BIOLOGY LIBRARY UNIVERSITY OF CALIFORNIA SCHOOL OF PUBLIC HEALTH 3563 LIFE SCIENCES BUILDING r. LEGGE, M. D. 6 ROBLE ROAD BERKELEY, CALIF. WORKS BY CHARLES F. BOLDUAN, M.D. PUBLISHED BY JOHN WILEY & SONS Immune Sera. A concise exposition of the main facts and theories of infection and immunity, Fourth edition, rewritten. By Charles F. Bolduan, M.D. 12mo, ix + 226 pages. Cloth, $1.50. TRANSLATIONS. The Suppression of Tuberculosis. Together with Observations concerning Phthisio- genesis in Man and Animals, and Suggestions con- cerning the Hygiene of Cow Stables and the Pro- duction of Milk for Infant Feeding, with Special Reference to Tuberculosis. By Professor E. von Behring, University of Marburg. Authorized Translation by Charles F. Bolduan, M.D. 12mo, vi + 85 pages. Cloth, $1.00. Manual of Serum Diagnosis. By Doctor O. Rostoski, University of Wurzburg. Authorized Translation by Charles F. Bolduan, M.D. 12mo, vi + 86 pages. Cloth, $1.00. Studies in Immunity. By Professor Paul Ehrlich. Translated by Charles F. Bolduan, M.D. Second Edition, Revised and Enlarged. 8vo, xi + 712 pages. Cloth, $6.00. IMMUNE SERA A CONCISE EXPOSITION OF THE MAIN FACTS AND THEORIES OF INFECTION AND IMMUNITY BY DR. CHARLES FREDERICK BOLDUAN BACTERIOLOGIST, RESEARCH LABORATORY, DEPARTMENT OF HEALTH CITY OF NEW YORK FOURTH EDITION, REWRITTEN AND ENLARGED FIRST THOUSAND NEW YORK: JOHN WILEY & SONS LONDON: CHAPMAN & HALL, LIMITED 1911 9tl 67 COPYRIGHT, 1907, 1908, 1911. BT CHARLES FREDERICK BOLDUAN THE SCIENTIFIC PRESS ROBERT DRUMMOND AND COMPANY BROOKLYN, N. Y. TO HERMANN M. BIGGS, M.D., LL.D. PIONEER IN SPECIFIC SERUM THERAPY AND SERUM DIAGNOSIS IN AMERICA, FOUNDER OF THE FIRST MUNICIPAL SERUM LABORATORY IN THE WORLD, THIS VOLUME IS DEDICATED AS A TOKEN OF GREAT ADMIRATION AND ESTEEM PREFACE TO THE FOURTH EDITION THE favorable reception accorded to the previous editions of this book, together with the fact that our knowledge of the subject has increased con- siderably in the past few years, has led the author to undertake a thorough revision of the work. In its first edition, in 1904, this book dealt only with certain antibodies whose discovery had aroused a great deal of scientific interest, namely, hsemoly- sins, cytotoxins, and precipitins. To this was added, in subsequent editions, a discussion of anti- toxins, agglutinins, and opsonins. All these topics were naturally embraced under the title " Immune Sera." In the present (fourth) edition, while the old title has been retained, the scope of the sub- ject matter has been greatly extended, so that now there is presented an exposition of the main facts of infection and immunity. It is but natural that any discussion of the immunity reactions should center about the ingen- ious side-chain theory of Ehrlich, which has domi- nated the work in this field. Its heuristic value vi PREFACE has unquestionably been very great. At the same time it cannot be doubted that some of the deduc- tions from the theory have led, here and there, to strained conceptions which apparently violate established biological facts. While presenting Ehrlich's views at length, therefore, the author has endeavored to bring out clearly just why and wherein certain other investigators differ. The aim of the book has been to present a broad, clear outline of the main facts and theories concerning infection and immunity, and while this may perhaps have led to the omission of some really excellent studies, it was felt best not to confuse the beginner with a mass of apparently contradictory observations. CHARLES BOLDUAN. NEW YORK, April, 1911. CONTENTS PAGE Antitoxins i HISTORICAL ; i PRESENT METHOD OF PRODUCING DIPHTHERIA ANTI- TOXIN 2 PRODUCTION OF DIPHTHERIA TOXIN 2 IMMUNIZING THE ANIMALS 3 COLLECTING THE SERUM 4 TESTING THE STRENGTH OF THE SERUM 5 EHRLICH'S THEORY FOR PRODUCTION 6 TOXINS, TOXOIDS 6 RECEPTORS 9 WEIGERT'S OVERPRODUCTION THEORY 10 EXPERIMENTAL EVIDENCE FOR EHRLICH'S THEORY... . 13 ANTIGENS OR HAPTINS 16 NATURE OF ANTITOXINS IN GENERAL 17 TOXINS AND OTHER POISONOUS CELL DERIVATIVES IN GENERAL 20 RELATIONS BETWEEN TOXIN AND ANTITOXIN 22 "L "and "L t " 23 PARTIAL SATURATION METHOD OF STUDYING TOXINS TOXONS, TOXOIDS 23 EHRLICH'S " POISON SPECTRA " 24 VIEWS OF ARRHENIUS, BORDET, AND OTHERS 28 Agglutinins 3 2 THE PHENOMENON 32 H^MAGGLUTININS 34 ISOAGGLUTININS 35 PURPOSE OF AGGLUTINATION 36 HISTORICAL 37 PFAUNDLER'S REACTION (THREAD REACTION). . 38 vii CONTENTS PAGE NATURE OF AGGLUTININS AND OF THE AGGLUTINATION REACTION ...................................... 39 AGGLUTINOIDS ...................................... 39 GROUP AGGLUTININS : ............................... 43 ABSORPTION METHODS FOR DIFFERENTIATING BETWEEN A MIXED AND A SINGLE INFECTION ................ 46 FORMATION OF AGGLUTININS ACCORDING TO THE ^IDE- CHAIN THEORY, RECEPTORS OF FIRST, SECOND, AND THIRD ORDER .................................. 47 Bacteriolysins and Haemolysins .................. 5 o HISTORICAL ........................................ 50 FFEIFFER'S PHENOMENON ............................ 51 HAEMOLYSIS ........................................ 52 NATURE OF H^MOLYTIC SERA .................... . . 54 ANALOGY BETWEEN THE BACTERIOLYTIC AND HVEMO- LYTIC PROCESSES ............................... 57 EHRLICH AND MORGENROTH ON THE NATURE OF HAEMO- LYSIS .......................................... 58 THEIR THREE CLASSIC EXPERIMENTS ................ 59 NOMENCLATURE ................................... 63 ROLE OF THE IMMUNE BODY ............. .- ......... 64 ON WHAT THE SPECIFICITY DEPENDS ................ 65 DIFFERENCE BETWEEN A SPECIFIC SERUM AND A NOR- MAL ONE ......................... .. ............ 66 DIVERGING VIEWS OF EHRLICH AND BORDET ........... 67 THE SIDE-CHAIN THEORY APPLIED TO THESE BODIES. . . 68 MULTIPLICITY OF COMPLEMENTS ...................... 70 THE BORDET-GENGOU PHENOMENON; NEISSER- SACHS BLOOD TEST ............................. 71 NORMAL SERUM, ITS H^MOLYTIC AND BACTERIOLYTIC , ACTION ........................................ 73 ACTIVE AND INACTIVE NORMAL SERUM ....... '. ...... 75 ACTION NOT ENTIRELY SPECIFIC .................... 77 MULTIPLICITY OF THE ACTIVE SUBSTANCES ........... 78 DIFFERENCE BETWEEN A NORMAL AND A SPECIFIC IM- MUNE SERUM ................................... 79 NATURE OF THE IMMUNE BODY PARTIAL IMMUNE BODIES OF EHRLICH. . 82 CONTENTS { x PAGE METCHNIKOFF'S VIEWS 84 SUPPORT FOR EHRLICH'S VIEW 85 ANTIH^MOLYSINS: THEIR NATURE ANTI-COMPLEMENT OR ANTI-IMMUNE BODY 86 ANTI-COMPLEMENT 88 FLUCTUATIONS IN THE AMOUNT OF THE ACTIVE SUB- STANCES IN SERUM 91 SOURCE OF THE COMPLEMENTS LEUCOCYTES AS A SOURCE OTHER SOURCES 93 STRUCTURE OF THE COMPLEMENTS COMPLEMENTOIDS. . . 94 ISOLYSINS AUTOLYSINS ANTI-ISOLYSINS 97 DEFLECTION OF COMPLEMENT 100 PRACTICAL VALUE OF ANTI-BACTERIAL SERA 104 Precipitins 107 DEFINITIONS 107 BACTERIAL PRECIPITINS 108 LACTOSERUM OTHER SPECIFIC PRECIPITINS 108 SPECIFICITY OF THE PRECIPITINS 109 NATURE OF THE PRECIPITINS in PRACTICAL APPLICATION 112 THE WASSERMANN-UHLENHUTH BLOOD TEST 113 IMMUNIZING THE ANIMALS 114 COLLECTING THE SERUM 115 THE TEST 116 APPEARANCE OF THE REACTION 117 DELICACY OF THE PRECIPITIN TEST 118 OTHER APPLICATIONS OF THE PRECIPITIN TEST , . . 1 18 ANTIPRECIPITINS ISOPRECIPITINS 119 Cy totoxins 121 DEFINITION, LEUCOTOXIN NATURE OF THE CYTOTOXIN ANTICYTOTOXIN 121 Nt v UROTOXIN 122 SPERMOTOXIN 123 COMMON RECEPTORS 124 CYTOTOXIN FOR EPITHELIUM 124 CYTOTOXINS BY THE USE OF NUCLEOPROTEIDS 125 Opsonins 127 HISTORICAL 127 X CONTENTS PAGE BACTERIOTROPIC SUBSTANCES 129 OPSONINS DISTINCT ANTIBODIES. 130 STRUCTURE OF OPSONINS 130 THE OPSONIC INDEX 131 TECHNIQUE 131 VALUE OF THE OPSONIC MEASUREMENTS 133 vSnake Venoms and their Antisera 137 THE VENOMS 137 ANTIVENIN'S 139 Anaphylaxis 141 HISTORICAL 141 THE PHENOMENON 142 SERUM RASHES 143 THEORIES OF ANAPHYLAXIS 144 ALLERGY 147 SUPPOSED RELATION TO PRECIPITIN ACTION 148 PATHOLOGY OF ANAPHYLACTIC SHOCK 150 RELATION OF ANAPHYLAXIS TO SERUM THERAPY 151 Infection and Immunity 154 INFECTION 154 THE INFECTING AGENT 154 ANAPHYLATOXIN 157 RESISTANCE AGAINST INFECTION 158 NATURAL IMMUNITY 159 ACQUIRED IMMUNITY 160 MECHANISM OF IMMUNITY 162 RELATION OF ANAPHYLAXIS TO IMMUNITY 163 IMMUNITY REACTION OF THE PART OF BACTERIA 165 ATREPSY 166 Bacterial Vaccines 169 HISTORICAL. . . 169 METHODS OF ACTIVE IMMUNIZATION 171 TREATMENT WITH VACCINES 173 THE VACCINES 174 DOSES :....' 175 RESULTS 176 CONTENTS xi PAGE Leucocyte Extracts in the Treatment of Infec- tions 177 THEORY 177 PREPARATION OF THE EXTRACTS 177 APPLICATION AND RESULTS 173 Principles Underlying Treatment of Syphilis with Salvarsan 179 PARASITOTROPISM AND ORGANOTROPISM 179 CHEMORECEPTORS 181 Appendices 185 A. THE WASSERMANN TEST FOR SYPHILIS 185 B. NOGUCHI'S MODIFIED WASSERMANN REACTION 200 C. BLOOD EXAMINATION PREPARATORY TO TRANS- FUSION 204 D. THE CONGLUTINATION REACTION 207 THE MUCH-HOLZMANN COBRA VENOM REACTION . .. 208 THE MEIOSTAGMIN REACTION 208 WEIL'S COBRA VENOM TEST IN SYPHILIS 210 ANTITRYPSIN DETERMINATIONS. .'.. . 212 IMMUNE SERA ANTITOXINS Historical. The researches of Buchner 1 in 1889 had shown that the serum of animals artificially immunized against a certain bacterium possessed marked bactericidal properties for that particular organism. In studying immunity on animals which had been successfully immunized against diphtheria infection, Behring, 2 working in Koch's laboratory was struck by the fact that in these animals living virulent diphtheria bacilli were often demon- strable in the scab at the site of. injection several weeks after the infection, and furthermore that the blood serum of the animals did not possess bacteri- cidal properties. In a study published in 1890 Behring showed that the serum of rabbits arti- ficially immunized against diphtheria was able to confer a specific immunity against diphtheria infec- tions in other animals. He also demonstrated that such a serum could be used therapeutically to cure an infection already in progress. Such a serum 1 Buchner, Centralblatt Bacteriologie, Vol. v, 1889. Archiv. f. Hygiene, Vol. x. 1890. 2 Behring & Kitasato, Deutsche med. Wochenschrift, No. 49, 1890. I 2 IMMUNE SERA was not bactericidal, and retained its therapeutic power for a considerable time. He believed that the action of the serum was effected by a neu- tralization of the bacterial toxin by an " antitoxic serum constituent." The action was strictly specific, an antitoxic serum obtained after a diphtheria infection protected only against diphtheria; one derived from a tetanus animal, only against tetanus. Subsequently Behring and Knorr showed that im- munization could be effected with bacterial-free filtrates of tetanus cultures and that the serum thus produced protected not only against tetanus infection but against poisoning by the toxic prod- ucts of the bacilli. After considerable experi- mental work Behring and his collaborators devised an effective method of immunizing Lheep and certain other animals against diphtheria and against tetanus and so produced antitoxic sera in con- siderable amounts. The following account taken from Park shows the present methods of producing diphtheria antitoxin. Production of the Diphtheria Toxin. A strong diphtheria toxin should be obtained by taking a very virulent culture and growing it in broth which is about 8 cc. normal soda solution per liter above the neutral point to litmus. The culture fluid should be in com- paratively thin layers and in large-necked Erlenmeyei flasks, so as to allow of a free access of air; the tem- perature should be about 35 to 36 C. The culture, after a weeks growth, is removed from the incubator, ANTITOXINS 3 and having been tested for purity by microscopic and culture tests is rendered sterile by the addition of 10 per cent of a 5 per cent solution of carbolic acid. After 48 hours the dead bacilli have settled on the bottom of the jar and the clear fluid is filtered through ordinary sterile filter paper and stored in full bottles in a cold place until needed. Its strength is then tested by giving a series of guinea pigs carefully measured amounts. Less than o.oi cc. when injected hypoder- mically should kill a 250 gram guinea pig. Immunizing the Animals. The horses used should be young, vigorous, of fair size, and absolutely healthy. Vicious habits, such as kicking, etc., make no difference, except, of course, to those who handle the animals. The horses are severally injected with an amount of toxin sufficient to kill five thousand guinea pigs of 250 grams weight (about 20 cc. of strong toxin). After from three to five days, so soon as the fever reaction has subsided, a second subcutaneous injection of a slightly larger dose' is given. With the first three injections of toxin 10,000 units of antitoxin are given. If antitoxin is not mixed with the first doses of toxin only one-tenth of the doses advised is to be given. At intervals of from five to eight days increasing injec- tions of pure toxin are made until at the end of two months from ten to twenty times the original amount is given. There is absolutely no way of judging which horses will produce the highest grades of antitoxin. Very roughly those horses which are extremely sensi- tive, and those which react hardly at all are the poorest, but even here there are exceptions. The only way, therefore, is at the end of six weeks or two months to bleed the horses and test their serum. If only high grade serum is wanted all the horses that give less 4 IMMUNE SERA than 150 units per cc. are discarded. If moderate grades only are desired, all that yield 100 units may be retained. The retained horses receive steadily in- creasing doses, the rapidity of the increase and the interval of time between the doses (three days to one week) depending somewhat on the reaction following the injection, an elevation of temperature of more than 3 F. being undesirable. At the end of three months the antitoxic serum of all the horses should contain over 300 units and in about 10 per cent as much as 800 units per'cc. Very few horses ever give over 1000 units, and none so far has given as much as 2000 units per cc. The very best horses, if pushed to their limit continue to furnish blood of gradually decreasing strength. If every nine months an interval of three months' freedom from inoculations is given, the best horses furnish high grade serum during their periods of treatment for from two to four years. Collecting the Serum. In order to obtain the serum the blood is withdrawn from the jugular vein by means of a sharp-pointed canula which is plunged through the vein wall, a slit having been made in the skin. The blood is carried by a sterile rubber tube attached to the canula, into large Erlenmeyer flasks and allowed to clot, the flasks, however being placed in a slanting position before clotting has commenced. The serum is drawn off after 4 days by means of sterile glass and rubber tubing, and is stored in large flasks in a refrige- rator. From this as needed small vials are filled. The vials and their stoppers, as indeed all the utensils used for holding the serum, must be absolutely sterile and every possible precaution must be taken to avoid contamination of the serum. An antiseptic may be added as a preservative, but is not necessary. Diph- ANTITOXINS 5 theria antitoxin, when stored in vials and kept in a cool place away from light and air contains within 10 per cent of its original strength for at least two months; after that it can be used by allowing for a maximum deterioration of 3 per cent for each month. Testing the Strength of the Antitoxin. This is carried out as follows: Six guinea pigs .are injected with mix- tures of toxin and antitoxin. In each of the mixtures there is 100 times the amount of a toxin (similar to that adopted as the standard) which will kill a 250 grams on an average in 96 hours. In each of the mixtures the amount of antitoxin varies; for instance, No. i would contain 0.002 cc. serum; No. 2, 0.003 cc. ; No. 3, 0.004 cc. ; No. 4, 0.005 cc -> etc - ^ at tne en d f the fourth day Nos. i, 2 and 3 were dead and Nos. 4, 5 and 6 were alive we would consider the serum to contain 200 units of antitoxin for each cubic centi- meter. When we mix only ten fatal doses of toxin with one-tenth of the amount of antitoxin used with 100 fatal doses, the guinea pig must remain well. The mixed toxin and antitoxin must remain together for fifteen minutes before injecting. Behring's publication was followed in the next two years by considerable work along these lines, valuable contributions being made by Aronson, 1 Roux, and Martin, 2 Wernicke, 3 Knorr 4 and -others. The statements of Behring as to the strict specifi- city of the antitoxins were fully confirmed. Certain 1 Berliner med. Gesellschaft, Sitzung, Dec. 21, 1892. Also Berliner Klin. Wochenschrift, 1893 and 1894. 3 Roux and Martin, Annal. Pasteur 1894. 8 Behring and Wernicke, Zeitsch. Hygiene, 1892. Vol. xi. Behring and Knorr, Zeitsch. f. Hygiene, 1893. Vol. xii. 6 IMMUNE SERA observations by Buchner 1 and by Roux and Martin threw doubt, however, on the correctness of Beh- rings view that the toxin was neutralized by the specific serum just as a base was neutralized by an acid. It was claimed, for example, that the specific serum acted mainly on the body cells causing them to become non-susceptible to the poison in question. Various theories were formulated to account for the production of the antitoxins, their specificity, etc., but of them all only one has at all maintained itself. This, is the so-called side-chain theory, which was formulated by Ehrlich 2 in 1897. Ehrlich's Side-Chain Theory. Originally the side-chain theory was applied by Ehrlich only to the production of the specific antitoxins, i.e., sub- stances in the blood, which act not only on the living bacteria, but also and especially on their dissolved toxins. Later on he extended it so as to apply also to the formation of specific bacteri- cidal and haemolytic substances in the serum of animals treated with living bacteria or with animal cells. Toxins Toxoids Special Function of the Side Chains. The basis of the theory is the fact that poison and counter-poison, toxin and antitoxin, combine directly in any given quantity. This combination always occurs in definite proportions 1 Buchner, Miinchener med. Wochenschrift, 1894. 1 Ehrlich, Klinisches Jahrbuch, 1897. ANTITOXINS 7 following the laws of chemical combination; and, still following those laws, is slower at lower tem- peratures than at higher, stronger in concentrate J. than in dilute form. Ehrlich could further show that each poison for which by the process of immun- izing one can develop a counter-poison possesses two groups which are concerned in the combina- tion with the counter-poison or antitoxin. One of these, the so-called haptophore group, is the combin- ing group proper; the other, the toxophore group, is the carrier of the poison. A poison molecule, therefore, might lose the one, the toxophore, and still be capable by means of its haptophore group of combining with antitoxin. Such a modified poison, which because of the loss of the toxophore group can hardly be called a poison, but which still possesses the power to combine with antitoxin, Ehrlich calls a toxoid. Toxoids may be produced spontaneously in old poisons through decomposi- tion of the poison molecule, or they may be pro- duced artificially by causing certain destructive agents such as heat or chemicals to act on bacterial poisons. The toxophore group is a very delicate one and much more readily decomposed than the combining (haptophore) group. Ehrlich reasoned that in order for a poison to be toxic to an organ- ism, i.e., in order that the toxophore group be able to act destructively on a cell, it is necessary for the haptophore group of the poison to combine with 8 IMMUNE SERA the cell. IS In every living cell," Ehrlich says, " there must exist a dominating body [Leistungs Kern] and a number of other chemical groups or side chains. These groups have the greatest variety of function, but especially those of nutrition and assimilation." The side chains, then, according to this author, toxophore group , POISON MOLECULE FlG. I are able to combine with the greatest variety of foreign substances and convert these into nourish- ment suitable to the requirements of the active central body. They are comparable to the pseudo- podia of the lower animals, which engulf food par- ticles and assimilate the same for the immediate use of the organism. In order that any substance ANTITOXINS 9 may combine with these side chains it is necessary that certain very definite relations exist between the combining group of the substance and that of the side chain. Using the well-known simile of Emil Fischer, the relation must be like that of lock and key, i.e., the two groups must fit accurately. Hence not every substance will fit all the side chains of an organism. It will combine only with those for which it possesses a fitting group. Receptors Weigert's Overproduction Theory. This doctrine of the chemistry of the organism's metabolism Ehrlich applied to the action of toxins and antitoxins. ' The toxin," he said, " can act only when its haptophore group happens to fit to one of the side chains," or receptors, as he now pre- fers to call them. As a result of this combination, the toxophore group is able to act on the cell and injure it. If we take as an example tetanus, in which all the symptoms are due to the central ner- vous system, the side-chain theory assumes that the haptophore group of the tetanus poison fits exactly and is combined with the side chain or receptors of the central nervous system. Other experiments, which we will not reproduce here, have shown us unquestionably that the action of the antitoxins depends on the fact that this com- bines with the haptophore group of the poison and so satisfies the latter 's affinity. Ehrlich, therefore, concluded that the antitoxin is nothing else than 10 IMMUNE SERA the side chains or receptors which are given off by the cells and thrust into the circulation. The way in which these side chains or receptors are thrust off as a result of the immunizing process, Ehrlich explains by means of Weigert 1 s Overproduction Theory. At the meeting of German Naturalists and Physicians held at Frankfurt in 1896, Weigert * in discussing regeneration, advanced an hypothesis the essential features of which are that physiological structure and function depend upon the equilibrium of the tissues maintained by virtue of mutual restraint between their component cells ; that destruc- tion of a single integer or group of integers of a tissue or a cell removes a corresponding amount of restraint at the point injured, and therefore destroys equilibrium and permits of the abnormal exhibi- tion of bioplastic energies on the part of the remain- ing uninjured components, which activity may be viewed as a compensating hyperplasia; that hyper- plasia is not, therefore, the direct result of external irritation, and cannot be, since the action of the irritant is destructive and is confined to the cells or integers of cells that it destroys, but occurs rather indirectly as a function of the surrounding uninjured tissues that have been excited to bio- plastic activity through the removal of the restraint 1 Weigert, Verhandlungen der Ges. deutscher Naturforscher iind Aerzte, 1896 ANTITOXINS II hitherto exerted by the cells destroyed by the irritant; and, finally, when such bioplastic activity is called into play there is always hypercompen- sation i.e. there is more plastic material gene- rated than is necessary to compensate for the loss. Ehrlich points out that owing to the combination of the toxin with the side chain of a cell, these side chains are practically lost to the cell ; that the latter or its fellows now produces new side chains to replace this loss, but that this production always goes so far as to make a surplus of side chains ; that these side chains are thrown off by the cell as unnecessary ballast, and then circulate in the blood as antitoxin. The same substances, therefore, which when part of the cell combine with the haptophore group of the toxin, enabling that to act on the cell, when circulating free in the blood combine with and satisfy this haptophore group of the toxin, and prevent the poison from combining with and damaging the cells of the organism. It does not follow from Ehrlich's theory that the antitoxin is produced by the same set of cells whose injury by the toxin gives rise to the particular clinical symptoms. Thus we might believe that although in tetanus the cells of the central nervous s}^stem give rise to the characteristic symptoms, cells entirely apart from these, e.g., in the bone marrow, might be the main source of the antitoxin. The 12 IMMUNE SERA fact that we appreciate symptoms from only one organ is, obviously, no proof that other tissues have been unaffected. It may be well here to call attention to another rather common misconception regarding the pro- duction of antitoxin, namely that the body cells have to become educated, so to speak, to produce the antitoxin. This, it is believed, is effected by giving gradually increasing doses of toxin. As a matter of fact the reason for this gradual increase in the dose injected is quite different. The object in view is the administration of an enormously large dose of toxin, one that will engage the recep- tors of many cells, The previous injections have brought about some production of antitoxin and this partially neutralizes some of the toxin in- jected, making it possible to give a larger dose than before. If one gives at the outset a large amount of toxin, partially neutralized by antitoxin, one will produce an amount of antitoxin equal to that ordinarily obtained in response to the same quan- tity of unaltered toxin given as the tenth or twentieth injection of a series. Park and Atkinson for example, injected a fresh horse with one litre of a toxin neutralized ij times for guinea pigs. At the end of a week the horse had produced a serum containing 60 units per cc. When the toxin was neutralized 6 fold no antitoxin whatever was pro- duced. ANTITOXINS 13 Experimental Evidence for Ehrlictis Theory. According to Ehrlich, then, the formation of specific antibodies must proceed in three stages: 1. The binding of the haptophore group to the receptor. 2. The increased production of the receptors following this binding. 3. The thrusting-off of these increased receptors into the blood. So far as the first point is concerned Wassermann * showed that with tetanus, in which, as is well known, all the symptoms are referable to the cen- tral nervous system, tetanus toxin was bound by central nervous system substance in vitro. A mixture of tetanus poison and normal central nervous system was innocuous to animals, showing that certain substances present in the central nervous system combine with and thus satisfy the affinity of the haptophore group of the poison. This of course prevents the latter from combining with any cells of the organism. Organs other than the central nervous system do not possess this property of combining with tetanus poison, just as the central nervous system is, on the contrary, incapable of combining with diphtheria poison, which clinically does not show any pronounced affinity for the central nervous system. Wassermann 2 also believes recently to have given 1 Wassermann and Takaki, Berliner Klin. Wochenschr, 1898, 1 Wassermann, New York Medical Journal, 1904. 14 IMMUNE SERA experimental proof of the second and third points, the increased production of the receptors and their thrusting off. For this purpose he employed a tetanus poison which he had kept for about eight years, and which was originally very poisonous. In the course of years, however, owing to the damaging action of light, of oxidation, etc., it had become so weak that it was no longer toxic at all. Injections of one cc. into a guinea pig produced no tetanus. Nevertheless the haptophore group remained intact, as could readily be proved, for this non-poisonous tetanus toxin was still able to bind tetanus antitoxin, i.e. thrust-off receptors. On injecting rabbits with this nOn-poisonous tetanus toxoid in increasing doses, and then examining the blood serum of the animal he found not a trace of tetanus antitoxin. This absence could have either of two causes: It might be that the toxoid no longer produced any physiological effect whatever in the organism; or although it still caused an increase in the receptors, these increased receptors remained in the organs (sessile) and were not thrust off into the blood. In order to decide this question Wassermann first determined the exact quantity of fresh tetanus toxin which constituted a fatal dose for guinea pigs. He reasoned that if he injected first the toxoid, and shortly after, say in one or two hours, the fresh toxin, he should in such an animal have to increase the fatal dose, ANTITOXINS 1 5 i.e. more tetanus toxin should be required to kill this animal than a normal one, because owing to the previous toxoid injection part of the cells sus- ceptible to tetanus toxin would already have been occupied. Provided Ehrlich's theory were correct, so that this binding of the toxoid really occurred, the conditions should be entirely different when, instead of injecting the toxin shortly after the toxoid, he waited somewhat longer, one to three days, and then injected the fresh tetanus toxin In that case Weigert's law should come into play and the receptors have commenced to increase in number, i.e. the organ should now possess more sensitive groups than before. This would manifest itself in such fashion that in contrast to the first experiment the fatal dose of fresh tetanus toxin could now be decreased ; in other words a small dose would now tetanize the animal in a shorter time. As a matter of fact Wassermann's experiments yielded exactly the results deduced theoretically. He injected a guinea pig with some of the non-- poisonous toxoid and then, an hour later, with tetanus toxin. He found that much more toxin was required to kill this animal than a normal guinea pig of equal size. When, on the contrary, he waited one to three days, it was found that then a dose of tetanus toxin which would not even tetanize a normal guinea pig was sufficient to kill this one. 16 IMMUNE SERA It will be seen that in the above experiments the completely non-poisonous toxoid, although it effected an increased production of receptors, did not cause their thrusting-off. The serum of the rabbit treated with toxoid contained no antitoxin whatever. Wassermann concludes from this and other experiments that the thrusting-off cannot be a function of the haptophore group, and that something additional is required. This " some- thing," he claims is a function of the toxophore group. It may be stated that Von Dungern has also published experiments (with majaplasm) point- ing to the existence of the second stage, the stage of sessile receptors. Antigens or Haptins. It has been found that it is impossible to produce any immunity against all poisons, e.g. strychnine or morphine. Accord- ing to Ehrlich these simpler chemical molecules do not enter into a true chemical combination with the tissues, but form rather a kind of solid solution, a loose combination with the cells, so that they can again be abstracted from these cells by all kinds of solvents, e.g. by shaking out with ether or chloro- form. The point can perhaps be likened to the difference between saccharin and sugar. Both sub- stances taste sweet, but despite this similarity in their physiological action they behave very dif- ferently toward the cells of the organism. Sac- charin simply passes through the organism without ANTITOXINS 17 entering into a firm combination, i.e. without being assimilated, and is therefore no food. Its sweeten- ing action is a mere contact effect on the cells sensitive to taste. Sugar, on the contrary, is actually bound by the cells, assimilated and burnt, and so is a true food. Until recently it was believed that the simpler chemical substances could not excite the production of antibodies. Ford and Abel 1 have however been able to show that toad stool poison, a true toxin, against which an anti- toxin can be produced is chemically a glucoside. As we shall subsequently see it is possible to immunize the animal body against a large number of substances, including not only such cell products as ferments, toxins and venoms, but also cells of the greatest variety, bacteria, dissolved proteids, etc. All these substances, therefore, must possess hapto- phore groups able to combine with the side chains or receptors in the animal body. Collectively, we speak of such substances as antigens or haptins. Nature of Antitoxins in General. But little is known concerning the constitution of antitoxins, for we do not know them apart from serum or serum constituents. It seems probable that they are proteid in character, but this has not been positively decided. It has been found that like the globulins they are quite resistant to the action of trypsin, but are acted on by pepsin-hydrochloric 1 Ford and Abel, Journal of Biological Chemistry, Vol. ii, 1907 1 8 IMMUNE SERA acid. In general they withstand a fair degree of heat, certainly far more than the toxins. Anti- toxins are to be regarded as inactive substances, effecting merely a blocking of the haptophore group of the corresponding toxin. They do not act on the toxins destructively. This is indicated by experiments of Wassermann on pyocyaneus toxin, and of Calmette and Morgenroth 1 on snake venom, which showed that in the toxin-antitoxin com- bination, the toxin could again manifest itself after the antitoxin had been destroyed. The antitoxins therefore are not ferment-like substances. As far back as 1897 attempts were made to determine the chemical nature of the antitoxins. In that year Belfanti and Carbone 2 found that the antitoxin was precipitated with the globulins of the serum by means of magnesium sulphate. Dieudonne 3 had previously shown that the proteids thrown out of solution by acetic and carbonic acids contained none of the antitoxin. In 1901 Atkinson 4 showed that the globulins increase markedly in the serum of horses as the antitoxic strength increases. The most recent work on this subject is that of Gibson, 5 who shows that if the ammonium sulphate precipi- 1 Morgenroth, Berlin, klin. Wochenschr. 1905. 2 Beifanti and Carbone, Centralblatt Bacteriologie (Ref.), Vol. xxiii, 1898. 3 Dieudonne, Arbeiten a.d. kaiserl. Gesundheitsamte. VoL xiii, 1897. 4 Atkinson, Jour. Exper. Medicine, Vol. i, 1901. 5 Gibson, Journ. Biological Chemistry, Vol. i, 1906. ANTITOXINS 1 9 tate (globulins, nucleo-proteids, etc.) is treated with saturated sodium chloride solution, practically all the antitoxic fraction passes into solution. Gibson's was the first really practicable method of concentrat- ing the antitoxin. By means of it solutions of antitoxic globulin could easily be made to contain 1500 units per cc. Continuing Gibson's work, Banzhaf discovered that if the antitoxic serum or plasma was heated to 57 for 18 hours, there was a change of a considerable portion of the soluble globulins (soluble in Nad solution) into insoluble globulins. The antitoxin remained unchanged. This procedure, therefore, permits of a still greater elimination of the non-antitoxic proteids. Gibson has recently studied the possibility of differentiating other antibodies by means of their precipitation characteristics. He believes that a differentiation of the antibodies into those precip- itated with the pseudo globulins and with the euglobulin fractions, according to the Hofmeister classification, is based on a misconception of the application of ammonium sulphate in separating proteids by their precipitation characters. While there seem to be some differences in the dis- tribution of the antibodies in individual specific sera in comparative experiments, this is not so absolute as maintained by Pick l and others. Gib- son's work on the fractionating of poly agglutina- 1 Pick, Beitiage z. chem. PhysioL u- PathoL, VoL i, 1901. 20 IMMUNE SERA tive serum shows that no separation of the several antibodies developed in an individual serum is possible. In the case of antitoxic sera both Gibson and Ledingham find that in goat serum the antitoxin is not invariably associated with the euglobulin fraction as maintained by Pick, but shows the same solubilities as that in horse serum. Toxins and other Poisonous Cell Derivatives, in General. Soon after bacteriology had demon- strated the etiological connection between bacteria and disease, the conviction gained ground that it was less the actual destruction wrought by the bacteria directly, than the injury produced by their chemical products that gave rise to the lesions in the infectious diseases. Brieger, especially, was one of the first to direct attention to the probable existence of specific poisons in the bacteria. He isolated a number of well defined chemical sub- stances called ptomaines, most of which were highly toxic. Subsequent study, however, showed that these were not the specific bacterial poisons. The latter, the true toxins are something quite different as we shall see in a moment. Still later other substances were isolated from bacteria, and these were termed toxalbumins. We now know that some of these were identical with the true toxins, but that others were entirely unrelated. What then are the true toxins? A number of pathogenic bacteria, when grown in pure culture, ANTITOXINS 21 produce dissolved poisons in the culture fluid. These poisons are neither ptomaines nor proteid substances; their chemical nature is still absolutely unknown. They are extremely sensitive to exter- nal influences, especially against heat, and in many ways are very analogous to ferments. Physio- logically the toxins are extremely poisonous, far beyond that of any of the ordinary' well known poisons, and this poisonous action manifests itself only after a certain latent period known as the period of incubation. Finally one of the funda- mental properties of the toxins is their ability to excite, in the organism attacked, antitoxins directed specifically against them, so that for every true toxin there is a corresponding antitoxin. In addition to these bacterial toxins we know of other poisonous substances possessing similar characteristics. Among these are the " zootoxins," - snake venoms, spider and toad poisons, the toxin of eel blood, and the " phytotoxins," ricin, crotin, abrin, etc. It may be mentioned that some of these are of somewhat more complex con- stitution than the ordinary bacterial toxins. Ricin, for example, appears to possess one haptophore group but two ergophore groups, a toxic and an agglutinating one. In the case of the snake venoms it is not yet definitely known whether they are haptins of the first order or of the second. (See page 49.) 22 IMMUNE SERA The Relations Existing between Toxin and Anti- toxin. The exact nature of the toxin-antitoxin reaction has long been the subject of study and has given rise to considerable discussion. For obvious reasons most of the work has been done with diphtheria and tetanus toxins and their antitoxins. In order to give the reader some conception of the diverging views of various authorities we shall devote a few pages to a brief study of ths diphtheria toxin-antitoxin reaction. During the earlier years of toxin-antitoxin in- vestigations the filtered or sterilized bouillon, in which the diphtheria bacillus had grown and pro- duced its " toxin," was supposed to require for its neutralization an amount of antitoxin directly proportional to its toxicity as tested in guinea pigs. Thus, if from one bouillon culture ten fatal doses of " toxin" were required to neutralize a certain quantity of antitoxin, it was believed that ten fatal doses from every culture, without regard to ths way in which it had been produced or preserved, would also neutralize the same amount of antitoxin. Upon this belief was founded the Behring-Ehrlich definition of an antitoxin unit. 1 The results of tests by different experimenters of the same antitoxic serum, but with different diph- 1 This unit was " ten times the amount of antitoxic serum necessary to just protect a 250 gramme guinea pig against ten fatal doses of the toxin " ANTITOXINS theria toxins, proved this opinion to be incorrect. Ehrlich 1 deserves the credit for first clearly per- ceiving and calling attention to this fact. He obtained from various sources twelve toxins and compared their neutralizing value upon antitoxin; these tests gave interesting and important in- formation. The following table gives the results in four of his toxins and well illustrates the point in question : Smallest num- Fatal doses re- ber of fatal doses of toxic quired to " completely Estimated bouillon re- neutralize " one L+ minus Serial minimal fatal quired to kill a antitoxin unit Num- ber. dose for 250 gm. guinea pigs. 2sogm. guinea pig within 5 days when mixed with one as determined by the health of the guinea pig remaining L(> in fatal doses. Remarks. antitoxin unit. unaffected. ("L t Ehrlich.") (" LO Ehrlich.") A o . 009 cc. 39-4 33-4 6 Old; deterio- rated from 0.003 ;o o. 009. B 0.0165 cc 76.3 54-4 22 Fresh toxin, preserved with :ricresol. C o. 039 cc. 123. 108. 15 A numbei of "resh cultures, grown at 37 C. four and eight days. D 0.0025 cc. 100 5 5 Tested immedi- ately after its x withdrawal. It was natural to suppose, as the early investi- gators did, that a just neutral mixture of toxin and 1 Ehrlich, Die Werthbemessung des Diphtherieheilserums. Klinisches Jahrbuch, 1897. 24 IMMUNE SERA antitoxin, would require the addition of but one fatal dose of toxin in order to regularly kill the test animal. In the above table, however, we see that this difference ranges from six to fifty fatal doses. Partial Saturation Method - - Toxons, Toxoids. Ehrlich obtained considerable additional informa- tion by means of his " partial saturation " method. Certain experiments had led him to believe that the original antitoxin on which he had based his " unit " determinations, while able to neutralize 100 fatal doses (per unit) really represented 200 " binding units," and that the toxic bouillon really contained several kinds of poisonous substances able to com- bine with antitoxin. He now believes that the diphtheria bacilli excrete at least two such poisons, " toxins " and " toxons ; " that these very quickly decompose to a greater or less extent forming various " toxoids." In the case of a hypothetically pure toxin Ehrlich believes that one antitoxic unit would correspond to 200 fatal doses or 200 binding units. If the entire amount of antitoxin, i.e. | is added to the amount of toxin in question, the result will be just complete neutralization. If the toxin is entirely pure, ^{jf of the antitoxin unit would neutralize all but ?hu of the initial toxicity and M&, or ?$% or ^A, etc. of the antitoxin added would permit correspond- ing degrees of toxicity to be demonstrated through animal inoculations. It was found, however, that neutralization according to this simple scale did not ANTITOXINS 2 5 take place. The results were complicated andEhrlich found it convenient to express them graphically in the form of the so-called "toxin spectra. " Without 10 20 30 40 Toxon 70 80 90 100 150 200 FlG. 2. going much deeper into the subject the point maybe illustrated by the appended diagrams or " spectra." Fig. 2 shows the simplest conceivable diphtheria poison. In this case the following values would be obtained. # cc poison (100 fatal doses) + f antitoxin units = o, i.e. absolutely neutral. # cc poison + iffft = Free toxon. # cc poison + *{} = Free toxon. That is to say, if the proportion of antitoxin added was Mti of the amount required for complete neutralization, it would be found that the poison thus uncombined was much less, and differently toxic than a corresponding amount of the original toxin. It was found that these fractions possessed a rather constant though low degree of toxicity with characteristic action. This consisted in the production of some local oedema, followed by a long incubation period, and finally the develop- ment of cachexia and paralysis. Ehrlich believes 26 IMMUNE SERA that this action is due to a separate poison excreted by the diphtheria bacillus which he calls a toxon. If we continue with the above poison we shall obtain these values: # cc poison + ^ = Toxin action (i fatal dose). # cc poison + sVu =30 fatal doses. x cc poison + sVk = 90 fatal doses, etc. That is to say, if we add only $fo units antitoxin, i.e. sitf unit less than in the JH mixture, we find that one fatal dose is set free. This relation would exist right to the end. The fact that in this experi- ment the toxins are liberated after the toxons, shows that the toxons have less affinity for the anti- toxin than have the toxins. As a matter of fact, however, conditions are prob- ably never as simple as this. In the process of toxin formation a double action is always going on that of toxin and toxon production, and that of their decomposition. As was pointed out on a previous page the poisons quickly change into non-poisonous toxoids, and these substances are still able to bind antitoxin. This is shown in the following " spectrum." Protoxoid 10 20 30 40 50 60 1GO FIG. 3. Toxon 150 160 ANTITOXINS Here we would obtain the following figures: 2 7 # cc poison -f lutely neutral. # cc poison + # cc poison + # cc poison -f oo poison + oc cc poison + antitoxin unit = o, i.e. abso- = Toxon free. = Toxon free. = Toxin free (i fatal dose.) - Toxin free (60 fatal doses. ) = Toxin free (100 fatal doses.) Now we come to the non-poisonous "prototoxoids" : # cc + inm = Toxin free(ioo fatal doses.) # cc + *oo = Toxin free (100 fatal doses.) x cc + sio = Toxin free (100 fatal doses.) We see here that after we have reduced the antitoxin to ^ no further increase of toxicity is brought about by any further reductions. Ehrlich calls these toxoids " prototoxoids " because they have such a high affinity for the antitoxin. But there are apparently still other toxoids, as is shown by the following spectrum: Protoxoid Syntoxoid Toxon ' 100 ' 10 ' '200 FIG. 4. Here we would obtain values as follows: x cc poison -f- f $% = o, i.e. absolutely neutral. x ce poison -f iol = Toxon. 28 IMMUNE SERA x cc poison 4- M