. Uygieoe, Vol, 39. II. The Method of Making Hsemolytlc Experiments. General Considerations. With a little practice the quantitative estimation of hsemolysis proves very simple. The two fundamental point*), entire bffimolysis (conaplete), and no hemolysis whatever (0), are usually very readily recognized. By "trace" we mean the occurrence of a faint zone of solution observed just above the cells by gently agitating the teat-tube. The estimation of complete hsmolygis only then offers difficulties if considerable agglutination has occurred, so that the fluid when shaken is clouded by the clumped stromata. Such cases in themaeU'es are poorly adapted for quantitative studies because at times the rapid agglutination may purely mechanically prevent the escape of the hiemoglobin and so simulate an absence of hemolysis. In this respect according to our experiences the greatest diffi- culties are presented by dog blood-cells and the specific immune sera (derived from goats) against these. This is still more the case in sueh sera derived from rabbits. It often happens, before even a trace of hemolysis has occurred, that the dog blood-cells are agglutinated and fall to the bottom of the test-tube, Goose blood and specific immune serum behave similarly. In these cases it is necessary by means of frequent shaking to separate the agglutinated blood-cells so that the bajmoglobin is given chance to escape. In those cases in which the usual method of describing the degree of solution does not suffice, and accurate quantitative determinations of the amount of biood-cells dissolved are desired, one makes use of a colorimetric procedure devised by Madsen in which a color comparison is always .made by dissolving blood-cells in water .^ Agglutination is usually easily recognized on shaking up the sedi- mented blood-cells. It becomes very evident when the specimens of blood are shaken and one then compares the rapidity with which the blood-cells settle to the bottom. This is always greater with agglutinated blood-cells. In general a 5% suspension of the blood-cells in 0.85% salt solu- tion has proven best adapted for hiemolytic experiments. 1 to 2 cc, of such a mixture in each test-tube is sufficient for most tests. When material is scanty one can use amounts very much smaller, though usually this will be at the expense of accuracy. In this case, of METHODS OF STUDYING HEMOLYSINS. 331 course, the test is made in very narrow test-tubes.^ The serum to be tested is added to the various tubes in decreasing amounts. The volume of fluid should be made the same in all the tubes by the addition of salt solution, for the total amount of fluid present may influence the course of haemolysis. We usually keep the tubes in a thermostat at 37® C. for two hours, frequently shaking them if necessary. They are then kept in the refrigerator at 8° C. overnight, which allows the intact blood-cells to settle. In the cases thus far examined by us this method has always sufficed to produce the maximum amount of haemolysis, though, of course, in a given case it may have to be modified to suit the circumstances. It should be mentioned that in testing any substances for haemoly tic action, the blood-cells must always be freed from serum by repeated washing, for the serum may in some instances (e.g. with solanin) give rise to a marked inhibitory action and so lead to errors. ni. The Technique of Immunization. So far as the production of hcemolytic amboceptors by means of immunization is concerned, only a few very general rules can be given, for thus far sufficient systematic investigations have not been made to determine the optimal conditions in any one direction. In immunization one always selects such animals whose serum by itself is not at all or but slightly haemolytic for the blood employed, for then the development of a haemolysin is most readily determined and the normal serum of this species always furnishes an ideal com- plement. If animals are immunized whose serum by itself already acts haemolytically on the blood used, it is necessary to make an exact preliminary determination of the haemolytic power of the normal serum, and also to make a simultaneous control with normal serum, when making the haemolytic experiments. In some instances it may be necessary to subject the blood to a preparatory treatment, for the purpose of removing the serum more or less completely. This is done by means of the centrifuge and is required especially in those cases in which intravenous injections are made, or if large amounts of a blood are employed whose serum * In certain cases the employment of very high columns of blood is indicated, for in that case the development of zones (colorless — feebly red — strongly red) permits of a very accurate estimation of the period of incubation of the poison, or of the different vulnerability of the blood-cells. See also Madsen, I.e. COLLECTED 8TUOIE8 IN IMMTJNITy. is liiglily toxic for the animal injected. If, for example, a rabbit is injected intravenously with 10 cc. of dog blood whose serum has not previously been renio\'ed, the animal will die acutely. By pre- viously heating the serum one also obviates the reactive production of serum coagulins and anticomplementa, both of which can at times hinder the estimation of hjemolysis. A general rule as to which mode of injection is to be chosen for immunization cannot be laid down. Larger laboratorj' animals are usually injected subcutaneously ; goats usually bear intraperitoneal injections very well. This mode of injection, ming blood-cells which have previously been dissolved with water, is used especially when a particularly marked " ictus jmmunisatorius " is desired, as, for example, in the production of isolysins. Hirds are injected into the large pectoral muscles or intraperitoneaUy. For rabbits and guinea-pigs the intraperitoneal injections are well adapted, since, if the malarial is not positively sterile, secondarj' injections (which in subcutaneous inoculations often lead to troublesome abscesses, especially in the rabbit) are most readily avoided. Injuries to the intestine are best avoided by holding the animals almost vertically, head down, and thrusting the needle into the abdomen in the median line a little above the bladder. The needle should not be too sharp, nor thrust in very deeply. (Personal communication of Dr. R. Krause.) The repetition of inlrarerums injections offer esjiecial difficulties, for after hEemolysin formation has once occurred the blood-cells introduced are rapidly dissolved, leading to the death of the animal from embolism. (Rehns.') Another thing which may lead to death from embolism is the formation of coagulins in consequence of a previous injection of blood which has not been freed from serum. These coagulins cause a rapid formation of precipitates within the blood circulation.^ 'ITie amount of blood used depends upon the size of the animal to be injected and upon the special conditions of the experiment. Up to a liter of blood, freed from must of its serum, can be injected ' Rehn.'i. Conip. rend, de In Soc, de Biol, 1901, No. 13; see alno similar observntionB made on man by Bier. Miinch. med. WochenscL. 1901, No. IS. ' Very likely the inejiplicable rei^iiUs obtained by Magendie ("Vor- leRiin^ii iiitcr das Blut," German translation by Kriipp. Leipzig. 1S39) were due to the formation of coagulins. Magendie found that rabbits which had tolerated two intravenous injection!i of egg albumin without any injury whatever inunedi* Bt«ly iuccumbed to a further injection made after a number of days. METHODS OF STUDYING BLEMOLYSINS. 333 into goats without injury. In rabbits of 2 kilos it will hardly be possible to go beyond 100 cc; and guinea-pigs, corresponding to their weight, proportionately less. According to our experience a single injection of 20-30 cc. sheep, goat, ox, or dog blood leads to a strong formation of hsemolysin, which can be still further increased by a subsequent injection of 40-60 cc. six to ten days later. We have found that further injections of the same or larger amoimts (80-100 cc.) have no advantage. We have occasionally observed that these were associated with a decrease in the amoimt of haemolysin. As a rule, the serum attains its maximimi power between the sixth and tenth day,^ but this is subject to individual variations, as is shown by the case described by Ehrlich and Morgenroth of a goat in which an isolysin developed critically on the fifteenth day (see page 29). The injections of serum lead principally to the production of antiamboceptors and anticomplements, in some instances also to that of hsemolytic amboceptors in consequence of the receptors present in solution in the senmi.^ The production of antiamboceptors necessitates a special selection of the animal species. Our own positive results are limited to the injection of goats either with the serum of a rabbit which had been immimized with ox blood, or with an isolytic serum. Since in these cases the inmiune serum is toxic for the goat, or, more particularly, acts destructively on the blood, it is necessary to commence with the injection of small amounts (10-20 cc.) and gradually, as the reaction subsides, go on to larger doses. As in the case of all inunimizations with toxic substances it is particularly necessary to keep a careful control of the weight of the animals; the rule always to be observed is that inmiunization can only then be proceeded with when the animal has again attained the weight it originally possessed. In order to produce anticomplemenis larger animals, such as sheep and goats, are injected with increasing amounts of normal serum, beginning usually with fairly large amounts — 100 to 500 cc. As a rule, when rabbits have been injected two or three times with guinea- pig, horse, goat or ox serum (commencing with 5 to 10 cc. and increas- ing to 20 to 50 cc), a plentiful supply of anticomplement will have developed in the serum. In many cases the injection of an inactive ^ See also Bulloch, Centralblatt f. Bact., Vol. 29, 1901. 'See Morgenroth (page 241, this volume) and P. Miiller, Miinch. med. Wochensch. 1902, No. 32. COLLECTED STUDlIiS IN IMMUNITY. serum, which had thus been deprived of much of its toxic property, woutd appear to be preferable, for, owing to the complementoids which it contains, this would cause the production of anticomplementa just as well as fresh serum. (See pages 79 et seq.) If it is desired by the injection of a certain serum to produce anticomplemeiits which are also directed against various other sera,^ it is necessary to repeat the injections several times in increasing amounts. While treating a goat with rabbit serum, Ehrlich and Morgenroth observed the development first of anticomplementa directed exclusively against the complement of rabbit serum (iso- genic anticomplementa) ; in course of time anticomplementa directed against the complements of guinea-pig serum (alloiogenic anticom- plements) also appeared. Here evidently we are dealing with partial complements, present in rabbit serum in small amounts, which require several repetitions of the injections in increasing amounts in order to excite the production of anticomplementa. In the production of serum eoagulina [precipitins] one proceeds as for anticomplcments. These serum coagulins have been shown to possess considerable value for the forensic determination of various species of blood, especially human blood, as has been shown by the researches of Wasscrmann and Schiitze, Uhlenhuth, and many others. In the preduction of milk coagulins one or two injections of 20 to 40 cc. of milk into a rabbit are usually sufficient. The milk can be heated to 60° previous to injection in order to reduce the number of germs present. In connection with the production of serum coagu- lins Uhlenhuth makes some interesting statementa (Deutsch. raed. Wochenschr. 1002, No. 37). Among other things he describes something we had also noticed, namely, the occasional failure of the reaction and the development of "alloiogenic " coagulins as the titer of the senmi increased, a fact which corresponds to what we have above described for the formation anticomplcments," IV. Determining the Hscmolytle Action. The fact that certain poisons of vegetable or animal origin, as well as nonna! sera and other body fluids, possess a hiemolytic action can be determined so readily that it will be superfluous to enter further ' See pages 1 1 1 et seq. ' Coaeemiag isogenic and alloiogenia antiooinpleiiieiitfl, see Uorgenrotli and Sachs, pages 258 et eeq. METHODS OF STUDYING HEMOLYSINS. 335 into the subject. In passing, however, it may be mentioned that for an investigation in this direction to be at all complete it is necessary to make use of as many different species of blood-cells as possible. The susceptibility of the cells can be extraordinarily diverse, so that certain poisons exert a marked haemolytic effect on some species of blood, while they fail to have any action whatever on other species. Thus the poison of the garden spider, studied by Sachs,^ is inert for guinea-pig or dog blood-cells, while it has strong haemoly tic powers for rabbit blood-cells. Crotin which dissolves certain blood-cells (e.g. rabbit blood) and agglutinates others (e.g. hog blood) behaves in similar fashion.^ In the case of the specific haemolysins produced by inununization the choice of blood, of course, is already indicated. But even here, extending the investigations to numerous other species of blood may lead to valuable information concerning a community of recep- tors such as exists between sheep, goat, and ox ^ and as has recently been shown by Marshall to exist between man and certain species of monkeys. In testing a serum for the presences of isolysins it ia necessary to use the blood of numerous individuals, for according ta our experience the sensitiveness of the blood, in the case of goats, is subject to the widest individual fluctuations. In this way one can easily be misled to assume that the experiment results nega- tively. It is advisable, when testing a fluid for hamolytic propertiea for the first time, to remove the serum by washing the blood-cells at least once. Under certain circumstances a slight degree of haemolytic action can be masked by an antihsemolytic action of the normal serum. This is seen to a high degree in the case of the haemolytic poisona of the organ extracts.* So far as the dosage is concerned one should select wide limits, especially in the first experiments. If one has once determined the presence of a haemolytic action, the quantitative estimation follows by means of a more or less finely graded series of experiments. Types of these experiments are found on pages 168, 270, 276, etc. In testing a haemolysin which has not yet been examined, it is * See pages 167 et seq. 'Elfstrand, tiber giftige Eiweissstoffe welche BlutkOrperchen verkleben* Upsala, 1891. ' See pages 93 et seq. * See Korschim and Moigenroth, pp. 267 et seq. COLLECTED STUDIES I.N IMMUNITY- important to determine whether the hemolytic agent is a haptin in the true sense. 80 far as the alkaloids, glycosides, etc., which act htemolyticaliy are concerned, they are generally readily identi- fied by means of the chemical methods devised for their separation, methods based on precipitations and shaking out with solvents. This is not true for the haptins; tlicy cannot be prepared by these methods. At the most, it is possible to precipitate them in con- junction with the albuminous bodies. Another distinction consists in this, that the substances wiiich are chemically defined are usu- ally thermostable, while the haptins in the great majority of caeea are destroyed by heat, especially by boiling temperature. One distinction above all, however, is the fact that only the liaptina are capable of causing the production of antibodies by immunization, and this makes a classification possible even in difficult ca-ws. Fre- quently the facts which we have already learned about a substance allow us to make definite conjectures. For example, if a vegetable extract possesses hemolytic properties which are not destroyed by boiling, and if it is foimd that the ha^molytic substance is soluble in ether, we can at once exclude this from the class of haptins. On the other hand, If one finds that the hEemolytic action of an animal body fluid is destroyed by heating to 56° C, this fact already argues in favor of a haptin. Other methods, including perhaps the immu- nizing reaction, would then be required to determine this positively. V. The Study of Complex Hsemolyslns. We now take up a question of paramount importance which arises in the study of every hsemolytic poison, namely, whether in any given instance we are dealing with a simple ha^molyain, or with a complex one consisting of amboceptor and complement. In determining the complex nature of a ha^molysin we now have the following methods at our disposal: 1. Separation of amlioccptor and complement by allowing the former to be tied by red blood-cells at low temperatures. 2. Removal of the complement or changing the same into the inert complementoid. (a) Alisorbing the complement by means of certain cells (e.g., yeast-cells, bacterial cells, cells of animal organs), or by means of porous filters. METHODS OF STUDYING HiEMOLYSlNS. 337 (6) Thermic and chemic influences, such as heating to 50-60° C, the action of alkalies and acids, digestion with papayotin. The separation of amboceptor and complement at low tempera- tures is of the utmost importance and has been used for the analysis of complex hsemolysins with considerable success. The conditions necessary for the successful operation of this method have been discussed in detail in a previous paper. A separation is only then possible when at low temperatures the affinity between the cyto- phile group of the amboceptor and the receptor is greater than that between the complementophile group of the amboceptor and the corresponding group of the complement. The degree of difference in the affinities would, of course, determine the degree of complete- ness of the separation. In some instances most peculiar relations are found, as is shown, for example, by the behavior of eel serum to rabbit blood. Attempts to effect separation at low temperatures fail in this case, first, because haemolysis ensues even at 0° C, and second, because the employment of higher concentrations of salt (up to 5%), which in other cases has afforded a means of loosening the combination of amboceptor and complement, does not suffice to prevent haemolysis. Naturally from this behavior we must not conclude that eel serum does not contain a complex haemolysin, but merely that in this case peculiar conditions are present which, owing to the insufficiency of the methods thus far employed, are still obscure to us. In those cases in which separation at low temperatures fails, a second method may be considered. This depends on the fact that a high degree of salt concentration, somewhat after the manner of low temperatures, can prevent haemolysis; concentrations which still permit the union of receptor and amboceptor preventing that of amboceptor and complement. The prevention of haemolysis by means of salts, first described by Markl ^ and erroneously ascribed by him to conditions of diffusion, is also due to this. Markl entirely overlooked the fact that in certain cases the combination of toxin and antitoxin (e.g. Tetanus toxin -f Antitoxin) is also prevented by salt. (Knorr.) For the application of this method see Ehrlich and Sachs, page 214. It is perfectly obvious that the cold method will fail absolutely in cases like the one described by Ehrlich and Sachs (page 217) in which the union of amboceptor and complement is the prerequisite » Markl, Zeitschr fiir Hygiene, Vol. 39, 1902. 33S COLLECTED STUDIES LV IBIMUNITY. for the union with the blood-cells. Such a possibility must always be borne in mind. The technique of this separation at low temperatures is very simple. The tubes containing the blood and the serum respectively are cooled to 0° by being placed in iced water or by packing in ice. Thereupon the serum, in amounts which are not far either way from the single solvent dose, is added to the blood. After being kept at 0° for two hours the mixture is rapidly centrifuged and the super- natant fluid quickly removed. If desired, the sedimented blood- cells can be washed with salt solution and then suitably suspended. The decanted fluid is again mixed with blood-cells. For this pur- pose, in order not to increase the total volume, one takes the blood- cell sediment- centrifuged from the required amount of the 5% sua- pension. If a complete separation of amboceptor and complement ha^i been efltected, it will be found that neither are the sedimented blood-cells dissolved nor is the decanted fluid able to dissolve the blood-cells added anew. It is then necessary to determine the pres- ence of complement in the decanted fluid, which is done by adding suitable amounts of serum inactivated by heating. Similarly the amboceptor anchored by the blood-cells at low temperatures is demon- strated by adding to the sediment the complement present in the decanted fluid. The second and simpler method is that of inactivating the hemo- lytic serum by means of heat and then activating the amboceptor by the addition of complement. In this the chief diSicuIty often consists in the fact that a certain complement required in a par- ticular instance is not contained in all sera, and further that the sera which contain this particular complement often in themselves dis- solve the blood-cells by means of a normal amboceptor. There are several ways of overcoming these difficulties. The neatest method and one which is applicable in many cases consists in selecting as the complementing agent the serum of that anitn&l species whose blood is being tested, as, for example, using guinea-pig serum as complement for amboceptors acting on guinea-pig blood. In such cases a solution of the blood-cella by means of the animal's own serum is, of course, precluded. In all other cases one must make use of complementing sera which are imrelated to the species of blood in question. One fre- quently discovers sera for this purpose which do not in themselves dissolve the blood-cells to be tested, aa, for example, in reactivating METHODS OF STUDYING HiEMOLYSINS. 339 the amboceptors for sheep blood or ox blood by means of goat serum. But it is often possible to complement an amboceptor with a serum which in itself dissolves the blood-cells, but which, in the amounts in which it is able to effect completion, has little or no hemolytic action. It is obvious that in such cases the solvent power of the serum by itself must be accurately determined by means of controls. While this method is often successful, the relation in these sera between the normal amboceptor and the complement is fre- quently so unfavorable that it is impossible to complement the foreign amboceptor. In such cases one can get rid of the normal amboceptor by anchoring it to blood-cells at low temperatures, as Flexner and Noguchi ^ have recently done in order to obtain com- plements for the haemolytic amboceptors of snake venoms. Or one can attempt artificially to increase the amount of complement con- tained in complementing serum, after the method of P. Miiller.^ This author succeeded in effecting a considerable increase in the complements of chicken serum, by injecting the animals with solu- tions of peptone. So far as the choice of the complementing sera is concerned it is obvious that, in amboceptors produced by immunization, whenever possible the preference will be given to those sera which are derived from the same species which jrielded the amboceptor. For the remaining cases the principle may be formulated that that serum is most useful which is derived from a species closely related to that furnishing the amboceptor, because often in distantly related species partial amboceptors present only in very slight amounts are com- plemented.^ Another point of considerable importance in the completion of amboceptors is the manner in which the sera are inactivated. As a rule inactivation is effected by heating the serum for half an hour in a water-bath. According to recent investigations special atten- tion must be paid to the degree and duration of this action.'* * Flexner and Noguchi, Journal of Exp, Medicine, Vol. VI, 1902. ' Miiller, Centralblatt f. Bacteriologie, Orig. Vol. 29, 1901. * Ehrlich and Morgenroth, see pages 110 et seq. * In order accurately to observe the temperature constantly maintained it is well to use thermometers with particularly wide divisions on the scale (1** C.-l cm.). These thermometers need only embrace a moderate range of degrees (about 40*^-80*' or 46'*-86*'). They can be obtained from A. Haak in Jena. 340 COLLECTED STUDIES IS IMMUNITY. For many years, o\>'ing to the valuable researches of Buchner, an inacCivation by means of temperature of 55-56° was regarded as practically a specific criterion for the alexins. We now know, however, that no general rule can be [ormulated in this respect. On the one hand there are complementd which are not at all influenced by the customary half-hour'a heating to 55° C. (thermostable comple- ments), and on the other there are amboceptors which are com- pletely destroyed by such heating. A complement belonging to the first cat^ory was first described by Ehrlich and Morgenroth • &a occurring in considerable amount in normal goat serum and in the eenim of a buck which had been immunized with sheep serum; and thermolabile amboceptors, especially in normal sera, are not at all rare. Thus the amboceptor above mentioned regularly present in horse serum and acting on guinea-pig blood, as well as one studied by Sachs ^ present in dog serum and also acting on guinea-pig blood, is completely destroyed by half an hour's heating to 55° C. Hence the first rule in the demonstration of the complex character of hiemo- lytie poisons by thermogenic inactivation is always to employ the lowest temj-ierature at which inactivation takes place within a short time (20-60 minutes).^ VI. The Quantitative Estimation of AmboceptorSt Complementa and Receptors, In special cases, e.g. during the course of an immunization, it is of considerable value to accurately determine the amounts of amboceptor and complement present in the serum. While referring to the studies of v. Dungem (p. 36), Bulloch (I.e.), Morgenroth and Sachs (pp. 226 and 250), we should like to emphasize that, in general, in determining the amount of complement it is necessary to make ' See page 13. ' See page 181 et seq. » According to the reaearches of Korscbun and Morgenroth (see pp. 267 et seq.) the hs>molytic subelunces of organ extracts are "coctostable." i.e., they arc not destroyed even by several hours' boiling. Hence we designate a substance as Thcrmolatrile, if it is rendered inert by heating to SS'-SG" C; Ttumnontabk, if it wilhstanda beating to 56° or over but is destroyed by boiling; Coiiostabk, if it resiste boiling at 100° C, In special caaes in order to still more closely characterize their behavior one can add temperature and duratiou of heat as an index. METHODS OF STUDYING H*:M0LYSINS. 341 two determinations, namely, one carried out with the single-solvent dose of amboceptor, the other with a high multiple of the same. The reasons for this procedure can be found in tiie study on the quantitative estimation of amboceptor, complement, and anticom- plement (page 250). So far as the estimation of the amount of amboceptor is concerned, this is effected according to similar principles, and lisually in such a way that one works with an excess of complement. A certain difE- culty is encountered in the fact that the amount of complement contained in the serum, e.g., rabbit serum, is variable. It is there- fore always necessary, in order to exclude tliis disturbing factor, to first determine the activating value of the complementing serum using a specimen of the immune serum in question as a standard serum. Directly after this test by which the amount of complement is strictly defined, the quantitative estimation of amboceptor in the new serum must be undertaken. It is also important to estimate the amount of receptor present in the red blood-cells: the measure of this is the binding of ambo- ceptor. Erhlich and Morgenroth (see pages 72 et seq.) have demon- Btrated that the binding capacity of red blood-cells varies to an extraordinary degree. While in many combinations the blood-cells combine with just that amount of amboceptor, which on the addi- tion of suitable complement leads to their complete solution {amho- ceploT unit), it was found that in numerous other cases the blood- cells are able to take up as high as 100 single-solvent doses of ambo- ceptor. Corresponding to the ambocejitor unit, the receptor unU, is that amount of receptor which combines with one amboceptor unit (see page 254). The combining power of the erythrocytes is determined by adding varying multiples of the amboceptor imit to the blood-cells, centrifuging at the end of about an hour and then allowing the various decanted fluids to act on fresh blood-cells in the presence of sufficient complement. The degree of haemolysis which occurs readily shows just how much amboceptor was still completely bound, (See page 75 and the protocols on pages 98 and 99.)» ' Concerning llie extraordinarily lai^e binding capacity of bacteria for ajtstlu- lininB and for amboceptora, aee the interesting communication of Tisenberg and Volk (Zeitscli. f. Hygiene, Vol. 80) and of Pfeifter and Priedberger (Berl. klin. Wftchensch. 1902, No. 26.) ■ klin. W 342 COLLECTED STUDIES IN IMMUNITY. Finally, in studying the complements of a serum it is often of considerable importance to determine their plurality. The methods leading to a differentiation of the separate complements have been described in detail in a number of [iluces, so that we can here con- tent ourselves by referring to the studies of Ehrlich and Morgenroth (pages 11-56, IIO), of Ehrlich and Sachs (page 195), and of Marshall and Morgenroth (page 222), VII. The Study ot Antlhaemolytlc Actions. The subject of antihtemolytic functions, which has only recently- been carefully worked up, has attained considerable importance for the comprehension of the mechanism of hEemolysins. Although at the present time the study of the influences inhibiting hiemolysis is not at alt complete, it is possible at least to indicate certain gen- eral principles. We shall begin with the simple hiemotoxins, which are character- ized by a cytophile haptophore group and a zy mo toxic group. (Analogous to these are the hiemagglutinins, also characterized by a cytophile haptophore group and an agghitinating group.) If we analyze the action of these hamotoxins, we see that this can be inhib. ited in two ways: (1) By means of an antibody which fits into the haptophore group and so deflects this from the receptor of the cell. (2) By means of substances which are capable of occupying tlie receptor of the blood-cell and so block this lor the entrance of the htemo toxin. So far as the first group is concerned, such antibodies are well known for a large number of blood poisons. We need only call to mind the antlhamolysins, such as anticrotin, antitetanolysin, anti- staphylolysin, antibodies against the hiemolytic venoms of snakes, spiders, and toads. Besides these there are the antiagglutinins, such as antiricin, antiabrin, anticrotin. These substances can be produced as antitoxins by means of immunization, but they also occur in normal serum, as, for example, antitetanolysin m horse serum (Ehrlich), aritiataphylolysin in serum from goats, man, and horse (M. Neisser and Wechsberg) , The second method of inhibition is effected by substances which occupy the receptors of the cells. Hence these must be substances which possess the same haptophore group as the htentotoxins them- METHODS OF STUDYING ELEMOLYSINS. 343 selves. This, however, at once leads to the idea that transformation products of the hsemolysin itself could exert this action. Ehrlich's researches on the constitution of diphtheria poison have shown that in toxins and related bodies the zymotoxic group is far less stable than the haptophore group. The bodies so derived, toxoids, still possess the property of combining with the cell receptors, they are still able to neutralize antitoxin, and to excite the reactive formation of antibodies, but they more or less completely lack any toxicity. This formation of toxoid, first described by Ehrlich, has since been demonstrated for a number of substances, hamotoxins (tetanolysin, snake venom, staphylolysin), as well as agglutinins and coagulins.^ Ehrlich in his first study already pointed out that an increase in the haptophore 's aflSnity, developing in the course of toxoid for- mation, was conceivable. The toxoid which was thus produced would then be able, owing to the increased alBSnity, to imite with the receptor of the cell even in competition with the imchanged toxin. In this way the toxoid would protect the cell against the entrance of the real poison, and of course, against the poison's injurious influence. For these toxoids Ehrlich has proposed the term fro- toxoids. Of course such a protective effect can also be produced in conformity with the laws of mass action by toxoids having the same affinity (syntoxoid) to the cell receptor as the toxin, whereas the protection will be slight or minimal if , as a result of toxoid for- mation, there is a decrease in the haptophore group's aflSnity (ept- toxoid). Recent investigations on the agglutinins of bacteria ^ and on coagulins have shown that by heating these substances, agglu- tinoids, which possess a higher affinity than the agglutinins them- selves, are developed in considerable quantities. These are, there- fore, termed proaggluiinoids. It is an easy matter in any given instance to determine experi- mentally which of these two inhibitory processes is present. If one is dealing with a certain particular serum which inhibits the action of the hsemotoxin, it may be regarded a priori as probable that the substance in question is an antibody in the ordinary sense. This becomes almost certainjtf - bacilli, horses are well adapted; goats very much less so; rabbits and guinea-pigs are very ' Miincli. med. Wochenach. 1900. No. 37. •ZeitMhr. f. Hygiene, XXVII, 1898. 'Deutsche med, WocheDBch. 1901. J TECHNIQUE OF BACTERICIDAL TEST-TUBE EXPERIMENTS. 349 ill-suited for this purpose. One should, of course, never forget to examine the normal serum for bactericidal powers previous to immunization. With a great many bacteria it has not yet been possible to pro- duce a serum bactericidal in vitro. Thus our experunents in this direction extending over many years were unsuccessful with staphy- lococcus pyogenes aureus (goat, rabbit) and with the diphtheria bacillus. Nor have we been able thus far to obtain bactericidal effects in vitro from Susserin and other similar sera which are effective in animal tests. The reasons for this behavior are not yet clear, and they are therefore still being studied. Bordet and Gengou have devised a method (Annales de ITnstitut Pasteur 1901) by the aid of which a bactericidal interbody pro- duced by immunization can be recognized even in those cases in which plate experiments fail (e.g. erysipelas of swine). This method depends on the property, said to be possessed by bacteria to which interbody has been supplied, of combining also with hsemolytic com- plements. This loss of complement, which can be readily detected, shows that the bacteria have combined with a bactericidal inter- body. Without entering into the theoretical significance of this interesting experiment we shall content ourselves by saying that in several cases in which we tested bactericidal immune sera in this way we failed to obtain satisfactory results. The method does not seem to us to be suited to a quantitative estimation of an immune serum. It need hardly be said that the first requisite for the success of bactericidal experiments is that all vessels, diluting fluids, as well as the sera employed be absolutely sterile. Great care is necessary, especially in collecting the blood. The method described in the preceding chapter for bleeding rabbits and guinea-pigs is sufficient to obtain sterile blood. For collecting smaller quantities of blood from the ear vein of rabbits it is necessary to first cleanse the ear with 70% alcohol and then thrusting a short sterile hollow needle into a vein. In many cases, to be sure, the blood can also be col- lected by making a short incision across the marginal ear vein with a sterile scalpel, and then, by holding the animal properly, allowing the blood to flow out without running over the ear. In bleeding pigeons and chickens by decapitation one cannot always coimt on sterile serum; hence it is well to lay bare the vessels of the neck. For repeated bleeding of guinea-pigs one must also 350 COLLECTED STUDIES IN IMMI'NITY. collect the blood directly from the vessels of the neck and then tie the vessel. It ia an easy matter to obtain very small quantities of sterile pigeon blood from the wing veins by first carefully removing the feathers, disinfecting the skin with alcohol and then after incising, touching the skin as little as possible. For purposes of collecting the serum, the blood is either allowed to stand overnight (see the preceding chapter), or by means of a sterile funnel is allowed to flow into a sterile bottle containing sterile glass beads or steel shavings. The bottle is then stoppered with a cork (previously burnt off), the blood defibrinated by shaking, and then centrifuged. As a rule, centrifuging does not injure the serum, especially if afterwards the upper layer of fluid is siphoned off. For absolutely certain sterility the spontaneous separation of the serum is to be preferred to defibrination and centrifuging. The active sera used for complementing are to be employed as fresh as possible, in no case more than two or three days old (refriger- ator). The immune sera, which are usually employed in the inactive state, will keep in the refrigerator for a long time. Even in these, however, a loss of power is observed. In the case of high-grade immune sera the addition of 0.5% phenol ia allowable for preserva- tion. In the small quantities in which the serum is used in experi- ments (about 0.01 cc.) this amount of phenol is without effect either on the bacteria or on the complements. Before commencing the experiment proper it is necessary to determine what amoimt sown gives the most favorable results. Thus in many experiments it may be of advantage to always sow Vsoo cc. of a one-day bouillon culture, whereas with another bac- terium sowing Viooo or Vioooo '"wp of i one-day agar culture will give more uniform results. It is further necessary to repeatedly convince one's self that the control plates regularly show a uniformly good growth, for only when that is the ease can uniform results be expected. For example, although the bacillus of hog cholera gows very well on ordinary slant agar, the control plates may result most irregularly. In that case one can make use of glycerine agar. Other bacteria again do not bear suspension in O.SS'^ salt solution at all well; in that case one must use bouillon cultures and make the dilutions with bouillon instead of with salt solution. The dilu- tion should always be managed so that the amount finallv sown is about 5-10 drops, for in sowing only I or 2 rlmps considerable varia- tions in the number of colonies may occur. In any case, however, the M TECHNIQUE OF BACTERiaDAL TEST-TUBE EXPERIMENTS. 351 plate sown must contain many thousands or an innumerable number of colonies. The bactericidal effect will then be distinctly shown by the reduction in the proper plates of this large number of colonies to zero or almost zero. The test-tubes most advantageously employed are the little- tubes 9-10 cm. long and 1,3 cm. diameter. The cotton stoppers are removed and all the different components filled into the tubes. Then the stoppers are replaced after being flamed. If the air is at all still one need not fear keeping the tubes open for this length of time. In testing an immune serum one commences by examining the immune serum in the fresh active state, and, of course, in the same manner that the serum of the animal in question was examined pre- vious to immunization. For this purpose a number of test-tubes are filled with 1.0, 0.3, 0.1, 0.03, 0.01 cc. of the fresh active serum. Finer gradations are useless in view of the lack of sensitiveness of the test- tube method. This we have already pointed out. The amount of culture to be sown is then added and all tubes filled up to 2 cc. with physiological salt solution. Finally three drops of bouillon are added to each tube. The addition of bouillon has proven to be of consider- able value, for it suffices to balance disturbing variations of the osmotic pressure. It is important to make the total volume of fluid the same in all the tubes by the addition of fluid. Besides this it is important to have a number of controls, namely, a control of the culture sown, second, a control testing the sterility of the maximum amount of serum employed, and third, a control, or better a series of controls, containing the culture sown plus the serum in an inactive form. Bv means of this last control one can see whether a thermostable complement is present or not. It also serves to show that the bacteri- cidal action is not simulated by the agglutinating power of the serum. The tubes are now kept in the thermostat for at least three hours, having previously, however, been carefully shaken. On being taken out of the thermostat they are again carefuUy shaken and then worked up into plates. For this purpose 5-10 drops are taken from each tube by means of uniform pipettes and made into plates in the usual way. The plates are placed in the thermostat upside do\Mi, and kept there until the following day. The growth is best and most rapidly described by means of approximate estimates, using a scheme somewhat as follows: 0 or almost 0, about 100, several hun- dreds, thousands, very many thousands, infinite number. A distinct bactericidal action is only then present if the controls result as they 352 COLLECTED STUDIES IN IMMUNITY. should, and if a rctluction of colonies from an infinite number or many tiiousands to 0 or very few has occurred. Furthermore the test can only then bo regarded as having a good result if the lower limits of the amount of active serum have been reached, i.e., when the last plates again show an increase in the number of colonies. A certain degree of control on the plate experiments is obtained in suitable cases by placing the tubes (from which a few drops were taken for sowing into plates) into a thermostat and ob8er%'ing them the next day. In this case the culture controls show a luxuriant growth, while in the other test-lubes, depending on the amount of serum, either a growth will occur or not. This test-tube experi- ment, of course, will only then show a result if the bactericidal power of the serum was large enough to kill even the last germ in the corresponding specimens. But if even only a few germs remain alive (in consequence, for example, of a special resistance), it will be found that these few, after the bactericidal substancea are used up, will again multiply enormously. Hence the test-tube method cannot give reliable results in spore-bearing bacteria. For the same reason it is important, in making plate tests, to keep the tubes in the thermostat for a certain particular time, which must be determined separately for each bacterium; for it must be borne in mind that the killing of the bacteria can be represented by a curve whose lowest point (lowest number of living germs) must be appro.ximately attained if marked results are desired. Either side of this point, unless this point be 0, the results will be correspondingly less. Smaller results, however, are worth- less for all these experiments, as is seen when we consider that agglu- tination, although it has so little directly to do with bactericidal action, is also able to cause a decrease in the number of colonies on a plate and thus simulate a decrease in the number of germs. This is one of the reasons why the control described above with inactivated senun, in which, of course, the agglutinin is still present. Is so im- portant. After the fresh active immune aemm has been tested as to its bactericidal power one proceeds with the examination of the inactive immune .serum plus complement, Inactivation is accomplished in accordance mth the principles laid down in the preceding chapter. For complement one chooses first the norma! serum of the .ajjecics from which the immune serum is derived. A preliminary trial will then be necessary to show what dose of this normal serum can be TECHNIQUE OF BACTERICIDAL TEST-TUBE EXPERIMENTS. 353 employed without causing bactericidal action by the normal serum itself. The dose of complement should be such that the plate containing only complement and the cultiu'e differs very little from the control of the cultiu-e sowing alone. Too large a quantity of complement should be avoided; certainly in no case should more than about 0.5 cc. complementing serum be used. The technique then is as follows: 1.0, 0.3,0.1,0.03, 0.01 cc. of inactive inmiune serum are placed into a series of test-tubes; to each of these is then added the same amount of the complementing active normal serum (e.g. 0.3 cc.) and the bacterial culture. All of the tubes are then made up to the same amount (2 to 3 cc.) with physiological salt solution, and finally each tube receives three drops of bouillon. The controls in this case must be still more numerous. The sterility of each serum must be demonstrated, as well as the fact that the inactive immune serum by itself and the active normal serum by itself are inert. The result of such an experiment is usually startling at first sight because the plates which had the largest amounts of immune serum show the largest number of colonies. One must therefore always bear in mind the deflection of complements in consequence of an excess of immune body. The paradoxical results caused by this deflection of complement is seen not only in the plates but also in the test-tube experiment. The various ways in which the comple- ment is deflected from its destination have already been discussed in a previous chapter. In bactericidal experiments the deflection caused by an excess of the amboceptors produced by immunization is especially important. In a mixture of bacteria, complements, and large amounts of amboceptor, the complement is bound not only by the amboceptors anchored to the bacteria but also in large measure by " free " amboceptors which are not anchored to bacteria. A portion of the anchored amboceptor therefore finds no complement at its disposal and is, therefore, imable to exert any bactericidal action. In this way there arises a relative lack of complement. This can occur especially if part of the amboceptors has become changed into an amboceptoid with increased affinity (Wechsberg,i E. Neisser and Friedemann^). In bactericidal experiments, how- ever, the cooperation of the amboceptoids has not yet been proved. The completion of amboceptors can be disturbed in another way. » Wiener klin. Wochensch. 1902. » Berl. klin. Wochensch. 1902. 354 COLLECTED STUDIES IN IMMUNITY. Thus complement^iverting groups pre-existing in normal serum of the species in question, und which have not, therefore, onginsted through immimization, may be present or may be act free by the inactivation (normal anticomplements, etc.). The question which arisea, namely, whether one is dealing with a deflecting body of nor- mal serum or with one produced by imrnvniiation, can, of course, be decided by the previous investigation of the normal serum of the animal in question, as well as by comparison with se\'eral other nor- mal sera of the same species In all of these cases, however, the plates with the largest amounts of immune serum will show the least bactericidal action, i.e., the lai^est number of colonies. From this it follows that one can err in judging the bactericidal power of a serum if only larger amounts of immune serum are used for the bactericidal test (about 1.0, 0.3). Thus in the beginning we overlooked the high bactericidal power of a dysentery serum (Shiga), for this became manifest only after we employed doses of 0.025 immune serum and still less. The deflection of complement just mentioned, by means of ambo- ceptors produced by immunization (or by amboceptoids) , permits of another method of t«sting by which also the serum can be shown to be a specific immune scrum. For this purpose one uses an active normal serum bactericidal in itself or a mixture of inactive immune serum and a complement. By means of a preliminarj' test one determines the amount of serum or serum mixture which completely kills the amount of culture sown. To such a dose of serum or senim mixture (bactericidal in itself) decreasing amounts of in- active immune serum are added, when it will usually be found that the phenomenon of deflection of complement again appears. Tliia manifests itself by the fact that the plates with the larger amounts of immune serum show a larger number of colonies, the number of these decreasing in proportion with the amount of immune serum added. In order to interpret the results of the plate tests correctly it is first necessary to be sure whether one is dealing with a normally pre-existing deflecting body or with one produced by inununization (see alwve). By means of combining experiments it must also bo shown whether the deflection is caused by amboceptors or aml>n- ceptoids. It is not difficult, by binding them to the corresponding bacteria, to remove the amboceptors produced by immunization. In most cases the addition of a moderate amount of bacteria TECHNIQUE OF BACTERICIDAL TEST-TUBE EXPERIMENTS. 355 fully killed (65° for ^-1 hour) and centrifuged will suffice. In these cases, however, the supernatant fluid must always be examined microscopically to make sure that all the bacteria have been removed by the centrifuging. For any such dead bacteria loaded with ambo- ceptor, which should remain in the fluid, would serve to deflect com- j)lements in the further course of the experiment. However, in many cases it is possible to remove all the bacteria by centrifuging. In that case it is easy to show that the bactericidal, as well as the com- pi cmcnt-de fleeting power of the serums has disappeared with the absorbed amboceptor. If only the deflecting power of the serum remains, while the bactericidal power has disappeared, and if the comparative test has shown that one was not dealing with a normal anticom- plemcnt or such like, we conclude that a complementophile ambo- ceptoid is present, one which has originated from the amboceptor j)roduced by immunization. In many cases in which a plate test, as it has previously been decribed, has seemed unsuited, another method has been used to overcome the difficulty. Thus after allowing the immune serum to act, instead of pouring plates, one can take a loop from each test- tui^e and make slant agar streaks. If one t"he nmerely regards ver>' broad results, such as no growth, luxuriant growth, one will obtain, by this simple means, useful comparative values. In this way Dr. Lipstein and I have several times determined the power of a gonococcus serum which we produced by immunization. XXXI. THE PROPERTY OF THE BRAIN TO ! NEUTRALIZE TETANUS TOXIN.' By Dr. £. Marx, Member of the lostitute. Wassermann and Takaki's* communication stating that it is possible by means of normal brain substance to decrease the toxicity of tetanus toxin, or even, in suitable closes to entirely neutralize it, was undoubtedly of great theoretical and practical significance. Their statement was confirmed by nmny different Investigators, Ransom,^ Mctchnikoff,* Marie ,^ lilumenthal,^ Milchner,' Uanyz* Ziipnik,* and others. These experiments were devised by Wasser- mann and Takaki as a test for the correctness of the side-chain theory-, according to which the cells, susceptible to the poison, possess recep- tors which anchor the same. They argued, if the theory were cor- rect, that the brain-cells which in vivo are susceptible to the poison should also be capable, at least in the fresh state, to bind the poison in vitro, i.e., it should be possible to neutralize solutions of tetanus poison with brain substance. As is well known the result of the experiments agreed with the theoretical premises and tliey were so interpreted by Wassermann, This interpretation was first denied by Metchnikoff. He as well as Marie had repeated Wassermann's experiments and conceded ' Reprint from the Zcitiii^h. f. Hygiene und Infectiona-Kraiikheilen, Vol. 40, 1902. ' Berl. klin. Wochensch. ' Deutsch.med. Wochensch. 1S98, No. 5 (communicated through v. Behring). • Annalea de I'lnstit. Pasteur, 180S, pp. 81 and 263. •Ibid. 1898. p. 91, 'Deutsch. raed. Wocheawh. 1898, No. 12. 'Ibid,. 1S98, No. 16. ■ Annalea de I'lnstit. Pasteur, 1899. ' Prager med. Wochensch. 1899. Nos. U and 15. TETANUS TOXIN NEUTRALIZED BY BRAIN SUBSTANCE. 357 their correctness, but on the basis of further experiments made by Marie, Metchnikoff was led to another interpretation of the results. Marie found that when poison and brain substance were injected separately, even large amounts of brain substance did not exert any protection. Metchnikoff, therefore, did not believe in any neu- tralization of poison by the brain substanc in vitro. He saw the cause of the apparent neutralization in mixtures of tetanus poison and brain substance in the leucocyte-attracting power of the brain substance injected with the poison. According to him the leuco- cytes were the agents which destroyed the poison, and the brain substance only the means for attracting these. It is hardly within my province to subject these experiments to a thorough criticism; that must be left to those directly interested. I should, however, like to mention two points which appear to me not to be sufficiently regarded. First, it must be remembered that with a dissolved antitoxin the success in neutralization on mixing antitoxin and poison in vitro is considerably higher than the thera- peutic success which the same dose attains in an animal. In the above experiments there is added to this the fact that we are not dealing with a dissolved antitoxin. On the contrary, the poison- neutralizing power is exerted by a mass which, from experience, we know is absorbed with great difficulty. Subsequently v. Behring, as a result of his combining experi- ments with brain substance, expressed doubts as to the correctness of Wasscrmann's explanation, without, however, positively taking either one side or the other. Basing his reasons on the experiments of Kitashima, v. Behring ^ stated his views as follows: " If an emulsion of fresh brain substance from a guinea-pig is mixed with a certain dose of tetanus poison, a dose whose power is exactly known, it will be found that with small amounts the poison will completely lose its poisonous property; with larger amounts there is a distinct decrease of this property. One would now sup- pose that large amounts of poison, whose poisonous property has been decreased by means of brain emulsion, would require less anti- toxin for their neutralization than before the addition of the brain emulsion. But this is by no means always the case. In the experi- ment— * V. Behring, Allgemeine Therapie der InfectioDs-Krankheiten, Part J, p. 1033. COLLECTED STUDIES IN IMMUNITY. O.OOS cc. poison solution No. 3, 0.2 cc. brain emulsion; one hour later: Viooo antitoxin unit — we not only found no excess of antitoxin, but found that the inji tion of such a mixture into mice caused death by tetanus." The result of this experiment led v, Behring to conclude that further study of the poison-neutralizing power of guinea-pig brain vould probably decide the question in favor of Melchnikofl's views as outlined above. A subsequent study from v. Beliring's instv tute demonstrated that a union evidently takes place when livii brain and tetanus ]ioison come together. Ransom ' studied the conditions found in the suI>arachnoid 8| after injections of tetanus poison or tetanus antitoxin. It lead us too far to recapitulate these brilliant experiments, and «hall, therefore, content myself by quoting Ransom's conclusii ■which are as follows: " These experiments strongly corroborate the assumption tetanus antitoxin is bound in the central nervous system; they a1 indicate that this union takes place somewhat gradually. There is surely no objection to our placing the^ experimeaj on the living brain parallel with those made on the dead brain, ■would be incomprehensible for a brain, removed at once from freshly killed animal, to be different in its property of binding tetani poison frora what it was a few minutes previously in the living animat 1 had just begun a study in thk institute dealing with these problems, but discontinued them on the appearance of Ransom's paper since that had so well covered the subject. Some time after this Kitashima's experiments were taken up Gruber,^ although without re-examination. In these experimeol Gruber saw further proof of the incorrectness, according to him. Ehrlich's Side-chain Theory. In resiionse to this, however, Paltauf ' very aptly demonstrated that a simple calculation wilt show that Kitashima's experiments cannot in any way be regarded as elusive. He expressed himself as follows: • Hoppe-Seyler's Zeitschrift tiir physiol. Ciiemie 1900-1901. Vol. XXX| p. 282 et seq. 'Munch, med. Wochensch. 1901, Nos. 46-49. ■Wiener klin. Wochensch, 1901, No. 51. TETANUS TOXIN NEUTRALIZED BY BRAIN SUBSTANCE. 359 "0.008 cc. tetanus poison No. 3+0.2 cc. brain; one hour later, ^/looo antitoxin unit. Tetanus poison No. 3 is very powerful. 1 cc. equals 5 million mouse. 15 mouse is a fatal dose for a mouse; in the experiment, therefore, 40,000 mouse or more than 2600Xthe fatal dose is employed, which quantity, to be sure, is neutralized by Viooo antitoxin unit. According to Wassermann, however, 1 cc. emulsion can at the most neutraUze 10 fatal doses; according to others, from 30 to 100 fatal doses. Usually 1/5 cc. suffices to neutralize not over 20 doses of poison, an amount which is very minute when 2600 doses of poison are concerned." It should also be mentioned that Blumenthal and Wassermann ^ opposed Gruber's view. Blumenthal called attention to the fact that when brain substance is added to a toxin solution it is possible by centrifuging to show that the original toxin solution has been robbed of its toxic power, a result which cannot be obtained with boiled brain. He also reminded his readers that he had shown how, by introducing the toxin in vivo, the power of the brain to neutralize poison had been diminished, as was seen on testing the same post- mortem. This diminution was due to the union of the brain sub- stance with the toxin, and was in proportion to the amount of poison injected. Wassermann too is still convinced that there is a chemical union. His view is also borne out by the fact that in the rabbit, in which, according to the researches of Donitz and Roux, an extensive dis- tribution of receptors capable of binding tetanus toxin was to be assumed, other organs besides the brain are also capable of neutraliz- ing the poison in vitro. This is in direct contrast to the guinea-pig in which only the brain possesses this power. In view of all this we determined to finally decide whether on the addition of brain to tetanus poison there is an actual union of poison, and whether if this is so there is a summation of neutralizing actions of brain and antitoxin. Our old studies were therefore again taken up. We began with the re-examination of Kitashima's experiments, but under such conditions that the errors which, independently of us, Paltauf had already pointed out, namely, the employment of too large doses of poison, were avoided. 1 Deutsche med. Wochensch. 1902, Vereinsbeilage, No. 3. COLLECTED STUDIES IN IMMUNITY. The Material Ewplotbd, and its Preparation. In these experiments b. great deal depends on the manner in which the brain emulsion is prepared. We shall therefore again describe the method in detail, although Wassermami and Takaki did so when they reported their experiments. Each guinea-pig brain was thoroughly mbced with 10 cc, 0.85% salt solution. In order to obtain uniform and good results it is neces- sary that the emulsion be as fine as possible. For this purpose the brain substance was crushed and the salt solutionadded.at first drop by drop, until a fine uniform emulsion resulted. It is well instead of using a mortar to use conical glasses, such as arc employed at the Rabies Inoculation Stations for preparing the fine cord emulsions for injections. These conical glasses are about 10 era. high and taper not to a point, but to a heraisplierical surface into which a ground- glass pestle fits. • This very fine emulsion is then forced through Herzberg funnels, such as are used in testing paper. If the emulsion is forced through the finest of these, fitted with wire-gauze with the smallest mesh obtainable, it will be found that the emulsion is actually free from macroscopically coarse particles. The poison 1 employed was a tetanus toxin preserved in the institute for diagnostic purposes. This poison, I may add, owing to the special method of preparation, difTered from Bchring's test poisons (at least from those which can be obtained in the market) in being free from spores. This fact may perhaps not be without significance, for, under the conditions which here obtain, a development of the spores with consequent production of poison in the animal can- not be denied oithand. This possibility must surely often be counted on. It was for this reason that Ehrlich long ago allowed only such tetanus poisons as were freed as much as possible from spores to be used for testing, and for exact experimental studies. I shall soon publish an account of the peculiarities of the procedure used in this institute for obtaining such poisons, and also describe a method for preserving tetanus poison permanently, which we have found very useful. The antitoxin used was also that preserved for testing purposes. 1 grm. contains 100 A. E. Bcbring. TETANUS TOXIN NEUTRALIZED BY BRAIN SUBSTANCE. 361 Method of Making the Experiments. The method employed followed exactly in principle that em- ployed by Kitashima. A 1 to 400 dilution of the normal solution of the poison was prepared. To each cubic centimeter of this, which represents forty times the fatal dose for a mouse of 15 grm., the desired number of doses of brain emulsion, or of a 1: 10 dilution of this emulsion was added, the fluid made up to 2.5 cc. by the addition of 0.85% NaCl solution, and the mixture thoroughly shaken. At the end of an hour 0.5 cc. of the dilutions of serum in question were added and after once more thoroughly shaking, i-cc. doses of this mixture were injected subcutaneously into white mice weighing 15 grm. It may be mentioned that in the controls containing only brain and poison the procedure was exactly the same except that 0.5 cc. NaCl solution were added at the end of the hour instead of 0.5 cc. serum. The control containing only poison and serum was treated in exactly the same manner and was injected in the usual way after the anti- toxin had been allowed to act in the toxin for thirty minutes. It may be added that no appreciable difference was observed if the mixture of poison -f brain -f serum was injected directly after the addition of the serum or if the serum was allowed to act on the brain + poison mixture for half an hour. Results of the Experiments. My results, obtained from over two hundred experiments on mice, do not furnish the slightest ground for assuming that the phe- nomenon found by Kitashima is the rule. On the contrary, from my experiments I can positively conclude that there is always a summation of the poison-neutralizing action of the brain and anti- toxin; furthermore that there is never any interference with the antitoxic action of the senun as a result of the previous action of the brain on the tetanus poison. This fact was constantly observed, no matter whether large or very small doses were employed. The series of tests with brain emulsions, as well as those with brain and poison alone without serum, do not, to be sure, proceed as smoothly as those with poison + senun; however, this is not at all surprising; on the contrary, it is quite natural that the particles suspended in the emulsion, even if they are very fine, cannot produce as uniform effects as a solution of antitoxin. 362 COLLECTED STUDIES IN IMMUNITY. The results of my experiments were bII the same and their sig- nificance is absolutely clear. From the large number of tests I shall therefore give but three, These will inciclently show the well-known fact that the power to neutralize poison is often very tliflerent in different cases. Degree □( DUutiua i Control TollD-l- 1:17500 1:16000 1:12500 1:10000 1: 8000 I: 6000 1: 4000 Control: only toxin and 1.5 cc. Drain moderately sick Degr« ol Dilution ftbe Control To»in +Senini. TheExperimenlrTnsin + O.act Bruin + Serum, 1:17500 t3 moderately sick 1:16000 t3 1:12500 t-* 1:10000 t4 very severely eick 1: 6000 (iverelyslok 1: 4000 moderately sick 1: 3000 I: 1000 well Control: onlytoxi 0.2 ce. bruin nand ) t4 Degrea ot Dilution of the eerum. Cflntro! The EsperinMinl, 0.1 «*. Br^nV 8erum. The Experiment. Benrall. ToiiD + O.aoc. Br«in + 8b mm. 1:17500 l:15O0O 1:10000 1: 5000 Control: only toxin + 0.1 cc.or0.2cc. brain 4 ■■4 moderately sick J ~ very sick moderately eick t4 very sick moderately aick t4 m TETANUS TOXIN NEUTRALIZED BY BRAIN SUBSTANCE. 363 All of these experiments show that the mice which received only toxin and brain died, whereas additions of antitoxin as did not by themselves suffice to neutralize the dose of poison were able to save the animals which received the doses of brain emulsion. Hence the action of the brain doses (which by themselves do not protect) adds itself to that of non-protecting doses of antitoxin and so forms a protective dose. Risumi. 1. The neutralizing effect possessed by guinea-pig brain on tetanus toxin is supplemented by that of antitoxin when tliese are allowed to act on the poison in vitro, 2. From this one can conclude that this neutralizing effect of guinea- pig brain on tetanus toxin and that of the antitoxin can be regarded as equivalent properties. XXXII. THE PROTECTIVE SUBSTANCES OF THE BLOOD.i By Professor Dr. P. Ehrlich. More than ten years have passed since the studies of Fliigge and of Buchner and of their pupils directed attention to the bac- tericidal substances present in normal blood serum and their rela- tion to natural immunity. Buchner es[jeeially assumed that the senim of each animal species contained a simple definite protective body, the alexin, which was able to kill off foreign eell.s, especially bacteria and the blood-cells of other species; that this acts somfr- what after the manner of a proteolj'tic ferment and leaves the cell elements of its cvn species iinscatheti. The recent development of the doctrine of immunity, inaugurated by v. Eehring's discovery of antitoxin, has also shed considerable light on the nature of pro- tective bodies preformed normally, so that it now seems advisable to subject the mutual relations existing between these to a closer analysis. There can hardly be any doubt that, in accordance with the principle enunciated by Virchow for the relation existing between cell physiology and cell pathology, the normal protective substances are subject to the same developmental laws as the artificially pro- duced antitoxic and bactericidal substances. It is obvious that with the artificially produced protective substances, especially with the antitoxins, it will be far easier to gain an insight into the mechan- ism of their development, for in tliis ease one possesses not only the exciting agent (as, for example, the toxin), but also the resulting specific product (the specific antitoxin), making it possible to study their mutual chemical relations. ' .\ddresa delivered in (he general HeRsion of the 73d Congres." of German Naturaliata and Physicians. Hamburg, Sept. 25. 1901. (Reprinted from the DeulBChe med. Wochenschritt 1901. Nos. 51 and 52.) THE PROTECTIVE SUBSTANCES OF THE BLOOD, 305 This, however, ia not possible ia the case of the substances natu- rally present, and, considering the complicated chemistry of the living organism, we shall probably long continue to be ignorant of the substances which act as the physiological excitants. Hence it is not a mere coincidence that the attempt to formu- late a theory for the development of the protective substances suc- ceeded first in connection with those artificially produced. Tliis is now well known as the aide-chain or receptor theory. According to my view this theory ia also of the highest significance for the con- ception of the nature of the alexins. I shall, however, first outhne my views on this subject as they are applied to the formation of antitoxin, as this is comparatively the simplest to study, There were, as you all know, cliiefly two views concerning the formalion of antitoxin, namely, the hypothetical metamorphosis ol toxm into antitoxin, and the secretion theory, which approaches somewhat the side-chain standpoint. The former was based on the ol>servation that the antitoxin excited by a certain to;:in acts only against just this toxin and against no other. This specific action is such a conspicuous phenomenon that it was at first believed that the intimate relation of toxin to antitoxin could only be explained by assuming the toxin itself to be the mother substance of the anti- toxin. So even to this day, Buchner maintains the view that the antitoxins and related substances do not correspond to preformed or even wholly newly formed constituents of the organism, but that they are non-poisonous transformation products of the substances introduced for purposes of immunization. In this ca'ie, therefore, the relationship of antibody to the substances exciting its produc- tion would be due to a similarity of the two components. In other words, there would be no antagonism such as exists between acid and base, but an attraction of like to hke, as is seen, for example, in poly- merization, in the attraction of crystallization, or in the structure of starch granules. Against this I should like to point out that this assumption can- not apply even from a purely chemical standpoint because the processes advanced as analogous occur in concentrated solutions, while neutralization of toxin and antitoxin takes place in extremely dilute solutions. The biological conditions, however, constitute the most serious objection to the assumption of a transformation of toxin into anti- toxin. First comes the enormous diff'erence in quantity which may 36G WtLLECTED STLiUIES IN IMMUNITY, exist between the toxin introduced and the resulting antitoxin. Ivnorr, for example, has shown that the injection of leUmus toxin into a horse is followed by the production of an amount of antitoxin which would neutralize 100,000 times the dose ol poison employed. Such an enormous disproportion cannot be reconciled with Buchner'a view. according to which each part of toxin would make an aotitoxin equivalent. This ratio can be explained only by a theorj- which makes the production of antibody more independent of the exciting agent. Another fact, which cannot be reconciled with a transformation of toxin into antitoxin, is the marked difference existing between Bo-called active and passive immunity. If, for example, by injecting an animal with poisons or bacteria an active immunity is produced, this immunity may in favorable cases persist for years, while in passive immunity the preformed antitoxin introduced into the organism exists but a short time, Such a difference cnuld not exist if the antitoxin were nothing else than transformed toxin, for m that case it should be absolutely immaterial how the antitoxin now in the organism had originated. The difference, however, deifcnds on the fact that in active immunity the tissues of the body con- stantly produce new antitoxm, keeping pace with the excretion of the same. This production of the antitoxin by the body-cells is furtlier- more confirmed by the interesting experiments of Roux and \'aiUard. and of Salomonscn and Madsen. They took an animal which liad been actively immunized, and whose serum showed a constant amount of antitoxin, and by means of repeated \enesection abstractttl a considerable portion of its blood. In case the antitoxin had been derived from the toxin introduced there should, now that the last traces of poison had disappeared from the body, have been a marked loss of antitoxin from the blood. On the contrary within a short time it was found that the amount of antitoxin had again reached its previous level. Another point in support of the assumption that the body-celts produce the antitoxin is an experiment of Salomonscn and Madsen, which shows that the amount of antitoxin present in tlie blood of an actively immunized animal is increased if the animal is treated with substances which increase the secretion of blood-cells in general, e.g. pilocarpine. This experiment was advanced by palomonsen and Madsen as absolutely opposed to the transfonnataon hypothesis and supporting their secretion theory. THE PROTECTIVE SUBSTANCES OF THE BLOOD. 367 There is one fact, however, by which the transformation hypothesis is especially refuted, namely, that antitoxins can occur in the blood of normal individuals. Thus diphtheria antitoxin is found in the blood of horses in about 20-30% of the animals examined, although diphtheria infection is surely a rare exception with these animals. Horse serum furthermore contains antibodies against one of the poisons produced by tetanus bacilli, tetanolysin, but not against the tetanizing poison of the same bacilli, the tetanospasmin, although the immune serum artificially produced contains both antibodies. Just these observations, which can easily be extended, show that even the normal organism can produce true antitoxins without the intervention of the corresponding bacterial substances. Hence these antibodies cannot be transformation products of the poisons injected, but are products of normal cell activity. The explanation especially of these normal processes constitutes one of the chief points in the side- chain theory. This theory is based primarily on a thorough analysis of the relations between toxin and antitoxin. It was found, by means of test-tube experiments with ricin and related bodies which act on red blood-cells, that it was extremely probable that toxin and antitoxin act chemically directly on each other, forming a new innocuous com- bination. It was now necessary to study the neutralization of these two substances in all directions in great detail. For this purpose 1 chose diphtheria toxin and antitoxin, because the guinea-pig organism furnishes such a uniform test object for this poison that exact quan- titative determinations, such as are used in physics and chemistry, are attainable in animal experiments. The limit of error in the titra- tion of diphtheria senmi titrations is not more than 1%, surely an astonishing result if we consider that we are dealing with substances which chemically aa yet are entirely unknown. The results which I obtained in the earlier years of my investiga- tions were really very discouraging, for they seemed to present an insurmountable obstacle for the chemical conception. In chemical processes when two substances unite to form a third substance, in accordance with the laws of stoichiometry, we must insist that these components act on one another in definite equivalent proportions. In the action of diphtheria antitoxin or toxin, however, this law seemed to be utterly disregarded. Thus in twelve different toxic bouillons I first determined the quantity which was neutralized by a constant amoimt of antitoxin; in certain instances by the official COLLECTED STUDIES IN IMMUNITY. standard unit of antitoxin. The figures thus obtained, as was to be expected, varied greatly: in one case the antitoxin unit neutralized 0.25 ec. toxic bouillon, in another case 1.5 cc. This is not in the least surprising, for it is well known that the an;iount of poison given off by the bacteria to the medium depends on the strain of the bacilli, on the preparation of the bouillon, etc., so that strong poisons and weak poisons arise. But, assuming that the toxin molecule follows chemical laws in its union with antitoxin, it was to be expected that in the different poisons the amounts neutralized by 1 1. E. (Immun £inheit= Immune Unit), and designated as Lq, would contain equal amounts of true poison, or in other words that the various poisons which differ in their Lg doses represent nothing more than more or less concentrated solutions of the same toxic substance. The amount of poison con- tained in a solution ia measured in poison units, i.e., that amount of toxic bouillon which just suffices to kill a guinea-pig weighing 250 grm. in four days, Thus if in a certain poison A we find the amount neu- tralized by 1 antitoxic unit, i.e., the Lq dose, to be 1 cc, and if we further find that 0.01 cc. of the same poison suffices to kill a guinea-pig, we say that in this poison the Lq dose represents 100 poison units. In accordance with the law of equivalent proportions we should liave expected that the Lo dose of the various poisons would contain the same number of poison units. As a matter of fact, however, the result was quite the reverse, tor we found that the number of poison units contained in Lq varied from a minimum of 10 units to a maximum of 150. According to the view held at that time that the antitoxin was bound only by the toxin, this wide divergence from the laws of equivalence could not help but cause the assumption that the relations existing between these two opposing substances were other than purely chemical ones. Finally by employing a method of study which has proved of considerable value in scientific investigations, namely, the genetic method, I succeeded in getting some light on this subject. Follow- ing this I subjected one and the same toxic bouillon to comparative tests at different times. I may be permitted to demonstrate this by means of a simple schematic example. In a freshly made poison we find that the quantity which is neutralized by 1 I. E., in other words the /-o dose, amnunts to 1 cc, and that this contains 100 [xiison units. If the same poison is examined at the end of about six months, it is found that the Lg dose is the same, but that this contains only 50, i.e., half the number of toxic doses. That is to say, the toxlo THE PROTECTIVE SUBSTANCES OF THE BLOOD. 369 bouillon still possesses the original neutralizing power but a weaker toxic action. Hence toxic action on an animal and combining power for antitoxin must be two different functions, the former remaining constant and the latter decreasing. If we regard these conditions from the chemical standpoint, we shall see that they are most readily explained by assuming that the toxin molecule produced by the diphtheria bacilli contains two dif- ferent groups, of which one, termed the haptophore group, effects the union with antitoxin, while the other, the toxophore group, represents the actual cause of the toxicity. These two groups also differ in their stability, for the toxophore group is very unstable the haptophore group far more stable. Modified poisons in which there has been a destruction of the toxophore group while the hap- tophore group has been preserved, and which have therefore com- pletely lost their toxic action, are called "toxoids." The presence of such toxoids fully explains the apparent devia- tions from the laws of equivalence which are observed in neutralizing tests with toxin and antitoxin. This furnishes new and, to my mind, incontrovertible proof for the chemical view of the process of neutral- ization. In diphtheria poison at least, for reasons into which I cannot here enter, it seems that the affinity of the haptophore group of the toxoid molecule for the antitoxin is exactly the same as that of the unchanged toxin. This indicates that the two functionating groups of the toxin molecule possess a certain degree of independence. I have tried further by means of refined investigating methods, such as partial neutralizations, to extend the views concerning the con- stitution of the poison molecule. My observations, so far as the facts are concerned, have been completely confirmed from various sources. Mention should be made especially of the excellent study (A Madsen on diphtheria toxin and tetanus toxin, and of the inter- esting experiments recently published by Jacoby on ricin and its toxoids. In studying the two groups of the poison molecule, we are con- cerned not only with a satisfactory explanation for the process of neutralization. The presence of these groups gives us an insight both into the nature of the poisoning and the origin of the antitoxin. So far as this last point is concerned, two facts in particular indicate that the haptophore group takes a leading part in the immunity reaction in the organism, viz., (1) the observation that 370 COLLECTED STtJDlES IN IMMUNITY. toxoids, which lack the toxophore group, are still capable of exciting the production of typical antitoxins, and (2) that toxins whose haptophore group is preoccupied by antitoxins lose, as a result of this procedure, their power to produce antitoxins. Now in order to understand the essential rdle played by the haptophore group in the formation of antitoxins and of the antibodies in general, it is neces- sary above all to study the other side of this question, namely, the junctions of the living organism in the formation of antibodies The demonstration that it is the haptopliore group of the toxin molecule that excites the production of immunity leads us at once to regard the process of assimilation of the living cells as most im- portant in our study. Since the beginning of medicine it has been, and still is, generally accepted that chemical substances can act only on those organs with which they are capable of entering into closer chemical relations. In his "Cellular Pathology," Virchow expressed this view in his usual clear and forcible manner: "Just as the single cell of a fungus or an alga abstracts from the fluid in which it lives as much and the kind of material as it needs for its vital processes, so also the tissue ce!l within a compound organism possesses elective properties by virtue of which it disregards certain substances and takes up and utilizes others." "We also know that there are a number of substances which have a special attraction for the nervous system when introduced into the body; that even among this group there are substances which pos- sess intimate relations to certain particular parts of the nervous system, some to the brain, others to the spinal cord or to the sym- pathetic ganglia, a few to certain special parts of the brain, cord. etc. 1 may mention morphine, atropine, curare, strychnine, digitalin. On the other hand we know that certain substances are intimately related to ceitain organs of secretion, that they permeate these secreting organs with a certain selective action, that they are ex- creted by them, and that when supplied in excess such substances cause an irritation in these organs." It is remarkable that this axiom was not re-echoed in the develop- ment of scientific pharmacology, and that only within the last ten years, thanks to the labors of Hotmeister, Overton, Spiro, Hans Meyer and myself, an improvement has taken place in this respect. According to these newer researches there is not the least doubt that the causes of this elective lodgment in certain celt domains are not all of the same nature. In general the modem pharmacological THE PROTECTIVE SUBSTANCES OF THE BLOOD. 371 school now believes that the substances ordinarily foreign to the organism, such as the indifferent narcotics, alkaloids, antipyretics, antiseptics, do not effect a firm chemical union with the body ele- ments, but that their distribution follows the laws of solid solutions or of the formation of a loose salt. In the case of the poisons acting on the central nervous system it is especially the fat-like substances of the nerve tissue, the so-called lipoids, which take up the narcotics, just as ether takes up the alkaloids in the Stas-Otto procedure of detecting poisons. There are a number of reasons in support of the view that the pharmacological agents in question are stored up un- changed in the cells or in certain constituents thereof, especially in those similar to tat. Naturally this does not deny the possibility that certain sub- stances foreign to the body may enter an albumin molecule by sub- stitution. Thus if protoplasma is treated with nitric acid the nitro group enters the albumin radicle, giving rise to a yellow color. Such substitutions, how^ever, in the conditions under which pharmacolog- ical actions can occur, will usually only be effected by combinations possessing high internal tension and for that reason capable of such addition reactions. This may perhaps be the case with vinylamin, which, according to Levaditi's experiments conducted in my labora- tory, produces necrosis of the renal papillae in a large number of animals, a phenomenon probably to be ascribed to such a chemical anchoring. The ordinary medicinal substances, however, are not so constructed that they can produce such energetic sections. In general we may assume that chemo-synthetic processes do not play a prominent part in their distribution. It may, however, be regarded as an absolute fact that synthetic processes play an important role in the life of the cell in another direction. If by boiling certain cell material with acids we are able to split off certain definite groups (such as those of sugar, etc.), this fact proves the chemical character of this combmation. As a matter of fact the two series of phenomena which we are here dealing with have long been separated by general custom. The term assim- ilabUiiy is reserved exclusively for those substances which are an- chored by the cells synthetically, and which in this way become con- stituents of the protoplasm. No one would think of speaking of morphine, or of methylene blue, substances which enter into certain cells and lodge there, as being assimilable 372 COLLECTED STUDIES IN LMMUNITY. These explanations will suffice to sliow that Uie t«rm assimit&- bility, as I employ it, is restricted somewhat more than is customiarj', for I reserve it exclusively for the specific nutritive substances of the living protoplasm. According to this view the process of cell assimila- tions is a synthetic one which presupposes the presence of two groups effecting the synthesis and having a strong chemical affinity for each other. Hence I assume that the living protoplasm possesses side-chains or receptors which possess a maximum chemical affinity for certain particular groups of the specific nutritive substances, and that they therefore anchor these substances to the cell. The receptor apparatus of the cells is highly complicated, the red blood-cell, for example possessing perhaps a hundred different types of receptors. If this view is accepted and it is recalled that in the toxin mole- cule it is the haplflphore group which effects the development of immunity, only a very small step is required in order to gain an insight into the nature of antitoxin formation. This is the very natural assumption that among the various receptors — perhaps by chance — the haptophore group of the toxin finds one which possesses an especial affinity for this haptophore prroup. It is not at all neces- sary that ever>- bacterial toxin should find fitting, i.e. toxophile, receptors in every animal species. On the contrary just this absence of ret^eptors constitutes one of the reasons why certain animal species are immune against certain particular poisons. Furthermore, all the facts indicate that the susceptibility, i.e. the receptiveness, of an organism for a certain toxin is associated with the presence of such toxophile groups of the protoplasm, a point which finds suitable expression in the term receptors. As a result of anchoring the toxin molecule by means of the haptophore group the cell is influenced in two directions. Primarily owing to the lasting influence of the toxophore group, it sickens, a condition which manifests itself by disturbed functions and possibly by pathological anatomical changes. liesides this, however, in a manner shortly to be discussed, a regenerative process ia begun which can lead to the formation of antitoxin. Since this regenerative process can be excited by toxoids lacking the toxophore group, as well as by the toxins themselves, we must assume that it is inti- mately related to the haptophore group. Hence the two parallel processes, antitoxin production and toxic action, are independent in that they are caused by two different groups. In harmony with this I THE PROTECTIVE SUBSTANCES OF THE BLOOD 373 is the fact that the two processes may interfere with one another; a marked pathological action can diminish the regenerative process or even prevent it entirely. This is shown, for example, by the fact that it is almost impossible in the case of certain iinimals highly susceptible to tetanus poison, such as mice and guinea-pigs, to pro- duce antitoxin by means of unmodiiied poison, while the result is easily attained by the use of toxoids. Coming now to the regenerative process, which leads to the pro- duction of antitoxin, it will be found by any one familiar with the fundamental principles formulated by Carl Weigert that there is nothing remarkable about the process. The receptor which has anchored the haptophoro group of the toxin or toxoid molecule becomes useless for the cell because of this occupation; it is no longer able to exercise its normal function, namely, the anchoring of nutri- tive substances. The cell has thus suffered a loss which must be replaced. In such processes it is very common to find, as Weigert's re- searches have shown, that the loss is not merely replaced, but that it is overcompensated. The same thing takes place in the methodical immunization when continued and ever increased doses of immu- nizing substance are introduced. Part of the newly formed re- ceptors still attached to the cell are occupied by the immunizing substance only to be replaced by a regeneration greater in degree than before. Owing to this increased demand the prolopksm to a certain extent is trained in one direction, namely, to produce anew a certain kind of constituent, the receptors in question. Finally, such an excess of receptors is produced that there is no longer room in the protoplasm for them. Then they are thrust off as free mole- cules and pass into the body fluids. According to this view the anti- toxin is nothing more than the thrust-off receptor apparatus of the protoplasm, i.e., a normal cell constituent produced in excess. From among the many facts already at hand I shall select merely a few to serve as proof of the correctness of this hypothesis, this "side-chain theory," as It is called. The first point deals with the demonstration in normal tissues of the toxinophile receptors assumed by the theory. Although such an anchoring of the jMJisnn by the organs had already boon demon- strated by the clinical course of the poisoning and by Donitz's thera- peutic experiments on animals poisoned with tetanus and diphtheria poisons, it remained for Wassermann to show that certain body 374 COLLECTED STUDIES IN IMMUNITY. elements anchor the toxin even in a test-tube and neutralize the toxin juat as does the antiUixin, If he added crualied fresh guinea- pig braiQ to tetanua toxin, he found that the brain aubatanee anchored the toxin in sucli a manner that not only was the supernatant fluid robbed of its toxic action, but that the brain laden with tetanus toxin also exerted no toxic effect. From this we can conclude that a chemical union has taken place between constituents of the ganglion cells and the tetanus toxin. This combination is so firm that it is not broken up on being introduced into the animal body; as a result the toxin remains innocuous. That this is really a specific reaction and not, for instance, merely an absorption is shown by the fact that boiled brain, in which the chemical groups in question are destroyed, is just as little able to exert this action aa the pulp of any other organ of the guinea-pig. In addition to this Ransom has shown that the brain of living animals pos.'iessea the same toxin-destroying power. In view of this it would appear that the objections made by Danysz, which refer to the divergent behavior of the decomposed brain pulp, possess no great significance. 1 will not deny the fact that the favorable result achieved in tetanua is evidently due only to the coincidence that the tetanophile receptors are present in large quantity in the brain. Such a coincidence, of course, need not obtain for every poison. If the organs endangered by the toxin contain only smsJI quantities of toxin receptors it will be found that with what are, at best, very coarse experimental methods these receptors escape detection. This is the case, for example, with botulism toxin and diphtheria toxin. Such confusing chance occurrences can, however, be avoided with certainty if one employs poisons artificially produced, poisons which, owing to their mode of production, are directed against cer- tain particular kinds of cells. The hemolysins producetl by injec- tions of blood, spermoto.xins, and numerous other cytotoxins may serve as examples. In all of these cases it can positively be proved that the toxin is anchored by the susceptible cells in specific fashion. The second point concerns that premise of my theory which states that the same organs which possess a specific affinity for the poison molecule are able to produce antitoxin. In this connection the very neat experiments made by Romer on abrin immunization should be mentioned. As is well known, abrin, the toxalbumin of jequirity beans, is able to excite marked inflammation of the con- THE PROTECTIVE SUBSTANCES OF THE BLOOD. 375 junctiva in man and animals. I have shown, furthermore, that it is possible, by means of conjunctival instillations, to actively immu- nize rabbits against abrin. Romer immunized a rabbit by means of rapidly increased doses into the right eye and killed the animal at the end of three weeks. It was then found that the conjunctiva of the right eye which had been the site of the inflammatory process was able, when ground up with a suitable amount of abrin, almost completely to neutralize the action of this poison, whereas the other conjunctiva, when similarly ground up with abrin, was unable to protect the animal from death. From this Romer rightly concludes that in this conjunctival immunization part of the antitoxin is fur- nished by the conjunctiva which reacts locally. Aside from its theoretical interest I believe that this demonstration of the local origin of antitoxin at the site of injection possesses great practical significance. In certain cases the possibility is thus given to trans- fer part of the antitoxin production from the vital organs to the in- different connective tissues. The third point concerns the thrusting-oflf of the surplus receptors. A prerequisite for this thrusting-oflf is that the receptors in question, which are normally firmly attached to the protoplasmal molecule, become loosened. In several favorable cases it has been possible to confirm this postulate of my theory experimentally, though to be sure these deal with immunization by bacteria and not with solu- ble poisons. Pfeiffer and Marx succeeded in showing that with a suitably conducted cholera immunization it is possible to find a period at which the blood is still free from protective substances, although the specific protective substances can be abstracted from the blood-forming organs by crushing them up with salt solution. In my opinion this can be due only to an extraction of receptors which, since it is just previous to their extrusion, are only loosely attached to the protoplasmal molecule. Almost simultaneously with Pfeiflfer and Marx, the same results were obtained by Wassermann with typhoid, and these were later confirmed by Deutsch. In all of these experiments the hsemato- poetic system represents the site of production of these antibodies. The significance of this circumstance for the immunizing process has been pointed out by Metchnikoflf's teachmgs. These few examples will suflSce to show that the side-chain theory has fully stood the test of experiment. During the many years of my experimental activity I have not met a single fact which con- 378 COLLECTED STUDIES IN IMMUNITY. tradicts thia theory and mipht serve to refute it. I may, there- fore, regard the theory as well established and proceed to discuss in detail several important points which follow from it. The side-chain theory explains in the mast natural fashion the specific relations existing between toxin and the corresponding anti- toxin. Furthermore the theory makes the immunizing action of the antitoxins perfectly comprehensible. When injected subculaneously into animals in the usual manner the poisons are brought to the organs possessing toxinophile receptors (susceptible organs) by means of the circulation. If, however, these poisous meet with free toxinophile groups in the blood, they will at once combine with the same and so be diverted from the susceptible organs, v. Beh- ring has expressed thia hypothesis as follows: "The same substance which when in the cells is a prerequisite and cause of the poisoning becomes the healing agent when present in the blood." To my mind we are here dealing with a general biological law which is not limited to the toxins but applies to a great many, if not to all, poisonous substances. I need only cite the saixmin poison- ing of red blood-cells. Ransom found that the blood-cells take up saponin owing to their content of cholesterin and are, as a result, subjected to the deleterious action of the poison, whereas certain sera, which exert a protection against saponin poisoning owe thia protective property to the same cause, namely, the presence of choles- terin in the serum. Furthermore the theory at once explains the fact that the tissues of an immunized animal are subject to the action of the jwison when in some way the action of the antitoxin contained in the serum is prevented. Thus Roux showed that rabbits immunized against tetanus become poisoned just as rapidly as control animals if the tetanus poison is brought into direct contact with the bmin-cells by means of intracerebral injections. This fact is demanded by my theory, for, just as in immunized animals, the ganglion cells contain an excess of toxinophile groups and are thus especially adapted to anchor the poison which injures them. It was a grave error on the part of Roux to suppose that this experiment controverted the side- chain theory, Rous thought that according to my view a consider- able amount of antitoxin had accumulated in the brain-cells and that therefore the immunized animals should possess a local brain immunity. There is evidently a misconception as to the term "anti- toxin," Just as we cannot term any mass of iron a lightning-rod, THE PROTECTIVE SUBSTANCES OF THE BLOOD. 377 but restrict this term to such masses of iron which deflect tlie liglit- ning from a particular point, so we must restrict the term antitoxin to those toxinophile groups which circulate in the blood and thus deflect the poison from the susceptible organs. The toxinophile groups present in these susceptible organs are not loxin deflectors but toxin atlr actors. The theory also explains why the property of producing antitoxins is restricted to certain products of metabolism of living cells. All experiments to produce antibodies by means of chemically well de- fined toxic substances, such as morphine, strychnine, saponin, etc., have failed. If we bear in mind that the distribution of these substances ia the organism takes place without chemical union and therefore with- out the intervention of receptors, the negative result of these experi- ments will not surprise us. The property of forming antitoxin is possessed only by such substances as possess a group able to unite with the side-chains or receptors which eflect assimilation. It must, be remembered that all the poisons which excite the production o£ antitoxin are highly complex products of animal and vegetable cells, which in their chemical properties approach the true albumins and peptones. In 1S97, by means of my theory, the production of anti- toxin and the binding of foodstuff were first brought into connec- tion. At that time nothing was known of the tact that even ordiruiry joadstufjs are capable of an analogous action. I have therefore been able to regard as an agreeable confirmation of my views the circum- stance that this consequence of my hypothesis has actually repeatedly been demonstrated within the past year, especially by Bordet. If animals are injected with milk, it is found that their serum gains the property of precipitating the milk in curds. This precipita- tion is also strictly specific, since numerous experiments show that the coagulating serum obtained by treatment with goat milk coag- ulates only goat milk, and not the milk of other species, as, for ex- ample, that of women or cows. The results are similar if animals are injected with other albumi- nous substances, e.g., with the sera of different species or with egg albumm. In this case in the serum of the animal there develop sub stances (termed coagulins or precipitins) which specifically precipitate the corresponding kind of albumin. Deviations Irom the law ol RpeciGcity ocoiir only in ao fai as the sera of closely related animal speciee coDtain subBtances more or less Bimilar Tbua ^■r'. 378 COLLECTED STUDIES IN IMMUNITY. the coitgulin obtained by teEling rabbits witfi human serum precipjiates only human serum sod tho serum of the ueureat related epecies. upes. This leactioo. which was developed especially by llie researches of [Ihlenhuth and of Waseer- maan, was thereiore proposed lor the forensic identification ol blood. From this we see that, entirely in harmony with my views, the injection of foodstuffs is followed by the production of typical anti- bodies, which combine with the exciting agent in a sjwcific manner. An analogous reaction takes jilace in the normal processes of cell nutrition and serves as the chief source of the protective substances present in normal blood in such great numbers. The conditions become much more complicated than those just described if, instead of the relatively simple soluble metabolic prod- ucts, living cell material is employed. This is the case, for instance, in immunization against cholera, typhoid , anthrax, erysipelas of swine, and many other infectious diseases. In these diseases under certain circumstances there develop many other reactive products beside the antitoxins produced against the bacterial toxins. The reason for this is that every bacterium is a highly complex living cell which, when it disintegrates in the animal body, gives rise to a large number of different components. Of these a great many are able to produce antibodies. Hence as a result of the introduction of bacterial cultures, in addition to the specific Ijacteriolysins, which cause a solution o[ the bacteria, we see substances develop, such as the antiferments {v. Dungern, Morgenroth, Hriot), the much discussed agglutinins (Gruber, Durham, Pfeiffer), and the coaguhns (Kraus, Bordet), which specifically precipitate certain albuminous substances that have passed into the culture fluid. The most interesting and important of the substances arising in such an immunization are undoubtedly the bacteriolysins, which have been studied especially by Pfeiffer and Bordet. At Brst it is highly surprising that the injection of cholera vibrios into the animal body should be followed by the formation of a substance which is able to dksolve the cholera vibrio, ami only (hv, 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 siib- etancea also. The solution of this problem offered considerable diffi- THE PROTECTIVE SUBSTANCES OF THE BLOOD. 379 -culties and did not succeed until the hsemolysins were used in the experiments in place of the bacteriolysins. Hsemolysins are peculiar poisons which destroy red blood-cells. iSuch 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 ot 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 hsemolysin directed againt the rabbit blood-cells. He showed further that such hsemolysins, 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 hsemoly tic 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 Pfciffer, MetchnikoflF, and especially by Bordet. In the case of bacteri- olysins it was found that serum freshly drawn from a goat immunized gainst cholera is able to effect solution of cholera vibrios, i.e., to give tlie 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- - 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. such 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: Schliisahelraclitum^en ; Erkrankiingen dea Ulute nagers Specielle Patbologie und Tberapie, Vol. VllI, Vienna. 1»0I Noth- 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), pyocyaneous 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 Robert has shown, abrin and ricin cause a rapid clumping of the er}'throrytes, 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 show^n 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- tetiinolysin (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 wliich 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 case of 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 hsemolysis. 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 conditionri are made more favorable by breaking up the clumps by shaking, one can easily observe the escape of the haemoglobin. 392 C0L1.ECTED STUDIES IN IMMfNlTY. by me in diphtheria poisons, depend on the separate d^^st^uction 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 it 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 Carlione. who injected animals with con- siderable amounts of foreign red Ijlood-cells (blood-cell immunization). They are readily separated from the hsemolysins developing simul- taneously by heating for halt an hour to 56° C As a result of this the action of the amboceptor lysine is destroyed while the agglutinins themselves are unaffected. To be sure if the temjierature 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 oj the second order. ^ ' Tlie agglutinins hero deBcrlbetl, in tontrast to ricin and abriu, give rise lo uo further injurious action on the discoplasma. ' In the lirxt part of " SuhluBsbeCraehtungon " I htkve diatinguisbed : i. Recrptura 0/ (Ae first urder, which concern Ihetnselvea with the aBsimilation of simple subetances (toxins, remienlfi. and other cell Beoratiooe). For Iliis purpose a single haptophore group Bufncea, When thruRt off into the blood in consequence of the inlroductioo of toxins, these receptors cDnstitut« the antitoxins (antiferroente). 2. Receptors of the seamd order, which in addition lo the haptophore group possess a second group which effects the raagulation. After they have been Fifl. 1.— The \ '., Receptors oj th i r I ih I i — llii tvpc i pictured in a. The portion e represents the huptuphore group ubiUt b represents a toxin molecule, wbii-'h possesses a haptophoro group c and a toxophoro group d. 'J'his represents the union of toxin and antitoxin or ferment a:id aatifer- ment, the union between antibody and the toxin or ferment being direct. . Receptors of the Second Order are pictured in c Here e represents the haptophore group and d (he z>iaophore group of the receptor, / being the food moTecufe with nhich this receptor combined. Such receptors arc posEiesiied by agglutinins and precipitins It is to be noted that the «ymophnre group is an mtegral part of the receptor. . Reeeplora oj the Thirl Order are pictiiiwl in III e being the haptophore group and g the complementophile group of the receptor. The com- plement k poBspssee a hnptophore group h and zymotoxic ^up »; wiulst / representi the food molecule which has heeome linked to the receptor. Such receptors are found in hipmolvsins. bacteriolysins, and other cytolvBins (he union with these cellular elements being effected by the amboceptor (n thniat-off receptor of this orderl. It is to be noted that the digesting body the complement, is distinct from the receptor, a point m nhich these receptors therefore differ from those of the preceding order 394 COLLECTED STUDIES IN IMMUNITY. Next we come W the very important substances in serum which j^uae hEemolysis. I have previously dwelt in detail on the fact that in this the action ia 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 er>'throcytes of other animal species. This is the case not only in distantly related types, such as 6sh and mammals, but, as was shown by therapeutic blood transfusions, occurs also in comparatively near relatives- Buehner was the first to appreciate the significance of this phenomenon, and assumed that the scrum contained a substance innocuous for its own body but acting destructively on foreign elements (bacteria and b lood -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 erj-throcytes 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 diiferent agents which injure the blood from a common point of view. Whether we are dealing with \'egetable 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 oS into the blood they constitute aggiutinius and precipitins. The toxins also lire to be regarded as receptors of the second order thrust oB by bacteria, 3. ReceploTt 0/ the Ihtrd order, which possess two haptophore groups, ooe of which effects the union with the loodstuff. whereas the other lays hold on certain substances circulating; in the blood plasma, the com piemen ts, which cause fertuent-like lurtions. After they are thrust off these receptors con- aitute the "amboceptors " THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS 395 same, namely j 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 theii: 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- am})oceptor only against a corresponding amboceptor. Hence in every species of blood-cell w^e 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 ricin, 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. Almast every day, however, 396 COLLECTED STUDIES IN IMMfMTV we are coming to know more such blood poieons; the Dumber of diflerent receptors which we can determine, therefore, continue to inciease. In this connection 1 should like to present the results which Dr. Moigenroth and I have obtained in atlempting to produce uuto- lysins b) immunizing goats with blood from the same species instead of blood from foreign species. In only one single instance were we succesdtul. 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. II 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 highlv 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 il can be shown that the isolysin consists oE the amboceptor which is anchored, plus a complement of normal goat &erum. 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 ot A and li; a second serum those of C and D; a third A and D, etc. V.y 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 lor 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 wc designate as the single lethal dose (L.D.) that amount of a cert^n amboceptor which when supplied with sufficient complement just suffices to completely dis- solvt a constant amount of blood, we tan, by employing different amounts oi amboceptor solutions inactivated by heat, readily deter- mine tow 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, therelore, we are dealing with a marked excess of these particular receptors. An analogous ease, by the way, has long been known as a result of VVosseiroann's experiment concerning the power ot brain substance THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS 397 to bind tetanus {)oison. 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 cf 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 tor 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.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 of 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 pheuomeiia of metabolism, while ihey are also at the same time the two phaaes of the vital process, " As a result of a large number of facte we have, as is well known, arrived at the concluaion, confirmed chiefly by Pfluger, that Che mid- point of metabolism is repreaenled by complicated combinations of egg albumin called by PHiiger 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, I have therefore proposed to replace the name 'living albumin molecule' by the term 'biogen molecule." The dccompon- tion and -prodvction oj the biogcns is OurefoTc the corncTslone of Ike mtat process in every living celt. The substances given oft by the cell are derived from the decomposition ol 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 giv-en 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 lood substances and 8o 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, 1SS5). 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 gronijs are attached to this central group as side-chains. These aide-thuirta are o/ subonli- nate jmponanee for the specific ceU function, but not so for the life ilsilf. I 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 oft oxygen, the other portion being thus consumed. On page II of this monograph I expressed myself as follows: "Tlie question as to the manner in which the side-chains constantly being consumed THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS. 39^ are regenerated must, of course, excite the greatest interest. It can be conceived that ceitain 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, ot the decades which have elapsed since Pfliiger's publication we have not advanced one step in our experimental knowledge of this sub- ject, a fact which is due to the endless diflSculties occasioned by the nature and instability of the living material. I hope that my theory is destined 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 inmiunization 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 r61e 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 STtTOiES IN IMMIINITV. of blood-cell possesses, it is not surprising that certain tvpea 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 numl>er of different erythro- cytes) are widely distributed in the animal kingdom. Side by side witli 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 tiiat the isolysins obtained by injecting goats with goat blood usually effected solution of sheep blood-cells, although to a less degree. In malting llie counter ex- periments, immunizing goats with sheep blood-cells, we obtuned in addition to sheep lysin the isolysin acting on goats. Besides this there are groups of receptors whicli 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 hiemolysins are formed, i.e.. ha?molysins 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 metalx)lic 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 si>ecies of animal as that others occur in exactly the same manner in man and in the frog. It is alw 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 [joint 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- ■ Wc have olitaioed entirely atialoguUH of blood serum, eg., with complemeata. 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 obtam 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 oflSce of these is to anchor a certain particular product of metabolism, 2. 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 ■i02 ('(H.LErTED STUDIKS IN IMMUNITY a temporary, met.aimlic proJurt. Perhaps this can be brought inS 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 ir 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. Tliis 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 tacts indicate that the species of receptors found in the erj-throcyles may also Im? 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 e.xample. found that serum of rabbits which had been treated with tracheal epithelium of oxen exerted a marked hemolytic action on ox blood in addition to its injurious action on epithelium. Metchnikoff'a objection that this was due to an error in technique (the injection of admixed blond- cells) was controverted by von Dungern, who showed that injections of cow milk, a material absolutely free from blood-cells, produced the same hsmolysins. It follows that certain receptors rouft 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-cell&. According to Miescher's comparison the red blood-cells serve ea 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 atTmity 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 RErEFrOH APPARATUS OF THE RED BLOOD-CELLS 403 crea.se of affinity would constitute a wonderful illustration ot how well the process of assimilation is adapted to its purpose. Note —Subsequent addition to paf^e 400: Calmette also bas recently reported (Compt. rend, de I'Acad^mie des ftciences, T. 134. No. 24, 1902) that the blood-cells of animals highly immunized witli cobra poison preserve their sensitiveness completely against the hspmolysin of the cobra poison. In a goat highly immunized with ricin. Jacoby fHof- meister's Beitr^e z. cbem Physiologic und Patbologie, Bd. 11, 1902) was unable to discover any increased resistance ot the red blood-cells agamst the action of the ncm. XXXIV. THE RELATIONS EXTSTTNO BETWEEN CHEM- ICAL CONSTITTTION, DISTRIBUTIOX, AND PHARMACOLOGICAL ACTION.' (An Address delivered iii the "Verein (ur mtwre Mediein," Dec 12, 1S98.) By Professor Dr. P Eurucii. Until recent years the relations between chemistry and medicine were in general confined to purely scientific cjueations. In Ihe last decade, however, a change has taken place. bUch oa has rarely been seen in the history of medicine. One is justified in saying that at the present time the chemical view constitutes (he axis about which the most important views in medicine turn, and that Ihe [wo poles are the synthetic construction of new thera[>eutic agents on the one hand, and the discovery of specific therapeutic products of living cells on Ihe olher. The contrast Itetween 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 jKiwers of living nature so infinitely well suited to Iheir purpose. A RreaKr contrast cannot be imaguied than tliat existing between the modern medicaments, whose constitution is known down to Ihe finest details, and diphtheria antitoxin, which we know only through its specific action and about whose chemical constitution we l:now al)solut«ly nothing. Thus far the genius of the mo.st eminent chemists has not availed to produce these bodies in a pure form and get an insight into their chemical nature. All that Ihi.s 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 ' lieprint \roai the v. Lejdeu Festschrift, Vol. I. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 40S As a result of this and other considerations the view haa 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 ix)ssibility 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 rurare-like action. Since that time a large number of ammonium baaee 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 curai-in is itself an ammonium base. He found that the cuiares contain a tertiary alkaloid, ciirin, which is of slight toxicity. If this curin was subjected to methylation an animoniimi base was formed which cor res I ion ded' 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 stutiied on many different combinations by a large number of investigators, among whom may be mentioned Nencki, Jaffe, Filehnc, 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 dune m 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 tact that quinine contains a hydrated chinolin derivative; by means of simpler combinations it was attempted to obtain the same end. Finally, after chinolin, 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 discoverj' 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 sultonal by Baumann has proven of great practical and theoretical significance. The same holds true of the production of the new ana«thetics (orthoform and eucain). which was closely con- nected with the discover)' 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 guaiaco] and formal- dehyd. These efforts, first undertaken by Nencki, seek by means of suitable combinations and cleavages to give rise to a gradual Uberation of the active component. While of great practical value they have but Httle interest in the cjuestion 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 ^hich 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-C6H4COCH3), the liberation of p-amido- phenol is prevented cannot be used as antip)rretics. 2. The discovery by Kendrick, Dewar, Filehne, that in the pyri- din series the hydrated products act more strongly than the parent substance. Thus piperidin, CsHiqNH, is a much stronger poison than pyridin, CsHsN. In this the transformation of the tertiary nitrogen atom in the imin group plays a certain rdle, 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, C6H5NHCOCH2SOJH, the carbonic and sulfonic acids of phenacetin, and the ethoxy-phenylglycin which is similar to phenacetin. p TT /OC2H6 U«4\NH.CH2C02H. 4. The demonstration by Filehne, Einhom, Ehrlich, and Poulson, of the anaesthesiophore 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 (Merling) and orthoform and nirvanin (Einhom). 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-(S0202Hs)z, and trional, CH3C2H6-C-(S02C2H5)2. Of the other hypnotics which owe their action in part to the ethyl group I may mention amy! en hydrate, C(CH3)3(C2H5) OH, and ethyl ui^ ethan, N'HiCOOCaHfi. The influence of the ethyl radical isfiirther- more clearly shown in another series of combinations. In an arti6cial sweetening sul>stance,dulein, which is about two hundred times sweeter than sugar, this influence is very evident. This substance is phenyl urea ethoxylatcd in the para |X)&Ition, C2H60-C6H4-NHCOXH2. Since neither simple phenyl urea nor the methoxy combination, CHs-O-TaHj-NH-CONHs, analogous to dulcin, possesses anysweet taste whatsoever, we are forced to conclude that this is due to a functiou of the ethyl radical. Of the remedies containing the ethyl radical there may still be mentioned phcnacelin, CjHs-OCaHj-NH-CO CHs, and two anassthetics, holocain, CaHsO-CeH* NH-CCCHa): N-CBHi-OCaHfi, 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 po-ssible, 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 aa the peripheral endings (dulcin, anesthetics). 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. Mirhaelis 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 (C2H5)2N| 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-saflfranin 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 diethylanilin 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 view 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 r61e 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. CWLLECTEU STLDIES IN IMMUNITY. n. One of the main causes which has made an insight into the rela- tion between conalitulion and action so dilFicuU to obtain is to be found in the fact that these reJations were considered to be murfi simpler than they really are, and in the further fact that purely chemical conceptions were applied arbitrarily to biological process**. In pure chemistry there is an abundance of materia! 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'a terminology, are "additive" and which "constitutive " by nature. The question arises what are the essential properties which are still found in the combmations. Evidently they are such as pet- tain to the subslaTice of the elements and are independent of the ajTatujtmenl of these. These properties accompany the elements m 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 neaiest approach lo them are perhaps the specific heat of solid com- binations, and m a less degree the refraction of organic substances uid their property to occupy space. In these, however, another factor becomes evident, namely, the arrangi-mcnt 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 o! those properties which are no longer in any way dejiendent on the nature of the substances but only on their arrangeoicnt, these are called "colligative " properties. To which group, then, do the properties of affinity, i.e., the jwwer of elements to effect chemical reactions, belong? Evidently tu 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 cimtain the same elements in the same piopta- tions by weight, yet they manifest entirely different reacting capaci- ties, Ilutyric acid and acetic ester are not only of the same con- CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 411 stitution but have the same molecular weight, yet their affinities 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 index. 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 determined, 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. l^>ut it is just because of this intermediate position that the chem- istry of dyes tuffs affords so good a point of vantage for our con- sideration. 1 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 O. 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 or ' Ostwald, Grundriss der allgemeinen Chemie. 412 COLLECTEU STUUIES IN IMMUNITY " chromophore " group. Concerning the devils ol the varioua types of chromophorea I refer the reader to the admirable work of Nietzki. I 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 combmations 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 ajtobenzol, which possesses the chromophore azo group, \ = 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 graups 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 thio it i» found 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, CH^NHi, CH,.N-(CH3)a, andOCHrf N {CH3).. From every chromogen, therefore, two series ot dyes may be de- rived, acid and basic, each acid derivative having an analogous basic one. Thus Acid BaEic Oxyazobenzol AmiduazulieDiol Dioxyazobenzol (reEorcin yellow) DlamidoaiobeDzoI (chrysoidiii) RoGolic acid . . . .... RoeaniljD Thionol Thionolin ApoBuflranoD ApoiiafTraoiD If several similar auxochromes are introduced into a chromogen it will be found that up to a certain point the intensity of Ihe shade and the affinity for the tissues increaaes with the number of groups in- troduced; thus, amidoazobenzol — yellow; diamidoazobenzol— orange, triamidoaKO benzol— brown. Witt's observations extended only to Ihe 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 eimply constituted dyes, possess a yellow eolor. He showed that the tint deepens not only with the mcrease in auxochrome groups just mentioned, but also with the accumulation ol carbon aluniB io CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 413 the molecule. In many eases 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, (COCeHs), 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 group, 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, 1 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 COLLEfTED STIIDIES IN IMMtlNITY Thus by allowing sulphuric acid to act on anilin, which, as is well known, is highly toxic, the toxicity ib completely destroyed, for the result- ing aiilfanilic acid can be taken in large doses without injury In like manner the amidobenzoic acids are non-toxic; so also the meta- and para-oxy benzoic acido derned from phenol, while the ortho isomer (salicylic acid) -.till exhibits the familiar toxic effects, although they are far loss intense than those of phenol. These surprising results cannot be ascribed to purely chemical effecta, as, for example, by assuming that the acid dcrivati\es are more difficult to oxidize thaa the original substance and that they therefore do not abstract oxyp;ea from the tissues. Certain observations, however, which 1 had made many years previously in connection with vital staining furnish a very simple explanation. I found that the power to stain 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 il 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 ot 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 b!ood destructive properties of phenylhvdrazin and benzidin are stilt present in their monosulfonic acids.' 'The action of tliese com bi nations ix not oa btrong as tbe original aub- stance, but this is probably due to tbe fact that the sulfonic acid radical (and even fbe neutral sulfonic radical) by itself reduces ihe toxic power of tbe amido group. This mitigating action explains why eiitfanilic acid which is derived from anilin Is no blood poison; tbia power of the sulfonic acid group, bon'ever. J CHEMICAL CONSTITirnON 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 disiribvtion 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 Stock vis, 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 insufficient to destroy the powerful NH*NH« group of phenylbydrazin, or the two amido groups of benzidin. 416 COLLECTED STUniES IN IMMUNITY, It can be carried out exactly with only a very small number of readily determinable substances, hence primarily with inorganic romhinations. 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 localiza- 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, nephritB, destruetJon of the blood, are always given. Nissl's investigations, however, demonstrated that exact histological studies on the central ner\'oiis 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, mast 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 easv 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 organi.sm remains unknown. J 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, e.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 dimethyl phenylene 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.^ ' It 18 possible that this pbenomenon can be fully explained by this that vve 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 lend, 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. \one 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 ilirough. 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 elands are not well adapted to the secretion of subst-ances 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 J iodide, rodan combinations, and the salts of mercury. In the arc- I matic series it is particularly paraphenylendiamin, dimethylpara- 1 phenylendiamin, trihydroparaoxychinolin. and related substances, which are excreted through the submaxillary gland of rabbits and there give rise to marked inflammatory changes (cedema, necrosis). The least important rflle is that taken by the sweat glands. So far as 1 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 J microscope. I need only call to mind the vital staining of nerve endings by means of methylene blue, a procedure which has found are bere dealing with large-moleculed fiubstuDcea which are soluble wiib diffi- culty and which tberefore must be regarded more like colloids In conttut to methylene blue, methyl violet, and many other dyes, beDzopurpunn is aEy Bolutely noD-dlfiufiibte According to the researches of Kraflt (Bericht der deutBcb. chem. Gesell. 1899) solutions of ben/opurpurin (raieiog of the boiling- poinl) showed an apparent molecular weight o! 3000 instead of 77A reckoned out from the fonnula. 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 ivell 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 1MMUNIT\ 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. W« find exactly the same conditions to hold with bacterial poisons. Tetanus poison, for example, as is shown by the experiments STL-DIKS I oxidized substances {the analogues of iodic acid) are no longer able to yield up their oxygen to oxidizabie combinations when pnissie acid is present. {One must think ot these highly oxidized substances as transmitters or carriers of oxygen.) Prussic acid poisoning la therefore an internal suffocation of the organs." This discovery of contart action constituted the first step Inward penetrating the mystery of the action ot drugs. This, however, afforded no explanation as to why the substances mentioned ex- hibited an elective action. That was because the link was mifeiog 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 1S87, in my article on " The Therapeutic Significance of the Substituting Sulphuric Acid Group" {Therap. Monatshefte. March, 18S7), I demonstrated that neurotropic stains are deprived of this property on the addition of the snlfonic-acid group. Even at that time I compared the localization of the dyes and ot the alkaloids in the brain with the principle of the shaking-out procedure devised by Stas-Otto, expressing myself as tollowa; "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 ot 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 ot 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 1 showed that fat tissue behaves like the brain, for a large part of the substances takeji up by the brain are taken up also by the fat tissue. In 1891 this question received a fresh impetus, for Hotmeister, Pohl, and abo Spiro, called attention to the significance of loose combinations which could readily be dis- sociated. Thus in 1S9I Pohl showed that the ability ot the red blood-celb to take up chloroform, a fact which Schmiedeborg had demonstrated in 1867, was due to the cholesterin and lecithin which CFIEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 427 the cells contain Both substances can be shaken out with chloro- form. He al!=o 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 sludy the Action of the above-mentioned substances in the brain. These substances, it will be seen, are most ail 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 basic 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 difTerent kinds of atomic groups possessing very strong affinities, and it was certainly very plausible when Low ascribed a leading rSle 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 rflle. 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 phenyl hydrazin, 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 ' ll IS impossible to do more ilian refer lo ihe great, advances made nine*" my address, especially thiougb llie lutturs of tluiiH Meyer and Overton. In three studies on the theory of alcoliol narcoaifl (Archiv f exiwnm. Palhologie 1899-1901). Meyer baa shown in the most eiact maimer lor a larRe number of chemical eubstitnces that tbe mode of action of the in difTerent narcotics ta not deoenctent on their other chetnical properties but is governed exclu- sively by the fwrtition coefficient which determines their distribution among water and certain iat-Iike subatances (brain and nerve fat). H Overton came to (he same conclusion regarding the causal relation between solubility in lat and narcotic action. Uia m vest igat ions, which have been gathered together in a work entitled "Studien iiber die Narkose," Jena, 1901. dealt eapecially with vegetable cells and small animalt preoent in the Suid. J 42S COLLECTTED 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 1 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 phenacetin, in which by the introduction of the methyl radical and of the acetyl group the powerful OH and NH2 groups of paramidophenol are occupied. All this has led me to conclude positively that Low's theory of the substituting action of therapeutic substances is untenable. Vy this I do not in the least wish to say that groups capable of reacting, 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 number of these, however, is still more limits if the 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. COLLECTED STUDIES IN IMMUNITY, Bro methyl amine = CH3 NHs + HBr» Since then, however, Marckwald has positively shown (1900- 1901) that this substance cannot, as was at first supposed, coDtain a double bond (ethylene combination), for it does not reduce per- manganate at ordinary temperature nor take up bromine. It can therefore only possess the constitution of a dimethylenimin; I CHj' r./ To view of this a complete analogy exists between the ethylenimja and the ethylenoxid: I >o In conformity with Bayer's tension theory we must ascribe ao extraordinary tension to the three-sided ring cont^ned 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 paas over into a substituted etbyl- amin of the chain series. Thus, as Gabriel showed, HCl is added witb the formation of chlorethyJamin, 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 showD 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 chlorethyl alcohol. 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 BomewhaC luodifyiiig tbe le: in acuirdaRce witb tbe positive advance of our knonriedge, the labors of Marckwald. t of ihia chapter rhich we awB to J CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 431 are especially interesting. Adnainistered to a great variety of ani mals (mouse, rabbit, dog, goat, guinea-pig, rat) in doses which cause death after 1^ 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 oedema. (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 neurin (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- appea. 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-NHa CgHuv allylamin with a double bond (ethylene radical): CH CH COLLECTED STtmiES IN IMMUNITY. and propargylatnin. which containB the acetylen group, C-H 1! Atl of these substances were found to possess the evanescent general symploms together with an absence of permanent organic injuries. Hence I believe that the chemical avidity of the double and triple combinations ia insufficient to effect substitutive reac- tions with the protoplasm. I am strengthened in this view by the CH fact that, pnissic 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 poMtesa 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 auxotozie properties, i.e.. that they are able to increase the toxicity when they enter into complexes which in themselves already possess certain toxic properties. 1 must emphasize tlie fact that all observations thus far niade 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 ah ' Neurin is tweuty tiuiCH as loiic us tboUn (trimelbyletbylammoaiuni hydroxide); allylalcohol fifty limes more toxic than piopyl alcoliol. cl n Low. Natiirlicbes Syaletn der Giflwirkiingen 1S93, page OS. 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 efforts 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 Ansemie, in Nothnagel's Handbuch, Vol. VIII, 1901, pages 655 et seq. 3 Die Schiitzstoffe des Blutes, page 364 of this volume. COLLECTED STUDIES IN IMMUNITY. VII. In the case of the chemieally defined poisons, drugs, and dyes discussed above, incorporation into the protoplasmic molecule does not, barring a few exceptions, Lake place by means of synthesis. Since, however, almost (he greater part ot all substances foreign to ihe body exhibit a typical selective action in the tissueo, il becomes neces- sary to study the reasons for this action. Here again we shall do best to begm with a consideralion of the phenomena which takes place in staining reactions. A cotton fibre placed in a dilution of picric acid ot one to a million takes up the dye, becoming intensely stained. Methylene blue introduced intra vitam into the organisnri is taken up by the nerve endings. In [loisoning by alkaloids certain nerve centres may react sjiecifieally and alone. All of these phenomena are obviously analogous in their nature. It seems net^essary, theieiore, to discuss briefly the views held concerning the nature of the stairung process. The purely mechanical conception which refers it all to physical processes, such as surface attraction and absorption, cuo 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 ihe fact that by treatment with alkalies an acid can be obtained — lanuginic acid derived from wool, and nucleic acid from nuclear substances — whieb possesses the property of precipitating the salts of basic dyestu& even out of very dilute solutions. Analogous conditions are found to a great extent in vital atainings. 1 need only remind Ihe reader of the investigations of Pfeifler. 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 in^^luble tannate of methylene blue. Naturally in the higher animals secretion substances present in tlie cells and constituting precipitants which form laky combina- tions can play a part in localization. The second theorj-, 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 ia 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: NH2 N N(CH3) 2 \/\/ \x Y CH, N /\y 436 aSLLECTED STUDIES IN IMMUNITY This substance has the property of staining the granules of celb most intensely, and the same holds true oi 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: NHa N N{CHa)3 I I I I CH3 N 1\ I OH CaHs In contrast to this, however, the combination in which one of the central amin radicals contains an ethyl gioup which gives to the group the character of an ammonium base, is absolutely unable to effect the staining. All phenaain 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 substJincea of the nervous system as well as the fat oi 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 Slas-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 evjiect that a single principle can be rigidly applied to the phe ' Tbia behavior baa been studied espec^mlly by OvertoD. He termB ibe substances of the brain wliich ser\e as extracting agents "Upoida" Chitl among these are choleaterin and lecithin Among Ilie alk&loida Overtoa dia- tinguiBhes feebly basic and more sl.rongly basic Kubstancea, Tbe loriiier can be shaken oiit^tor example, (lie indifferent narcotiCJd; nticreas the more sltvngljr basic unite with constituents of the cell tu lorm salt-like cumbmationa which are very easily diissociaied, .According to Overton's conception therefore Knecht'a explaoation would apply at one tinie 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 (Uber physikalische und physiologische Selection, Habilitationsschrift, Strassburg 1897). In these, although starting from entirely different stand- points *be author reaches many of the views held by me. At the time of my address I was unaware of this study, as it ib not to be had in the bookshops. ■438 COLLECTED STITDrES IN IMMtTNITY of substances foreigu to the lx>dy synthetic processes play practirally no r6Ie whatever. If we take methylene blue as an example, we see .at once that we ean easily find a large number of different duids which are able to shake it out. On the other hand, we know of a large number of acids, like picric acid, phoaphomolybdic acid, hyper- sulphuric acid, which are able to precipitate the methylene blue ia 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 oi^anism. 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 foodstiifTs, in assimilation, and in the growth of living matter, are cnnnerted 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 spccificaHy to each other (like lock and key: E. Fischer). The eagerness with which the living protoplasm lays hold of the foodstiifT which it re- quires is in marked contrast to the manner in which it resists taking up substances foreign to itself. This was observed e^'en in the l)egin- ning of histology, for at that lime it was regarded as an axiom that living cells could not possibly he stained. Gerlach, for example, had shown that an am»rba 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 eresyl 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. '^Tiat practical conclusions can be drawn from these considera- tionsr 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 grou[)s 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 CULLECTEU STIUJIKS IN IMMl'NITY. behavior, namely, in mice they ail produce a peculiar foam-like degeo- eratioB of the liver-cells which I have observed only in substances belnnging to this series. From this it follows that all bodies of the cocaine series are alike so far as the liver is concerned. CoDsiderini 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 an33sthctic action we shall have to assume that the rest of the molecule is only the carrier which brings the benzoic acid radical to the proper place, (The anaesthesiophore character of this group had already been made very probable by the earlier investgationa of Filehne.) Let us go back to our illustration of the mosaic in order to get this idea clearly before ua. In order for a piece to help complete a given figure it is first necessary that it possess a particular jorm, 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 tlie 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'a "tropeins," e.g., benzyltropein, salicyJtropein. phenylglycoltropein (homatropin). A comparative study of the these substances showed that for mydriatic purposes aromatic oxyacids were the most favorable — and esiwcially those m which the hydroxyl is in aliphatic combination, as in tropic acid and phenylgly colic acid. In cocaine, Einhom and I attempted to determine the function of the benzoyl group by introducing various side-chains. It waa found that the introduction of a nitro group in the meta position had a marked influence on the anesthetizing 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 aniEsthetizing property bad dis- appeared, the toxic action on the liver decreased. Meta-amido cocaine waa entirely inert. J 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 anajsthesiophore 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 U desirable that chemists and pharmacologists work together in some definite plan. That is one reason why I have gone into such detail x;oncerning 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 Bechboki and Ehrlich on the relation of chemical constitution to disinfecting power. .(Zeitschrift fur physiol. Chemie, Vol. XL VII, Noe. 2 and 3, 1906.) XXXV. A STUDY OF THE SUBSTANCES WHICH ACTIVATE COBRA VENOM.' BY Dr. Preston Kyes. Dr Hans Sachs. Associate in Anatomy. University of Assistant at the Royal Institute Chicago, Fellow of the Rockefeller for Experimental Therapy, Institute for Medical Research. Frankfurt-on-Main. I. Concerning the Activation of Cobra Venom by Means of Compiements. In a previous study ^ one of us has shown that cobra venom is activated not -only by^-ewtain active sera but afaa by lecithin and certain complement-like, substances of the red blood-cells called **endocomplements." This, of course, harmonizes with the ambo- ceptor_nature oi the poison which had been demonstrated by Flexner and Xoguchi .3 In view of the wide distribution of lecithin in the organs and tissues it seemed advisable to penetrate deeper into the mechanism of cobra- venom haemolysis, especially in order to deter- mine if the assumption of complements and endocomplements is not superfluous and the presence of lecithin in the red blood-cells and serum sufficient to explain the complement action. It is true that certain sera whicb_activate cobr.^ yenom. lose -this property when they are Heated^ to 56° C. for half an hour, and the endocomplements produced by dissolving the red blood-cells'iri water are inactivated by heating to 62° C. Considering the great ease with which lecithin combines with albuminous bodies, etc., it was possible that the thermolability of the activating factors was simulated by a com- bination of the lecithin with other substances. An important fact which speaks strongly against this view, however, is one first brought * Reprint from the Berlin kiin. Wochensch. 1903, Nois. 2 and 4 * P. Kyes. See page 291. * Flexner and Noguchi, Snake Venom in Relation to Usemolysis, Bacterid olysis, and Toxicity, Journ. of Exp. Medicine. VoL VI, No. 3. 1902. 443 » 444 COLLECTKD STUDIES IN IMMUNITV out by Calmette,' namely, that almost all sera after being heal 65" C. and higher usually show even an increased activating power This we could explain only by ascribing it to the lecithin set free through heating (Kyes, I.e.)- It thus appeared that heating wasmort likely to effect a splitting off than a combination of the lecithin. Our further studies have dhown, however, that this view is not correct in all cases.^ To begin, we exaramed the complementing proj)erties of senan, choosing for our analysis the combination ox blood + cobra venom + guinea-pig serum. The activating property of the fresh guinea-pig serum was destroyed by halt an hour's heating to 56° C, and hewt was apparently not due to the presence of lecithin, but to some other complement-like subatance. Subsetjuent investigations have CM- firmed us in this opinion. 1~he general course of the hxmolrsit through snake venom is markedly different when lecithin ot Genun is used for complementing. Ijccithin effects rapid solution; with large amounts of cobra venom this is almost instantaneous. ^Vben serum is used as complement a longer or shorter period of incubatioo is observed, such us we are accustomed to see with the hsmoIvtJe sera. Furthermore, ha-molysis with cobra venom + lecithin ocfUts even at CC, whereas the action of cobra venom + serum as comple- ment requires a greater degree of heat. That the activating substance of the serum belongs to the cbs ot complements was further demonstrated by the fact that it wis destroyed by digestion with papain. Following the method of Ehrlkl) and Sachs,^ in order to digest the complement, 5 cc. guinea-pig eeruo were mixed with 1 cc. 10% solution of papain, digested for li hours and then centrifuged. The decanted fluid was used to activate the cobra venom. Table I shows that this property was almost com- pletely lost. The serum treated with papain had thus almost completely \asl its activating property, whereas a solution of lecithin similarly treated preserved its activating property unchanged. (See Table II.) ' CalmeUe, Sut Taction bftnolytique ".?'"• 0 0.25 ° H ■ 0 1 0-05 0 0 ^ This threw some light on the inactivation of the blood solution at 62° C, a fact which made the complement character of the acti- vating substance seem exceedingly probable. In contrast to the native blood solution we find that the suspension of stromata remaios unchanged when heated to 62° C. SUBSTANCES WHICH ACTIVATE COBRA VENOM 451 The activating substance itself is therefore thermostable. If, however, the decanted haemoglobin solution is again added to the stro- ma ta and this mixture heated to 62® C. inactivation will again ensue. (See Table VIII.) TABLE VIII. Amountfl of Ox Blood +0 01 cc 1% Ck>bra Venom + a, 6. and c. cc. (a) Guinea pig Blood Stromata Suspennion. (6) The Suspension Heated to 62« C. (c) The Suspensions Decanted Fluid (Hspmo- globin) Heated to 62<'C. 10 0 5 0 25 0.15 0 1 0.025 complete < ( trace 0 complete < < < t strong trace 0 0 0 0 0 0 0 From this it appears that the inactivation of the native blood solution depends on this: that on heating to 62° C. the active substance combines with the haemoglobin in such fashion that it is no longer able to combine with the cobra amboceptor. Hence in view of the readiness with which lecithin combines with albuminous substances, etc., we believe that the activating property of dissolved blood-cells which we previously described as an "endocomplement action" is really due to the presence of lecithin or lecithin-like substances in the stroma.^ We have convinced ourselves of the correctness of this assump- tion also by the fact that lecithin is bound by crystallized horse haemoglobin by heating for half an hour to 62® C.^ An experiment of this kind is reproduced in Table IX. A solution of haemoglobin heated for half an hour to 62® C. is also able to inhibit the activating property of lecithin when digested with this for half an hour at 37® C. The lecithin character of the activating substance present in the red blood-cells is confirmed by a number of other observations which deal with the analogous character of cobra-venom haemolysis on the I We were able to completely extract the activating substance from the stromata suspensions by means of alcohol. Besides this, in activating with stromata in the presence of excess of cobra venom, one observes an inhibition of haemolysis due to the deflection of the lecithin. ' We are much indebted to Prof. Hiifner of Tubingen for this hemoglobin. 452 COLLECTED STUDIES IN IMMUNITY. addition of lecithin and of blood solution. These characteristics are as follows: 1. The haemolytic activity at 0°. 2. The comparatively rapid course of haemolysis. 3. The marked inhibitory action of cholesterin. (See Table X.) TABLE IX. Amounts of the Hffimoglo bin-Lecithin Solution. cc. Ox Blood + 0.01 CO. 1% Cobra Venom + Haemoglobin- Lecithin Solution.* (a) Native. (6) Heated for One- half Hour to 62« C. 1.0 0.75 0 5 0.35 0.25 0.15 complete ( t little trace 0 0 0 0 0 0 0 * 5 cc. hsemoglobin X 5 cc 0.0125% lecithin solution. TABLE X AmountH of the Cholentenn Solution. cc 1 cc 5% Ox Blood-l-0 01 cc 1% Cobra Venom + (a) 0.25 cc. Solution of Guinea-pig Blood.t (6) 0.25 cc. 0 01% Lecithin, t 0.025 0.01 0.005 0.0025 0 trace moderate complete 0 0 0 complete t =" complete .solvent dose It will be remembered that in these three points guinea-pig serum exhibited exactly the opposite behavior, a fact which led us to ascribe its activating power to true complements. We have therefore come to the conclusion that solution by means of blood solutions is only a property of the lecithin contained in the blood-cell stroma, and is not due to true complements. We know that according to Ehrlich's^ conception the stroma ta of the red blood-cells are to be looked upon as living protoplasm. In this * Ehrlich, Zur Physiologie und Pathologie der Blutscheiben, Charity An* nalen, Vol. X, 1885. SUBSTANCES WHICH ACTIVATE COBRA VENOM. 453 respect the demonstration of lecithin in the stroma would appear to be of special interest, for just this substance is regarded as particu- larly important for the functions of the protoplasm.^ A further problem, to be sure, is whether this lecithin exists free in the red blood-cells. We have a number of reasons for believing that this is not the case. It was first shown that in yolk of egg only a small part of the lecithin can be shaken out with ether, whereas by extracting with alcohol the entire amount can be obtained.^ The reason for this is that the greater part of the lecithin is conjugated with the vitellin of the yolk. This combination can be obtained as a globulin-like body which is soluble in salt solution and precipitates on dialyzing.^ The lecithin is obtained free, however, only after extraction with alcohol, by which the vitellin also changes and becomes insoluble in salt solutions. In demonstrating the presence of lecithin by means of cobra venom we too have observed that the serum and the red blood-cells yield no lecithin to ether, or if they do it is only in faint traces. On the other hand, the active power of the alcoholic extracts at once led to the recognition of the presence of lecithin. From this point of view some of our earlier observations can easily be explained. We stated that solutions of certain species of blood-cells were strongly activating, while others showed this property to a far less degree or not at all. The alcoholic extracts of all species of blood, however, contain nearly the same amount of lecithin (demonstrated by the activation of the cobra venom). This apparent contradiction is readily explained by the fact that in the different species of blood the lecithin is conjugated with different substances of the stroma ta and, furthermore, that the firmness of this combination varies extensively. Thus in goat blood the union is so firm that the avidity of the cobra venom does not suffice to separate the two components; the consequence is that there is no activation with a solution of goat blood. On the other hand, in ^ It has long been known that lecithin is a constant constituent of the red blood-cells; for many species of blood-cells this content has even been worked out quantitatively. Nothing, however, was thus far known concern- ing the localization of the lecithin. 'iSee Hoppe-Seyler's Handbuch der physiologisch- und pathologisch-chem- ischen Analyse, Seventh Edition, edited by H. Thierf elder, Berlin, 1903, page 157. * Ibid., page 369. 454 COLLECTED STtlDIES IN IMMUNITY. guinea-pig blood, for example, the lecithin ia so lotwely combined that this blood can be used for activation. Hence, in si^eaking ol the lecithin action of nnimal tissues or juices, we refer only to the lecithin which is /rcc (available) in the senae just described, pait or all of the lecithin present may escape detection by means of the activation of cobra venom. Ilie fact that relatively slight alterations can cause the rombioa- tion of lecithin to be either looser or firmer may be of some interest in another direction. We have seen that the lecithin of many species of sera becomes free only at 65° C, while the haemoglobin, on tbe other hand, anchors the lecithin at 62" C. It is possible that during life slight variations in the physical and chemical properties of the tissues (variations which have heretofore been undetected) play an important r6le in the sense that they properly regulate the exchange and trans- portation of the lecithin so im[Xirtant for the vital functions. Dieu- donn^'s' researches show that the albuminous bodies with which the lecithin is combined (principally in the form of lecithalbumin) are demonstrably modified, even at temjieratures still quite distant from their coagulation point. This author showed that B. eoli, for example, when inoculated into a serum lactose solution causes a distinct precipitation even at 45° C, while this does not occur at 37° C. In the ease of serum albumin, therefore, the temperature at which this modification takes place is very near the temiwrature which occurs in the living organism under pathological conditions In view of this and of the evident dependence of the physiological behavior of the lecithin on the integrity of the albumin molecule one is tempted to see n cau.sal relationship between febrile processes and disturbances in lecithin metabolism. 111. The Inhibitory Action ot Choteitterln. The marked inhibitory action which many sera exert on hemolysis with cobra venom and lecithin was described some time ago (Kyes. I. c.) and the opinion then expressed that this protective action of the serum was probably not due to a single substance but was the resultant of several factors. Evidently we are here dealing with certain rela- tions which exist between serum constituents and the lecithin, making ' DJeudonnfi, Ubcr das Verliallen dea Baci toll i Wn Ciweiss. liyg. Ituadsch 11X)2, No. 18. dcnatuiit- SUBSTANCES WHICH ACTIVATE COBRA VENOM. 455 it impossible to demonstrate the existence of the latter by means of cobra-venom haemolysis.^ Having thus learaed that cholesterin exerts a marked inhibiting effect on the action of lecithin we shall probably not err if we assume that part of the serum protection is due to the choles- terin present in the sera. One thing which agrees perfectly with this assumption is the fact that often this protective action is still present after heating the serum to 100° C. The marked inhibition of hsemolvsis on the addition of choles- terin, an inhibition which applies also to the haemolysis produced by lecithin alone when in large quantities, points to an mteresting antagonism between lecithin and cholesterin, to which a few words may be devoted.^ In this case the cholesterin probably has a rela- tion to lecithin which is similar to that of saponin in Ransom's well- known experiments.^ In both cases we seem to be dealing with the effect of a kind of solvent affinity between cholesterin on the one hand and lecithin and saponin on the other, by means of which affinity the presence of cholesterin within the blood-cells gives rise to toxic action, and outside of the erythrocytes exerts a protective action. It is possible that the protection observed by us in haemolytic test-tube experiments with cholesterin is in some way connected with the protective action of cholesterin against snake venom in the animal body described by Phisalix.* Another fact may be men- tioned in this connection, namely, that the haemolysis of washed ^ On the other hand the specific protection exerted by Calmette's snake- venom immune serum is not an antileclthin effect, but, as was to be expected, one depending on the action of the antibody produced by immunization (anti- amboceptors) on the amboceptors of snake venom. When varying amounts of lecithin were employed the protective action of Calmette's serum remained constant, always neutralizing the same amount of cobra venom ' We may add that, like Noguchi (The Antihsmolytic Action of Blood Sera, Milk, and Cholesterin upon Agaricin, Saponin, and Tetanolysin, etc., Univ. of Penna. Med. Bulletin, Vol. XV, No. 9, 1902). we observed a very marked choles- term protection against the action of tetanolysin. (0.00025 cc. of our stock solution, which certainly contains not more than 1% cholesterin, protects against the complete solvent dose of tetanolysin (0.05 cc.)*) On the other hand, choles- terin is absolutely without effect on the hsBmolyses due to staphytolysin and arachnolysin. In connection with this we might mention the fact so inter- esting biologically, that even so indifferent a substance as neutral olive-oil dis- solves the red blood-cells. This haemolysis is likewise inhibited by cholesterin. ' Ransom, Saponin und sein Gegengift. Deut. med. Wochenscb. 1901 ^Phisalix. Compt. rend de la Soc de Biologie, 1897 436 CULLhCTKlJ STUDIES IN IMMUNITY. guinea-pig blood-tells, in themselves susceptible to cobra venom alone, is also inhibited by fholesterin. To be sure, rather large quan- titiea ot the latter are required, but in view of the lecithin character of the substances which functionate as endoactivators, this is to be expected. (See Table XI.) TABLE XI Amounta ol lim ChaJcBtgnn BdLu- 1 EC B% Guinea pi« Blood xcouas CO. 1% fobw V.Qom. 1.0 0.5 0.25 0.1 0.05 0.035 0 0 Uttle marked almost complete complete On the other hand, as already remarked, cholesterin exerts littie or no protection against cobra-venom hemolysis when senira com- plement is used for activation. This agrees entirely with the nega- tive findings on the protective action of choleslerin recently reported by Flexner and Noguchi in an interesting paper on the amboceptor, toxoids, and separate constituents of snake venom.' The apparent deviations are probably to be explained merely by the different conditions of the experiments, for, as it appears to us, these authors made their experiments only on unwashed blood- cells or by the addition of serum. In both cases, however, one is deaUng with an activation with complement, against which we aJso failed to delect any marked protection with cholesterin. IV. The Quantitative Relations ExlstfnK Between Cobra Venom and Lecithin. So far as the mechanism of cobra-venom-lecithin hsmolysis is concerned, we assume that the lecithin acts after the manner of complements, being anchored by certain definite groups of the poison molecule. This has previously been described by Kyes, 1. c. Cobra venom and lecithin accordingly combine just like am- boceptor and complement in serum hsemoiysins, and it was there- tore to be expected that the quantitative relations which exist be- ' Mexner and Noguohi. The Constitution ol titiake Venom and Snake Sera, Univ ol PenDa. Med, Bulletin, Vol. XV, No 9, 1903. SUBSTANCES WHICH ACTIVATE COBRA VENOM. 457 tween amboceptor and complement would be very similar in this case. In our studies in haemolysis due to cobra- venom-lecithin we have therefore been able to observe the same mutual dependence between amount of amboceptor present and the complement re- quired which the researches of von Dungern,^ Gruber/* and Morgen- roth and Sachs ^ showed to exist in their experiments. The rela- tion between these amounts is such that when large amounts of amboceptor are present, smaller doses of complement suffice for haemolysis. To be sure, when an inordinately large amount ot cobra venom is added the amount of lecithin required for complete solution is also larger, as has already been mentioned by Kyes. This is evi- dently explained by assuming that when the amount of amboceptor is excessive the distribution of the lecithin is such that part of the amboceptor loaded with lecithin is deflected and does not come into action. If, however, the amount of cobra venom is decreased^ results will be obtained which, within wide limits, agree with those observed by Morgenroth and Sachs (1. c.) with serum haemolysins- The more cobra venom one adds the less lecithin will be needed to effect complete hcsm^lysis, and, conversely, in adding larger amounts oi lecithin the minimal complete solvent do^e of the cobra venom is constantly decreased. This is well shown by Table XII. TABLE XII. A. 1 cc. 5% Ox Blood. B. 1 cc 6% Ox Blood. Amounts of the 1% Solution of Cobra Venom. The Amount of Leci- thin Solution (0.025%) Necefl«ary for Com- plete Solution. Amounts of the 0 025% Lecithin Solution. The Amount ot Cobra Venom (1%) Neces sary to Effect Com- plete Solution 0.01 0 001 0.00025 0.0001 0.00001 0.035 0.05 0.075 0.1 0.5 0.3 0.06 0.03 0.00001 0.0001 0.005 From these experiments we see that the quantitative relations which exist between cobra venom and lecithin furnish an additional * von Dungem, page 36. ' Gruber. Wiener klin. Wochensch. 1902, No. 15. ' Morgenroth and Sachs, pages 233 and 250. 4oS COLLECTED STLTJIES IN IMMUNITY. argument for the view that cobra venom and lecithin amboceptor and complement. V. The Susceptibility of tbe Red Blood-cells. These observations show that in comparing the susceptibility ol the various species of blood to cobra venom the limit of activity of the venom must be determined with the optimum iiuantity of lecithin. The values thus obtained may be regarded, so to speak, as the '"abso- lute susceptibility" of the blood-cells. In Table XIII the minimal complete solvent dose is determined for several species of blood on the addition of an abundant quantity of lecithin (0.2 cc. of a 0.025% lecithin solution). TABLE XIIL 'F-.-™ Amount of t«.^iihiD. '*™V«tm^;'"'" Guinea-pig, . . . Ox Rabbit Man 0.2cc.ota0.025%8o!. 0.00000005 0 OOOOOOI 0.00000025 0 0000005 0.000001 If we compare these values with the susceptibility of the variotia blood-cella with cobra venom alone (see Table XIV) we shall see that when the latter is used the amount of venom necessary for complete hEPmotysis is many times greater than when a sufficient amount of lecithin is added. Thus the absolute susceptibility of guinea-pig blood against cobra venom + lecithin is 500 times greater than that obtained without the addition of lecithin. This shows also that although guinea-pig blood heads the list in cither case there are marked deviations, so far as the other bloods are concerned, from the results obtained on tbe addition of lecithin. Ox blood, for example, which is not at all susceptible when lecitbio is lacking, is more susceptible than either rabbit or human btood when lecithin is present. Yet the two latter species of blood are dissolved even without the addition of lecithin. We thought it would be especially interesting to study the sus- ceptibility of human blood-cells to cobra venom in various diseases. In the few cases thus far observed (several healthy persons, tm-o casa SUBSTANCES WHICH ACTIVATE COBRA VENOM. 459 of diabetes, one of pneumonia, and one typhoid) we were unable to discover any essential difference in susceptibility.^ TABLE XIV. Susceptibility of Various Species of Blood to Cobra Venom Alone Species of Blood (1 co. 5% SuBpennion ). Amount of Cobra Venom Reauired for Com- plete Unmolysia. Froflf 0.00001 0.000025 0.000025 0.00C05 0 00025 0.00025 0.00025 0.0005 0.001 0.001 not susceptible DoK Guinea-Die Man Rat Pig * 'o Mouse Goose Rabbit Horse Ox, sheep, goat As a result of our extensive researches we must continue to uphold the view that blood species are clearly divisible into those directly susceptible to cobra venom alone and those not susceptible under those conditions. This follows also from the above table. In this respect our observations are at variance with the recent state- ments of such excellent workers asFlexner and Noguchi. It may be well therefore once more to point out a few possibilities by which this difference can be explained. Flexner and Noguchi observed that, in general, after cbpious washing, the blood-cells were not dis- solved by cobra venom, or at least were only partially dissolved. In spite of repeated washing of the blood we were unable to discover any decrease in susceptibility. If Flexner and Noguchi insist on such a thorough washing (6-10 times) it appears to us that it can no longer be a question of removing the serum complements. The small quantities of serum which are contained in the 0.05 cc. blood employed in each tube in the test- tube experiment (1 cc. of a 5% suspension) are entirely too small, according to our experience, to exert a demonstrable complement Mt is possible that investigations in other diseases will lead to positive results. We are not in a position to apply our observations to more extensive clinical material, but shall be glad to supply cobra venom for this purpose to any one applying for the same. 460 COIXECTED STUDIES IN IMMUNITY action after one or two washings We are therefore more inclined to assume that the insusceptibility observed by Flexoer and Noguclj is due to a washing out of the activating substances present in th» blood-cell. One of ua has already reported such extraction phenom- ena (Kycs, 1. c); we have, however, been unable to repeat the a- ' penments. It is possible, as has already been stated, that tbe ' divergent results are due to minute differeucea in the experiment, differences which for the present at least cannot be analyzed. It is also possible that a certain degree o( racial divergence in the blood- cells of animals of the same species used by Flexner and NogucM ' and by uh gives rise to what at present is an inexplicable differeoo,] In the blood-cells employed by u.'* the activating substances couldl not readily be washed out. This is shown by the fact that the acti-l vating substances are so firmly bound to the protoplasm that tbef ' are not separated even in preparing the stromata. Attention is also called to the antagonism which is so oftes observed between blood-cells and their own serum. Tbis has already been pointed out by Kyes. Thus rabbit blood-cells are dissolved by cobra venom, and this action is intensihed by the addition o* rabbit blood-cells which have been made laky. In spite however, the active serum of the same rabbit inhibits cobra- hfemolysis (see Table XV), In this case, therefore, adherent trac^ of serum cannot possibly effect autoactivation of the rabbit blood- cells. TABLE XV. dition 0* j of th».J ra-vennml :nt trawa I ,4"x^1^- Inj. 5% R.bbil Blood* Cobra Veoon Alone. ^%M^^.VniJ:^'^ Cobra Vennm-fOJUM. Ribliil-MoadScUu- O-I 0.05 0.025 0.01 0.005 0-0025 almoBl 0 0 0 0 0 0 0 0 0 0 0 complelo ^^^^cept There is another point of considerable interest in connection wil these questions, one very important for the technique. The em ceptibility of the washed blood-cells can readily be overlooked i SUBSTANCES WHICH ACTIVATE COBRA VENOM. 461 many cases owing to the occurrence of a marked inhibition of haemolysis due to the presence of an excessive amount of cobra venom. Kyes (1. c ) has already discussed in detail the fact that in hae- molysis with cobra venom alone a phenomenon can occur which is analogous to the deflection of complement described by M. Neisser and Wechsberg.^ In rabbit blood we have observed extensive indi- vidual differences so far as this deflecting phenomenon is concerned. We have often found animals whose blood-cells remained undissolved in the presence of even a very slight excess of cobra venom, so that it was necessary to have just the right amount of venom in order to effect haemolysis. Table XVI shows several examples of this. TABLE XVI. Amounts of 1% 1 cc 5% Rabbit Blood. Cobra Venom Rabbit Rabbit Rabbit Rabbit cc I, II. III. IV. 1.0 0 — _ _^ 0.5 faint trace — — 0 25 little — 0 1 complete 0 trace complete 0 075 almost 0 complete — 1 1 0 05 0 moderate marked n 0.025 0 0 complete f ( 0.01 0 0 trace If 0 005 0 0 0 strong 0 . 0025 0 0 0 trace 0.001 0 0 0 almost 0 0 0005 0 0 0 0 The marked deflection which is observed in the blood of rabbits I, II, III is evidently caused by a relatively slight amount of activating substances present in and at the disposal of the red blood-cells. On the other hand the different behavior of other bloods, as in rabbit IV, shows how the amount of free lecithin contained in the blood-cells can vary from case to case. It might pay to examine the blood of different rabbits for this purpose. See page 120. COLLECTED STUDIES IN IMMUNITY. VI. A Few Cbemlcal Considerations. Finally, wc should like briefly to discuss some of our experiencw with the power possessed by certain other substances to activate cobra venom. In view of its content of lecithin, it will not surprise us to know that bile activates cobra venom. It may be interesting, however, to learn that goat milk acquires activating properties only when it ha.1 previnui^ly been heated to 100* C. This behavior corre- sponds entirely to that of certain species of sera whose lecithin does not become available until after they have been heated to 65-1(10°, Among chemical substances we have found a number of fatty acids and their soaps, chloroform, and olive oil able to activate to a cer- tain degree. All these substances by themselves, however, dissnh'e the blood-cella to a greater or less degree ' and the increase of this action is so slight that it is doubtful whether we can here speak of pure activating phenomena.^ According to our experiences only one more substance, namely, the lecithin-like cephalin, possesses marked activating properties. (Cerebrin does not possess them.) For this cephalin we are indebted to Waldemar Koch of Chicago, who made it from sheep's brain. According to him, it is a dioxyatearylmonomethyl lecithin.^ The cephalin (which is insoluble in alcohol) and the lecithin (which is soluble in alcohol), both made by Koch from sheep's brains, further- more two other preparations of lecithin (one from Riedel in Berlin, the other kindly placed at our disposal by Dr. Bergell), all these manifested a hcemolytic action {if at all) only in 500-600 times the amount sufficient to activate the cobra venom. A preparation of lecithin derived from leguminous seeds, for we are indebted to Prof. Schulze of Zurich, showed leas dif- ference between activating power and ha-molytic action, but evea ' It ia po)uiibte that the coclostable lieiniolysina (solulile in a Icofaol-etfaer) of the orgnD extracts belong in the same class with these siibstaaces (see Korscbun and Morgenrolh, page 267) ' It must slwayfi lie bnrne in mind that the activating property of tlieae suh- etanccB may possibly only be an indirect one, the presence of the subatanoe sufficing to make available the lecithin always prcisetit in the blood-cella in combination. ' W Koch, Zur Kenntniss des !.«cithina. Cepbalins iind Cerebrins Gubstan)!, Zeitsch. I physiol Chemie, Vol. 36, Nos 2 and 3, 1903. SUBSTANCES WHICH ACTIVATE COBRA VENOM. 463: in this the ratio was still 1:200. A lecithin obtained from E. Merck behaved similarly. Nevertheless all of these preparations were exactly equal in their activating power. It is hard to say whether possibly the cholin radical or the fatty-acid radical represents the active toxophore group of the combination formed by the union of the lecithin with the cobra venom. It may be mentioned, however, that neutralized cholin exerts no haemolytic effect, and that sinapin ^ (the sinapic acid ether of cholin), despite the cholin radical which it contains, possesses no activating power. We are therefore inclined to believe that the toxic action is caused by the fatty-acid radical in the lecithin molecule. This also agrees with the haemolytic action observed by us in neutralized stearylglycerophosphoric acid and in the above-mentioned fats and fatty acids. We shall report on further researches in this direction at a subsequent period. In conclusion we may be permitted to discuss briefly a few inci- dental observations. Among these is the fact that hydrochloric acid not only causes no destruction or weakening of the cobra venom, but even exerts a marked protective action on the same. A venom solution which completely loses its activity by heating to 100° C. for twenty minutes can be heated for half an hour to 100° C. without losing its haemolytic property if it contains ^/\%n HCl. Not until the poison containing the acid is heated for two hours to 100° C. is destruction complete. Possibly the protection exerted by the acid may indicate the basic character of those binding groups of the cobra-venom molecule which are here concerned. So far as the influence of other agents on the cobra venom is concerned we shall only mention that all procedures which prevent the action of the cobra venom in the animal body - (an action due mainly to the neurotoxic components of the poison ^) also destroy the haemolytic action of the venom. Examples of this are powerful oxidizing sub- stances (potassium permanganate, chloride of lime, chloride of gold, soda lye, etc.). * For this we are indebted to Gebeimrath Schmidt in Marburg. ^ See especially the detailed and excellent investigations of Calmette. Annales de rinst. Pasteur. T. VIII. 1894. ' See Flexner and Noguchi, 1. c. COLLECTED STUDIES IN IMMUNITY. 1. The property of certain sera to activate cobra venom, a erty which is lost by heating the sera to 56° C, depends on the preseoce ol complements in the restricted sense, 2. Tlie activating property of blood solutions depends on the lecithin contained in the red blood-cells; this also gives rise to the susceptibility of the blood-cells against cobra venom done. The lecithin which cornea into play is a constituent of the stroraata. 3. The tact that blood solutions are inactivated by heating to 62° C. is due to the combination at this temperature of the lecithiB with the btemoglobin; suspensions of blood stromata are not inacti- vated at this temperature. 4. Choiesterin inhibits to a high degree hipmolysis by means oJ cobra venom alone, and of cobra- venom-lecithin. When serum complements are used for activation, choiesterin exerts little or no protective action, 5. Choiesterin does not inhibit haemolysis due to BtaphylolynB and arachnolysin, but very markedly inhibits that due to tetfr- nolysin and to ohve-oil. 6. The quantitative relations between cobra venom and lenthln correspond to those of amboceptor and complement; the more cohta venom present the less lecithin will be required for hsemolysis. and vice versa, A deflection of lecithin does not occur unless verv large amounts of cobra venom are used. 7. Most species of blood are susceptible even to oobra venom alone. The "absolute susceptibility" determined with the optimum addition of lecithin may be many times that obtained without tbe addition of lecithin, 8. Hydrochloric acid exerts a marked protection on cobra venom agajnst destruction through high temperatures. Potassium wr- manganate, chloride of lime, chloride of gold, soda lye deatroy cobn venom (experiment with blood -f- lecithin). 9. Bile activates cobra venom; milk (goat) only after it has prc- I viously been heated to 100° C, I 10. Fatty acid, soaps, chloroform, and neutral fats have a hsemo- I lytic action. The haimolytic action is somewhat increased on tie I addition of cobra venom. I 11, Lecithin and cephalin, on the other hand, exert a hremolytic SUBSTANCES WHICH ACTIVATE COBRA VENOM. 465 action on the ordinary species of blood only, if at all, when 200 or 600 times the amount is used which suffices for activating the cobra venom. 12. In the poisonous combination formed on the union of cobra venom with lecithin the fatty-acid radical may, with a certain degree of probability, be regarded as the active group. XXXVI. THE ISOLATION OF SNAKE VENOM LECITHIDS.' Special interest attaches to the study of snake venoms be- cause of the analogy which exists between their peculiar charactef and that of bacterial toxins. All investigators who have worked ftiih this subject have been struck by this analogy, and Phisalix - has dis- cussed it in a special monograph. The analogy between snake venonis and bacterial toxins consists, above all, in the fact that neither are crystallizable, that their constitution is unknown, that both are highly virulent specific products of poison-formiDg cells, and both possess the power to excite the production of antibodies in the organism. This last fact we know from the fundamental researches of Calmette.3 A further analogy between snake venoms and the toxins is the fact that the poisonous properties of both are destroyed bv heat, and that the non-toxic substance thus formed is able to excite the production of antibodies just as well as the original substance. In other words, in both poisons there is a formation of toxoid. Snake venom has accordingly played an important role m the theoretical doctrine of immunity. Martin and Cherry,* for example, by their well-known filtration experiment were able to prove that snake venom and sjrecific antitoxin unite to form a new non-poisonous combination. This experiment is based on the principles first formulated by Ehrlich ' Reprint from the Berliner Win. Wochensch. 1903, Nos. 42 43. ' Phiaatix, Etude compare dea toxines microbiennes et dea -venins, L'Annfe biologique I, 1805, ■ Calniette, Ann. de I'lnatitut Pasteur No. S, 1804. ■ Martin and Cherry, Proceeding of the Royal Society, Vol. LXIII. 1808- 466 i^^^^H THE ISOLATION OF SNAKE VENOM LECITHIDS. 467 in his studies on ricin and antiricin, and the results are entirely similar to Ehrhch's.^ Still another important analogy between snake venoms and bac- terial poisons consists in their plurality, a fact which has been demon- strated for a number of poisons. In the ordinary well-defined chem- ical poisons we are accustomed to regard the diverse toxic phenomena as due to the action of one and the same substance on different organs. (In poisoning with corrosive sublimate, for example, the diverse toxic phenomena which are produced in the various organs.) The toxins, however, have to a large extent shown a different behavior^ the action on different organs being ascribed to different kinds of poisons, which frequently possess different haptophore groups. The possibility of correctly and sufficiently analyzing these poisons de- pends in a large measure on Ehrlich's theory of the combination of these poisons. In this way it has been shown that tetanus toxin consists of at least two components, tetanospasmin and tetanolysin,^ to which, according to Tizzoni, a third poison must be added, one which gives rise to the cachexia. In snake venom the conditions are entirely similar, the different effects which it produces in the animal body being due to the presence of different poisons with different haptophore groups. The late lamented Myers ^ showed that the haemolytic property of snake venom is to be separated from its neurotoxic property; and recently Flexner and Noguchi* have shown that the oedematous swellings produced by injections of snake venom are due to the presence of a third toxic component acting on the endothelium. For some years I have closely studied cobra venom, and especially that constituent of the same which causes solution of the red blood- cells. Part of these researches were conducted conjointly with Dr. H. Sachs.^ I was able to confirm the interesting observation of Flexner and Noguchi ® that the snake venom, as such, did not act on certain blood-cells, but that haemolysis occurred only when a second substance is present which acts after the manner of a complement. ' Ehrlich, Fortschritte der Medizin, 1897. ' Ehrlich in Madsen's paper. Zeitschrift f . Hygiene, Vol. XXXII, 1899. ' Myers, Journal of Pathology and Bacteriology. 1900. VI, 405. * Flexner and Noguchi, Univ. of Penna. Medical Bulletin, Vol. XV, No. 9^ 1902. * Kyes, see page 291 ; Kyes and Sachs, see page 443. * Flexner and Noguchi, Journal of Exp. Medicine, Vol. VI. No. 3. 1902. 468 COLLECTED STUDIES IN IMMUNITY. By following up a very important observation made by CA- mette,' that the complemeniing action of a serum, in conlrasi 4 what IS seen with ordinary complements, is still preserved after heatinf to 62° C, we succeeded in discovering what this complementing was, and proved that lecithin was able to activate the cobia venom amboceptor. Especially were we able to show that the di' behavior of the various species of blood-cells (some of them, ox blood, goat blood, sheep blood, are not dissolved by cobra venom alone, while others, such as guinea-pig blood, rabbit blood, human blood, dog blood, are dissolved imder these circumatancea) is due exclusively to the lecithin, only those blood-cells being dissolved in which Iht lecithin is so loosely bound that it is available for the activation d the cobra -venom amboceptor.^ An exact study of these activating phenomena by means of lecithii seemed to us to be of the highest importance for one of the fundamoicai problems of immunity, namely, the mode of action of coinplemenl& Every one who has had any large experience with the activation d ordinary hemolytic amboceptors by means of complements, ani who compares this activation with that of cobra venom by means of lecithin, will be surprised at the complete similarity of both processes, and will not doubt that essentially the same mechanism must contrcl both. For some years the schools of Bordet and Ehrlich have had a sharp conflict of opinion concerning the explanation of the furnl*-' mental facts observed by Ehrlich and Mnrgenroth, that the ambocvptv is anchored by the red blood-cells, thus making the blood-cells si ceptible to the action of the complements. For numerous which arc given in the earlier studies, Ehrlich's school assumes thil ' Calmette, Compt. rend, de I'Acad. lies Sciences, T. 13-J, No. 24. ]902. ' Some time ago we confirmed the ol>8ervation8 of Flexner and Nogiicb^ thai liltiod-cellB utiausceptible to cobra venom alone can be activated by c«ruia fresh sera, aod that this activatibiilty ta then loat on heating the sera to 56° C In conformity with these autbots we assumed that the cobra venor alao be activated by true complements. At present, bowever, we have rather skeptical as to the correctness of this explanation. We cannot at t dismiss the assumption that the action is an indirect one, the action o( •eriim cauaing the lecithin combination in the red blood-celU to become loo eo that then this substance can exert its activating power on the amboceptoE This iind" further support in several observations which we have m&de o the favoring influence of certain indifferent substances (oils, pure fa I ty acid on hiemolyais with cobra amboceptor In these cases the cause of the sdl tion can only be a change in the churucter of the lecithin combinalioa. THE ISOLATION OF SNAICE VENOM LECITHIDS. 469 complement and aralwceptor unite to form a new poisonous combina- ' tion, and this is the view which I also lake, Bordet, however, even in his latestatudy' asumes that there is nodirect affinity between com- , plement (alexin) and amboceptor, but "que la sensibilatrice modifie I'fl^ment de manifire i iui faire acqu^rir le pouvoir de fixer directement I'alexine avec beaucoup d'^nergie." The Frankfurt Institute has furnished a number of important argu- ments tor the direct union of amboceptor of complement. Because of the lability and great number of the complements as well as the impossibility to isolate the active product chemically it was out of the question to furnish direct chemical proof for these views. Nor is there in the present state of scientific knowledge any hope that this problem will bo solved in the near future. For this reason we rejoiced in the discovery that in lecithin we had found a substance pHJssessing com plement -I ike properties, and which because of its chemical behavior would serve to settle this dispute. ' In other words, it was to be seen whether or not the cobra am- boceptor combined directly with the lecithin to form a new hiemolytic l combination. If it did not do so, it would help sustain Bordet'a view that the union of the cobra venom amboceptor serves only to give the lecithin access to the blood-cell. From the following studies it will be seen that the decision which we had been led to expect as the result of our biological experiments is confirmed by chemical means. One thing especially argued for the correctness of our conception, namely, the fact that it is possible to inhibit the cobra venom hiemolysis by employing very large amounts of cobra amboceptor. In that case susceptible blood-cells which can be dissolved by a certain definite amount of cobra amboceptor are no longer dissolved if many times this amount is employed. This corresponds to the phenomenon which we observe in certain bactericidal sera, and which according to Neieser and Wechsberg is due to a deflection of complement through an excess of free amboceptor. The result is comprehensible only on the assumption of a direct chemical affinity between amboceptor and complement. For this reason we felt that it would be of the greatest interest to gain a clearer insight through ' Mode d'aclion et engine dea aubatnncefl actives, des e*rutns prSventifa et des adrums antiloxiquea. Rapport pr£seDt£ par Jp Bordet au CoDgrte de ■ t tomographic. 1903. J 470 COLLECTED STUDIES IN IMMUNITY chemical means into the analogous deflection of complement observed with cobra amboceptor. I. Preparation ot Cobra Leclthlds. Owing to the tenacious character anil the slight solubility of lecithia in water it was, of course, impossible to ulteinpt to effect the dcsirBd combination by direct mixture of the a<]ueou8 solution of eobn venom with lecithin. On the contrary it was necessary to adopt ■ common chemical procedure, "shaking out," whereby, through the agency of an appropriate solvent, the lecithin could combine wilh the cobra venom. After a number of trials wo found the best solvent for this purpose to be chloroform. In our experiments we employed dried cobra venoms which bad kindly been placed at our disposal by Dr. I.amb and Dr. Grtig ol Bombay, and Prof. Calmette ot Lille. The lecithin used was the so-called "I>ecithol" nf Ricflel, and later on "Agfa-lecithin" of the Actien-Gesellachaft fiir Anilin-Fabrication. Both of these proved tO' be excellent. Special emphasis must be laid on a sufficient purity of the lecithin. For our purposes this is best recognized by tfsting, it against red blood-cells. 0.5 ce. of a 1% solution of the ledthin; should not dissolve red blood-cells. If the contrary is the case the lecithin should be purified by precipitating it once or twice witfc ace ton. 1 Forty cc. of a 1% solution of cobra venom in a 85% salt eolutios are mixed with 20 cc. of a 20% solution of lecithin in chloroform. The mixture is placed in a bottle holding about 100 cc. and thor ! oughly shaken tor two hours in a shaking apparatus. Thereupon the mixture is centrifuged for three-quarters of an hour in an elecinc centrifuge making 3600 revolutions per minute. If the procedure' has been successful the chloroform layer must then be distinctly separated from the watery portion, only a very slight compact,' cloudy, intermediate layer being present. It the lecithin is not ' fiufBciently pure this separation will not take place. The watery portion is separated from the chloroform layer by carefully pipetting off the former. The chloroform layer, usually measuring about 'We were iilao able lo activate cobra amhooeploi with a broiii-|pcit]>i|) n'hicb Dr. B«rKell kindly placed at our disposal. This preparation proved torn active than kcichin but it evidently posaesscs the power to unite wUb oobi* amboceptor to form a iecithid. THE ISOLATION OF SNAKE VENOM LECITHIDS. 471 19 cc, is then mixed with five times its volume of chemically pure €thcr which has been distilled over sodium. A precipitate forms consisting of the desired cobra venom lecithid, while the lecithin remains dissolved in the ether. Precipitate and fluid are separated by means of the centrifuge, the original volume of ether again added, shaken, and the mixture once more centrifuged This is repeated at least ten to twenty times in order to remove any adherent lecithin. The substance thus ob- laincd IS the cobra venom lecithid. The product can be kept for a long time under ether, apparently undergoing little or no change; or it can be carefully dried, through which, however, it suffers some change, affecting especially its solu- bility but not its action. The yield of dry substance is quite large, 1 grm. of dry cobra venom yielding about 5 grms. dry lecithid.^ After having worked out the best method for obtaining the cobra lecithid it was next necessary to determine by biological means whether the product isolated by us showed itself through its specific action to be the cobra-amboceptor-lecithin combination sought for. That this was actually the case could be proven in two ways, namely : 1. By showing that the extracted watery fluid has lost its haemo- lytic property, and 2. By showing that this property is now present in the chloroform- lecithin solutions (see Table I). So far as the behavior of the aqueous solution is concerned it can actually be shown that the single treatment with chloroform-lecithin removes all but traces of the haemolytic power, and that a repetition of the procedure suffices to remove all of the hemolytic agent from the watery solution Corresponding to this we find that the haemo- lytic power of the watery solution has been transferred completely to the chloroform-lecithin portion, a fact which shows that the leci- thin has united with the cobra venom (Table I). ' If one has but very little of the primary substance at one's disposal another method of prepanng the lecithid can be tried, one which will answer at least for preliminary examinations. 1 cc. of a 4% aqueous solution of cobra venom is mixed with 1 cc ot a 20% solution of lecithin in methyl alcohol, the mixture is kept in an incubator for several hours and frequently shaken; then 10 cc. absolute ethyl alcohol are added and the precipitated albuminoids separated by filtration. On precipitating the clear filtrate with ether, one obtains the lecithid. COLLECTED STUDIES IN IMMUNITY. 1 CO. 5% Ox Bu)OD+0.2 cc. 0 1% Lecitbw. B B c &)b™ Veoon. Cobn Lecitlud Native 0.001 ^„ 8h!il™0™ ^^^^^i te.ai°;fK:- cc. (Coatr^olJ."" Chloroionn Lecithm -£L .„„_,. 1.0 0.75 0.5 com^plete complete nU completa <■ 0,35 0,25 0.15 0,1 ftlraost com p. 0.075 marked 0.05 little almost oompleta 0 035 trace almost com p. 0 025 almost, oil moderate httle 0 01 nil little 0 0075 trace BlmoBi nil 0.005 almost nil nil 0.0035 nil 0.0025 0 0015 " — •• Number of aorvent doses reckoned on the total original vol- ume o( 40 cc 268,000 to 267,000 800 0.0 266.000 to 267.000 Percentage of hEemoly- ainsineuehHolulion. 100% 0.003% 0.0% 100% The asaunoption that the cobra amboceptor is extracted by chloro- form alone is refuted by control teste. The neurotoxic action of the native poison ia entirely absent in the cobra lecithid. Relatively large quantities of the lecithid in aqueous solution can be injected subcutaneously into animals with- out producing constitutional symptoms. For example, an amount of lecithid which sufficea to destroy 200 cc. mouse blood can be injected into mice weighing 15 grms. without causing any further symptoms than infiltration at the site of injection. In like mann^ rabbits can be injected subcutaneously with 10 cc. of a 1% solution of the lecithid without causing any constitutional symptoms. In thia case, however, the local reaction ia extensive, the infiltrated area often including a considerable portion of the abdominal surface. J THE ISOLATION OF SNAKE VENOM LECITHIDS. 473 According to this, therefore, the second constituent of cobra venom does not pass into the chloroform-lecithin. In this reaped, however, we have been able to demonstrate that the watery portion which has practically been freed from the ha^moyltic amboceptor still possesses its toxic properties in animal experiments. (See Table II.) The essential difference between the hamotoxin and the neurotoxin, first pointed out by Myers, is thus confirmed by direct chemical means. Comparative Tbbt op Venom (a) Bbfohe ^ 3 Nbohotoxic Action o» a I (b) AfTER Shaking the < ra Chlohoform-Lbcithin. 0.01ft Venom. (a) Native Venom. tfr) E.t™=tecl Venom. 0.5 0,35 0 25 0.15 0 12 0 1 t after 2 hours t '• 2^0"™ I :: ^! :: t '■ 30-40 hours living ' aft«r 1 hour " 1) hours ■• ihour " 8 hours ■' 30-40 hours living II. The Properties of Cobra Leclthids. In the description of cobra lecithid we shall do beat to keep to the product obtained by the method above described, the last traces of lecithin having been removed from the ethereal precipitate by repeated washing with ether, and the main portion of the ether in turn removed by pressing the precipitate between two folds of filter- paper. This primary product is insoluble in aceton and ether, but soluble in chloroform, in alcohol (cold), and in toluol (on heating). The solutions in chloroform and in alcohol are precipitated by the addi- tion of ether and aceton. When stiil moist with ether it dissolves very readily in water, a point of some importance. Even if the ether which the product contains is rapidly evaporated by means of a current of air and the product then dissolved in water an abso- lutely clear, light-yellow solution will be obtained. These facts show that the primary product is absolutely different from the two substances from which it was derived, cobra ambo- ceptor and lecithin. It differs from lecithin particularly in its insolu- 474 COLLECTED STUDIES IN IMMX7NITY bility in ether and its ready solubility in water; from cobra venom amboceptor in itH fiolubility in the above-mentioned organic solvenu. alcohol, chloroform, toluol. Cobra venom does not give up even x trace of cobra amboceptor to these solvents. It has been found that the watery solution of the primary coIks lecithid obtained from cobra venom and lecithin, as described ttbo\"e, undergoes spontaneous modification which leads to the formation of an insoluble substance. If the watery solution is allowed to stand at room temperature it gradually becomes cloudy, and in the course of a few hours a whitish precipitate is formed. On removing this pre- ciliitate, either by filtration or by centrifuge, a precipitate will again form in the clear fluid. The sediment Ls microcrystalline, white, trans- parent, and very refractile. It can easily be shown that this sediment is nothing but a modified form of the lecithid, for after thoroughly washing the precipitate which has been separated by the centrifuge, it will lie found thai this still exerts its full ha;molytic action. In accordance with this, the original solution of the primary product shows a proportionate loss of power. In one experiment which we followed rather elosclr we found that in course of time about two-thirds of the lecithid had separated out in solid form, while one-third was stil! left in solution. The aecondani lecithid produced in this way is, as already stated. almost insoluble in cold water; on the other hand, it is readily soluble in warm water, although it again separates on cooling. This Ix- havior constitutes the chief difference between the primary and the secondary lecithid; the behavior of the two substances toward the above-mentioned organic solvent is identical. Owing toitscharacter the secondary lecithid is particularly adapted for chemical investigations, and one of the foremost authorities has already commenced work on this substance. Some imiiortant results which have already been obtained will be mentioned later on. For the present we shall merely mention that the product gives no biuret reaction even when in concentrated solutions. We are reserving for future study the chemical study of the above lecithids, as well as the investigation of the neurotoxin obtained in purified form by means of the method above described. The formation of the secondary lecithid also occurs if the ethereal precipitate is dried at incubator temperature. It is then easy to see that such a product has more or less completely lost its solubility in water, especially if it has remained in the thermostat for several days. THE ISOLATION OF SNAKE VENOM LECITHIDS. 475 In its properties the insoluble portion corresponds entirely with the secondary lecithid precipitate from aqueous solutions. To obtain the secondary lecithid as a pure product, however, the method first mentioned seems preferable, namely, that which starts with the aque- oas solution of the primary substance , the result seems to be a lighter- colored product. It is natural that this lecithid when finished differs in its action in many ways from the cobra amboceptor. It can readily be under- stood that the cobra lecithid acts on the blood-cells of all the species thus far examined, no matter whether these cells possess available lecithin or not. One fact of considerable interest has been discov- ered, namely, that the absolute quantity of lecithid necessary for hirmolysis is the same for the blood -cells of different species. We found that an amount of lecithid which corresponded to about 0.003 mj^. dry cobra venom was able to dissolve 1 cc. of a 6% suspension of blood -cells of different species (guinea-pig, rabbit, man, ox). This quantity, we may add, corresponds to the amount of cobra venom which causes solution of the blood-cells in the ordinary test when a large excess of lecithin is present. An observation which is also of considerable interest is a com- parison of the time necessary for the action of the cobra lecithid and the amboceptor, with and without the addition of lecithin. In our previous papers we pointed out that when cobra venom is allowed to act on susceptible blood-cells, solution occurs after a considerable period of incubation, so that in case a minimal quantity is employed 12 to 18 hours (two hours at 37°, then at 8°) elapse before complete solution is effected. Even if a suitable excess of cobra venom and the most susceptible species of blood are employed, at least 10 to 30 minutes will usually elapse before solution is completed. Similar differences are observed if, as previously described, we allow cobra venom and lecithin to act on unsusceptible blood-cells. In that case, again, with minimal quantities of lecithin and ambo- ceptor 6 to 18 hours are necessary to effect solution; this time is decreased if large excesses are employed, but solution is never instantaneous. In contrast to this a marked decrease in the period of incubation is observed if the finished lecithid is used, solution being instantaneous on the employment of cencentrated solutions. The shortening in the time necessary for solution becomes particularly marked when small doses of the lecithid are used, solution commencing at once 476 COLLECTED STUDIES !N IMMUNITY and bDing completed within !5 to 20 minutes. In other words, Ihe increase lo the rapidity of the prot-ess is about twenty-fold. This behavior is sigaificant, for it shows that in this case the period of incubation is due not to a slow action of the anchored toxophore group (lecithin), but exclusively to the slow development of the real toxic agent, the lecithid. The difference in the time of action in the case of small and large doses is in accordance with the well-known law that the reaction (in this case the union of cobra ambocepUir and lecithin) proceeds more rapidly in concentrated solutions tbaa in weak ones, TABLE IIL I oa. 5% Ox Blood AlDDUDI* Ol ^b^«'u''B.rf™i.'t'"^ Solution ■ ""bliu^ltSn?™?"' Doana wilh the Addi- L«ilhid.Kbaui tion oi Ledithin. Do«i. 1) SolVBDl OoKS 0.1 0 0 0 0 075 0 0 0 0.05 0 0 o 0 035 0 0 0 0 025 0 0 0 0015 0 almoBt 0 0 01 0 trace 0 0075 0 little Utile 0 005 almost 0 moderate moderate 0.0035 irace marked marked 0 0025 little almost complete almost compJel* complete 0 0015 iMjmplela 0 001 marked 0 00075 0 0005 0 00035 0 00025 almost complete 0 00015 complete " 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 explaioed J 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 -f 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 III opposite.) IV. The Leclthlds of Several Other Potoons. 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 lanceolcUus; 2. Daboia Rvssellii; 3. Naja haye; 4. Kerait; 6. Bungarus fasciatus; 6. Trimeresurus anamalensis (Hill viper) ; 7. Trimeresurus Rivkivxirvus (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 47S COLLECTED STUDIES IN IMMUNITY. Botlirops poison is ten times weaker, and that of Trimcresitnu anavia- Itnsis twenty-five times. This observation made the formation of a lecitliid 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 hiemoiytic power of the poisons.* Hence we believe that in general ail hjemolytic snake venoms are of the -un- Ixiceptor type and [wssess a leeithinophile group, the occupation of which by lecithin gives rise to the hemolytic action. In fact it seems ^ though in the last analysis the factor which determines the type of the hiemoiytic action of snake venom was principally this lecithinophilc group. A fact which goes to support this view ia 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-cellB. Thus f-amb^ has shown that the Daboia amboceptor, unlike the cobra amboceptor, is not inhibited in its action by Calmette's serum. The same ia true for Bothrops, Crolalus, and Trimeresurits Rivkiuanu*. whereas the poisons of Bungarus and .VajVi haye are aimilar 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 haptophorc group, white the characteristic lecithinophilc group is identical in all cases. It was important to see whether in animals other than snaks poisons are present which are capable of forming lecithids. We therefore next studied the poison nf the scorpion, choosing thi» 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 hosmolylic 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 Bothropi poison fields only a l«ntb the lecitbid obtained Erom the other poisons, and Ibe poison of Trimtrt»%Bru» anamoltTtrnt aniy one twenty-fitth. ■ Lamb, Scientific MemoirH. Medical and Sanitary Depts., Govt, of India, 1903, No. 3. ' Calmette, Ann. de I'lnstit. Pasteur, 189.'i, No. 4. ' For this scorpion poison we are much indebwd to Prof. Treub. Director o( the Botanical Garden in Buiteniorg. THE ISOLATION OF SNAKE VENOM LECITHIDS. 479 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 Scorpion Poison wfth and without the Addhion of LscrrHiN.. AmouDtB of the 0.2% 1 CO- 5% ( Ox Blood. Solution of Scorpion Poison cc. + 0 2oc.0.1% Control without Lecithin. Lecithin. 1.0 complete 0 0.75 0 0.5 0 0 35 0 0 25 0 0.15 0 0.1 0 0 075 0 0.05 0 0 035 0 0 025 0 0.015 almost complete 0 0 01 moderate 0 0.0075 little 0 0 005 trace 0 0.0035 ti 0 0 0025 faint trace 0 0.0015 0 0 Corresponding to this behavior we succeeded in actually pro- ducing a typical lecithid from scorpion poison by following the usual procedure. 1 All this leads us to the view that the essential character of the hacmolytic 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, Trackinus draco (see Briot, Journ. de Physiol, et de Pathol. g6n. 1003, No. 2), is also capable of forming a lecithid; at least a statement of Briot speaks in favor of this, namely, that the hsemolytia agent in the Trachinus poison can be activated by a serum which has been heated to more than 60^ C. 480 CX)LLECTED 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 torong 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* formed in the cell, due to the anchoring of the cobra amboceptor. XXXVII. THE CONSTITUENTS OF DIPHTHERIA TOXIN.* By Paul Ehrlich. 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. Wochenschr. 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 4S2 COLLECTED STUDIES IN IMMUNITY &a authority as Arrhcnius recognizes my method as correct in prin- ciple aad proceeds along the same lines. The study of .\rrhenius and Madsen deals principally witK tetano- lysin, the hspmolytic poison discovered by me in tetanus toxin. Telaoo- lysin and tetanospasmin differ from each other in their baptupboie groups, as a result of which each possesses a particular antitxxJy in the tetanus serum of the market. Madsen studied this letano- lysin in my Institute, and found that when it is gradually nentrahzcd with increasing amounts of antitoxin, the same definite amounts of antitoxin 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 thU several poisons of different affinities were present. On taking up these studies in tetanolysin Arrhenius and Madges obtained practically the same results, and these authors succeeded in constructing a formula for the action of an ti tetanolysin on telanfv _ lysin which conforms to the law of Guldljerg-Waa^e. Ba^ed oo I this they next attempted to determine similar relations in the rase I of very simple blood poisons. This had already been done by Danyat, ■ but the method was open to criticism. Arrhenius and Madsen i-Uoee a weak base and an acid (ammonia and boric acid) as haimolysin and antihffmolysin. It was found that in these the neutralizaliMi . phenomenon is very similar to that of tetanolysin and antih'sin, I from which they concluded that in the neutralization of toxins and! antitoxins we are dealing with reactions between simple substa,ncetw«B.' a strong add and base. In accordance with this also I am convincrd' 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 i> < impossible to apply the results of the neutralization of boric acid ftcd j ammonia to every combination of acid and base, so it is impossiUc I to apply the experiences with tetanolysin to the doctrine of tonus I ia general.^ t ' Wben, then, years ago, in apite o( these unfavorable conditions, I propooej the study of tetanolysin to Thorvald Mndsen, Ihia was but a niakediift nrtrt J sitat«d by the lack, at that lime, of suitable hicniolyslnK. At present a numtMH of Buch Bub.'jtances are availablo. xuch aa arachnolysin and Miiuke venom. llbMO are very stable and far better suited for exact determlDation since the factdfl ot decompoaition is absent. ^l ' 1 should like to mention that recently Dr. Kyes has discovered that ia snake venom alNo the neutralization with antitoxin proceeds with high afGnitJM ' and in a straiight line. ~ ' THB CONSTITUENTS OF DIPHTHERIA TOXIN. 485 If, therefore, the affinity between diphtheria toxin and antitoxin is so great, we shall have to ascribe the irr^ular course of the neutraliza- tion process to other factors thai2 those assumed by Arrhenius and Madsen. Diphtheria 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 officia 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 (Lo)» ai^d corresponds to the quantity of poison which is completely neutralized by 1 I. E. The other is limit death (Lf) and corresfX)nds 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 x L. D. present, 1 I. E. neutralized 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 would have ex- pected would be that in different poison solutions, the volume in 4S6 COLLECTED STUDIES IN IMMUNITY. which a given number of L. D. were contained would vary from ease 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 w have been analyzed the figures have fJuctuated between 15 and 160. Since it had been shown, especially by myelf, that the neutraliKation 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 ablr 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 comparaliv analyses. In the course of these it was found that the non-toxic Bubstanees, which still jwssess combining proi^erties, increase as [be toxic bouillon ages, and 1 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 ' that my results were derived majnlv from a study of decomposed poisons might readily be misconstrued and give one the impression that in my investigations I had not been espt- eially careful. I may at once add, however, that my most valuable results were obtained by studying the course of this deconiposttio but this, nf course, corresponds entirely with the methods of chet 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 decomimaition 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- ligently carried out, and a careful study of their products. Of couts« the decomiKJsition must not give rise to secondary reactiona whiefa could obscure the results; this might be the case if strong acids or a. high temjierature were employed. The decomposition must be quantitative and of moderate intensity. The following obserra- tions will show that this is especially the case in the sjmntaneotis THE CONSTITUENTS OF DIPHTHERIA TOXIN. 487 attenuation of the toxins, which occurs at room temperature and without any further chemical manipulation.^ // 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 Lf 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. -t!S« COLLECTED STUDIES IN IMMUNITY. far less basic trimetbylamin, methyl alcohol being split off in the process. Let ua 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, u 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 nercaaary for neutralizing the tertiary amin. In other words, there has beeo a change in the position of the neutral point, although the niimtw of basic radicals remains the same. This necessarily follows from llie decrease in affinity brought about by the transformation. The reverse will take place if a weak base is transformed into » 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 anotht-r fact, which affords concluare 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 dealing 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 po.s.sesses consideralde resistance :<■ llmt 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 aa 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 (I>, 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 Lt dose contained 160 L, D. But even this poison, as I shall show later,' merely approached the character of a pure poison. > It is especially important that even diphtheria paisons which have 1 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 (recrystaJlization, 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 Lt. 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 L^ reached. 490 COLLECTED STUDIFiJ IN IMMUNITY time 1 gained the impresBjon that a pure poison must oe so consti- tilled that one 1. E. fully neutralizes exactly 200 L. D.' Later on 1 shall show that by means of the "speiitrum" analysis I ha\e sue- ceede neutraliz(?d by each wmbining unit of aii> toxin. Such a reproduction possesses so many advantages over tk curve used by Arrhenius and Madsen that 1 shall not hesitate a momenl in retaining the s[>ectrum method for diphtheria jjoison. By ill means one obtains a view of the enlire process of iieutralijatioo.* It may be well at this point, by means of a tiuitable chemin) illustration, to elucidate the influence which such admixtures of toxoid exert in the titration of alkaloids. In doing this it will i» best to proceed on the following assiunptions. An alkaloid arts iacrat- lytically when in the form of free base, but not when in the fatmd & salt.* The base would then correspond to the toxin. The an* logue of the toxoid would then be an alkaloid which e.xerta no delfr teriouu action either as such or in the form of a salt. The antitoxift ■would be represented by any acid, e.g., hydrochloric acid. L'bdff these conditions the mixture of the two alkaloids c«n be titrated hifr logically (by determining the hs'molytic power at any ix>int) hy 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 i possesses so strong an affinity for hydrochloric acid that neutralize tion is effectt^d to within a very small fraction. A solution of « nmlt' cules .4 would then corresjwnd to the pure toxin, while mixtures rf ' It is self-evident that each toxin-combining unit can be replaced br equivalent amount of lesa toxic or non-toxic subBtances poasei«Eing combinia properties (toxones, Ujxoids). 'The poison studied by Madsen, tbereCore, vbich contained 160 L. D.& Lt. corresponded to a purity of four-fifths. ' See also page 552. * This is probably the cose with soknin, whose hn-tnolytic power ia by the addition of acid sella (Fohl) or ol free acids (HMon, Basbford). J THE CONSTITUENTS OF DIPHTHERIA TOXIN. 491 A and 5: -+^ 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 pioceed in a straight line only if we are dealing with the pure alkaloid. In ail 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 equilibrium /iC = 2.03. 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 neutralized 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 itselt entirely uniform. If another combination is taken, in which the second alkaloid possesses a weaker affinity, so that iiL = 10, 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 obtam 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 alkaji 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 hmitations, it is possible to learn the true conditions if, as I have done, one does not confine one's 492 COLLECTED STUDIKH L\ IMML'NITV. ► self to a single mixture, but analyzes a great many different mixiims in wljich the relation of toxin-alkaloid and toxoid-alkaloid varies.' It is all the more eurprising that in the analysis of the constitu- tion of jjoisons Arrhenius and Madsen have not studied the tjuestjon from this point of view because they do not at all neglect the exisl- ence of toxoids. Apparently this is because of a slight misunder- standing, for these authors proceed exclusively on the assumption thai in toxoids one is dealing with protoxoids, i.e., with toxoids wjiich possess a higher affinity for the antito.xin than does the toxin. In fact, one can easily observe that the formation of prototoxoids affect* the end point of the titration but little. This I had predicted in my first study on the evaluation of diphtheria serum. Let lis 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 add will be neutraliaed first, after which the neutralization of the prussic acid will procwd 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 Madscnj I have calculated the relation of proto- toxoid and toxoid to toxin. In doing this 1 have regarded exclusively the Lf dose, and so eliminated the toxons which would otherwise still more increase the toxoid figure. ' In the very simple example of two BlkaloidH just mentioned two determina- tions of different mixtures would permit the calculation. In my opiiiion do definite conclusions as I« the constants of the toxin can bo drawn from Uw analysia of one particular toxin containing toxoid. Arrhenius und Mnds«it analyzed two different tetanus poiBona, one of which had undergone toxcnd mod ilicB lions through years ot preservation as a dry substance, while the olber had suffered Himilai niodifi cat ions through several days' stiuidint; ol the solu- tion. The authors calculated from their experiments that in the one case tlie constant of disaociation had been increased 50%, in the other ten tinice. In what has just been stated this calculation, which leaves out of aecount the presence of toxoids, cannot be regarded as conclusive. The divci^encv o( the constants could easily be due exclusively to the presence of Toxoids uid these, in view of the diHerent methods by which the [>oiFons wore allcnimteid could lie different in the two c mation of diphtheria loxine 1 an remain do not suffer any change ii I may also add that in the toxoid for convinced that the toxin groups which tfaeir affinity. THE CONSTITUENTS OF DIPHTHERIA TOXIN 493 Poison. For 100 Parts of Toxin there are Pro to toxoid. 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 by 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 antitoxin, such as is seen in the tritotoxoid 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 I shall select a poison which I have already described in my publication on the constitution of diphtheria poison ^ 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 Lf dose 0.25 cc, that is, just twice as much. The L. D. was 0.0025 cc.,so that the I^o dose contained exactly 50 L. D. and the Lf 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 difi^erent 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. Woehensch. 1898, No. 38. COLLECTED STUniES IN IMMTJNITT, frnra a dirert tie termination , it follows that the first phase must h„^ represented a pure liemitoxin which rearhed exactly to 100 (see illi» tration). Accordingly each ^Jj^ 1- E, ( = 1 combining ur sively added to the L dose takes away i L. D. from the fatal doas ■ 170 1« L» -• ) 130 140 ISO 16Q ITO 1 1[l PhoGein „ a* JO w 00 70 HI K 100 no m 130 i« iw leo iro i « IM 3 ID Phase IV 71 ai ^'"" y" ■';7y ■//'/■''^ ' 1_ 10 JO 0 in : :fl 1 *> \-fii (.« I'll 31 contained inLo. anti '^'i-'' »" occurs within the first hundred antitoxin doses added. Amounts of antitoxin beyond this have no further influence on the toxin (death, necrosis), but afTeet only the toxon . A fact to which I attach particular significance is that the hetni- 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 II. Here we see a simultaneous increase of the Lt 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 cf the Lt 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 — toxicity instead of the hemitoxin. In order to reach the Lt dose in this 210 20*^ case we must multiply the Lq dose by ^tjti and not by ^-r^, as was the case with hemitoxin. On carrying out this calculation, Lq being nior: ,0125X210 ^^^_ J 0.125, we get rrrjr — = 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 emptu^iud 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 arc due exclusiveiy » the fact that a part of the toxin has become transformed into toxoids; in fact into toxoids which arc neutraUzed after the main portion ot the toxin, and which, therefore, must [wssess less affinity. If we woe to represent this phase by means of a curve according to the metlnd of Arrheniua and Madsen, we should observe a marked f]att«iuDf of the curve in the tritotoxoid zone. This, however, is not the expfff- aion of the weak affinity of the diphtheria toxin, or of the nciilraiut tion dependent thereon. It is to be ascribed with absolutiQt which speaks against a condition of equilibrium ia the fact that through artificial influences— heat, chemicals — any desired proportion of toxin and toxoid can be produced. Only one other explanation therrfnre 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 assunie the existence of ■prejormtd varieties of toxins. As a result of my «c- periments I must emphatically deny the assumption that the phr- nomena observed by me in diphtheria poLson are only the cxpressiao of a weak affinity between diphtheria to.xin and antitoxin. I have demonstrated that the observed deviations can only be duo 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 Madeen 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 ihe errors in tho interpre- tation of neutralization phenomena when dissociation is difiregarded, My results were obtained by the long and tedious experimental method. I can assure the reader that the experimentji 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. U. 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 ioxons. On testmg a diphtheria poison and determining the two hmits, Lq and L|, we should expect that the difference, L|-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., Lf should contain (a 4- 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 Lq is much greater. In the poisons de- scribed in my first conmiunications 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 as indicating the existence of a toxin derivative of very Httle 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.^ , -. 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 hmited to diphtheria bacilli. According to the observations of Sciavo on animals infected with anthrax it is highly probable that anthrax bacilli also produce poisons having a toxin-like action. 504 COLLECTED STLTJIES l\ LMMUNITY the toxons. According to them the long-drawn-out toxon zones the expression of the incomplete combination of toxin and antitoxin, the neutralization of which they bebeve follows the ammonia-boric acid type. There are, however, a number of weighty reasons wh/ ■ this view cannot be accepted. I It was but natural at iirst to ascribe the toxon stage to phenomena I such as Arrhenius and Madsen now have in ^iew. It had already ' been noticed by others that often a considerable interval exists be- tween Lt and I^. Ivnorr, in refernng to this, had spoken of "un- neutralized poison residue," The a^umption, 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 ibe course of my investigations. This was Poison No, 10 (of my series), ■ whose Lq and L^ values were very close together. L^ contained 27.» I and Lf 29.2 L. D. Hence D= 1.7 L. D., which is a close approach to I the figure demanded by a simple diphtheria poison. ' The loUowing conside rations will show thuC this vaJue, 17 should be ecu- reeled so as lo be Blill lower. The original cakulalioos were based on my earlKt tksaumplion that toxics and toxoids are imiComily mixed. This however htf been superseded by the spectrum method of representing the neuirBlixation of poisons . Experience has taught ub that such deteriorated poisons usiiaUj oonsist of a small zone of hemitoxin and a more or less pronounced zone of tritotoxin-toxoid, in which at a rule nine toKOid equivalents fall on une toxin equivalent. Several times 1 have observed tritotoxm -toxoid zones containing V„ toxin, and Madsen also has deBcnt>ed such a poison. As can be seen fnnn our calculations given above, the theoretical change from L. to Lt is iriflu«Dc«l solely by the tritotoxoid zone. If we therefore assume that our poison po«- sessed a tritoto toxin -toxoid portion whose strength was '/,, (and this is extt«melT probable) we shull find that by a little calculation that the poison prohafalj contained no toxon whatever. Very likely the tritotoxoid zone reached to ■ the end (200) of the spectrum. On the assumption of a '/,< trilotoxin-toxoid. | if we multiply L, by "°/„ we shall obtain Lt'28.n L. D. This agrees very I well with the figures obtamed experimentally, Lt = 29.2 L. D. 1 We may therefore very well assume that we were dealing with » poison free from toxon or one which contained only very small tracea of toxon. This fact is hard to reconcile with the theory o( Arrhenius and Madsen, for if toxin and antitoxin neutralized each other like ammonia and boric acid, all poisons should show a long zone of iti' complete neutralization. The independent existence of the toxons is further corroborated by the tact 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 0 per cent to 300 per cent. Hence 1 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 L^, 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 I^o has increased from 0.15 to 0.20. After this an insignificant increase brought this to 0.21 ; from then on Lq 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 -fl 1. E. killed in six days and 0.275 -fl I. E. in three days. Lf, which accordmg 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 Lf 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 Lt 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 im^^S toxon zone become considerably greater. In thia case, however, wa see that the toxin zone remains constant while the toxon zone il reduced to one-Efth.' Determination of L, Dose. ■OfPoiKOl a«i.«.pi«,„™ I„i«c.d .i,b I 1, E..V.rvin,A„ou.. o( r.i^ ..^. July .. 2. » ' * 6 10 0.125 0.1275 0,13 0.14 0,15 0 1() 0,17 0.18 0.19 0,2 0,21.5 0,2:i faint trace 0 almost 0 slight hut distinct just neutral slight but distinct little sl^hl flight cedema oedema almoit □eutrsl (cdema mark«d "Famt truce," "slight," etc. dcno to the degree of infiltralion. It is difficult to say a priori what has become of the to.Yon 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. 1 The enliro course of the decompoEition. in whirh from day to day we could observe the toxon becoming weaker and wealicr speaks against the poasibilllj (in itself very remote) that the varying composition of the bouillon is respon- cible for the variation in the numbet of toxons in the individual poisons, la the poison here described the deoom position has taken place in the saine bouiUoa and in so short a time thai very great alterations in the bouillon uppeu MlH excluded. ^■fl 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, necl-osis 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, J, 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 oedema 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 «ervstiatt My very extensive but unpublished studies made at that time convinced ow that this property is not common lo all diphtheria poieons, for I al«a (ami some in which the I^ dose was ejiactly the same in rabbits and in guinea-^)ip This fact furthermore refutes the assumption that the phenomenon ~ ~ * L 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 imder- 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 ihe curve obtained by the authors, is here reproduced (Fig. 3. Phase II). From this we see that a zone of hemttoxin in the beginoing of the spectrum is followed by a zone of almost pure toxin, and this in tuni by a zone of tri to toxin -toxoid. Then cornea the very long toxin fraction. To one employing this mode of representation, such a spectnuo not only pictures the present constitution of the poison but also frequently permits him to reconstruct its 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 stago. According to these figures I would assume that in the first phase the poison contained a pure toxin in the initial zone. In the sewud phase, the period at which the poison was studied by Dreyer aiul Madsen, this had become transformed into hemitoxin. In the thiri phase it may become pure prototoxoid. A fourth phase n-ould then show the transformation of the pure toxin in the above spectra into hemitoxin and the jmison would then have reached the point which we have so frequently observed in other poisons. The spectra of these various phases is as follows (Fig. 7) : I shall now present the figures which Madsen and Dreyer ol>- tained when they startetl with double the 1^ dose (0.1 cc. poison). In the first phase, their statement that the lethal dose was 0.0015 ce. shows that 0.1 cc. jxiison contEuns 66 L. D. Calculation from the spectrum gives 65 L. D. The second phase, of course, agrees entirely with the statcmenU of the authors, since the sjx^ctrum 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. eorresponding to 37 L. D. in 0.1 cc. Calculating this from my spectrum I obtaia 35 L. D., which is but 2 L. D. smaller than would oorre3[>ond to the final stage. Perhaps tliis stage had been nearly but not j-et completely attained. It is probable that if the examination had been made a little later the figure woiild have been exactly 35. The figures obtained from my reconstructed spectra harmoniie so well with those obtained experimentally by the authors that it seems almost impossible to doubt the correctness of my assumptioiu concerning the constitution of the poison and the process of its trata- 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 believe it will be seen from my explanations that my mode of procedure in the study of diphtheria poison has been exceedingly Phase I lio io m u iM 110 1^ ite i4o iGd tlo ito tto ito soO' 50 ia -ta Oi A IAD iio liD i^ tio IN ita it« ite ite ^ Phase IT careful, and that the objections raised against my results do not apply. I roust therefore continue to maintun my original standpoint, and deem it well therefore to once more define my views concerning the poison of diphtheria. 512 COLLECTED STUDIES IN IMMUNITY. 1. The diphllieria bacillus produces Beveral kinds of poisoBs, especially toxins and toxons. 2. The affinity of diphtheria toxin to the antitoxin is verj- gmt. 3. The deviationa from a straight line as they manifest themselves in the graphic representation of the neutralization of the poistm cannot be explained by the assumption of a single poison possessing a weak affinity. They are rather the expression of the fact that tbe poiFKin bouillon contains admixtures of various kinds of substani-eB of a toxoid character. 4. The varied affinity of the toxoids cannot be explained hy Ihe assumption that a simple toxin when transformed into toxoid aiifferB a change in affinity cither positively or negatively. Rather d<« this indicate that the toxic bouillon contains, preformed, various toxins of different affinities. 5. There is no change in tbe haptophore group in tbe formatioD of toxoid. 6. The absolute number of combining units contained in the immune unit or in the l^ dose of poison is 200.' I have finished. If the results of the first encounter of two such different methods of study as the mat hem ati co-physical and the bio- logical have not shown complete agreement we should not be at all surprised. The natural aim of physical chemistry must alwaj's be ' Bonlet has recently ntteropted to explain Ibe toxon pbcDonieoa by tbe Bsaumption that tbe toxin molecule can combine with Eintitoxii) in varyini proportionH. Oue would accordingly have to asaumc that the toxin mol«cul* contains BGvenU haptophore groups. The complete occupation of these ftroupt causes the toxicity to be entirely loat. whereas partial saturation causes a itr> crease in toxicity. That is to say, amounts of antitoxin which do not coni- plcCely neutrohzo tbe toxin would weaken it in Euch lashion that it would exeft a different action. It ia atrange that so eminent un invcBtiitator as Bordtf shiiuld not have attempted to convince himself of the correclncss of tbis hy- pothesis by mesDS of tbe experiment. He would then have foiitid that i^ facta are irreconcilable with such an assumption. We have Bhowm at grot length that the toxon actions are nothing les.* than constant phenomenB and have called attention to the great extent of tbe quantitative variations (0-3001. If one were to follow Bordet it would then be necessary to assume an ciir>rmoiM multiplicity of haptophore groups in the toxin molecules, and this would lead to a hypothesis fat more comphcated than mine, although the lat<«r banuo- niies all the experimental results. In support of his conception Bordet relBM to experiments with complement and anticomplenienl. I must say, lio«re\«, that in these we are dealing with such complicated relations that it is not raissible to apply the conclusions drawn from them to the far simpler rehukm* 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 w^ork 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. XXX\in. TOXIN AND ANTITOXIN:* A REPLY TO THE LATEST ATTACK OF GRUBER. By Paul Ebrucb. In a domiun that is open to experiniental investigation it ii neither easy nor without danger for one to express criticism menh as a result of literary studies. This is especially true in that most difficult field in the entiw etudy of immimity, namely, the subject of toxins. Only one who has devoted years of unprejudiced study at the laboratorj- Ubie to this subject and gathered a host of observations and experienns will be in a position to orientate himself in the confused mass of true and false statements contained in the literature. The outsidH will find it very difficult to correctly analyze all this material. Hence it is all the more remarkable that Gruber' should clioose the subject of toxins for the main portion of his attack upon roe, for according to his own admissions that is the fipid which he know merely from literary studies. Against such critics I am in the unpleas- ant position of a man who is compelled to discuss colors with ibt blind. Nevertheless I cannot well escape the thankless task ol replying, at least to the main points in Gruber's polemic, for it is indisputable that this attack, addressed chiefly to those withoiB special training in this field, is capable of causing wide-spread con- fusion, owing to its positive tone and its severitv- Gruber'a first important error lies in the assumption that a rw troversion of the plurality of poisons, to which I hold, nignifiM the downfall of the side-chfun theory without further ado. The stSe- chain theory, however, proceeds from the assumption that the toxin- ' Reprinted from the Miinch. nied. Wochensrh. 1903, Nob. 33 t^ad W, ' M.Gruber and CI, v. Pir(|uet,Toiin und Antitoxin. Munch, med. Wndff&Mfc. 1903. Nob. 2S and 2fl. 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^ 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 th*» 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 Deutsche med. Wochensch. 1898, No. 38. 'Annaies de I'lnstitut Pasteur., T. 13, 1899. * S. Arrhenius and Tb. Madsen. Physical Chemistry applied to Toxins and Antitoxins, Festskrift ved. indvielsen af Statens Serum Institut, Kopenhagen, X902; German in Zoitsch. f.ir physiol. Chem. 1003. 5ir> COLLECTED STUDIES IN IMMITXITY. tenfold whereas the Lo value will remain unchanged ; thU ia Ek- lich's hj'[)uthesis. If Vio the toxin moieculea had lost their tonotji, without there being any formation of toxoid^s capable of combitunj with antitoxin, the I-o value would be increased ten times. H, hw- ever, simuHaneously with the loss of Vio the toxicity, ihc fluid were to lose ^/lo the reaction rapidity for antitoxin, so that tit constant of the reaction would be decreased ^/ig, it would be found that the I^ value would manifest itaelf unchanged." Gruber would have done better to have made some of these con- paratively simple experiments himself than to aenn)«u which constitute, in fact, the very beginning of the technique rf testing poisons. Thus, when in 1897' I formulated the law Ihu the combination of poison and antiljody takes place more npiiU in concentrated solutions than in weak solutions, it was as the tesnh of just such studies made on diphtheria and tetanus toxin. In tlw* Btudies I convinced myself that the affinity between diphtheria snu- toxin and diphtheria toxin is far greater than that between I "tun" antitoxin and tetanus toxin. The union of diphtheria toxin add its antitoxin is effected very quickly, so that at the end of five It ten minutes one may be sure that complete union has taken plaa, It ia entirely immaterial whether one is dealing with fresh potsou or with poisons poor or rich in toxoids. I shall here reproduce u exireriment which I have recently made because Danvsz ^ tnaislfd that the neutralizing power of the diphtheria poison changes wba the poison is allowed to stand for some time. The e.\periraent was performed with the standard serum and standard toxin used in the official standardization. Both substaons had therefore been very accurately titrated, The mixture wb allowed to stand fifteen minutes and twenty-four hours and ih result showed that in this lime not the least change had taken pla« in the constant. In the experiments of Danysz, therefore, somf error has probably crept in. In any event there is no change i" the reaction time on the decrease of toxicity of the diphthenn toxm. Ciuinea-pig 1 receives 1 I. E. serum+0,78 cc. poison (Lf) fiflu* minutes after mixing. It dies on the fourth day. Guinea-pig 11 receives the same mixture twenty-four hours att-.i How false such a conrluaion is can be seen from the fact ihit when I devised the aide-chain theory I believed the diphtheria poison to lie a simple substance. My later studies, however, convinrKi me that the poison consist** of several modifications: prololown, deuterotoxin, trilotoxin, and toxon. It can easily bo seen tiwm my publications, however, that I ascribe the same combining group to all of these; they differ merely in their toxnphore gmune. lo the production of diphtheria antitoxin all of these mndificatioos act in exactly the sanic way. It shows a deplorable lack of mat- prehension, therefore, when Gmber says that the refutation of tk plurality of toxins will " give this side-chain-theory spook lU 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 nwide of reasoning would at once be apparent to the specialist. Now thit Gruber, however, returns to this subject I think it may l>e wdl u discuss the facts somewhat in detail. In the majority nf poisons it is probably a fact that the toxtdir depends upon the animal species, a certain poison being more loxi* for one si^cies than for another. In chemically definite poisons, alkaloids, etc., this behavior is usually a constant one, so that ~m text-books on toxicology the fatal doses per kilo of bodv wHclit 'Arrhenius and Hadsen (I.e.) in their verj- interesting study hiive imb- tioned whether the phenomena of neutralization, which I drscrilied aiid nfemd to B. plurality of poisons, are due to a difference in the iKiisoiis or nhethet ■• they think probable, they are merely the expression of a neut rati tat ion betnvn two substancea ot weak alfinities. For the preaenl 1 shall merely poiol «ie that my own statements refer only to diphtheria toxin, which fHissesaM « taoA higher affinity tor the antitoxin than does tetanus toxin. The in^-psticBCfwtf of theae esteemed authors have disclosed one Bource of error whirh (H>ukl ^u3r creep into neutralisation experiments. Nevertheless I Mieve that their roa- ception does not apply to the tonin ot diphtheria which I have studied so i-JonJr I shall go into these questions more fully elsewhere, und hope th^n to dM« ihat the stjindpoint ninintained 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 w^ere 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- ])hore 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 different 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 provei not to be the ease, for v. Behring demonstrated that his tetanus poison when injected into rabbits in large quantities suffers a conadcrable diminution in toxicity. On testing the properties of the poisoo contained in the serum of the poisoned animals he found that ths residual poison possessed the same constanta as Tizzoni's poiion. From this it follows that v. Behring's poison contained also a cer- tain proportion of the Tizzoni variety. The Marbuig culture must therefore have produced two varieties of poison at the same lime. Naturally by mixing the two poisons one erni obtain new poirana which, while they manifest the same action on mice, will have any desired relative toxicity for rabbits; this, of course, n-ithin certain limits. If one were to take the time and trouble lo examine a large number of native poisons from different lalwratories, coirespondiog 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 animab, and then consider the behavior of tetanus toxins as just described, we shall conclude that bacterial poisons of different origin, whifh manifest a variation in their relative toxicity, are not of simple con- stitution, but are made up of several different constituent*. It ahowa 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 t Ms., i.e., whose unit of volume kill* a like number of grammes of mouse in four daj"8, may have an entirdy different content of t rabbit, t pigeon, tgoat, and f horse. This at once disposes ot Ehrlich's conclusions." It is just such phenomena which argue in favor of the plurality of poisons; they do not spe^ against it. Gruber bases another of his objections on the interesting obser- vations made by Madsen and Dreyer on toxons (Zeitsch, f. Hypene, Vol, 37, page 251). In his dictatorial manner he says that " theae observations demonstrate conclusively that Ehrlich's method of itnalyzinp toxins is absolutely useless. Only a person ignorant of chemi.'itry could maintain that the different results in guinea-pigs and in rabbits are sufficiently explainett by the different suscepti, bility of the animals to the toxins." To begin, Gruber's premise is absolutely misleading, when be says: " But if the poison \e neutralized it will be without effect even J 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, how^ever, indicate that the affinity of the tissue receptors can become increased. I 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 few 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 affinity 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,' many years later lai entirely independent of me, reached exactly the same conclufioB as I had. His very interesting study was based on experimeDti with diphtheria-immune horses. Following his usual tactics. Gnibe will, of course, draw the conclusion that the increase in the tiaaw 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, ntti hardly be told that it is impossible for chemical groups attarlie^ to living protoplasm to maintain their affinity unchanged as though they were made of stone; especially is this true if we consider tb* varying function of the protoplasm. Let us take anilin as an example, and determine the combinini heat of the NHa group for a certain acid. We shall then find thit nearly all substitutions of the Ixinzol 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 indiffennt 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 ab&olutdv inconceivable and beyond the pale of discussion. Since Gniber has given only that part of Madsen and Dreyw's experiments which fits into his j.x)lemic, it will be necessarj- (or bk to supplement this with some additional data from their atudv. These authors employed a diphtheria poison of which the fatal dose for a guinoa-pig of 250 grammes was 0.009. and for rahbite of 1200-1600 grammes, 0.0070. Calculated per kilo this shows that tlie rabbits wore about six times as ^uaceptible as guinea-pi^ 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 1 conceive it. refers exclu- sively to guinea-pigs, since according to my experiences this is the only animal in which, thanks to the peculiar susceptibility, ihe con- slants of the poison can accurately he determined. In the serum mixture I^ all the constituents of the poison, toxin, and toxon we completely neutralized, so that not only the single amount but ako TOXIN AND ANTITOXIN 523 high multiples of this can be injected into guinea-pigs without causing a trace of local or general reaction. If the same amount of poison, 0.6 cc, was mixed with ^r^ 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.^ //, however, the quantity of antitoxin mas 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 ^^ 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 + ^7^ 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 ^z-^ 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, sa^ ten times this mixture, be injected, this will contain ten times 1/10 fatal dose. 524 COLLECTED STUDIES IN IMMUNITY tralized doses, which (like the mixture ;^\ possess & considerable I excess of antitoxin, are absolutely innocuous for guinea-pigs md can be injecteci in any desired quantity. In fact, owing to tbe exrass of antitoxin, such mixtures furnish the animal with passive ImmumlT and protect it, provided suitable amounts have been injected, agaiosl diphtheria poison and diphtheria bacilli. If such mixtures, bow- ever, are still toxic for rabbits, only one possibility remains, namely that the diphtheria poison in question contams a substance wbirh is non-toxic for guinea-pigs, but still toxic for rabbits. This is my toxonoid.' So tar as the behavior of partially neutralized mixtures is cod- cerned. the investigations of the two authors show that naistures which exert only toxon effects on guinea-pigs cause death and symp- toms of diphtheria poisoning in rabbila. In my opinion the phe- nomenon described can best be explained by the assumption tlut at least three varieties of poison are to be distinguished, posi different affinities and different actions. Such an assumpti believe, will best harmonize the actual facts. These poisons are: 1. Toxin, possessing the greatest affinity, kills rabbits and guints- pigs acut-ely, but is much more toxic for the former. 2. Toxon, killing rabbits acutely and guinea-pigs witli paraiytie 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, esiif- cially in the sludy of toxins. Thus heroin, an acetyl derivative ' Almost at the oulael of my investigations and long pnor to Madsen and I Dreyer 1 obtained resulta entirety similar M these. My utipublistied but vtij I extenaive Ktudies showed that ttiiit property ik not possesHcU by all diphlherM poisons, for I also encountered poisons in whieb Ihc L, dose was exactly Uie same in guinea-pigs and rabbila. Ttiis tact controverts the assumption tial perhaps the described phenomenon is due to an incomplete neutralixatioa. '. Biich as Arrhenius and Mudsen have iicmonBtral«d in the union of boric nai a and ammonia, and In that of t«tanolysin and antilysin. If this were th« »M'l one would expect the phenomenon to be present in alt diphtheria poisons to I the fiame 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 1 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 1 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. Wocbenschr., No. 27, 1903. 526 COLLECTED STUDIES I\ IMMLINITY. to practically zero. In this case then, just as Gruber stales, thf water has acted as an antitoxin. On the addition of more Ksltr to the mixture, however, the iron is again attacked. In fact tlw more water now added the stronger becomes this action. We ihus obtain the curious result that in small doses water acts as antitoxiD. while in large doses it increases the action oE the poison, surely to interesting problem for Dr. Phantasue! This is merely one of the special instances of the fact thus br unexplained, that the different hydrates of sulphuric acid, or ihcir mixtures, manifest a most extraordinary variation of propenies- ! may refer the reader to the minute and fundamental study of Knietsch,' in which the variations of the properties of sulphiinc acid at different concentrations have been represented in the fnrm ot a curve for many of these properties, thus specific heat, elerlnf resistance, boiling point, vapor tension, viscosity, capillarity, action on iron, etc. A glance at this chart gives one the impreasioo ot 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-G ruber -Pi rquet are absolutely worthlca. This is especially true in Gniber's case, which deals with an objure reaction in which oxidation, abstraction of water, cleavage and sul- phurization take part- Hence I deny that crude experiments ot this kind can be used to gain an insight into such an entirely diffenul subject, or that the conditions there observed can even be com- pared to the minutely differentiated processes of toxJn-anti toxin combination. We shall next take up Gruber's experimenta which deal with the hemolytic 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 hipmolvtic toxins. The experiments are sup- posed to show that water is composed of an infinile number of differ- ent poisons. I.*t us listen to Gruber for a moment: " Pure water exercises a very great osmotic pressure on wd blood-cells, leading to their swelling and to the escape of hxmnglobin. 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 osmolitf pres.sure. is thus gradually decreased." 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 the 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 a^ain 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- S2S COLLECTED STUDIES IN IMMUNITV lutely incorrect, and that they contradict all that is thus far knoin concerning solution of the red blood-cells. Gruber states that a a 1/10 isotonic solution, one containing about 0.07^. NaCI, about one-fifth of the blood-cells remain undissolved. All other autbon, however, have found that even in a solution of 0.3% NaCI. ihe blood-cella of all warm-blooded animals are still completely de- eolved, so that the solution appears uniformly lakj', and microscopidl examination shows not even a trace of red-blood corpusclej. la Gruber'a spectrum, however, we find that with this percentage more than half of the blood-cells remain undissolved. Tliis indicate that in Gruber's experiments the grossest sort of errors abouml. With Salt WithSugar HDmoly» Id rermu BmuoijMlu rcrceut FlQ What ca Isotonieity Poison spectnim ' of n Isotonicity r accordiTiR to Gruber. 1 we deduce from these spectra? The fact that a c«- tain amount of NaCI can be added to the " poisonous " wat-er with- out inhibiting hjemolysis, would lead authors holding Gruber's view to conclude that this " poisonous " water contains a prototoxotd whose neutralization has no effect whatever on the toxic actioiL A single glance at the detailed literature on this subject should, how- ever, have convinced these authors that their curve, as such, bit nothing whatever to do wifh toxic actions, but m merely the expiw* BOn of the specific differences in the red blood-cells. It is well knom TOXIN AND ANTITOXIN 52U 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 vulnerability according to its age. Are Gniber- Pirquet entirely unaware that an important and much-employed procedure for determining the resistance of the blood rests on just With Salt With Sugar r Pig. 2.— "Poison spectrum" of i Isotonic) ty T according to Ehrht^ this principle? Every text-book on hseniatology 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 hia 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 IMMONITY. If one conducts experiments that have nothing to do with tbf problem under discussion, further, if the method of th«^ expei- menta is grot^ly at fault, and it, finally, the results thus ohtuiMl are given an utterly false interpretation, it is not surprising Hot the mojst fantastic res-ults are obtained. Finally (.'ruber describes one more experiment which he ilhi;- trates by means of a curve. Accordiiiji to him this too demnnstnia that my theory is untenable. The experiment shows that the hxmul- yais of ox blood, bj means of a certain quantity of specific )uE-nii>ltDr serum within hall an hour, is dependent on the dilution. I dmiI hardly remind my reailers that 1 have always laid stress on the cbtisa- cal nature of the toxin and antitoxin combination. 1 can assnrr them that the factor of the degree of concentration has ever bea sufficiently regarded. If Gruber will refer to my first study on thk BUbject, " Die Werthbcmessung des Diphlherieheilserums," he «il find the statement: " that the union of poison and antibody [i» ceeds much more rapidly in concentrated than in diUite sohitiont.* and further also " that heat hastens the union and cold retaidi the same." The behavior which Gruber describes is all the lews surpiisof since he is dealing with a complex process depending on the aclJM ot the amboceptor -complement combination. How readilv tb» combination is diaaoeiated has repeatedly been ]»ointed out by », Perhaps Gruber thinks that this experiment is new to me; ^vbj one versed in the subject, however, knows that we are here dtJ- ing with the most commonplace phenomena, with which every bt^inos is well acquainted. 1 should like to point out, however, that thit phenomenon, namely, that dilution with water inhibits the actia of ha^molysins, is not at all constant. On the contrary ii is hmitnl to those cases in which tho affinity between amboceptor and nJ), or between amboceptor and complement ia relatively slight. H one employs iKiiaons in which the affinity between receptor sod cell is great it will be found that wilhin the limits mentioned the addition of water is practically without effect. Thiis, in woriciog with cobra venom, I found that a given quantity of this iMiscm exerted exactly the same effect whether the volume of water usrf was 1 or 15. It would lead us too far to enter into all the distortions and ^B^ conceptions contained in Grul'ier'a polemic. To do this would rrawte almost a complete reprint of all my articles, as well as of tnanv othos TOXIN AND ANTITOXIN. 531 emanating from the Institute — with all of which Gniber seems quite unfamiliar. I 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 neutralized 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. a'Si COLLE(TED STL'DIES IN IMMLI.MTY had apparently autreeded in immunizing against solanin. ftin* then, however, the researches of Bashford ' and of Besredka ' hs*i shown that it is impossible to produce antibodies against r^\ha solanin or saponin. Pohl himself no longer maintains the exi-iairf of a specifjo antisolanin Of the vari.ius poisons, which fevtati to promise the best for successful immunization, morphine shoud be mentioned first. Recently Hirachlaft ^ clsimed actually In hsn produced an antimorphine serum. Morgenroth,* however. wa.s ^' to show that the results obtained by Hirschlaff were merely appan-ni, not real, and that they depended on the fact that the do^e^ of p* son employed bv Hirschlaff were not surely fatal, especially oiiu to the increased resistance of the animal foliowinR the ewno injection. Hence the statement still holds true that all ptiuwi' chemically well defined do not possess the property of produmtf antitoxins. So far as other differenees between ordinary poisons and ti^u: are concerned, I mav refer particularlv to niv detailed articles ■ von Leydens Festschrift" and tn the excellent monograph by Oib-' ton.^ From these it will be seen that the action of the chemifsllf defined poisons, alkaloids, ghicosidea. etc., on parenchyma result of a solid solution or of a loose salt formation. In acconlan' with the loose character of the coml>ination, the action of iIm poisons is a transitorv' one. The firm union and prolonged peculiar to the toxins is entirely absent. Besides this the paioJ of incubation is wanting in most ordinary poisons, although lh« are a few exceptions like arsenic, phosphorus, tartrate of tin ul sodium, and vinylamin. In the toxins, on the other hand, a prriJ of incubation is the rule. Entirely in accordance with the views of Emil Fischer k ing ferments, I have aHcril>ed the specific combining proceese d toxins to certain stereochemical groups of atoms (haptophore grou|^ These unite only with such other atomic groups which fit to Ih* as does a key to a look. The ordinary chemical groups of orgMl chemistry po8se5a affinities for a large number of other groups. ' ' Artliives Internntionales de PharmncodvnamiCH. Vols. S aod 9, 'See Metchnikoff L'Immunitf. Paris, 1901, ' Berliner klin. Woohenschrift 11102. Mbid., 1903. No 21 • Von Leydens Feat .TCh rift. .^iiRiist HitsrhwBlcl, Berlin. 1902. • Stiidien liber die N:irk.>se -Iciiu iml. 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 limited, 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 aflfinities 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. 1 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 passan^e from Ebrlich and Morgenroth's First Communi- cation Concerning Hemolysins (see page 7 of this volume), a passage which Wechsberg has already called to Gruher's attention (Wiener klin. 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 STirDlES ]N IMMUNITY laid down the diPtiiin, " There is po dissociation by raeana of rold.' It seems not to have mattered to him that his statement is oppoed to even the most elementary principles of chemistry. As a matter of fact we have always paid due attention to dim- cistion and to the reversibility of the reactions. I should like b) call Gruber's attention to the fact that the sentence: " In the mrm of the amlK)neptors we are dealing with a reversilile process" ocnits in one of Morgenroth's studies = from this Institute. Further thn this such questions do not afTect the Side-chain Theory, as such. TTk whole discussion is evidently designed to hide the fact that Grubo-'i position is really based on my theory. So tar as the mode of action of the toxins is concenwd Gruber's standpoint and mine are es.sentially the same. Thie Gruher states that: "All poisons must be 'anchored tq- ibt cells and the anchoring group of atoms is probably always different from that group which gives the substance its toxicity " I sitent many years in establishing this view and it is now everywhere aireptf^ as axiomatic. I defy Gruber to show me the text-books of toii- oology in which, previous to ray work, this conception apjieais, i conception which dominates the laws of the distribution and action of poisons, If he should again refer to S. Friinkel'a book ^ 1 cm only remark that while the account of my views is very admirable. it is nothing more than a rfeumS of the points which I had previoudv developed. Perhaps I can even aid Gruber's memory and let him apeak for himself. A year before his declaration of war he spoke of " the brilliant hypothesis of ihat genius Paul Ehrlirh, the greaint of living pathologists.' In a little work^ published at that timr and quite enthusiastic over my theory he states: '■ According to Ehrlith only such substances are poisons which unite chemtcallv with ."ajme constituent of the organism." And yet this eamc Grulw to-day says: "These are merely new words for what has long brcB known," I should not like to deprive the reader of hearing still aaotiier tainif that my view of llie production .amboceptOT and complement are firmly united, is abaoiulely incoroi ' Miinch. med. Wochenscht. 1901, No. 48. ■ Ibid.. 1903, ' Die Arziieiiiiitteliiyntbese, Berlin. 1901. ' Max Gruber, Neuere Forechungen iiber erwarbene Immunilgt 1900. ding to wUA 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 thstt 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 aflSnities I must emphatically deny. The studies of Donitz^ and of the Heyman school 3 show that the injected toxins * Deutsche med. Wochenschr. 1898. ' Ibid.. 1897. • Decroly et Rouse, Arch, de Intemat. de Pharmat^odynamie, Vol. VI. 536 COLLECTED STUUIfa IN IMMUNITY disappear from the circulation in a, few minutes. It is ihereim idle to talk of a slow union such as would correspond to weak affim- ties. But, says Gruber, " it is impossible to understand why the toxophore groups, after they have been brought into proximiiy the protoplasm, do not at once commence their activity, but alwin stop to consider the matter for several hours," One cannot seriou^ discuss the subject with such a questioner. Gruber might ju&l a ask that all chemical reactions proceed rapidly, and deny the poai- bility of a slow reaction. The slow action of the toxophore group is not at all remarknUt, especially in the domain of toxins. This is particularly true if «t remember that with certain poisons (e.g. botuhsm toxin), one p»B of toxin to 500 million parts of body weight suf!tce3 to cause deMK and that the rapidity of action is dependent to a high degree ot the amount of the active substance. Is Gruber possibly of the opinion that in the paralysis of dijib- theria, which as is well known usually develops after the lapse d weeks, the toxon courses about free for twenty days or more befon entering the tissues and then suddenly exerts it.s action? To llx unprejudiced critic the importance of the separation of toxin biml- ing and toxin action for the proper understanding of the period ti incubation, is conclusively demonstrated by Morgcnroth's ' experi- ments with tetanus in frogs. Courmont and Doyon, as is well known, discovered that the frog is susceptible to tetanus poison only )t higher temperatures, and not when the animal is kept cold. Mor genroth was able to show that at low temperatures the tetanus poima is bound, but exerts no toxic action. Frogs are injected with lelanw toxin and then kept on ice for days. If then they are subjected to higher temperatures, it will be found that they behave exacttr as if they had just been inoculated. And yet the toxin has been bound by the centra! nervous system even at the low temperaturr; for if after several days at low temperature the animal be injected with an amount of antitoxin, even much more than sufficient neutralize the poison, tetanus will still develop if the frog is subject«d to a higher temperature. But this is not all. If frogs, after toeing injected with tetanus, are subjected to a high temperature for one day, and then placed in the refrigerator, they will not become sicL But on bringing the animals back into higher temperatures aft« 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 n. 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 .. in the main, in others I 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- . elusion. 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 hasisted 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 I formu- lated, not Gruber. As far back as 1898, Weigert ^ rightly pointed out that my demonstration ^ of antitoxin production through non- poisonous toxoids was sufficient to demonstrate the independence of antitoxin production and toxic action. Furthermore I have repeatedly pointed out that the development of antitoxin depends on the haptophore group. Over IJ 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. Wochenschr. No. 49, 1901. 538 COLLECTED STiroIES LN IMMUNITY \b) " Certain animab no n -susceptible to certain toxins newr- tbeleas produce antibodies " needs no further explanation accMdia| to my theory. Certain species of animals may possess suiUhli receptors For binding the toxin and producing antitoxiu <to0 their cells are insensitive to the action of the toxophore group, Kwxi- ing to Metchnikoff this seems often to be the case with tetanus lau in crocodiles. As already pointed out years ago by Weigert' sctont' ing to my theory, the production of antitoxin need not at tJI fc* preceded by any injury in a clinical sense. In fart, loo slronjia injury may cause the cell to lose its power of regeneratioo. oirat to the toxic action on the vital group [I^istungskern]. I"or etfltnpia, if a specific nerve poison is anchored by a fitting receptor of an iailiSe- ent cell (liver) we should expect the production of an antibodv bf the liver, even if the liver-cell does not become tctanized. address at Hamburg' before the Congress of Naturalists I poinid out that the local origin of antitoxin, which Romer deducfs his splendid experiments with abrin, will often make it possible n transfer part of the antitoxin production from the vital ore&nsV the indifferent connective tissue, by means of subcutaneous ipjer tion of poison. Gruber's next statement is; (c) " Despite a plentiful production of antibody, the subo?' tibility to the poison may remain, or even increase," I have already discussed the principle of hypersensitivenoi and mentioned the fact that this objection restrained me for h time from publishing my theory. But even these phenomena wot satisfactorily explained in accordance with the side-chain theoq', by the assumption of an increase of affinity and a rupture of ih toxin-antitoxin combination. To be sure it is possible that explanation touches but part of the subject, and that in reality lie phenomena are far more complex. But this is no reiisnn for ing to overthrow the theory; to do so would be to completely apprehend the purpose of a theory. Surely one cannot that a theory will at once explain all the complex phenomena rf so difficult a subject as this. A theory ought primarily to posem heuristic value, pointing out new paths into a complex subject; i should smooth the way. The actual research must be left to tie' 8cienti6c investigator. Science can be advanced only by TOXIN AND ANTITOXIN. 539 of experimental analysis, and not by high-flown words of a mis- leading dialectic. {(l) **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 1 have never yet claimed that this is the only means by which the organism can defend itself against deleterious influences. 1 would call attention particularly to the Sixth Communication on Haemolysins,^ 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 wav 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 i)oison new receptors may be formed which are so firmly imited to the protoplasm that they are not thrust off. Such receptors Morgenroth and 1 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. 2 Schlussbetrachtungen in Nothnagel's Handbuch., Vol. VIII. F 540 CULLECTED STnilES IN IMMl'NITV. Another possibility of cell immiinily is that the proiojik-aii rf cells which are ordinarily susceptible is no longer affei-ted In* to- tain poisons. This kind of immunity, whieh to be sure I i^oad? very rare, would correspond t« mithridatism or acquired lolenin in the old sense. A fourth possibility, finally, is the adaplalion d the phagocytic apparatus in Metchnikoff's sense. It IS obvious, of course, that all the sevarious subordinate HiA of immunity occur alone as well a.** in manifold combinations. This, as already mentioned, immunization with cc! blood i^ followed b; antitoxin immunity and tissue immunity. In the lower aninut. however, which as Metchnikoff has shown are but little ad&vud to the production of antitoxin, other defensive contrivam-es Jeodiif to cell immunity will predominate From this ]x)int of view there- fore the condition described by Gruber. namely, that frogs can be immunized against abrin without their showing any antitoxin, no difficulty. So far as the frog is concerned the only question ii which kind of cell immunity is present, i.e., whether there is a ife- appearance of receptors, or whether there are sessile receptors, et*. In view of the detailed statements given above I presume 1 neti add nothing to the following passage in Gniber's conclusion (c) "The production of antibodies takes place at entirely differait localities than does toxic action." The discerning reader will at once see that this statenoent doe not in the least contradict my views. In fact it is merely anotbtr way of expressing what is really the nucleus of mv theory. Thf generalization, however, is false, that the production of anttbotti necessarily takes place in localities different from those in »hid toxic action occurs. If Gruber therefore believes that this riddln my theory it is evident that he understands the principles under- 'Gruber citea, as a seriouH objection to my theory, that Madsc-n uti«n' and imnnmin Gruber himself has found that this theory is constantly gaimti ground, whiSe I am gratified to see it treated in detail in the bri text-books as well as in excellent digests compiled by a large nun?- ber of my colleagues.^ In addition to this hundreds of separate studies have been based on the side-chain theory so that I may vfH believe that it best serves to explain the fact« already* obsen'ed e well as to allow new facts to be predicted. Gruber'a appeal,^ there- fore, that '■ Ehrlich's theorj' is a great mistake, and is bound soon » disappear from the scientific arena," has had but little sucte&s; in fact it aeems to have had the contrary' effect. The large number of investigators, who are constantly eagerly working on the pnA- lems of immunity know what ia best for them, and will not be dictat«il to against their own experience and conviction by one who iL. 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 ttus poison) human pathology tiad 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 tiarmonized so perfectly, the probability became almost a certainty It has been found that the toxicity of mixtures whose toxin content lies between Lq and Lf is not quantitatively diminished, but is actually 560 COLLECTED STUDIES IN IMMUNITY. different ijualilatively. Guinea-pigs injected with such mixtures tiicken, after a long period of incubation, with typical paralyser and show no louai reaction. The hypothetical toxic constituent which gives rifle to these paralyse sis termed "toxon," Why then is it ini|X)ssible to demonstrate the action of the toxoo in native diphtheria poison? This is readily exphuned by the relative I oiicentration of toxin and toxon in the toxic bouillon. Quantitati^-fi 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 live long enough to manifeet toxon effects asually contain too little toxon to produce the typicsl paralyse). If, however, a large amount of poison is so far neutralized mth serum that all the toxin, with the higher affinity, is just bound »nd the toxon is still free, a mixture will be obtained which practically represents a pure toxon solution, for the neutral toxin-atititoxiii raoleculea play no rflle 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. Aa a matter of fact, we find tliat the number of lethal doses contained in the different Lf — Lo varies enormously, and so far as the toxon content is cerncd the variations were from 0 to 300^ figured on the basis of the toxin content. It will be well to enter somewhat more into a study of these t wo extremes, for these striking exceptions to the tyiijcd conditions argue strongly in favor of the views here presented. On« of the poisons in question was studied by Ehrlich. and was remarkable in that the difference Lt — Lo represented only 1.7 lethal doses, 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, tht 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 descril>ed by Dreyer and Madfen. The constants of this showed that it contained three times as mucb 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 abo**e^ this is readily understood, the relative concentration of toxon in this ease was so great that even sublethal do.ses sufficed to make the toxon effects manifest. In most native poisons this demonstratwo 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 suflSciently 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 poison 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 inEhrlich'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 acide (ammonia and boric acid). Tim similarily constitutes the basis for their mathematical work, which leads them to conclude that toxin and antitoxin are simple substances I whose reaction is reversible. This reaction finds its expression in the curve just mentioned. Let ub examine their conclusions and I Bee whether they are justified. The two graphic methods referred to are equally correct. Never- theless it cannot be denied that the one employed by Ehrbch, 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 representatiDn of the differential quotienta 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 antitoxm causes no decrease in toxicity (pro to toxoids), so that the neutralisa- 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 poison spectrum, by rejiresenting 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 mm 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 aetuallyexist, or whether a gradual transition must be inter- polated, it is certain that the changes should always occur in the Bsme 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 irre^ilar ' The sole object in employing tbia poison is U) illuatrste ttie two mol 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 irr^ular 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 s t o 10 9 8 7 6 5 4 8 2 1 0 o.. PROTOTC KOID 6., HCMITOX N c. PURE Ta I UN TOXON OM 0.1 0j5 OJl 0.85 0.8 0.85 0.4 "V '^ y "^ r ^ AHTTTOWIi Fio. 1. — Poison spectrum according to Ebrlich. 0.45 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 6 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 (a) 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 deuterotoxin in its affinity. The constituents of the 554 fOLLECTED STUDIES IN IMMUNITY. poison can thus be arranged as proto-, deutero-, , tritotoxiii, eK,, 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 undecomposn) constituents of the poison from the outset, is demonstrated by the genesis of tosnid formation. Thus if one is in a position to study a very pure poison in its various stages of decomjmsition, 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 pro to toxoids 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- I crease the toxicity the most. 5 Nevert.heleas a numlxsr of experi- e ments were published by these e authors (Madsen, with diphtheria S poison, and Madsen and Wal- baum, for ricin) in which the prolo- 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 isdueto "change- Fjo 2.-.\eutraliwtion curve accond- ments minimes dans le milieu am- ing to An-hemuaaDd Madaeo. biant," or by saying that tlie proto- toxoid zone is "of little interest." In order, therefore, to elimli 0 \ \ \ \ \ \ \ \ \ s 'v *■ X II. It lUttJ nledl eath j these prototoxoids, so annojing for their formula, thej' have discanled the well-tried criterion for a fatal dose of diphtheria poison (dttth 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: RetatDioi the definition of a fatal dose, they believe it possible to calfruUie the fraction or multiple of the fatal dose employed, from the tittle the animal's death or even from the resulting loss of weight. Such 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 i or ^ 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 had 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 in diphtheria poison by so excellent a worker as Theobald Smith. 556 COLLECTED STUDIES IN IMMl-^vITY. will then be governed by the law of mass action formulated by Guldberg-Waage, namely, (toxin). (antitoxin) = A(toxln-antitoxin), in which the brackets denote the concentration, and k the constant o( equilibrium to be determined for each poison.' All the calculations of Arrhenius and lladsen 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 t«st«d 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 sam* amount of antitoxin we add at intervals fractional parts of the toxin, we shall find that the resulting end product is considerably more toxie 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 la^t fraction in the former case. Von Dungeni 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 alwa>-s 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 ihe reverse of what is actually observed.' Hence all those poisons in which this phenomenon of ' In their recent publications Arrhenius and Madeen assuine that one mole- cule toxin combines with one molecule antitoxin, not to form two molecuka of the toxin-antitoKin combioation.as the above formula would show, but that two different substaaces are formed, toxinan and titoxin. To be sure aa Uw equation then reads, (toxio) (antitoxinj — fc (toxinan] (titoxin), one objection to the above formub is done away with, but a new hypotheaia, lacking all evi- dence whatever, is thus introduced merely tor the sake of the formula. ' The phenomenon in question therefore shows exactly the reverse of vbat Arrhenius and Madsen's theory demand. For this reason the limit of «nw need not be considered, although. owing to the enormous quantitative ditTeieocM, it would play no r61e in judging the result. Nor can Arrhenius extricate hinw self from the predicament bysuggeating that we are dealing with alowly progiw» 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 Arrhenius 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 affinity 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 toxin, 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 Dimgem. 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 Dungem and Sachs, the increased toxicity is abeady 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 h^emolytic 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 line. 558 COLLECTED STUDIES IN IMMItNITY. poison constituents, is out of the question. On the contrary we most assume that the union of these substances with the antitoxin is subse- quently tightened. This tightening is also borne out by other obsena- tions, both old and recent, If the toxin-antitoxin reaction vcre reversible, it should be possible, by removing the supposedly free toxin residue, to constantly change the equilibrium, so that the toidn could all be recovered. Nevertheless, although toxin can be filtered through gelatine and antitoxin cannot, it is impossible either \>y gelatine filtration (Martin and Cherry) or by gelaline diffusion ivan Calcar) to obtain free toxin from neutral toxin-antitoxin mixtuna.' In addition to this one cannot help being surprised that the calcula- tions of Arrheniua and Madsen entirely ignore the cells' toxin -binding receptors which effect the poisoning. In accordance with their views, these receptors should represent an imimrtant element in the equilibrium; and yet they appear to have entirely overlooked ihis 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 htemolytic test-tube experiments. This illustrates the danger of & 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 maj' be able to represent the resultants of the processes which bring about the phenomena, but in that ease the formula is nothing more thaa an interpolation formula. Corresponding to this, therefore, we we 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 onlv lo 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. la perfectly evident that toxin can be obtained from fresh toxin-Antiioni a by diffusion through gelatine, and this has recently been deanonslnUil by Madsen and Walbaum. According to Morgenroth such mixtiucs rcnuin at least twenty-four hours for the union to liecome complete. Henc« Ihe slaie- ment by Madsen and Walbaum that the mixtures must be fresh in order to demonstrate what tliey regard us 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 WerthbemessuDg des Diptberieheilserums. Klinisches. Jahrbuch. The samCi Zur KeDntniBS der Antitoxinwirkung. Fortschritte dcr Medizin. 1898. The samei Ober die ConstitutioD des Diphtheriegiftes. Deutsche med. Wochenschrift. 1899. Th. Madben, Uber Tetanolysin. Zeitschr. fiir Hygiene, Vol. 32. 1902. S. Arbhenius and Th. Madben. Physical chemistry applied to toxins and antitosdns. Festskrift ved Indvielsen af Statens Serum Institut. 1903. The same, Anwendung der physikalischen Chemie auf das Studium der Toxine und Antitoxine. Ztscbr. f. physik. Chemie, Vol. 44. P. Ehrlich, Uber die Giftcomponenten des Diphtherietoxins. Berl. khn. Wochenschr. E. YON DuNOERN. Bindungsverhftltnisse bei der Pr&cipitinreaktion. Centralblatt f. Bacteriol., Vol. 34. Th. Madsen. La constitution du poison diphth^rique. Centralblatt f. Bacteriol., Vol. 34. 1904. S. Arrhenius, 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 Absftttigung von Toxin und Antitoxin. Berl. klin. Wochenschr. E. VON DuNOERN, Beitrag zur Kenntniss der Bindungsverh<nisse bei der Vereinigung von Diphtheriegift und Antiserum. Deutsch. med. Wochenschr. S. Arrhenius, Die Anwendung der physikalischen Chemie auf die serum therapeutischen 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. Walbaum, Toxines et Antitoxines. De la ricine et de I'antiricine. Centralblatt f. Bacteriol., Vol. 36. W. Nernst, Cber die Anwendbarkeit derGesetze des chemischen Gleich- gewichts auf Gemische von Toxin und Antitoxin. Ztschr. f . Electro- chemie, Vol. X, No. 22. 560 COLLECTED STUDIES IN IMMUNITY. J. MoKOENROTH, UntersuchuDgen uber die Bindung von Dipbtberie- toxin und Antitoxin, eowie iiber die Constitution des Dipbtben^ giftes. Berlin, klin. Wocbenschr. in detail in Zeitschr. f. Hygiene. Vol. 48. H. KoppEi Zur Anwendung der physikalischen Chemie auf dai Studium der Toxine and Antitoxine und das Lackfarbenwerden rotber Blutscheiben. Pfliiger's Arcbiv, Vol. 103. An address entitled '* Die Serum tberapie vom pbysikalisch-cbemiMfaeo Standpunkte/' by Sv. Arrbenius. Discussion by Ehrlicb, Nerast, Arrbenius. Zeitscbr. f. Electrocbemie, Vol. X» No 35. E. YON DuNOERN, Bemerkung sum Vortrag von Professor S. Arrbeniui: "Die Serumtberapie vom pbysikaliscb-cbemiscben Standpunkte." Zeitscbr. f. Electrochemie, Vol. X, No. 40. Th. Madsen. Toxins and Antitoxins. Britisb Medical Journal. R. P. VAN Calcar, Ober die Constitution des Dipbtberi^ftes. fied. klin. Wocbenscbr. 8. Arrbenius et Th. Madsen, Toxines et Antitoxines. Le Poa dipbtb^rique. Centralblatt f. BacterioL, Vols. 36 and 37. H. Sachs, t)ber die Bedeutung des Danyss-Dungemachen nebst Bemerkungen uber Prototoxoide. Centralblatt f. BacteiioL, Vol. 37. L. MicHAELis, A collection of studies on tbis question together with • critical review. Biocbemiscbes Centralblatt, VoL VI, No. !• XL. THE MECHANISM OF THE ACTION OF ANTIAMBOCEPTORS.i By Prof. Paul Ebrlich 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 individiuil 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 morphological 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 Win. Wochenschrift, 1905, No. 19. ' P. Ebrlich and Morgenroth, Haemolysins. See page 88. 561 562 COLLECTED STUDIES IN IMMUNITY. complementophile group of the complement. Each of these twD haptophore groups mil 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 hspmolyain, 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 haptopbon groups of the lysin, can frustrate the action of the lysin." In other words, according to the amlxiceptor theory two difTemit an ti amboceptors are at once conceivable, either of which would inhibit the action of the amboceptor One would act by prevenliflg the union of amlxiceptor and cell, the other by preventing the comple- ment from uniting with the amlxiceptor. Originally the antiambo- ceplors 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 existenw of antibodies for the complementophile group should be demoo- 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 verj' neat confirmation of this, Bordet finds that antianiboceptors can be produced not only by immunization with hfemolytic immune serum, but also with nonuil serum of the same species, even though this normal eenim contain no corresponding amboceptors. He treated guinea-pigs with trarmd rabbit serum which contains no hipmolytic amboceptors for M blood, and obtained an immune serum which yet was able to in- hibit the action of the amboceptors derived by immucizirg will ox blood. That, certainly, is a discovery which cannot readilv be »■ plained in harmony with Bordet's sensitization theory. Accordii( . to Bordet, as we know, these immune bodies {his "sensitiaeia") possess the one property of combining with the susceptible aS and thus rendering this ^^Jlnerable to the action of the complenmt. This being the ease it is incomprehensible how a serum which ' P. Ehrlich, On Immunity, Proceedings Royal Society, 1900. ' Ehrlich and Morgenroth, VI. Communication, page 88. ' J. Bordet, Leg propn^tfs des antisensibila trices et les ttiteri^B de riramunitfi, Annal. de I'lnstit. Pasl«ur, 1904, No. 10. MECHANISM OF THE ACTION OF ANTIAMBOCEPTORS. 563 lo seDsitizers whatever for the species of cell in question can ^et excite the production of antibodies directed against them, rhe matter takes on an entirely different aspect if we regard this >henomenon from the standpoint of the amboceptor theory. Ac- »rding to what has been said above we at once see that two func- lionally different types of antiamboceptors are possible. In Eordet's jase the normal rabbit serum possessed no amboceptors (i.e., no cyto- >hile 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 anii" miboceptors of the complemerUopkile group. The facts brought for- ward by Bordet all go to confirm this. If such antiamboceptors are to be produced, the only requisite B that the serum used for immunization must contain the corre- iponding complementophile groups. Is this the case in normal tibbit serum? Every normal rabbit serum, as Eordet admits, con- ains a large number of different amboceptors. If, by immunizing vith a given species of cell, a new specific amboceptor develops in he serum, the new element in the receptor apparatus is really only the ytopkile group, which is produced in response to immunization. The »mpIementophile apparatus need not suffer the least change quali- Atively; in fact according to our conception it usually does not ;hange markedly, there is merely an increase in the complemento- )hile groups corresponding to the formation of the additional immune x)dy. We have already expressed this opinion in a previous paper.^ 'In my judgment we shall arrive at a correct conception if we pro- teed from the standpoint that in general the specific amboceptors ixhibit a uniform structure so far as their complementophile portion 3 concerned, while their c)rtophile groups, which physiologically ire concerned with the absorption of food, differ most widely." It must not be thought that this uniform constitution of the com- >lementophile portion ^ contradicts the assumption of a multipUcity * P. Ehrlich, Betrachtungen iiber den Mechanismus der Amboceptorwirkung md seine teleologische Bedeutung. Koch Festschrift, Jena, 1903. * For the present we cannot say whether the complementophile complex 3 really uniform throughout or whether, perhaps, certain partial groups do lot differ in the individual amboceptor types of the same animal species. Such k condition is easily conceivable. In any event we must assume that the com- plementophile apparatus of the amboceptors of a given species is identical kt least in some essential part of its haptophore functions, and that this char* bcterizes it as coming from the animal species in question. 664 COLLECTED STUDIES IN IMMUNITY. of complements. Naturally the different cotDplements must lun dItTerent com piemen to phile groups corresponding to them. But, waa stated in the Sixth Communication on Hemolysins,* an imnii body, in addition to a particular cy tophile group, contains two, three, or more complementophjle groups. In a later paper Ehrlich and Marshall offered experimental evidence for just this joint; bedda this, Bordet's experiments, according to which an amboceptor after having combined with cellular elements is able almost completdf to rob a serum of its complement, also support this \iew.* We must therefore conceive the amboceptor to be structurally a polyceptor, and assume further that the amboceptors of a distiMl species are all supplied with a large number of complememophib groups which vary considerably in detail but in their entirety r^ sent a uniform complex. This complex is reproducf?d in all tl amboceptors of the same serum. In general the amboceptors M different and specific only so far as the cytophile group is concenied This being so it will at once be clear that an ti amboceptors directed against the compiementophile groups, and obtained through imiui zation with any particular amboceptor, will act against all amborqt tors of the same animal species no matter whether thcs9 amlA ceptors are normally present in the serum or have been produr«d by immunization. For the compiementophile amboceptor appanti^ is the same for all types of amboceptors of the s ; si>ecie8, Afii result of this, an immune serum obtained through immunixalii* with normal serum contains, thanks to the normal amboceptor* is the serum, antiamboceptors directed against the artificiaily produi amboceptors of the same species. This explains also the earto observations made by Pfeiffcr and Friedberger^ that ant.iamboce[>im obtained by immunizing with cholera serum act also against ty[>hni4 serum;* it also explains the recent experiments made by Bordet. Wt ' Ebrlich and Morgearoth. Sec page 88. 'P. Ehrlich and 11. T. Marshall, t'ber die complementophilen tier Amboceptorcn. Berl. kliii. Wochenschr. 1902, No. 23, 'R. Pfeiffer and E. Friedberger, Wcitere Beitrflge zur Frage dr-r Anl und deren Beziehungen n) den bacteriolylischen Amboceptoren. CeatnDU f. Dacteriol. 1904, Vol. .17; also 1903, Vol. 34. 'Naturally the statement made liy Ehrlich and Morgenroth (B«il I Wochenschr. 1901, No. 21) that "it Bcema improbable, imless in a «fMi a tortunat* coincidence intervenes, that anti-immune bodies will be «bu directed against the bactericidal immune bodies" cannot apply to the antiunte ceptors directed againat the com piemen tophile groups. That statement aj^ 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 Keififer 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 against 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-blocking" 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, "Uber 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 tbftt tl snti amboceptor aciion is easily inhibited by iiormal rabbit senita Naturally the normal amboceptors, whose completnentophile groufi excited the production of the antiamboceptor, will combine »it this antiamboceptor and so be able to deflect it from the ambocepH acting in the given case. Since we regard the antiamboceptor i the sense of a complementoid, thia phenomenon corresponds in prii ciple to that described by Neisser and Wechsberg as <]eflectioD complement.' The entire complex of phenomena just discussed shows rant Btrikingly that our a-ssumption harmonizes best with the obeencd facts. We assume that in Bordet's antiamboceptors we are deaiiD| with antibodies directed against the com pi em en top hile groups, "[}» existence of such antiamboceptoi-s again demonstrates that t) amboceptor theory is correct. According to Hordet's seositiiatic* theory only such antiamboceptors are conceivable which prntit the amboceptor's union with the cell. But if there are other kidl of antiamboceptors, as the findings just discussed show, we assume that the amboceptor has other affinities besides those for tit cell, and this leads us at once to the conception which we Ian defined under the name amboceptor. The sensitization theory therefore be abandoned. The next question which arises is whether or not it is by means of immunization with amboceptors to produce antitunbl this, to effect an inhibition of haMiiolyais by Biibsequenlly adding &ntiaa ceptor. It seems to ua that thia aimplificd procedure is more coHvJBcin*. it will hardly be claimed that the guinea-pig Hemm is a better auspending mtdi than physiologicul suit solution, and that it therefore, in contrast to the lit leaves the blood-cclla intact, Furlhennore, J gum a-pig ( inhibits the liseinolyais of ox blood by anilioceptor and complciAenl igiiim pig). Hence when guinea-pig serum ifl present the tiuestion trfaether iht i aence of bffmolyBiB is due to an antiamboceptor or not ia left undecided. ' In contraat to Bordet, however, we were unable by meaaa of Donnal « ceptor to effect the subsequent breaking of the union between aDIiamboetfH and sensitized l)lood 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 substanceg 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 antiamlioceptor action. Furthermore, since this inhibition a only brought about by large quantities of the semm. this substaoee 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 foUcws: We must assume that this sub- stance's point of attachment is a co m piemen to phi! ic auxiUar>~ group in the amboceptor. The occupation of this group bo affects ibe amboceptor molecule that the simultaneous presence of antiaoibo- cept.or no longer prevents the combination with complement. Sucb a behavior would be analogous to an observation published by Ehr- lich and Marshall.'' At that time, by means of a different iatiif method made available for one particular instance ^ by MarsfaiU and Morgenroth, it was shown that the amboceptor anchored to the cell, althoughit could deprive native guinea-pig serum of all its complement functions, was unable to absorb the non-dommaol complements if the dominant complement had 6rst been neutralii«il by the partial anticomplements of Marshall and Morgenroth. In ] other words, an anchoring of the non-dominant complements wm ' only possible after the corresponding com piemen tophile group of the amboceptor had combined with the dominant complement In our case we would be dealing with an influence entirely similu in principle, except that here the influence is reversed, i.e., the affini^ of the amboceptor to the antiamboceptor is reduced by the occup* tion of the auxiliary group. We believe that we can show direct!] that the antiamboceptor is bound in either case, but that where tb auxiliary group is occupied, the union of amboceptor and antjamb* ' We can oE course assume a, priori that an antiamboceptor serum (UrMW against the com piemen tophile groups will poasesa a multiplicity of puttf antiamboceptors, for tbe amboreptora which take part in the inunuoiatva i possess a targe number of different com piemen top bile groups, and wwat each of these a particular antibody b conceivable, * Ehrlicb and Marshall, 1. c. 'H. T, MarebatI and J. Morgenroth, Uber DifFereoKierung von Comi menten durch ein PartJalanticom piemen t. Centralblatt [. Bact. 1902 Vol. 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 p; the eflfective antiamboceptor corresponding to group a is a, the antibody fitting group ^ is 6. 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 /} Loose Union Fio 1. — a and ^: Complementophile groups of the amboceptor, a and b are Partial Substances of the Antiserum, a is the effective Antiamboceptor; b 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 diflficult, for it is but a 572 COLLECTED STUDIES IN IMMUNITY. natural consequence for us to assume that normal rabbit eenim con- tains the corresponding counter-group ^ in such high concentration that even small ^mounts 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 witli a, or whether, while the reaction takes ]>lace, 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 Dormal s 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 h 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 ANTI AMBOCEPTORS. 573 (0.25 cc). 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. Id active Normal Rabbit Serum. cc. Amount of Hsmob^. 0.01 0.006 0.003 0.0015 0 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 ^ of the antiserum constituent b by subsequently digesting them with small amounts of normal rabbit serum, thus a^ain 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 )9, 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 aipboceptor 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 b, once it is bound, rapidly be- comes firm, thus permitting only a partial dissolution by means of free p. In any event this experiment 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 amlxiceptor, that this influence will most likely become manifest with large amounts of antiamboceptor. Further- more, it can then happen that the conditious obtaining are those of the zone in which the curative action obtained with smaller doses ia concealed, owing to the excess of antiamboceptor. This may perhaps account for Morgenroth's negative findings;' 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 (hat Llordet 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 HiEmolysins.^ Chir 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 riecessary that the absence of the antiamboceptflrs' 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 Si.xth 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 amboceptois, OM of which acts only on ox blood, the other also on goat blood; ud that immunization with goat blood has exactly analogous renne ' J. MorRonrolh. Deflection of Complement by Means of Hemolytjo , oeptors. Cenlralblatt Bact. 1904. Vol. 35, No. 4. 'Ehrlich and Horgenrolh. See page 88. MECHANISM OF THE ACTION OF ANTI AMBOCEPTORS. 575 results. The plurality of amboceptors is further demonstrated by :be results of the isolysin experiments published by Ehrlich and Vloi^enroth,* for in these experiments the presence of antibodies icting against the complementophile group of the amboceptor can 36 excluded. The fact that we have drawn an incorrect conclusion •rom one single experiment certainly does not justify Bordet in deny- ng the existence of a plurality of antibodies (especially amboceptors) n a given immune serum ; the correctness of our view is established Dy a number of incontestable experiments. Borders arguments concerning deflection of complement by an jxcess of amboceptor may be answered in the same manner. Even granted that Morgenroth's view 2 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 Imctericidal sera. In these experiments absolutely no antiambo- jeptor 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 th^rie de la deviation du complement par Tambocepteur est une l^gende." Needless to say this will have little effect on our view. It is thus seen that Borders 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. Borders attack on the receptor theory has thus ' Ehrlicb and Morgenrotb, Third Communication. See page 23. ' J. Morgenrotb, 1. c. ' M. Neisser and Wechsberg. See page 120. « A. Lipstein, Centralblatt f ur 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 welcomo^ as supplementing the arguments supporting the amboceptor theory ^ The mistake contained in our previous conception of antiamboceptoi that they were antibodies directed against the cytophile group, is essentialW- one regarding the situation of the point of attack. In this connection we ma»^ look upon certain chemical substitutions as furnishing ready comparison; for •example, the different substances resulting when the benzole nucleus is substi* tuted in the ortbo, meta, or para positions. Considering how difficult these problems are, it is not surprising that a statement concerning localization wiU now and then be made which subsequent deeper study shows must be corrected Even so high an authority as Kekul^ once erred in defining a compound, and yet this did not in the least affect his fruitful hypothesis. In our case after tbe way had been cleared by the demonstration of the ''blocking of complemento" (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 oonect interpretation and transfer the site of the antiambocepter's action to tbe compfe- mentophile group. It is at once clear that this merely fulfills an old postulate of the side^hain theory. It would therefore be interesting to see bow 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 l>e demanded of immunization products of ''complementophile groups." XLL A GENERAL REVIEW OF THE RECENT WORK IN IMMUNITY.! By Paul Ehruch. Two yeaiB 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 rfeum6 of the literature. I may at once say, however, that very Uttle 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 Dungem, 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 bodies.^ ' This chapter is written expressly for 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 677 1578 TOiSs IN IMMITNITY. In view of the extraordinary success which physical chemistry IS st:ored, 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 tlie new field ot immunily. 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 tiiat 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 tliey can merely represent certain experimental resull* in the form of interpolation formulas, Neither do I believe that the phenomena observed in toxins and antitoxia'j bear any relation to the processes of colloid chemistry. The attempt which has been made t« interpret tlie 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 feare 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 stiidies, 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 ld 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 com[X)nents of the neutral toxin-antitoxin combination can be restored. This author succeeded in completely j recovering the two components of a neutral mixture of cobra venom J (Berl. kiin. Wocbenscbr. No. 8, 1906) have been effectuuUy anawerod by \ Calcar by means of Home additional eicperimeota, and by the denionstnilioiJ that the membranes employed by ROmer were unsuitable (Berl. klin. Woe No. 43, 1905). GENERAL REVIEW OF THE RECENT WORK IN IMMUNITY. 579 ^*d antitoxin by means of an ingenious method. But even here we ^^-te not dealing with a reversible reaction, for it requires certain ^^^anipulations to disrupt the neutral combination; thus, in the case ^Df cobra venom, the addition of hydrochloric acid is necessary. The :jieutral cobra-venom-antitoxin combination therefore behaves like a ^lucoside, 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,^ 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 dia^otizing 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 haemolytic inunune 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 Borders views. On the other hand, they were readily explained in accord- ance with the side-chain theory by assuming that the complement was deflected by an excess of amboceptor. But even if this expla- > Centralbl. f. Physiologie, Vol. XIX, No. 23. ^■€80 COLLECTED STUDIES IN IMMUNITY. ^H nation is not the correct one, as Gay has recently stated, it would 'a^'^^ ^M no way affect Ibe soundness of the amboceptor theor>'. The exist- ^ V ence of amboceptors is con6rmed by so many experimental consider- ^ ■ ations that it is no longer a postulate of the theorj', but is practically ■ the direct expression of observed pheuomena. The terra amboceptor, ■ of course, is used merely to express the two-sided affinity, to the I 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 I direct union of amboceptor and complement is confirmed by a host I of decisive observations. Of these, it will suffice to mention the teat-tube demonstration of complementoids which occupy the com- plementophile groups of the amboceptor. This demonstration haa ; been effected in other ways (Fuhrmann, Muir, Browning, and I Gay), so that the existence of complementoids is no longer evidenced \ merely by the possibility of producing anticomplements by means oE inactivated serum, but is demonstrated primarily by the unmLstak- I able interference of the complementoids in hiemolytic teat-tube experiments. It is not necessary that complementoids should always ' exert an inhibiting action on hiEnioh*sis; for it is obvious that (changes I in affinity may occur in consequence of external influences, physical, chemical, or chronological in nature. I believe that changes in affinity, I «ther positively or negatively, are of the highest imjwrtance in cor- rectly understanding the course of immunity reactions, although I do not deny the influence of certain catalytic factors on these proc- (von Behring, .Morgenroth, Otto, and Saeh.s). However, no general rule can be laid down. Experiments are constantly bringing forth surprises, but by diligent empiricism it is usually ]H>EsibIe to bring the many different observatjons into harmony with a single point of view. The original assumption, that amboceptor and complement (at least in the case of hjemolysins) exist free side by side, and that tlie 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 complcmentophile group), — this assumption has not proven ten- P.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, dkcovered 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 \ GENERAL REVIEW OF THE RECENT WORK IN IMMUNITY. 581 '^•^s the affinity of its cytophile group increased, so that now it is able unite with the cells. Thus far, such observations have been made ^yon normal amboceptors; and this fact explains why the numerous Itempts of various authors to separate normal hemolysins, by means f absorption at low temperatures, have failed.^ The amboceptors btained by immunization, on the other hand, regularly possess a 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- ^on 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 hemolysin, 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 m 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, tot 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 hsemolytic autoamboceptors in the serum of a patient suffering from paroxysmal luemoglubinaria. 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 am bo- 5S2 COLLECTED STUDIES IN IMMTINITY. which, as a result of biological experiences, I had always looked forward to. The correctneaa of the amboceptor theory formulated by Motgen- 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 antilylic antibodies were possible. In addition to the substances which act as anticom- plements, we could conceive of antiamboceptora 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 ia evident that the demonstration of antiamboceptors directed against the complementophile group ia by far the most important; for, owing to the mode of origin, the devel- opment of cytophile groujis 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 fumLihed the demonstration that the anti amboceptor developed with an immune, or with a normal serum, is usually directed against the complementophile group. This discovery verv' prettily demonstrates that the mechanism of hEemolysin 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 anti amboceptor. We know that it is possible to produce antiamboceptors by im- munizing with normal serum, and Pfeiffer and Friedberger hara shown that the action of the antiamboceptor serum extends to all the amboceptors of the animal species whose serum was used for inmiunization. These tacts are only apparently a contradiction of the specificity of amboceptors, for the specificity of the amboceplora 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 complementophile ocptor. Thia of course destroyB the foiindat are ba^ed on the luisiiniption that the react certain statements made by Bredig. of iVoguchi'a calculatioae, which a raveraible; it also dispoaea ol 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- nificance " (Koch Festschrift, 1903): "In general, the specific ambo- ceptors possess a miiform structure in their complementophile 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 Hemolysins, 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 ])y 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 overthro^^^l. This conception is supported by so many arguments of a different kind that the existence of partial amboceptors can })e 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 eo-called group reactions showing that the receptor apparatus and the antisera poBsess a highly multiple constitution. This fact, as is well known, has here lieen of great practical value. We see, therefore, that the plurality of the amboceptors, bo far aa the cytophile group is concerned, is an assured fact; the differentiation by means of anti amboceptors directed against the cytophile group can therefore very welt be foregone. The production of anti amboceptors against the cytophile group seems to encounter particular difficulties, for the complemen tophi le 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- piementophile 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 applies: (1) To the receptor employed in immunization, and this mani- fests it«elf in the configuration of the haptophore group; and (2) To the animal species from which the amboceptor is deri\'ed. The latter kind of specificity shows itself in the structure of the cora- plementophile apparatiLS, which, as we know, consists of a large number of individual complementophile groups. To this plurality 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 hajitophore 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 dispeased 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 alwolutely conclusive. Recent studies by Gengou. Moreschi, and Gay have shown that in the immunization with serum, antibodies directed against the albuminous constituents istituents ■ A GENERAL REVIEW OF THE RECENT WORK IN IBIMUNITY. 585 formed which, by uniting with the corresponding albuminous todies, possess the property of exerting anticomplementary effects. Cn this case, therefore, the anticomplement action is brought about oy the interaction of two components, one present in the serum of bhe 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 futiu^ 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 inununiz- 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 Schutze and of Uhlen- huth, one class of antibodies against dissolved albumins, namely, the precipitins, has been used, as is well known to differentiate albuminoas bodies of various origin. These have thus come to be successfully COLLECTED STUDIES IN IMMUNITY. employed in Lhe forensic demonstration of the origin of blood-atains. The same thing, of course, was possible In the case of the albunun amboceptors. This fact has recently been taken advantage of by M. Neisser and Sachs,* who have devised a procedure by which, by deflecting hipmo- lytic complements by means of albuminous bodies loaded with am- boceptor, they diagnosticate human blood, etc. The studj- of im- munity thus furnishes two biological methods for deciding a [xjint of vital importance in forensic medicine, namely, the origin of blood- stains. Considering the extreme importance of tests of this kind, 1 am convinced tliat hereafter it will be well to use this method in addition to the well-tried Uhlenhuth-Wassermann reaction. This brief r&um^, I believe, covere the chief points wliich have recently come up tor 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 in^'estigtlr 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 tliat these views are not so much a theory as a necessary abstraction of the observed facta, an abstraction which is necessaPi' 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, klin. Wochenschr. No. 44, 1905, and No. 3, 1006. SHORT-TITLE CATALOGUE OF THE PUBLIOATIONS OF JOHN WILEY & SONS, New York. Lokdok: chapman & HALL, LiMmD. ARRANGED UNDER SUBJECTS. Descriptive circulars sent on applicatiun. Books marked with an asterisk (*; are sold »t net prices only, a dnub!e asterisk (**) bonks sold under the rules of the American Publishers* Association at ttet prices subject to an extra charge for postage. All books are bound in cloth unless otherwise stated. AGRICULTURE. Armsbj's Manual of Cattle-feeding xamo, Sx 75 Priociples of Animal Nutrition 8vo, 4 00 Budd and Hansen's American Horticultural Manual: Part L Propagation, Culture, and Improvement. x3mo, x 50 Part II. Systematic Pomology. lamo, x 50 Downing's Fruits and Fruit-trees of America 8vo, s 00 Elliott's Engineering for Land Drainage lamo, i 50 Practical Farm Drainage lamo, i 00 Green's Principles of American Forestry lamo, i 50 Grotanfelfs Principles of Modern Dairy Practice. (Woll.) lamo, 3 00 Kemp's Landscape Gardening lamo, a so Maynard's Landscape Gardening as Applied to Home Decoration lamo, x 50 ^ McKay and Larsen's Principles and Practice of Butter-making 8vo, z 50 Sanderson's Insects Injurious to Staple Crops lamo, x 50 Insects Injurious to Garden Crops. (In preparation.) Insects Injuring Fruits. (In preparation.) Stockbridge's Rocks and Soils 8vo, 3 so Winton's Microscopy of Vegetable Foods 8vo, 7 50 WoU's Handbook for Farmers and Dairymen x6mo, z 50 ARCHITECTURE. Baldwin's Steam Heating for Buildings lamo, a 50 Bashore's Sanitation of a Country House lamo, x 00 Berg's Buildings and Structures of American Railroads 4to, 5 00 Birkmire's Planning and Construction of American Theatres 8vo, 3 00 Architectural Iron and Steel 8to, 3 50 Compound Riveted Girders as Applied in Buildings 8vo, a 00 Planning and Construction of High Office Buildings 8vo, 3 50 Skeleton Construction in Buildings 8vo, 3 00 Brigg's Modem American School Buikiings 8vo, 4 00 Carpenter's Heating and Ventilating of Buildings 8vo, 4 00 Freitag's Architectural Engineering 8vo, 3 50 Fireproofing of Steel Buildings 8vo, a 50 French and Ives's Stereotomy 8vo, a 50 1 Guhaid'B Gulds lo Saollu; BouK-iiiip«ticui ibi Theatre FliM and PwiUt lai ■Gleeoe's SlructunlMettunics . 8 BoUf't Caipenleis' and Joineis' Handbook i8c Johnson's Stalici hy Atscbcaic aad Graphic Utthodl. 8 Kiddei'sAichiiecti'andBulldtn'Pocliet-baak. KenitteD Edition. tCmo.D II Build in) neUUic 't Stair-building. : Their Occi 'THtise on Poandatian. , . Ric bey's Handboo Bimia's Modern Slonc-cuttinf and Masonry. ..,-.-....... 8^ icipHi Species ol Wood .8». 't Graphic Sutics with Applications (o Tmun, Beams, and Arcb □d Arehitccluial Juiiipruilencg . IS Preliminarr to Conslruclloa li md Elfdrotfiii ot Iron and Slee ARMY AND NAVY. Btmidou's Smokeless Pi lufi's Teil-boDk OTdi le Tlwotr ol the Ctllutew ChaK'i Screw P ClDkc's Gunne Craic's Aiimul s Poiariiini Pbolo-chronocraph. . j| Law le Hililary Law of United Slates. . De Brack's Caialrr OutposB Dutiet. Dieti's Soldier's First Aid Handbook . • Dredge's Modern French Artillei? Durnnd's Resistance end Propulsion c ■ Djtfs Handbook ol Light Artillery. EitBlrr's Modern High Eipktsivei. . . . • Fiebcger'B Teil-book on Field Foi '■ Catethim 'sEleoK of Probleiu o Elettroni gnetic Phcnonsoa. Volt. I. and n. .8n>. a rli- 16100, pioro. *< Cott of lUanfactarw—Aad tht AdminiftnliaB of Workaboyi. .8to. 5 00 Ortaonco and Otmnonr. a vok. lamo. 5 00 ly^i lafuitry Drill Roguktioiis. i8mo, faptr, 10 I's AdihttuttB* Mamtil. 24mo, i 00 ibo4y't HoTml Architoctara 8to. 7 so ** Plio^i Pncdcal Ifarino Sanreying. 8td, a 50 PoiwoITi Aniij Oflcor't Kwminfrr lamo, 4 00 Sharpo't Art of Snbtifltiiig ArmiM in War i8mo, morocco i 50 ^ Tupm and Foolt't Kanual of Bayonet Ezercitei and Xiaslcetry Fencing. 34mo, leather, 50 * Walka'e Lecturei on EzploaiTet. Sro, 4 00 * Whealer'e Siege Operationa and Military Mining Bro, 2 00 Winthrop'a Abridgment of Military Law xamo, 2 50 WoodhnU't Hotea on Military Hygiene x6mo, x 50 Tonng't Simple Elementa of Navigation x6mo» morocco, 2 o « ASSAYING. Fletcher*! Practical Instructiona in Quantitative Aasaying with the Blowpipe. xamo, morocco, Forman'a Mamial of Practical Aasaying Sro, Lodge's Notes on Aasaying and Metallurgical Laboratory Experiments .... 8vo, Low'a Technical Methods of Ore Axialysis Sro, Miller^ Manual of Assaying xamo, Minet^ Production of Aluminum and its Industrial Use. (Waldo. ) xamo, O'Driscoll's Notes on the Treatment of Gold Ores 8to, Ricketts and Miller's Notes on Assaying 8to, Robine and Lenglen*s Cyanide Industry. (Le Clerc.) 8to, Ulke's Modem Electrolytic Copper Refining 8vo, Wilaon'a Cyanide Processes xamo, Chlorination Process xamo, ASTRONOMY. Comstock's Field Astronomy for Engineers 8to, Craig's Azimuth 4to, Doolittle's Treatise on Practical Astronomy 8to, Oore's Elements of Geodesy 8vo, Hayford's Text-book of Geodetic Astronomy 8vo, Merriman's Elements of Precise Surveying and Geodesy 8to, * Michie and Harlow's Practical Astronomy. 8vo, * White's Elements of Theoretical and Descriptive Astronomy '. . xamo, BOTANY. Davenport's SUtistical Methods, with Special Reference to Biotogical Variation. 1 6mo, morocco, x as Thom<< and Bennett's Structural and Physiological Botany x6mo, a as Westermaier's Compendium of General Botany. (Schneider.) 8vo, a 00 CHEMISTRY. Adriance's Laboratory Calculations and Specific Gravity Tables. xamo, x as Allen's Tables for Iron Analysis 8vo, 3 00 Arnold's Compendium of Chemistry. (Mandel.) Small 8vo, 3 50 Austen's Notes for Chemical Students xamo, x 50 Bemadou's Smokeless Powder.— Nitro-cellulose, and Theory of the Cellulose Molecule xamo, a 50 * Browning's Introduction to the Rarer Elements 8vo, i so 8 X SO 3 00 3 00 3 00 X 00 a 50 a 00 3 00 4 90 3 00 X 50 X so SO SO 00 50 00 SO 3 00 a 00 m ClMseo's Ouamilati" Chemieiil Analysis by ElecUoljiii. (tollnooil.). 8to, QD Cohn's Indialors and T»st-papeis. , . . . IJmo, Teit»«niiRMEents. .. .,.. Bn, Cr.ft.-s Short Course in OuBlit.li». Ch.miul An.lyii.. fSc1l«dI«,). tJOio. SO Dolezalik'B Tbcoij of Ibc Liad Accomuliler sical Ubonlaiy Bipeilmenli. .. .8vo. Low's Technical Hetbod at Ore Analysis. . . . Byo. , Lunie'a Techno-chemical AnatyBi.. iCotin,) umo * McKsy und Lacsen's Principles and Practice o( Batlu-awkiDg 8*0. JO Mandel's Handhooti for Bio-chemiial Laboratory . . ijnro. • Manin-E Laboratory Guide to OuaUtalive Analysis with Ihc Bloin»pc. ilmo, 6a jd Edition, Rewrillen. ".. Bvo. E«aininationoIWat' timo •Fiebeger'g TrmtiK on Civil EniinKring 8yo ?lainu'« PhDIolapogiapbic Mettiodi and Inilnunenti. Std FolweU'E Sewerace. (Dnignini and Uaintenaace, ) Sre Fnltai'i Arcbileclunl Enfineiriag. id Edltfon. RewritUo 8to Francb and Ivei'i SltieotonJi , tro Goodbue's MuniclfMl Improvemenls. , . . .lama Goodrich's Economic DiipoHl ol Towni' RefuK Svo Gnre'i Elemeols of Qeqde»» 8»o HuTlord'i TtMt-booli ol Geodetic ABiionomy. 8vo Howe'i ReUinIng Walls lot Esith. iima JobOKjo'i (J. B.iTbeoiT and Praclice of Sur*eTiti|. Small 8to Jobnson'i ng4, SaDin-a Induslrial and Aninic Techaolciio' of Paints and Taraliii... . , Bvo \Z Snow's Principal Species of Wood '.*.*,*.*,'.'. .".'.'-*.'. .".".'-.*.'.','.'.".*.*. .Bvo Spalding's Hydraulic Cement iimo TeEl-book 00 Roads and PavefDcnta. ...-...,-.........-.,--,. .umo i" TburstOD's Materials ol Engineering. J Pails.. ..... Svo Part I. Bon-meialUc Materials of Engineering and Metallurgy. ... Bvo Part II. Iron and SteeL Bvo Pan m. A Treatise on Braises, BroDZei, and Other AUox and thel Thurston's Teit-hook of the Materials ol Conitiuction .Svo ;E Tinson's Street Pavements and Pavioa Materials. Bvo Waddell's De Pontihus. (A Pockel-book for Bridge Engin«r».). .i6n». mor Specificstions (or Steel Bridges unw 1 »s Wood's (De V. ) Treatise on ihe Resistance ol Materials, and an AwcBdii 0 the PreservBtion of Timber Bvo Wood's (De V.I Elements o( Analytical Mechanics Bvo Wood's (M. P.) Rustless Coatings: Corrosion and Electrotysls of Iron on ^H ^'"^ "" *oo ^^^^M RAILWAY ENGUfEERING. 1 IS ^F Berg's Bt-lldings and Structures ol American Railroads 4to ^H Brook's Handbook of Street Railroad Location , i6mo, mofocto 1 so ^H Bull's Civil Engineer's Field-book. . . ibmo. morocca ^M CrandsH's Transition Curve i6mo. moroeto 1 so 1 50 ^H Sawson-B "EoBineering" and Electric Tracilon Pocket-book . iGmo, morocco S 00 k i Dredge's Higtory of the Pennsyhrania Railroad: (1879) Paper, 5 00 ^ Drinker'i Tnniielliiic, EzploeiTe Compouads, and Rock I>ri]!i.4to, half mor., as 00 Tfsher^ Tahle of Cnhlc Yards Cardboard. 25 Godwin's RaihtMul Engineers' Field-book and Explorers' Guide. . . i6mo. mor., 2 50 Howard's Transition Cunre Field-book i6mo, morocco, i 50 Hudson's Tables for Calculating the Cubic Contents of Excavations and Em- bankments. 8to, I 00 MoUtor and Beard's Manual for Resident Engineers. x6mo, i 00 Nagle's Field Manual for Railroad Engineers x6mo, morocco, 3 00 Philbtick's Field Manual for Engineers z6mo, morocco, 3 00 Searles's Field Engineering z6mo, morocco, 3 00 Railroad Spiral i6mo, morocco, i 50 Tayfer's Prismoidal FormuUe and Earthwork 8vo, i 50 * Trautwine's Method of Calculating the Cube Contents of Excarations and Embankments by the Aid of Diagrams 8vo, a 00 The Field Practice of Laying Out Circular Curves for Railroads. - xamo, morocco, a 50 Cross-section Sheet Paper, as Webb's Railroad Construction z6mo, morocco, s 00 Wellington's Economic Theory of the Location of Railways Small 8vo, s 00 DRAWING. Barr's Kinematics of Machinery 8vo, * Bartlett's Mechanical Drawing 8vo, * " " " Abridged Ed 8vo, CooUdge's Manual of Drawing 8to, paper Coolidge and Freeman's Elements of General Drafting for Mechanical Engi- neers Oblong 4to, Durley's Kinematics of Machines 8vo, Emch's Introduction to Projective Geometry and its Applications 8vo, HilTs Text-book on Shades and Shadows, and Perspective 8vo, Jamison's Elements of Mechanical Drawing 8vo, Advanced Mechanical Drawing 8vo, Jones's Machine Design : Part I. Kinematics of Machinery 8vo, Part n. Form, Strength, and Proportions of Parts 8vo, MacCord's Elements of Descriptive Geometry 8vo, Kinematics; or, Practical Mechanism 8vo, Mechanical Drawing 4to, Velocity Diagrams 8vo, MacLeod's Descriptive Geometry.. Small 8vo, * Mahan's Descriptive Geometry and Stone-cutting 8vo, Industrial Drawing. (Thompson.) 8vo, Moyer's Descriptive Geometry g\ro. Reed's Topographical Drawing and Sketching 4to, Raid's Course in Mechanical Drawing 8vo, Text-book of Mechanical Drawing and Elementary liachine Design. 8vo, Robinson's Principles of Mechanism 8vo, Schwamb and Merrill's Elements of Mechanism 8vo, Smith's (R. S.) Manual of Topographical Drawing. (McMillan.) 8vo, Smith (A. W.) and liarx's Machine Design. 8vo, Warren's Elements of Plane and Solid Free-hand Geometrical Drawing, zamo. Drafting Instruments and Operations zamo, Manual of Elementary Projection Drawing zamo. Manual of Elementary Problems in the Linear Perspective of Form and Shadow zamo. Plane Problems in Elementary Geometry zamo, 0 2 50 3 00 50 00 50 00 50 00 50 00 50 00 00 CO 00 50 50 50 50 00 00 00 00 00 CO 50 00 00 as 50 00 as d PcrtpKti«e. WaiTBn'i PrimaiT GeamalrT ElomMiU of DcBcripUiB GeDmeDj'i Stud Gsncral Problcnu of Shid« and Shidav ElemenU of Hachinc Conitruction and Dnwloi. Problcnu, Theoiinu, and Etamplcs in DMctiptive CaoraaliT. . Wcblwch't Kinemalica .aad Power of Tni Whtlplt]''* Pnctic*] Imlructioii la the Ait of Latter EB(H*iDg. . Wilaon'i (H. M.l Topogiaphie Surroyine. Witaon'l IV. T.) Free-hand PeripBcli™... Wilion'atV. T.) 'b ElcmeDtarf Count la Oocrlptivs Ggometty Lubb Svo ELECTRICiry ABD PHYSICS. If s Tail-book of Phnici. (Uagie.' .... otes on the Theory ol Elect BottmMd.KSvo. -modjnamicB CliaSEd'g Quantitative Cbemlcal Analj^i by Electrolysii. Crehon aad Squier'i PoluiiiDS Ptaoto.chronograph ., . Svo Damon's "EnsioMfioi" and Eliciric Traction Pocket-book . ifimo, morocca Doleialek*! Theory of the Lead Accumulator (Slarace Baiter^). iVai Ende.) Iimo iDd Chcmislry. [Buckch.) Svo Id Ibe Heasuremenl of Power. . . ;...,... .iimo Gilberfa De Magncic. iMottelay.) Svo Hanchetfs Allernaling Cuntnu Eiplaiaed .Iimo Bering's Ready Relerence Tables (Conveninn Faclera) tSnu. maraeco Hohnan's PteciBion ol MeasurementB. . . .8yo Teleuopic Uirror-aule Hctbod, AdjiulmeDle, and TmM !«[■■ 3to Kinzbruiuier'g Teitiai of Continuoua^uireal Hachinei. 8*0 Landauer'i SjKctrua AoalyBls. (Tinile.) , .Svo LcChatelletiHiEh-iempfiacureUEasuienienU, (Boudouanl — Burgen.) iitno. Lob's ElecDochemislry of Organic Compouoda, iLoreni.l... . . • Lyoni'i Tintise on Electro magnetic Phenomena. Vols. L and n, 8*0. • Hichie's Element! of Ware Motion ficlatlns ID Sound and Liibl.... Hiaudct'i Blemenuiy Tiealise on Electric Batteries. IFiabbsck.). . . . i • RoMnberi't Electrical Engineering. (Haldane Qe«— Kiuibniniui.). . [ie'a Electrical Urehloeiy. Thur ■i Elemi todem Electrolylic Cop LAW. if United StalM. .Svo lioni PreUmlnary to Cooitruction in Bntiowrinc u • « MAHUFACTURES. Bcniadou't Smoktlen Povdtr— Nitro-ceUulow and Th»orj of the CeOiiloM Molecule lamo, 2 50 BoUuid's Iron Founder. lamo, a 50 founder," Supplement zamo, a 50 >pedui of Founding and Dictionary of Foundry Termt Used in the Practice of Moulding xamo, 3 00 * Eckel's Cements, Limes, and Plasters 8to, 6 00 Eissler's Modem High Explosives. 8vo, 4 00 Elfronfs Enzymes and their Applications. (Prescott) 8to, 3 00 Fitzgerald's Boston Machinist zamo, z 00 Ford's Boiler Making for Boiler Makers. z8mo, z 00 Hopkin's Oil-chemists' Handbook. 8vo, 3 00 Keep's Cast Iron. 8to, a 50 Leach's The Inspection and Analysis of Food with Special Reference to State Control Large 8vo, 7 50 * McKay and Larson's Principles and Practice of Butter-making 8to, z 50 Matthews's The Textile Fibres. 8to, 3 50 Metcalf's SteeL A Manual for Steel-users. zamo, a 00 Metcalfe's Cost of Manufactures — And the Administration of Workshops. 8to, 5 00 Meyer's Modem Locomotire Construction 4to, zo 00 Morse's Calculations used in Cane-sugar Factories z6mo, morocco, z 50 * Reisig's Guide to Piece-dyeing 8to, as 00 SaUn's Industrial and Artistic Technology of Paints and Varnish 8to, 3 00 Smith's Press-working of Metals. 8vo, 3 00 Spalding's Hydraulic Cement zamo, a 00 Spencer's Handbook for Chemists of Beet-sugar Houses. .... z6mo, iziorocco, 3 00 Handbook for Cane Sugar Manufacturers z6mo, morocco, 3 00 Taylor and Thompson's Treatise on Concrete, Plain and Reinforced 8vo, 5 00 Thurston's Manual of Steam-boilers, their Designs, Construction and Opera- tion 8vo, 5 00 * WaDce's Lectures on Explosives. 8to, 4 00 Ware's Beet-sugar Manufacture and Refining Small 8vo, 4 00 West's American Foundry Practice zamo, a 50 Moulder's Text-book zamo, a 50 WolfTs Windmill as a Prime Mover 8vo, 3 00 Wood's Rustless Coatings: Corrosion and Electrolysis of Iron and Steel. .8vo, 4 00 MATHEMATICS. Baker's Elliptic Functions 8vo, z 50 * Bass's Elements of Differential Calculus zamo, 4 00 Briggs's Elements of Plane Analytic Geometry zamo, z 00 Compton's Manual of Logarithmic Computations zamo, z 50 Davis's Introduction to the Logic of Algebra 8vo, z 50 * Dickson's College Algebra Large zamo, z 50 * Introduction to the Theory of Algebraic Equations Large zamo, z as Emch's Introduction to Projective Geometry and its Applications 8vo, a so Halsted's Elements of Geometry 8vo, z 7S Elementary Synthetic Geometry 8vo, z so Rational Geometry zamo, z 7S * Johnson's (J. B.) Three-place Logarithmic Tables: Vest-pocket size. paper, zs zoo copies for $ 00 ^ Mounted on heavy cardboard, 8X xo inches, as zo copies for a 00 Johnson's (W. W.) Elementary Treatise on Differential Calculus. . Small 8vo, 3 00 Elementary Treatise on the Integral Calculus Small 8vo, z 50 11 Johnsoa-i (W. W.) CutTO Tmcing in Canoiin CiwiidiMtei ijido , SmaU Std Johnson'. 1 W. W. ) Tbwry of Error, ud the H.thod of Leml Sqiuuei i jno , 8.Woodw«d 0CU.O, Mth I Ko. 3. Synthetic ProjcEtlve GcomelrT. by George Bruce BbIiIhI. 4 bolie Functions, by Jamet HcM.hon. Nd. S- H«monic Func- * tion.. bT WiUiiun E. Byerlr. Ho. 6, Gniuninn-a SpMC Ajulr.ie. bT EdwKd W. Hyde. Bo. T. Prob.bility md Theory of Errors. by Alenndrr Mactirlane. No. 0. Differential Equalioas. by . . by ThDmu S. Fidu. 4 Mjniinan'B Method of Leasl Squares ,. 8ni DiHmntiil and Integral Calculus. 1 vola. in on. SniU Byo I MECHANICAL ENGmEERIWG. UATBRIALS OF EHGIHEEIUnG, STEAH-EKOIIfES tXD BOIL£R& *.- Abfidied CuriKnter's EiperimenUl Engineering &^ Heating and Ventilating Buildingi Si Cory's Smoke Suppreuion in Pliali u«inc BltuminoB* CoaL (In Prepua- Uon.l Clerk's Gas and Oil Engine SniaU 8to ts of General Drafting tor Hedunical En- gineers --.-.-,-,...,. Oblong 410, Cromwell's Treatise on Toolhed Gearing TreatiK an Belt* and Pullers Durley's Kinematic* of Uachlnes Flalher'a Dynamo meters Hope Di Id Fuel lalysis r EnglnHT*. ■ . Button's The Gas Encine 8vo. Jamison's Mechanical Drawing 8vo, Jones's Machine Design : Part L Blinematics of Machinery 8to, Part IL Form, Strength, and Proportions of Parts. 8vo, Kent's Mechanical Engineers' Pocket-book i6mo, morocco, Kerr's Power and Power Transmission 8vo, Leonard's Machine Shop, Toob, and Methods 8to, * Loreni's Modem Refrigerating Machinery. (Pope, Haven, and Dean. ) . . 8to, MacCord's Kinematics; or* Practical Mechanism. 8to, Mechanical Drawing 4to, Velocity Diagrams 8to, MacFarland's Standard Reduction Factors for Oases. 8to, Mahan's Industrial Drawing. (Thompson.) 8to, Poole's Calorific Power of Fuels. 8vo, Reid's Course in Mechanical Drawing 8to, Text-book of Mechanical Drawing and Elementary Machine Design. 8vo, Richard's Compressed Air zamo, Robinson's Principles of Mechanism 8vo, Schwamb and Merrill's Elements of Mechanism 8vo, Smith's (O.) Press-working of Metab 8vo, Smith (A. W.) and Man's Machine Design 8to, Thurston's Treatise on Friction and Lost Work in Machinery and Mill Work 8vo, Animal as a Machine and Prime Motor, and the Laws of Energetics. z2mo, Warren's Elements of Machine Construction and Drawing 8to, Weisbach's Kinematics and the Power of Transmission. (Herrmann — Klein.) 8vo, Machinery of Transmission and Oovemors. (Herrmann — Klein.). .8vo, Wolflf's Windmill as a Prime Mover. 8vo, Wood's Turbines 8to, 5 oo a 50 I 50 3 oo 5 oo 3 oo 4 oo 4 oo 5 oo 4 oo z so z 50 3 50 3 00 2 00 3 00 z 50 3 00 3 oo 3 00 3 00 3 00 z 00 7 50 5 00 5 00 3 00 a 50 MATERIALS OP ENGINEERING. * BoTey's Strength of Materiab and Theory of Structures. 8vo, 7 50 Burr's Elasticity and Resistance of the Materiab of Engineering. 6th Edition. Reset 8vo, 7 50 Church's Mechanics of Engineering 8vo, 6 00 * Greene's Structural Mechanics 8vo, 2 50 Johnson's Materiab of Construction 8vo, 6 00 Keep's Cast Iron 8vo, 2 50 Lanza's Applied Mechanics 8vo, 7 50 Martens's Handbook on Testing Materiab. (Henning.) 8vo, 7 50 Maurer's Technical Mechanics 8to, 4 00 Merriman's Mechanics of Materiab 8vo, 5 00 Strength of Materiab zamo, z 00 Metcalf's Steel. A manual for Steel-users Z2mo, a 00 Sabin's Industrial and Artistic Technology of Paints and Varnish 8vo, 3 00 Smith's Materiab of Machines zamo, z 00 Thurston's Materiab of Engineering 3 vols., 8to, 8 00 Part II. Iron and SteeL 8to, 3 50 Part III. A Treatise on Brasses, Bronzes, and Other AUoys and their Constituents 8vo, a 50 Text-book of the Materiab of Construction 8to, 5 00 Wood's (De V.) Treatise on the Resistance of Materiab and an Appendix on tue Preservation of Timber 8vo» a 00 13 1 ,8«. .8>o 8mo 8to Sto .Svo .8to .Bto ,4lo uno .8.0 Sto 8»o Sto .S10 Sto .Bto .Svo .Bto Sto 81c 8to .8vo .Bm .8to Svo .evD 8.0 1 1 I 50 1 JO 10 at > so 1 so a so 1 OS 1 50 I 50 3 SO I so 5 00 1 so i ■ Wood-1 <«. P.) RiuUaa CoUiatt: Corroiioo .od EleeltolTiU o» In. ^^H steah-engihes akd boilers. Camof* Hiflettioiu on ihc Halivt Pontr of He»l (ThoraWo.) Da»H>a-| ■■ Eoginecrinc " und Electric IrmclioD PodMl-book, . . . iGow Ooss's LMomoIiv* Sjaiks. , Beo«nwBr's Indic.for Pmciice and Sl>un-«iiciM Eeoaomr Tsblo of the Ptopertiei of Siiunted Steun «D« Otbai Vapon . . . Pisy's Twenty Yttrt with Uu lodicalor. , . . Lari (Oslirbere.t Thonue's Sltam-turbinea .... . . Tliut»lDn-s H»ndr T.biM. . P«I I, BistDTT. Slmcture. ■nd Tlmory Banitbook o( Engine and Boiler Tiiali, and tbe Uh of UU bidlcatc Manual of Steam-hoile™. theL. DeMgns. Consiniciion, and Opatatlon , . , Weisbach's Heat. Sleam. and Steam-engiEM. (Du Boil.) Whilham'i Slcain-eniine Dusign MECHAHICS AHD MACHlUfeRY. Notes and E.ampiti in Hechaoici ComploD'. Firm Leuoqi in Metal- working. ... Complon and De Groodl't Tbe Speed Lathe , 1 ^'' Cromwell's Treatite on Toothed Grearing zamo, i so Treatise on Belts and Puneys. iamo» ' so Dana's Text-book of Elementary Mechanics for Colleges and Schools. . lamo, i 50 Dingey's Machinery Pattern Making lamo, a 00 Dredge's Record of the TransporUtion EzhibiU BuiMing of the WorU's Columbian Exposition of 1893 4to half morocco» 5 00 Da Bois's Elementary Principles of Mechanics: VoL L Kinematics 8to. 3 50 VoL II. SUtics 8vo, 4 00 Mechanics of Engineering. VoL L Small 4to, 7 50 VoL IL Small 4to, 10 00 Durley's Kinematics of Machines. 8to. 4 00 Fitzgerald's Boston Machinist i6mo, i 00 Flather's Dynamometers, and the Measurement of Power xamo* 3 00 Rope Driring xamot a 00 Goss's Locomotive Sparks 8to, a 00 * Greene's Structural Mechanics. 8to, a so Hall's Car Lubrication. lamo, z 00 Holly's Art of Saw Filing z8mo, 75 James's Kinematics of a Point and the Rational Mechanics of a Particle. Small 8to, a 00 * Johnson's (W. W.) Theoretical Mechanics. lamo, 3 00 Johnson's (L. J.) Statics by Graphic and Algebraic Methods. Sto* a 00 Jones's Machine Design : Part I. Kinematics of Machinery 8to, i 50 Part n. Form, Strength, and Proportions of Parts. 8to, 3 00 Kerr's Power and Power Transmission. 8vo, a 00 Lanza's Applied Mechanics 8to, 7 50 Leonard's Machine Shop, Tools, and Methods 8vo, 4 00 * Lorenz's Modem Refrigerating Machinery. (Pope, HaveUf and Dean.). 8to, 400 MacCord's Kinematics; or, Practical Mechanism. 8to, 5 00 Velocity Diagrams 8vot i 50 Maurer's Technical Mechanics 8to, 4 00 Merriman's Mechanics of Materials Svo, 5 00 * Elements of Mechanics lamo* z 00 * Michie's Elements of Analirtical Mechanics. 8vo, 4 00 Reagan's Locomotires: Simple, Compound, and Electric xamo, a 50 Reid's Course in Mechanical Drawing 8to, a 00 Text-book of Mechanical Drawing and Elementary Machine Design. 8vo, 3 00 Richards's Compressed Air lamo, i 50 Robinson's Principles of Mechanism. 8to, 3 00 Ryan, Norris, and Hoxie's Electrical Machinery. VoL 1 8vo, a 50 Schwamb and Merrill's Elements of Mechanism. 8to, 3 co Sinclair's Locomotire-engine Running and Management. lamo, a 00 Smith's (0.) Press-working of Metals 8to, 3 00 Smith's (A. W.) Materials of Machines xamo» z 00 Smith (A. W. ) and Marx's Machine Design. 8to, 3 00 Spangler, Greene, and liarshaL's Elements of Steam-engineering 8to, 3 00 Thurston's Treatise on Friction and Lost Work in Machinery and Mill Work 8vo, 3 00 Animal as a Machine and Prime Motor, and the Lawc of Energetics. zamo, X 00 Warren's Elements of Machine Construction and Drawing 8vo, 7 50 Weisbach's Kinematics and Power of Transmission. (Herrmann — Klein. ).8vo, 5 00 Machinery of Transmission and Governors. (Herrmann — Klein. ).8vo, 5 00 Wood's Elements of Analytical Mechanics. 8vo, 3 00 Principles of Elementary Mechanics. zamo, z as Turbines. 8vo, a 50 The WorU's Columbian Exposition of z8q3 4to, z 00 15 METALLURGY. Eticsten's UeUUurgr <>( Silver, Gold, oi •• IIm's LmiJ- Kecp-a Co. KuDhardt' Iron. .olOi LeChitellii'iHlgh- HetcalTB StecL A HuuKl tc Minel'! Production ol Alumli Robins and Unslcn's Cyaold Smiih-B Uaiectils ot Uachloe I of Engii PoiUie a ccali additiDiuL). . , : Drtning la Euioix SKcV-UHn im and ill lodutuial Uh. (Wilde.). . Induttrj. (Le Clerc.) Pari II. Part HI, Com Ulko-i Hodcll In Three ParlB. 8 A Treailse oa BiasuB, Broniei. and Olhtr AUon and tl ilutnW.., . S Eleclialj^ic Copptr ReflninB Bto. MINERALOGY. si Virignia. -Pockrl-lKioliti Sto, 'e Uintniatj. (PenReld.). . Clieiter's Caialopje of Minetali Clolb, DicIlonaiT of the namei of Uinet Daca'i Syiitm of MinrtaloBy. . . Fitir Apjirnilii lo Dinn's Hew " S Teit-book of Mineralogy. Mlnitato and How lo Sludy Then Catalogue of American Loraliiln of Hinerali. .Large Sio. Uannal of Mlreralagy and Pe ttognphy cal Addreates on Technical Subtecti. . Eakle'i Mineral Eeleiton's Calnlc i[ Mine ock-foTmlni Mine Their Occurricce ■h. ISaith.).Sm«U»i * Penfleld'i Holea on Delerminalire Hlnerj Ro«obii»ch's Microscopical Phytiogiaphy c (Iddinge.l • TlUnjau'i Teil-book of lioporlanl Minerals a ! Sock-making Hlncr*^ fieard't Ventilallcm ol Hlnei umo. Boyd's Reaoureei of South»«t VlrginU f *o, Map of Southweal Virginia Pocket-book (oim Douglas's ODlechnlcAlAddreSKBonT«hnlcalSubJMt(. ,11100 • Drlnker'a Tunneling. Eiplmive Compounds, and Roc i Drills, 4U,lil. mat., Eisslir'a Modern High EiptoiiTes. 8*s IB SO J 00 I Goodyear's Coal-mines of the Western Coast of the United States. iamo» a go. Ihlsenc's Manual of Mining 8to, 5 00 ** nes's Lead-smeltinc. (Poetafe 9c. additional) lamo, a 50 Ktinhardf s Practice of Ore Dressing in Europe 8vo, i 50 O'DriscoO's Hotes on the Treatment of Gold Ores. ^o, a 00 Robine and Lenglen's Cyanide Industry. (Le Clerc.) 8to, 4 00 * Walke's Lectures on Ezplosires. 8vo, 4 00 Wilson's Cyanide Processes. xamo, z 50 Chlorination Process. ! zamo, i 50 Hydraulic and Placer Mining lamo, 2 00 Treatise on Practical and Theoretical Mine Ventilation. lamo, i 35 SANITARY SCIENCE. Bashore's Sanitation of a Country House lamo, Folwell's Sewerage. (Designing, Construction, and Maintenance.) 8vo, Water-supply Engineering 8vo, Fowler's Sewage Works Analirses lamo, Fuertes's Water and Public Health lamo, Water-fihratiop Works lamo, Gerhard's Guide to Sanitary House-inspection i6mo, Goodrich's Economic Disposal of Town's Refuse Demy 8to, Hazen's Filtration of Public Water-supplies 8to, Leach's The Inspection and Analysis of Food with Special Reference to State Control 8vo, Mason's Water-supply. (Considered principally from a Sanitary Standpoint) 8to, Examination of Water. (Chemical and Bacteriological.) i2mo, Ogden's Sewer Design lamo, Prescott and Wlnslow's Elements of Water Bacteriology, with Special Refer- ence to Sanitary Water Analysis i2mo, * Price's Handbook on Sanitation i2mo, Richards's Cost of Food. A Study in Dietaries X2mo, Cost of Living as Modified by Sanitary Science z2mo, Cost of Shelter 1 2mo, Richards and Woodman's Air. Water, and Food from a Sanitary Stand- point 8vo, * Richards and Williams's The Dietary Computer Svo, Rideal's Sewage and Bacterial Purification of Sewage Svo, Turneaure and Russell's Public Water-supplies Svo, Von Behring's Suppression of Tuberculosis. (Bclduan.) z2mo, Whipple's Microscopy of Drinking-water Svo, Winton's Microscopy of Vegetable Foods Svo, WoodhuU's Notes on Military Hygiene i6mo, * Personal H/glene i2mo, MISCELLANEOUS. De Fursac's Manual of Psychiatry. (Rosanoff and Collins.). . . .Large X2mo, 2 50 Emmons's Geological Guide-took of the Rocky Mountain Excursion of the International Congress of Geologists Large fvo, i 50 Fcrrel's Popular Treatise on the Winds Svo 4 00 Haines's American Railway Management i2mo, 2 50 Mott's Fallacy of the Present Theory of Sound i6mo, x 00 Ricketts's History of Rensselaer Polytechnic Institute. X824-1894. .Small Svo, 3 oc Rostoski's Serum Diagnosis. (Bolduan.) i2mo. i 00 Rotherham's Emphasized New Testament Large Svo, 2 00 17 I eo 3 00 4 00 a 00 X 50 a 50 z 00 3 50 3 00 7 50 4 00 I 25 2 00 I as X 50 I 00 I 00 I 00 2 00 I 50 3 50 5 00 I 00 3 50 7 50 I 50 I 00 Steel's Treatise on the Diaeuet of the Dog. 8to. 3 50 The World's Columbian Exposition of xSga 4to, z 00 Von Behring's Suppression ot Tuberculosis. (BoUuan.) lamo, z 00 Winslow's Elements of Applied Microscopy xamo. z 50 Worcester end Atkinson. Small Hospitals. Establishment and Maintenance; Suggestions for Hospital Architecture: Plains for Small Hospital. lamo* z 25 HEBREW AlTD CHALDEE TEXT-BOOKS. Onen's Elementary Hebrew Grammar. zamo, i as Hebrew Chrestomathy 8to, 2 00 Gesenius's Hebrew and Chaldee Lexicon to the Old Testament Scriptures. (Tregelles.) Small 4to, half morocco, 5 00 Letteris's Hebrew Bibte. Svo. a as 18 LANE MEDICAL LIBRARY STANFORD UNIVERSITY MEDICAL CEPfTER STANFORD. CALIFORNIA 94J0J FOR RENEWAL: PHONE 723-6691 DATE DUE APR Z 1 ZOIIO MAR 3 0 2W[