Principles of immunology

THE PRINCIPLES OF
IMMUNOLOGY

THE PRINCIPLES OF
IMMUNOLOGY

BY

HOWARD T. KARSNER, M.D.

PROFESSOR OF PATHOLOGY, WESTERN RESERVE UNIVERSITY, CLEVELAND

AND

ENRIQUE E. ECKER, PH.D.

INSTRUCTOR IN IMMUNOLOGY, WESTERN RESERVE UNIVERSITY, CLEVELAND

ILLUSTRATED

PHILADELPHIA AND LONDON
J. B. LIPPINCOTT COMPANY

'•• •

(Qf

COPYKIQHT, 1921, BY J. B. LIPPINCOTT COMPANY

Electrotyped and Printed by J. B. Lippincott Company
The Washington Square Press, Philadelphia, U.S.A.

DEDICATED
TO

CHARLES KARSNER
WILLIAM C. KARSNER
CHARLES W. KARSNER

DANIEL KARSNER

AND

JAMES H. M. KARSNER
DOCTORS OP MEDICINE

694119

PREFACE

THIS book has been prepared in the hope that a concise state-
ment of the facts and more important hypotheses concerning resist-
ance to infection may serve to provide a clear understanding of a
subject of the utmost importance in modern diagnosis and treat-
ment. Designed primarily for students of medicine and for those
practitioners whose duties have made it impossible to digest a large
mass of publications on the subject, the scope of the book is
restricted to fundamental principles. The plan throughout is to pre-
sent on an experimental basis the demonstrated facts and to supple-
ment these with brief discussions of the practical and theoretical
bearing of the phenomena upon resistance and disease in man. A
few illustrations have been inserted, but it must be recognized that
technical details can only be fully comprehended on the basis of
actual work with the methods. The usual diagrams of the side-
chain theory have been omitted because of the belief that they
serve to confuse rather than clarify the conception of processes
whose fundamental basis lies in the field of physical chemistry.
Certain material concerning the practical application of immunology
to the prevention and cure of disease has been collected in three
appendices. These have been added in order to explain the basis of the
practical methods rather than as an exact guide in their application.

Knowledge progresses from the known to the unknown, from
the simple to the complex, and if the brevity of the book serves to
implant essentials in such a way that the reader not only grasps the
facts, but finds himself stimulated to seek further information and
discussion in more comprehensive works, the most compelling aim
of this book will have been achieved. For this purpose books
which we have used with considerable freedom are recommended:
Zinsser, " Infection and Resistance " ; Wells, " Chemical Pathol-
ogy " ; Kolmer, " Infection, Immunity, and Specific Therapy " ;
Kraus and Levaditi, " Handbuch der Technik und Methodik der
Immunitatsforschung " ; Muir, " Studies on Immunity " ; Kolle and
Wassermann, "Handbuch der pathogenen Mikroorganismen " ;
Metchnikoff, " Immunity in Infective Diseases "; Bordet, " Traite de
rimmunite dans les Maladies Infectieuses " ; Besredka, " Anaphy-
laxis and Antianaphylaxis " ; Bordet and Gay, " Studies in
Immunity " ; Gay, " Typhoid Fever " ; Browning, " Applied Bacteri-
ology"; Craig, "The Wassermann Test"; Noguchi, "Serum
Diagnosis of Syphilis " ; Zinsser, Hopkins, and Ottenberg, " Lab-
oratory Course in Serum Study." The names of those who have
contributed to the literature are given in the text, but precise refer-
ences have been omitted, since the articles can be found by refer-
ence to such bibliographic journals as the Index Medicus, The Index

vii

viii PREFACE

Catalogue of the Surgeon General's Office, and in particularly available
form in the Quarterly Cumulative Index of the American Medical
Association. Every effort has been made to give credit where it
belongs ; if omissions or errors have been made they are due to the
vast amount of material that has been accumulated on this subject
rather than to intentional oversight.

Our thanks are due to our colleague, Doctor Maurice L.
Richardson, for extremely valuable aid in the revision of the manu-
script, to Mr. E. L. Miller for three important microscopic draw-
ings, to Miss May E. Treter and Miss Catherine E. Lennon for
faithful and painstaking clerical work. Mr. W. T. Brownlow, of
Cleveland, has made the line drawings and Mr. E. F. Faber, of
Philadelphia, the drawings of the lungs in anaphylactic shock. We
have taken materials from certain journals and make grateful
acknowledgment by reference in the text.

January, 1921.

HOWARD T. KARSNER,
ENRIQUE E. ECKER.

CONTENTS

CHAPTER. PAGE

PREFACE vii

LIST OF ILLUSTRATIONS xi

INTRODUCTION xiii

I. VIRULENCE OF ORGANISMS i

II. GENERAL CONDITIONS OF INFECTION AND RESISTANCE n

III. THE GENERAL PHENOMENA OF IMMUNITY 16

TYPES OF IMMUNITY.

THEORIES OF IMMUNITY.

SITE OF ANTIBODY FORMATION.

IV. TOXINS AND ANTITOXINS 37

BACTERIAL TOXINS AND ANTITOXINS.

DIPHTHERIA.

TETANUS.

DYSENTERY.

BACILLUS BOTULINUS.

GAS BACILLUS.

BACTERIAL HEMOTOXINS
PHYTOTOXINS.
ZOOTOXINS.

V. AGGLUTININS AND PRECIPITINS 78

BACTERIAL AGGLUTININS.

HEMAGGLUTININS.

PRECIPITINS.

VI. CYTOLYSINS 115

HEMOLYSINS.
CYTOTOXINS.
BACTERIOLYSINS.

VII. CELLULAR RESISTANCE 151

PHAGOCYTOSIS.

OPSONINS.
OTHER FORMS OF CELLULAR RESISTANCE.

VIII. COMPLEMENT FIXATION 173

THE BORDET-GENGOU PHENOMENON.

IX. APPLICATION OF COMPLEMENT FIXATION TO THE DIAGNOSIS OF DISEASE. 186
THE WASSERMANN REACTION.
COMPLEMENT FIXATION IN TUBERCULOSIS.
COMPLEMENT FIXATION IN GONOCOCCUS INFECTIONS.
OTHER COMPLEMENT FIXATION TESTS.

X. HYPERSUSCEPTIBILITY 208

ANAPHYLAXIS.

ANAPHYLACTOID PHENOMENA.
RELATION OF ANAPHYLAXIS TO IMMUNITY.

XI. HYPERSUSCEPTIBILITY IN MAN 228

SERUM DISEASE.
ANAPHYLACTIC SHOCK.
NATURAL HYPERSUSCEPTIBILITY.
THE TUBERCULIN AND SIMILAR TESTS.

XII. DEFENSIVE FERMENTS 245

THE ABDERHALDEN TEST.

APPENDIX.

A. THERAPEUTIC EMPLOYMENT OF BLOOD SERUM 252

B. PROPHYLACTIC VACCINATION .' 272

C. VACCINE THERAPY 296

LIST OF ILLUSTRATIONS

FIG. PAGE

1 . Apparatus for Filtration through Porcelain 43

2. The Rosenau or Reichel Syringe for Injecting Toxin-antitoxin Mixtures. . . 47

3. Wooden Box for Holding Rabbits 79

4. Method of Obtaining Blood from the Rabbit's Ear 80

5. Method of Complete Bleeding from the Femoral Vessels of the Rabbit. ... 81

6. Collection of Serum in a Flask 82

7. Method of Drawing Up Measured Volumes of Fluid into a Graduated Pipette 83

8. The Wright Tube for Obtaining Small Quantities of Blood Serum 85

9. Coiled Pipette for Taking Up Small Quantities of Fluids 85

10. Microscopic Drawing of Bacterial Agglutination 84

11. The Nipple Pipette 96

12. Hemolysis in the Test Tube 1 18

13. Quantitative Relations of Amboceptor and Complement in Hemolysis 119

14. Method of Obtaining Blood from Guinea-pig 128

15. Stages of Lysis in Cholera Vibrios 144

16. Microscopic Drawing of Phagocytosis 154

17. Microscopic Drawing of Guinea-pig Lung in Anaphy lactic Shock 214

1 8. Blood-pressure Tracing from Dog in Anaphylactic Shock 216

PLATES

PLATE I. Positive Schick Reaction of Moderate Severity Seventy-two Hours
After the Intracutaneous Injection of One-fortieth the Minimal
Lethal Dose of Diphtheria Toxin. Patient's Blood Serum Was

Found to Contain No Antitoxin 54

PLATE II. Colored Drawing of Guinea-pig Lungs in Anaphylactic Shock 212

INTRODUCTION

THE history of immunology as a science is distinctly modern and
in the investigation of details dates back only as far as the time of
Louis Pasteur. Jenner's work on smallpox vaccination represents
most painstaking and thorough investigation; it was epochal in
character, and of the utmost importance in practical results, but was
not immediately followed by any general application to other dis-
eases, probably because of the limitations of technical methods.
Observations of the phenomena of immunity were, however, made
in ancient times and the resistance to second attacks of such dis-
eases as measles, scarlatina, variola, varicella must have been com-
mon knowledge from the earliest days of the human race. Whilst
many of the earlier students of medicine recognized a certain simi-
larity between poisoning and infectious disease, yet Hippocrates
could see no such resemblance and his theory of the four humors
was dominant throughout the Middle Ages. With minor exceptions
this belief held sway until well into the Renaissance. In 1548, how-
ever, Fracastore proposed the theory that infection was carried
from person to person " per contactum " or " per fomites," and from
this time dates real progress in the investigation of infectious dis-
ease. This led subsequently to the establishment of two schools of
thought, the one believing disease to be due to substances of basic
or acid principle, and the other believing disease to be due to para-
sites. The development of the latter idea was forced to await the
discovery of means to view minute parasites and, as a matter of
fact, was delayed much longer, because the invention of the micro-
scope by Kircher in 1659 and van Leeuwenhoek in 1675 far ante-
dated the connection now established between minute parasites and
infectious disease. Nevertheless, Plenciz in 1762 expressed a belief
in the direct etiological connection between certain forms of disease
and microorganisms, and established the conception of the " con-
tagium vivum." This idea was revived by Henle and by
Brettoneau, but attracted no permanent attention.

As perhaps the first observation leading up to our present con-
ception of infectious diseases, and therefore to immunity against
them, was the discovery in 1837 by Schwann that certain forms of
fermentation are due to the presence of yeasts, an observation made
at about the same time by Cagniard-Latour. Although at this time
there was little, if any, thought that this discovery had any impor-
tant bearing on infectious disease, yet within the succeeding decade
favus, thrush, and pityriasis versicolor had been demonstrated to be
due to specific fungi. Nevertheless, the possible similarity of fer-
mentation and infectious disease had been considered in a more or
less philosophical way, and Robert Boyle had said : " He that thor-

xiii

xiv INTRODUCTION

oughly understands the nature of ferments and fermentations shall
be much better able than he that ignores them to give a fair account
of diverse phenomena of certain diseases (as well fevers as others),
which will perhaps be never properly understood without an in-
sight into the doctrine of fermentations." In the further develop-
ment of the origin of infectious disease in living organisms perhaps
the work of Rayer and Davaine on anthrax was of the utmost im-
portance. They reported in 1850 that in the blood of anthrax vic-
tims " are found little thread-like bodies about twice the length of a
blood-corpuscle. These little bodies exhibit no spontaneous motion."
In 1863 Davaine showed that the blood containing these rods could
transmit the disease while blood free from them did not transmit
the infectious agent. Davaine suggested at this time that the
manifestations of the disease might represent the results of the
specific fermentation produced by these bacilli. Such a parasitic concep-
tion of disease was further supported by the discovery in 1873 of the
spirillum of relapsing fever by Obermeier. Subsequently the work of
Louis Pasteur, Koch, and the great school of early bacteriologists gave
the final evidence in support of the " contagium vivum."

Although the essential development of the science of immu-
nology necessarily awaited the critical study of infectious disease,
as can be seen from the foregoing summary of the development of
the knowledge of the cause of infections, yet throughout the ages
there had been speculations as to the nature of immunity running
hand in hand with hypotheses as to the nature of infection. Im-
munology took its most important step forward more than a half
century before the work of Schwann had reached its fruition in the
studies of Davaine, Obermeier, and Pasteur ; namely, in the master-
ful experiments of Jenner. It is almost certain that for at least a
century before Jenner's publication there had been practised, in the
far and near East as well as in certain parts of Europe, including
England, the inoculation of smallpox during full health in order to
produce a mild attack of the disease and thus protect against later
more severe or fatal attacks. It is indeed possible, as claimed by
Carburi, that such a procedure originated in Europe as early as the
sixteenth century and was carried to Constantinople and thence to
the far East. Similar attempts to produce mild attacks of other
diseases were tried, but with little success, as, for example, the work
or Vesepremi in 1755 with plague, of Home in 1757 with measles, and
of Turenne in 1844 with syphilis. It seems unlikely, however, that
any of this work had any direct bearing on the discovery of Jenner.
Sprengell states that for many years before Jenner's time the pro-
tective influence of cowpox against smallpox was known in certain
districts of Ireland, Holstein, Brandenburg, Switzerland, Catalonia,
Peru, and the East Indies. Similar observations had been published,
as, for example, the statement of Bose in 1769, that persons who
had suffered cowpox were not subsequently attacked by smallpox.
Jesty in 1774 had inoculated some members of his family with cow-

INTRODUCTION xv

pox and reported that they remained free from smallpox. In 1791
Jensen and Plett practised protective inoculation with cowpox and
reported good results, as did also Penster in 1765. None of these
studies, however, bore critical scientific examination, nor did they
serve to stimulate active work along this line. Indeed, it seems un-
likely that they influenced Jenner in any way. Jenner brought to
bear the critical method of the experimental investigator and proved
the point. The method was rapidly put into clinical practice,
spread over the British Isles and Europe and stood the test of
time and wide application. With very slight modifications it stands
to-day, in spite of our great advances in the study of immunity^as
the most effective method we have to guard against infectious dis-
ease. Jenner vaccinated a boy on the arm with cowpox virus ob-
tained from a lesion on the hand of a dairy maid, and subsequently
inoculated the boy with fresh smallpox virus, which failed to pro-
duce the disease. He also reported an attempt to inoculate small-
pox unsuccessfully in ten persons who had had cowpox nine
months to fifty-three years previously. In 1800 Waterhouse in
Boston repeated the experiment of Jenner on his own son, and in
1802 performed a more extensive and even more critical experi-
ment, in which he vaccinated nineteen boys with cowpox. Twelve
were inoculated with smallpox virus three months later and failed
to develop the disease, the same virus being inoculated at the same
time into two unvaccinated boys, producing well-developed small-
pox. The virus from these latter two boys was later inoculated into
all the iiineteen vaccinated boys without, results. Thus began the
period of experimental investigation of the phenomena of immunity.
Further progress of importance was not made until 1880, when
Pasteur announced his results in vaccination against chicken
cholera. No brief review such as this can do justice to the stimulus
to modern biological science furnished by this man and his asso-
ciates, and the reader is referred to the interesting and intimate
view of the life of Pasteur written by Valery Radot, his son-in-law.
At the beginning of Pasteur's work the theory of spontaneous gen-
eration was still generally accepted by the scientific world, and be-
fore he was compelled to cease his active investigations not only
had this theory been overthrown, but also the ideas of chemists in
regard to crystallization and to the rotation of light by bodies in
solution had been completely revised, the silk and wine industries
of France, and indeed of the world, had been entirely rejuvenated,
the bacteriological cause of numerous diseases conclusively proven,
and the science of immunology put on a plane where its progress
must be uninterrupted. His first contribution to the science of im-
munology was in connection with his work on chicken cholera.
Although he did not offer it as such, nevertheless, this incident well
illustrated his doctrine that " chance favors the prepared mind." He
had saved some old cultures of this bacterium and later found that
they were avirulent. He subsequently tried to cause the disease in

xvi INTRODUCTION

animals which had been inoculated with this virus, using the second
time a culture which was virulent for untreated fowl. He showed
that the inoculated fowl were immune to the virulent culture. In
1881 he demonstrated with his collaborators, Chamberland and
Roux, that this was not an isolated fact, but that essentially the
same thing had been accomplished with anthrax. The virus of
anthrax could not be attenuated by the same simple method as for
fowl cholera, because the bacillus anthracis preserves its virulence
by the formation of spores. They showed, however, that they could
prevent the formation of spores by growing the bacillus at 42° to
43° C. At this temperature growth of six to eight days sufficiently
attenuates the organism for protective inoculation. The proof of
the vaccination was given publicly before the Society of Agricul-
ture at Melun. For this phenomenon Pasteur used the term vac-
cination, and in London in 1881 said: " I have lent to the expression
vaccination an extension that I hope science will consecrate as a
homage to the merit and immense services rendered to humanity
by one of the greatest men of England — Jenner." In 1882 Pasteur
and Loir confirmed Thuillier's observations on the cause of swine
fever and then successfully vaccinated pigs against this disease.
Then in 1885-1886 came the final brilliant chapter in the work with
rabies, in which vaccination was practised without definite knowl-
edge of the etiological agent. The work with rabies was of further
importance in that it led to the discovery of the fact that a virus
may be increased in virulence, a phenomenon quite the reverse of
the earlier discovery of the possibility of attenuation.

In his studies Pasteur had worked almost entirely with the active
organisms causing disease, and the next step forward was the dis-
covery that the products of bacterial growth and activity can be
utilized in the development of immunity. Salmon and Theobald
Smith published in 1886 their studies on the immunization of hogs
against hog cholera by the use of the products of the specific organ-
isms. This idea had been suggested by LoefHer in 1884, but not
proven. Before he had made any conclusive experiments the sub-
ject had been taken up by numerous other investigators. Behring and
Kitasato in 1890 had discovered tetanus toxin and Roux and Yersin in
1 888- 1 889- 1 890 had published their discovery of diphtheria toxin.
These workers showed that the symptoms of the special diseases
studied could be reproduced by the soluble products of the causative
organisms and by their later work that one of the important phases of
immunity is due to the development of substances capable of neu-
tralizing these products. It became evident with further work that
this principle does not apply to all pathogenic organisms, and the
work of Pfeiffer with cholera in 1891 led to the differentiation of
exotoxins and endotoxins.

The antagonistic action of blood and body fluids on putrefaction
had been noted by John Hunter, Traube, and Lister, but Grohman
in 1884 was the first to publish well-founded experiments upon the

INTRODUCTION xvii

inhibition by fresh plasma of the actual growth of bacteria. Fliigge
and Nuttall in 1888 demonstrated under the microscope the destruc-
tion of bacteria by blood, and Buchner in 1889 showed this property
to be present in the serum. At about the same time the work of
Richet and Hericourt and of Babes and Lepp showed that an
immunity artificially produced against pyogenic cocci and against
the virus of rabies could be transferred from one animal to another
by means of the blood serum. These studies were followed almost
immediately by the discoveries of Behring and Kitasato that the
serum of animals immunized to the toxins of tetanus and of diph-
theria bacilli not only could produce immunity in other animals, but
that the specific disease could be cured by the use of the respective
sera. These discoveries led immediately to the development of serum
therapy, and in 1894 diphtheria antitoxin was being marketed in
Germany. Contemporaneously with these developments Metchnikoff
conducted his observations and experiments upon phagocytosis, and
in 1883 published his " Recherches sur la digestion intracellulaire."
He studied various lower forms of life, such as echinoderms, and
found that during metamorphosis the atrophic cells of the larvae are
devoured by other cells, either leucocytes or other phagocytic cells.
These studies were later extended to include reparative conditions,
such as the healing of wounds and resistance to infection. The out-
come was a series of brilliant discoveries of the part phagocytosis
plays in combating bacterial invasion, and ultimately the practical
application in the use of bacterial vaccines for prevention and treat-
ment of infectious disease. The discovery of the various forms of
immune bodies and of the substances which might lead to the pro-
duction of such immune bodies followed with considerable rapidity,
but the details may best be left to the study of the particular immune
bodies concerned, which include agglutinins and precipitins, cytoly-
sins, and other complement binding substances. " That a plague
of diarrhea in a poultry yard, studied by a professor of chemistry,
should be the seed from which has grown the vast development of
later years is a strange fact, but fact, nevertheless " (Adami).

THE PRINCIPLES OF
IMMUNOLOGY

CHAPTER I
VIRULENCE OF ORGANISMS

MUTUAL RELATIONS OF PARASITE AND HOST.
PARASITISM.

VIRULENCE.

METHOD OF DEMONSTRATION.
THE BASIS OF VIRULENCE.
ANIMAL PASSAGE.

CAPSULE FORMATION.
AGGRESSINS.

POISONOUS SUBSTANCES OF BACTERIAL ORIGIN.
PTOMAINS.

TRUE TOXINS (EXOTOXINS).
ENDOTOXINS.

POISONOUS BACTERIAL PROTEINS.
ALTERATIONS OF VIRULENCE.
INCREASE OF VIRULENCE.
DECREASE OF VIRULENCE.

Mutual Relations of Host and Parasite. — The existence of in-
fectious disease depends fundamentally upon the invasion of a
plant or animal by some infective agent. The infective agent is
usually a microorganism either bacterial or protozoan in nature,
although infestations by larger organisms, such as worms within
the body or various forms of pediculi upon the surface, are some-
times spoken of as infections. In addition to bacteria the vegetable
world includes parasites, such as yeasts and fungi, which are cap-
able of producing disease. The actual production of disease depends
fundamentally upon the interrelationship between the infectious
agent and the invaded body. Bacteria are widely distributed in
nature, but the greater number of varieties have no capacity for the
production of disease. Those which produce disease are spoken of
as pathogenic, and those which do not produce disease are spoken
of as non-pathogenic. There are forms, however, which although
they ordinarily do not produce disease, may, under certain circum-
stances, develop this character. Animals and plants possess cer-
tain factors of resistance to the invasion of pathogenic organisms,
and the pathogenic organisms possess certain characters which
favor invasion. Both animals and plants live in constant associa-
tion with microorganisms, and apparently in many instances both
are benefited by this association. It is well known that certain
plants require for favorable development the association of the nitri-
fying bacteria. The intestinal canal of man, although free from

. V : ;liRE PRINCIPLES OF IMMUNOLOGY

few days of extra-uterine life, soon becomes in-
habited by large numbers of organisms, which produce no deleteri-
ous effect under ordinary circumstances and, in fact, appear to aid
in the process of digestion. Animals may adapt themselves to
organisms even of the pathogenic varieties, as, for example, in the
condition known as the " carrier state," in which virulent diphtheria
bacilli or virulent typhoid bacilli are harbored without apparent
harm. This capacity is due to certain changes which take
place in the body, so that the organisms and their products do no
damage. In the carrier state the organisms themselves have prob-
ably developed a state of resistance against substances produced in
the host which ordinarily would destroy the organisms and neu-
tralize their toxic products.

Parasitism. — The parasite is a living organism which carries on
its existence within or upon its host, and derives its nutrition there-
from. Parasitic bacteria include both pathogenic and non-pathogenic
forms. Bail has classified bacteria in three forms: (i) Pure sapro-
phytes, which do not develop within living animal tissue, but derive
nutrition from dead material; these may be pathogenic, provided
they produce poisonous substances which may be absorbed, as is
the case with the bacillus aerogenes capsulatus; (2) pure parasites,
which live entirely within tissues, including such organisms as the
anthrax bacillus ; they may exist in a vegetative form for long
periods of time outside the body; (3) half parasites, which may be
pathogenic if introduced into the animal body, but do not possess
the invasive character and the necessity for life within tissues ex-
hibited by the pure parasites. Most of the bacteria pathogenic for
man belong in this last group, as, for example, the bacillus typhosus
and the cholera vibrio. Such organisms as these may live and grow
for long periods of time in water, and in foods of various kinds, may
vegetate for a certain period under unfavorable conditions, but upon
introduction into a susceptible host produce local lesions and in
some instances may become moderately invasive for the entire
organism. Symbiosis has relatively little significance in human
medicine, but certain instances occur, as, for example, the apparent
symbiosis of the fusiform bacillus and the spirillum of Vincent's
angina. Certain parasitic protozoa, such as the endameba histolytica
of dysentery, require the associated presence of bacteria, but these
latter are not necessarily pathogenic and the phenomenon is not
that of symbiosis, because the endamebse live at the expense of the
bacteria, and the organisms, therefore, are not mutually advantage-
ous to the existence of each other.

Virulence. — By the term virulence is indicated the capacity of
an organism to produce disease. The degree of virulence may differ,
not only between different species of organisms, but between strains
within the same species. It is probably true also that individual
organisms in the same culture possess different degrees of virulence.
Furthermore, the virulence of a species or of a particular strain

VIRULENCE OF ORGANISMS 3

may be altered by favorable or unfavorable conditions. Virulence,
however, does not depend entirely upon characters inherent in the
infectious agent, because the production of disease is an exhibition
of reaction between invading organism and host. We may, there-
fore, say that virulence depends upon two groups of factors, those
inherent in the invading organism and those dependent upon the
resistance exhibited by the attacked individual. This resistance on
the part of the host is represented by the condition of immunity and
will be discussed subsequently. The capacity of the infecting organ-
ism in the production of disease depends upon certain inherent ele-
ments of virulence which are not well understood, upon the capacity
of the organism to protect itself against the defensive mechanism of
the host, upon the capacity to produce certain substances which aid
invasion, and upon the development of poisonous bacterial products.

Demonstration of Virulence. — Inherent virulence of organisms
may be demonstrated by the administration of accurately measured
doses of the organism and observance of the effects upon susceptible
animals. Ordinarily the dose is measured in the form of certain
quantities of fluid culture. Growths on solid media may be meas-
ured by the use of a platinum loop so standardized as to take up
approximately 2 mg. of the organisms. Such growths may also be
measured by suspension in a suitable menstruum. If 5.0 c.c. of salt
solution are added to a slant agar culture, fractions of the resulting
5.0 c.c. suspension contain equivalent fractions of the total surface
growth. The most accurate method is that of Barber, who has developed
a technic in which the use of a capillary tube permits picking a single
organism out of a suspension. Of importance in considering viru-
lence from this point of view is not only the quantity of organisms
injected, but also the length of time they have lived upon artificial
media, inasmuch as prolonged cultivation leads to deterioration of
virulence. If a culture is maintained for a period of time without
transplantation considerable numbers of the organisms die, and
therefore may constitute a part of the bulk injected, at the expense
of living organisms. This is not a true decrease of virulence and
constitutes a factor of error. The route of injection is also of im-
portance, because certain organisms may be virulent by one route
of injection and not so by others. For example, the cholera vibrio
may produce disease by introduction into the intestinal tract and is
entirely without pathogenic effect when introduced subcutaneously.

The Basis of Virulence. — The studies of pathogenic bacteria have
shown that they may acquire or in certain instances may lose viru-
lence by passage through animals, and that they may lose virulence
by cultivation upon artificial media. The method whereby they
acquire virulence has been extensively studied. It is well known
that the pneumococcus possesses a capsule when growing in ani-
mal tissue, but that it loses its capsule after artificial cultivation.
This is true of certain other organisms, and it has been demon-
strated that the protection afforded by the capsule makes these

4 THE PRINCIPLES OF IMMUNOLOGY

organisms resistant to the defensive phenomena, phagocytosis and
agglutination, as will be discussed in subsequent chapters. There-
fore, capsule formation may well constitute an aid to invasion.

Aggressins. — It was found by Koch that tuberculous animals
injected intraperitoneally with fresh cultures of tubercle bacilli suc-
cumb soon after the injection, and that a considerable amount of
exudate appears in the peritoneum. This phenomenon seems to
have been the basis of Bail's aggressin theory. Bail injected tubercle
bacilli, together with sterile tuberculous exudate, into healthy
guinea-pigs and found that the injected animals died in the course
of twenty-four hours, while control animals inoculated with the
exudate alone did not show any appreciable effect, and control
animals which received tubercle bacilli alone died only after the
lapse of several weeks. He argued from this that the sterile exudate
must contain a substance or substances which are responsible for the
increased virulence or aggressiveness of the bacilli. He named this
substance " aggressin " and believed that during an infection the
organisms secrete certain substances which have power to inhibit
or destroy the protective powers of the host. These bodies are sup-
posed to be formed by the living bacteria in the living body only,
and the pathogenicity of bacteria is said to depend, in part at least,
upon their ability to produce aggressins. Bail believed further that
the germicidal activity of body fluids in natural immunity had been
overemphasized. He had noted with Petterson that animals highly
susceptible to anthrax often possessed sera which had marked bac-
tericidal powers against the anthrax bacillus. If such animals were
inoculated with a few hundred organisms, a number easily destroyed
by their sera, they nevertheless rapidly succumbed to the disease.
Bail also showed that the peritoneal fluid of guinea-pigs dying after
a fatal injection of typhoid or cholera organisms possessed the
ability to increase the virulence or infectivity of particular strains
that would otherwise have been harmless. Experiments of this
kind were also performed in dysentery, chicken cholera, pneumonia,
and staphylococcus infections, and the results obtained were identi-
cal with those observed in the case of tubercle bacillus. Heating
the exudate to 60° C. instead of inhibiting, increased the aggres-
siveness of the organisms. Small doses appeared to act relatively
more strongly than larger doses. In tuberculous animals the tissues
seemed to be saturated with this body, and when fluid collected in
the body cavities, as happens on injection of tubercle bacilli, these
fluids contained large quantities of aggressins capable of inhibiting
phagocytosis by preventing the migration of polymorphonuclear
leucocytes. Bail, however, was not the first to observe this par-
ticular phase of bacterial offense. Salmon and Smith, as early as
1884, noted that bacteria multiply in the tissues of their host be-
cause of a poisonous principle which is produced during their
growth and multiplication. Kruse maintained that the organisms
secrete ferment-like bodies (referred to as " lysins ") which have

VIRULENCE OF ORGANISMS 5

the power of inhibiting the bactericidal activity of the blood serum,
thus allowing the invader opportunity for further invasion. By re-
peated injections of aggressin exudates into animals, Bail suc-
ceeded in immunizing these animals against various infections, thus
producing anti-aggressins. These rendered the bacteria defenseless
and permitted unhindered phagocytosis. The agglutinative power
of the sera of such animals was markedly enhanced.

Wassermann and Citron, and many others, soon opposed Bail's
aggressin hypothesis, by pointing out that the phenomenon can be
explained without assuming that a new type of immune body is
concerned. Wassermann and Citron, Wolff, Sauerbeck, and also
Doerr found that the action of the so-called aggressins can be ex-
plained by the fact that exudates contain extracts of the bacteria.
Artificial aggressins were prepared by making extracts of bacteria
in vitro. It thus seems probable that the aggressins are nothing
more than endotoxins which have a negative chemotatic influence
and a non-specific action. Citron was able to show by means of com-
plement-fixation that the exudates contain free bacterial receptors,
which by absorbing immune bodies, tend to neutralize the destruc-
tive power of these antibodies. Levy and Fornet showed that fresh
twenty-four- to forty-eight-hour culture filtrates of bacillus
typhosus, paratyphosus, pyocyaneus and proteus possess non-
specific aggressive powers and, according to Ikomikoff, aggressins
of bacillus coli, staphylococci, and vibrios will act interchangeably,
thus showing the non-specific nature of these substances. From
Zinsser and Dwyer's experiments these bodies appear to be practi-
cally identical with anaphylatoxins (see page 218). The addition of ana-
phylatoxin to bacteria will change a sublethal dose into a lethal dose.

Closely related to the aggressins are the " virulins " of Rosenow.
This author found that freshly isolated cultures of pneumococci
were not readily phagocyted, but this property was lost on repeated
subculture. He prepared salt solution extracts of the virulent
strains. Upon treating avirulent strains for twenty-four hours or
more with these extracts, the avirulent organisms became virulent
for animals, and at the same time resistant to phagocytosis. The
substance contained in the salt solution extracts capable of render-
ing the organisms virulent was named virulin. This substance ap-
pears to be essentially the same as the aggressin prepared in vitro
by Wassermann and Citron. In our opinion, these extracts, whether
prepared in the form of exudates or as extracts, contain poisonous
bodies which augment the invasiveness of the organism. They may
be non-specific bacterial proteins or protein products, such as are
probably contained in so-called anaphylatoxin. They may be
in part endotoxins. The anti-aggressins of Bail are agglutina-
tive and are probably called forth by the injection of the ex-
tracted bacterial proteins in the exudates or extracts. It seems
probable also that the effect of these anti-aggressins may depend
upon their agglutinative capacity. The subject is confused and in-

6 THE PRINCIPLES OF IMMUNOLOGY

tricate, and whilst at present we are disposed to regard the aggressins
as extracts of the bacterial proteins and their split products, as well,
perhaps, as exotoxic in nature, further study may offer more com-
plete and satisfactory explanation of the problem.

Production of Poisonous Substances.— The virulence of organ-
isms depends, to a certain extent, upon the poisonous substances which
they produce. Nevertheless, virulence is not necessarily parallel to
the capacity for production of these toxic substances. The poison-
ous bacterial products may be divided into four groups, namely, the
ptomains, which are the result of decomposition of the media upon
which the bacteria grow; the exotoxms or true toxins, which are
soluble poisons produced by the life activities of the bacteria and
easily absorbed and diffused in the body of the host ; the endotox'ms,
which develop within the bodies of the bacteria and are liberated
probably only upon the death and disintegration of the bacteria ; and
poisonous bacterial proteins, which result in large part from the break-
ing down of the protein molecules which go to constitute the
bacterial substance.

The ptomains are formed from the decomposition of the media
upon which bacteria grow, provided these media are nitrogenous in
nature. The ptomains are basic substances formed not from the
bacteria themselves, but from the decomposition products of those
media which contain nitrogenous material, especially proteins whose
nitrogen is in the form of amino-acids. Most ptomains are combina-
tions simply of carbon, hydrogen, and nitrogen, and they may be
divided into three groups, the first of which includes methylamine,
dimethylamine, and trimethylamine ; the second group somewhat
more complex, contains putrescin and cadavarin; the third group,
the so-called cholin group, contains, in addition to cholin, neurin,
muscarin, and betain. The cholin group are derivatives of lecithin.
Cholin itself is found in extremely minute amounts in body cells and
has a relatively low degree of toxicity. It is a substance which has
been the subject of much experiment and hypothesis, but there is
no very good reason for believing that it has any great pathologic
importance. Neurin may be transformed from cholin, and although
somewhat similar chemically, it is highly poisonous. Muscarin is a
crystalline alkaloid obtained from poisonous mushrooms, but is also
formed by the decomposition of fish; its chemical composition is
very closely similar to that of neurin, and it may be prepared syn-
thetically from cholin. Both neurin and muscarin produce definite
toxic symptoms in man following subcutaneous injection of i to 3
milligrams, but when given by mouth approximately ten times this
amount are required, indicating that probably the liver breaks up
and detoxifies that which is absorbed from the intestine. Betain is
a constituent of plant tissues and has a toxicity from one-tenth to
one-twentieth that of neurin and muscarin. The simpler ptomains
are not extremely toxic. The ptomains as a group are not specific in
any sense, except in so far as they are dependent on the chemical

VIRULENCE OF ORGANISMS 7

composition of the media upon which the bacteria grow, and any
differences in constitution of ptomains are differences due to varia-
tions in medium rather than variations of bacteria. In this respect
they differ from toxins. Furthermore, it is not possible to produce
immune substances against ptomains. Ptomains are not to be con-
fused with toxins produced by bacillus botulinus, by bacillus enteri-
ditis, or other members of the " food-poisoning " group, which are
true toxins and are capable of inducing the formation of antitoxins.
Food poisoning may, therefore, be due to the decomposition of food
with the production of ptomains which are absorbed and produce
toxic symptoms, or may be due to the presence in food of toxins
produced by the bacillus botulinus and similar organisms. In addi-
tion to the ptomains which contain C, H and N, a fourth group
contains also oxygen, as exemplified in the substance sepsin ob-
tained from decomposing yeast cells. This is closely related to
cadavarin in its chemical composition and acts as a powerful dilator
of intestinal capillary blood-vessels from which diapedesis may occur.

The true toxins or exotoxins are soluble and diffusible poisonous
substances produced by the life activity of bacteria. They may be
produced when the organisms exist in a parasitic state or when they
grow upon artificial media and the nature of a toxin for any given
species is not determined by the medium upon which the organisms
grow, except in so far as certain media favor the production of
greater amounts of toxin than do others. The diphtheria bacillus
produces the same toxin regardless of the medium upon which it is
grown, although nutrient veal broth is the most favorable for toxin
formation. The same general statement is true of the tetanus
bacillus and those other organisms which produce toxins. Toxins
are unlike ptomains in that they have not a definite chemical com-
position and in that they serve to induce antitoxin formation. They
have certain resemblances to enzymes, but are probably not identi-
cal with enzymes. The nature of toxins, their action, and other
details are considered in the chapter on toxins and antitoxins.

The endotoxins develop within the bodies of bacteria and are not
secreted into the surrounding medium. They apparently are only
liberated upon the death and disintegration of the organisms. It is
not certain that they can be differentiated absolutely from the
poisonous bacterial proteins, and it is extremely difficult to induce
antitoxin formation by their use. If they are injected into an ani-
mal the animal may produce agglutinins and precipitins, but not
antitoxin. This subject also is discussed subsequently.

Poisonous Bacterial Proteins. — The whole protein of certain
bacteria is poisonous, and the work of Vaughan and Novy shows
that the split products of bacterial proteins produced by treatment
with alkalinized alcohol are extremely toxic. These substances
apparently are not specific as regards the bacteria from which they
originate, but owing to their poisonous properties they may add to
the virulence of the organisms. Similarly toxic split products may

8 THE PRINCIPLES OF IMMUNOLOGY

be obtained from other proteins, such as those of cheese and milk.
The poisonous effect is in some way connected with the foreign
character of proteins. In some respects these substances resemble
ptomains, but they are certainly not of the same constitution. They
are obtained from bacteria regardless of whether these produce
toxins, endotoxins, or ptomains, and are fatal for animals in very
short periods of time. The methods for the production of endo-
toxins are such as may lead to splitting of bacterial proteins, and at
the present time no satisfactory differentiation can be made. The
chapter on anaphylaxis and hypersusceptibility will present a dis-
cussion of the poisonous substance called anaphylatoxin, which may
also be related to the general group of poisonous split products.
The influence of toxin on invasion by certain bacteria is illustrated
by the recent work of Bullock and Cramer. They found that
bacillus aerogenes capsulatus, vibrion septique, bacillus edematiens,
and often bacillus tetani, when completely freed of toxin by washing
or by heating to 80° C. for one-half hour do not produce the special
disease upon injection into the rat or guinea-pig. The usual de-
fenses of the animal, such as bacteriolysis and phagocytosis, are
sufficient to rid it of the bacteria in the absence of toxins. A point
of further interest in this work is the discovery that if a small dose
of a soluble ionizable calcium salt be injected before or at the same
time as the spores or toxin-free bacteria, the defenses are broken
down and the special disease results. The experiments showed that
this is not the result of action upon the bacteria, but is due rather
to some influence upon the host. Bullock and Cramer suggest the
name " cataphylaxis " for the rupture of defense. Other salts have
no such effect, and it is possible to demonstrate the antagonistic
action of magnesium upon calcium in similar experiments. It is
difficult to find a series of experiments showing more clearly the
delicacy of balance between resistance and infection.

Alterations of Virulence — Increase of Virulence. — As has been
indicated above, virulence may be increased by the passage of organ-
isms through animals, and this method is commonly employed in
laboratory work. The increase of virulence of the pneumococcus by
passage through mice is an excellent example of the process. The
organisms are injected intraperitoneally, recovered upon the death
of the animal, cultivated for twenty-four hours, reinoculated, and the
process repeated until a satisfactory degree of virulence is obtained.
The degree of virulence is usually measured in terms of the bulk of
broth culture which will kill an animal in a given period of time.
In the case of some bacteria an increase of virulence by animal
passage is only effective for the animal concerned; and the fact
that an organism exhibits increased virulence for a guinea-pig does
not necessarily presuppose that the same increase will apply to other
animals. Not only is this true of direct animal passage but, as has
been shown by Danysz, cultivation of an organism upon media con-
taining rat tissue may increase the virulence for the rat but not for

VIRULENCE OF ORGANISMS 9

other animals. The importance of proper selection of the animal
species for increasing bacterial virulence is emphasized by the work
of Hussy, who found that the passage of streptococci through
mammals and fish increased the virulence, but their passage through
birds decreased the virulence. In the increase of virulence by means
of animal passage, the organism apparently may develop a mechan-
ism of resistance against the protective activity of the animal body,
as has been discussed above. Another method of increasing virulence
is to place the organism in collodion sacks. These are planted in
the peritoneal cavity of an animal and apparently the slow diffu-
sion of the animal fluids into the sack permits the organism to
acquire resistance to the antagonistic substances of the animal and
thus increases its virulence. A third method of increasing virulence
is to grow organisms upon media which contain blood serum or
other animal fluids. By several transfers upon such media the
organisms may acquire resistance similar to that obtained in the
other methods. A fourth method has been applied, depending upon
the separation of the more virulent individuals in a culture from
the less virulent at the height of phagocytosis. A culture is in-
oculated into the peritoneal cavity of an animal, such as the guinea-
pig, and by removing small quantities at regular intervals the time
of greatest phagocytosis by the peritoneal cells is determined. The
entire exudate is then withdrawn and slowly centrifuged, so as to
throw down the cells, leaving the unphagocyted bacteria in the
supernatant fluid. The organisms in the supernatant fluids are
cultivated, and if they are not sufficiently virulent the process may
be repeated until a satisfactory culture is obtained.

Decrease of Virulence. — The virulence of pathogenic organisms
may be decreased by removing them from the favorable environ-
ment of the animal host and growing them upon artificial culture
media. As they become accustomed to this type of existence they
usually lose considerably in virulence. As has been indicated above,
there are instances where animal passage may decrease the virulence
of certain infective agents. Whereas the virus of rabies increases
up to a standard maximum on passage through rabbits, similar pas-
sage through monkeys will decrease its virulence. It is probable,
also, that the natural passage from dog to dog decreases virulence.
It is now generally accepted that cowpox is the same disease as
smallpox, yet the inoculation of cowpox into man produces a very
mild form of disease. Therefore, it is to be presumed that the pas-
sage of smallpox virus through the calf reduces the virulence. A
similar example is found in the work of Hussy on the streptococcus
quoted above. Decrease of virulence by animal passage is not
clearly understood. It may be due to the same factors that influ-
ence virulence in artificial culture media, whereby the organisms in
an unfavorable environment lose their ability to combat the resist-
ance of the animal host, or it may be due to a direct lowering of
virulence as the result of more or less successful attacks of the pro-

io THE PRINCIPLES OF IMMUNOLOGY

tective mechanism of the animal body. The influence of heat on the
virulence of organisms is now well known. The degree of heat and
the time of exposure must be so adjusted as to reduce virulence
without causing actual death of the organisms. Similar reduction
of virulence or attenuation may be accomplished by growing the
organisms at temperatures which are not optimal. The first ex-
ample of this was Pasteur's work in the attenuation of anthrax
cultures by growth at 42° to 43° C. The attenuation by means of
drying was practised in the classical work of Pasteur on rabies.
The virus contained in the spinal cord of rabbits was subjected to
desiccation, and it was found that the longer the time of desiccation
the less potent was the virus. Chemical agents, such as phenol,
acids, iodine and its salts, potassium bichromate, and others may
also be used in proper concentrations and for proper periods of time
to produce attenuation. Physical agencies, such as growth under
pressure, the influence of light, etc., have been employed for pur-
poses of attenuation. Of interest in connection with attenuation is
the fact that certain organisms, when introduced into the body, vary
in virulence, depending upon the route of introduction. For ex-
ample, the virus of rabies may be injected intravenously into goats
and sheep without producing rabies. This injection, however, serves
to confer a certain degree of immunity upon the animals. As has
been mentioned before, the organism of cholera may be injected
subcutaneously without producing disease, and within certain limi-
tations aids in the protection against invasion by these organisms
through the intestinal canal.

CHAPTER II

GENERAL CONDITIONS OF INFECTION AND
RESISTANCE

THE PRODUCTION OF INFECTIONS.

ENTRANCE OF THE INVADING ORGANISM.

TYPES OF INFECTIOUS DISEASES.
FACTORS FAVORING THE INVADER.
FACTORS INHIBITING THE INVADER.

FACTORS OPERATING AGAINST RESISTANCE OF HOST.
FACTORS FAVORING THE HOST.
THE COURSE OF ACUTE INFECTIOUS DISEASE.

The Production of Infection. — The widespread dissemination of
bacteria in nature is such that they have ready access to plants and
animals. Invasion by pathogenic forms may set up- infection.
Whether or not the infection may lead to disease depends upon the
final relationship established between the invader and the invaded
body. There is probably no condition under which animals or
plants fail to exhibit some degree of resistance to the invading
organism, and similarly the latter attempts to accommodate itself
to the conditions found in the invaded host. If the resistance be not
sufficient to overcome the invader, infection results. The produc-
tion of disease, however, depends upon the superior powers of the
invader over the resistance of the host. Occasionally a mutual
adaptation appears, under which circumstances an animal may be
infected by an organism, but shows no symptom or sign of dis-
ease. Not infrequently the trypanosoma Lewisi is found in the
blood stream of rats, the rats continuing to live an apparently nor-
mal existence. A similar mutual adaptation is found in the " carrier
state," wherein man may harbor virulent diphtheria bacilli or other
organisms without any evidence of disease. Mutual adaptation is
not attained without a struggle on the part of both invader and
host, and infectious disease results when the invading organism
triumphs. This does not mean permanence of infection, because
even although disease is established, the defenses of the host
continue to operate, and often are augmented in such a way that
ultimately the infection disappears. This accounts for the self-
limitation of most of the acute infectious diseases. The increase in
defensive powers may in certain diseases become permanent and
immunity thereby be established. In all cases of recovery from
acute infections immunity of some duration appears, although it may
be limited to a few weeks or a few months.

Entrance of the Invader. — The entrance of the invading organ-
ism may be due to an interruption of continuity of those surfaces of
the body which ordinarily are impermeable to bacterial invasion.
These surfaces include skin and the mucous membranes of the re-
spiratory, alimentary, and genito-urinary tracts. The interruption

ii

12 THE PRINCIPLES OF IMMUNOLOGY

of continuity may be due to trauma or may result from profuse
growth of bacteria on the surface with the elaboration of poisonous
products which may kill the epithelial cells. The former condition
is exemplified in infected wounds and the latter in infection by
diphtheria bacilli, streptococci, and fungi, such as produce favus,
thrush, and pityriasis. Entrance may be favored by changes in the
character of secretions, as, for example, the reduction of acidity of
the gastric juice in certain forms of chronic gastritis. The bacteria
may be implanted in some site which favors their multiplication, as,
for example, in the crypts of the tonsils, in the crevices between
unclean teeth and in hair follicles. Multiplication in these situa-
tions favors the production of poisonous products which may by
destruction of cells serve to interrupt surface continuity. Somewhat
similar is the fact that extensive destruction of tissues may provide
dead material in which saprophytes may develop, and if this mate-
rial is so deep as to be excluded from the access of air, conditions
favorable to the development of anaerobes are produced. Infec-
tion may be favored by the movement of cells and fluids. For
example, although leucocytes may take up bacteria, they do not
invariably destroy them, and the migration of such leucocytes may
lead to the dissemination of organisms by the subsequent death of
the leucocyte. The movement of lymph may favor invasion as is
seen not uncommonly in those cases of infections of the hand by
streptococcus, wherein the lymph flow carries the organisms so as
to set up infections of the lymph-vessels and the lymph-nodes, and
even of the blood stream. Gaining access to the blood, the circula-
tion of this fluid tissue may deposit bacteria in numerous foci
throughout the body. The route of invasion depends somewhat
upon the type of organism, those of typhoid fever, dysentery,
and cholera, gaining access to the intestinal canal through the
mouth. Their implantation upon the skin is of no significance,
except that they may thence be transferred to the mouth. The
gonococcus produces no lesions of the intestinal canal, but implanted
in the genital tract, the eye, or the endocardium leads to serious
results. If plague bacilli be inoculated subcutaneously in rats a
large percentage of the animals survive, but if implanted in the
lower respiratory tract small doses suffice to produce fatal infec-
tions. The pneumococcus appears to infect man only through the
respiratory tract. This phenomenon probably depends in part upon
a local susceptibility to the organisms.

Types of Infectious Disease. — The types of infectious disease
are differentiated according to the method of invasion and dissem-
ination. An organism may grow locally and produce only local
manifestations, as seen in a small abscess. It may grow locally and
produce marked general disturbances, as is the case in diphtheria, in
which instance, although organisms may enter the blood stream,
they are usually confined to some focus, such as the tonsils. They
elaborate in that situation poisonous substances which are absorbed
and set up general manifestations of intoxication. Certain other

INFECTION AND RESISTANCE 13

diseases may produce marked local manifestations and rapidly in-
vade the blood stream, as is true of typhoid fever. This organism
enters the lymph-nodes of the intestinal tract, produces enlarge-
ment, softening, and necrosis. The diarrhoea in these cases is largely
if not wholly due to the local lesions, but the severe general mani-
festations are due principally to the entrance of the organisms into
the blood stream. Other diseases may show little local manifesta-
tion, as is true of tetanus, but even with slight local disturbances
profound general symptoms occur as the result of absorption of
toxin. Other diseases, such as anthrax, may show little local mani-
festation, but rapidly exhibit generalized infection through the blood
stream. Infection then simply signifies successful invasion. Bac-
teremia signifies the presence of organisms in the blood. Septicemia
signifies blood infection associated with the production of toxic
substances. Pyemia indicates that bacteria are present in the blood
stream and because of lodgment in numerous situations produce
multiple abscesses. Sapremia indicates absorption of toxic products
from the growth of saprophytic organisms. Primary infections are
those which occur without any decrease of resistance due to another
infection. Secondary infections occur in individuals already suffer-
ing from an infection of another nature. Such an infection is well
exemplified in the secondary infection of a tuberculous cavity of the
lung by staphylococcus. Terminal infections are those which occur
near the fatal termination of some other disease, whether that other
disease be of bacterial nature or of some other origin. Infections of
this type are seen in the terminal broncho-pneumonias and sep-
ticemias which occur in the course of certain chronic diseases.
Mixed or multiple infections are not rare and it is sometimes diffi-
cult to determine which infection is of greater importance. There is no
doubt that one infection influences another existing at the same time and
usually in a manner deleterious to the patient. Infection with measles or
lobar pneumonia may excite latent tuberculosis into activity. Duke
reports the lighting up of latent syphilis by an attack of typhoid
fever and of latent gonorrhea by an attack of tonsillitis. The re-
moval of one chronic infection may favorably influence another, as
seen in the relief of certain cases of pyorrhea alveolaris by the re-
moval of infected tonsils and in numerous other instances of mul-
tiple chronic infections.

Factors Favoring the Invader. — The small size of pathogenic
bacteria and protozoa aids in their avoidance of detection, favors
transportation, and aids in penetration. The rapidity of multiplica-
tion of such organisms is of considerable importance to their patho-
genic powers. Those bacteria which form spores resist destructive
agents and can resume activity when favorable conditions present.
Certain of the protozoa, more particularly the endamebae, are cap-
able of forming cysts which are more resistant to unfavorable en-
vironment than the active organism. Either in the active state or
in the vegetative state, organisms may persist for a long time in the
so-called carriers, in intermediate hosts, or living as saprophytes.

i4 THE PRINCIPLES OF IMMUNOLOGY

The microparasites, therefore, can be said to have a ready adapta-
bility to varying environment and to be aided in propagation by
their ability to derive nutrition from food-stuffs which possess
wide differences in constitution. Certain bacteria apparently can
produce their own protein from amino-acids and have no diffi-
culty in deriving nutrition from whole proteins. As has previ-
ously been indicated, those factors which go to increase virulence
of organisms, such as capsule formation and the production of toxic
substances, aid materially in invasion.

Factors Inhibiting the Invader. — Although rapid multiplication
aids invasion, nevertheless, the brief life period which most of the
microparasites exhibit is an influence operating against rather than
in favor of invasion. Many pathogenic organisms are susceptible
to the destructive influence of light, heat, desiccation, etc. In cer-
tain instances the life of organisms outside an animal body operates
to reduce virulence and therefore to inhibit the capacity for inva-
sion and production of disease.

Factors Operating Against Resistance. — The animal host is sub-
jected to the attacks of invading organisms because of the multi-
plicity of contacts with the environment. The large body surface
and locomotion of the body are influences favoring approximation
of the invader. Certain living activities, such as the ingestion of
foods and water, coitus, and the ready availability of superficial
orifices, such as the nose, ears, mouth, anus, genital orifices, all aid
invasion. The fact that most animals have a constant body tem-
perature and that their tissues are continually moist, provides con-
ditions favorable to the invading organism. Although light rays
beyond the violet end of the spectrum have a certain capacity for
the penetration of tissues, yet ordinary sunlight exhibits very little
penetrability; therefore, the construction of the body is such that
the inhibitory effect of light is not brought to bear upon organisms
that have already gained entrance. The anatomy of the body pro-
vides certain structures which are relatively inactive, such as the
appendix vermiformis and the crypts of the tonsils where organ-
isms find moisture, warmth, and darkness, suitable for their de-
velopment. In chronic infections, particularly by the tubercle
bacillus, necrotic tissues, or actual cavities may exist in contact with
surfaces and with the outer air, and both conditions operate to re-
duce resistance by providing favorable places for bacterial multipli-
cation. The circulation of lymph and blood may operate against
the host if organisms are particularly virulent. Inspiration of con-
taminated air may also serve to aid invaders. The resistance of the
host may, in a manner as yet unexplained, be decreased in general
by bodily fatigue, exposure to heat and cold, poor hygienic sur-
roundings, deleterious gases, or improper diet. The extremes of
life, childhood and age, are associated with reduced resistance..
Drugs, operative procedures, improper diet, and similar conditions
favor infection.

Factors Favoring the Host.— The possession of intelligence by

INFECTION AND RESISTANCE 15

the higher forms of animal life aids in the detection and elimination
of infective organisms. Not only may this be accomplished by vol-
untary movement, but the purposeful action of involuntary re-
flexes may similarly aid the host. The body possesses a variety of
defenses in the form of structure, secretions, chemical substances,
cellular activity, all of which serve to aid in its protection in connec-
tion with natural and acquired resistance to disease. These will be
discussed in the next chapter. Plants produce certain diastases,
aromatic products, aldehydes, and other substances which create in
the plant a state deleterious to germination of harmful invaders.
Pigments such as chlorophyl may destroy toxic substances and
even bacteria in a manner somewhat similar to the action of
bile pigment.

The Course of Infectious Disease. — The exact moment of inva-
sion of an infectious agent is difficult to determine, but in cases of
infectious disease, the time of exposure to infection can usually be
stated to have occurred within the limits of a few hours. Following
the moment of invasion there occurs a period of incubation during
which the host exhibits no symptom of infection. This period of
incubation in some diseases is extremely variable, whereas in others
it is relatively fixed. In diphtheria incubation may apparently vary
from twenty-four hours up to nine or ten days,, and certain other
diseases show similar variation. In scarlet fever, on the other hand,
the incubation period is very commonly five days, and numerous
other diseases show similar fixity of incubation time. Following
the period of incubation the less violent infectious diseases show a
short period of prodromal symptoms in which headache, malaise,
and other minor manifestations may appear. The next period, that
of onset of disease or so-called invasion, may be frank or insidious.
Lobar pneumonia may develop within a period of a few hours and
exemplifies frank onset. As a contrast, typhoid fever is likely to
occupy a week or ten days between the period of prodromal symp-
toms and the full development of disease, thus illustrating insidious
onset. That period during which the disease is at its height is called
the fastigium or acme. Following the fastigium comes the period
of decline or defervescence. This may be by crisis or lysis. Crisis
is seen in approximately half the cases of lobar pneumonia, in
which the decline occurs in a period of a few hours. Deferves-
cence by lysis is seen in a large number of infectious diseases and is
particularly well exemplified by typhoid fever in which several days,
a week, or more, may be consumed. Convalescence indicates that
period during which the symptoms of disease have practically dis-
appeared and the patient gradually recovers and is restored to nor-
mal. At any period the infection may become so overwhelming as
to cause the death of the individual. Chronic infectious diseases
exhibit no such regularity of development and decline. In con-
trast to the acute infections, these are not likely to be self-limited,
but progress until they have reached a point of such great severity,
or of such complete exhaustion of the host that death ensues.

CHAPTER III
THE GENERAL PHENOMENA OF IMMUNITY

TYPES OF IMMUNITY.

NATURAL IMMUNITY.
SPECIES.
RACIAL.
FAMILY.
INDIVIDUAL.
INHERITED IMMUNITY.
ACQUIRED IMMUNITY.

NATURALLY ACQUIRED.
ARTIFICIALLY ACQUIRED.

ACTIVE ARTIFICIALLY ACQUIRED.

INOCULATION OF LIVING VIRUS IN HEALTH.
USE OF ATTENUATED VIRUS.
USE OF DEAD BACTERIA.
USE OF BACTERIAL PRODUCTS.
PASSIVE ARTIFICIALLY ACQUIRED.
THEORIES OF THE NATURE OF IMMUNITY.
THE EHRLICH SIDE-CHAIN THEORY.
THE EHRLICH CLASSIFICATION OF IMMUNE BODIES.
CRITICISM OF THE EHRLICH HYPOTHESIS.
THE SPECIFICITY OF IMMUNE REACTIONS.

NON-SPECIFIC THERAPY OF INFECTIOUS DISEASES.
THE SITE OF ANTIBODY FORMATION.

PRODUCTION OF ANTIBODIES AT SITE OF INJECTION.

Types of Immunity. — Resistance to disease may be natural or
acquired. If natural it may be of a species, race, family, or indi-
vidual character. If acquired it may be naturally acquired, as seen
in the immunity following an attack of infectious, disease, or it may
be artificially acquired. If artificially acquired it may be the result
of active immunization or of passive immunization. Artificially
acquired active immunity is such as may follow the injection of
various antigens, such as toxins, bacteria, and numerous other sub-
stances. Artificially acquired passive immunity is the result of
transfer of active immunity from an immune animal to a normal
animal, which latter becomes passively immunized.

Natural Immunity. — Although the term immunity may be consid-
ered as equivalent to the capacity for resisting disease, nevertheless,
in common usage it often implies an increase of resistance. In esti-
mating an increase of resistance a normal degree must be presup-
posed and the determination of the normal is extremely difficult.
In considering natural immunity the term is used in contrast to sus-
ceptibility and is not comparable to a normal level of resistance.
Natural resistance to disease is favored by structure, movement,
fluids, and secretions of the body. Structurally the skin is practi-
cally impermeable to bacteria. In a general way this is true of
mucous membranes, although we know that certain organisms may
pass through mucous membranes of the intestinal tract without any
16

GENERAL PHENOMENA OF IMMUNITY 17

lesions of the surface. Crypt-like structures, such as hair follicles,
sweat glands, crypts of the tonsils, gastro-intestinal glands, urethral
glands, may serve as foci where bacteria are able to multiply, and
may thus determine penetration by the organism. Accessory struc-
tures of the skin, such as the hairs of the anterior nares and the
cilia of certain parts of the respiratory tract, aid in either filtering
the air or in propelling lodged organisms toward external orifices.
The nature of certain secretions may be antagonistic to the growth
of certain bacteria either by virtue of chemical substances, such as
the hydrochloric acid of the gastric juice, normal alkali of the saliva
and upper intestinal tract, or by virtue of digestive ferments which
may act deleteriously upon bacterial growth. The movement of
secretions, as, for example, that of the conjunctival sac, may favor
the elimination of organisms. Bodily movement is of considerable
value in resistance to infection, whether it be the simple process of
wiping away irritating substances or the more intricate process of
bathing either with water or with definite anti-bacterial fluids. Re-
flexes such as coughing, sneezing, and vomiting are definitely pur-
posive in protection. The movement of materials in the intestinal
canal serves to prevent any too great bacterial activity, and if in
spite of normal intestinal movement irritative substances are formed,
the response by diarrhea serves a useful purpose in elimination.
Internally the fluids of the body, more particularly the blood, con-
tain definite anti-bacterial and anti-infective substances. In addi-
tion to these the non-specific ferments of the body fluids aid in
combating infection. The acidity or alkalinity of fluids within the
body, as well as certain substances of unknown nature, may serve
to retard or prevent bacterial invasion. Of great importance in pro-
tection is the reaction of inflammation. In the course of this process
fluids and cells are exuded from the vessels. The exudation of fluids
upon surfaces aids in washing away bacteria, as, for example, the
profuse exudation of fluid in acute coryza and acute enteritis. Accu-
mulation of fluids may serve to dilute bacterial poisons and by diffu-
sion and absorption aid in the elimination of these poisons. The
cells which form part of the exudate possess, as characteristic func-
tions, the capacity of taking up bacteria by phagocytosis and de-
stroying them. The formation of fibrin in the exudate, as well as
the subsequent proliferation of fixed tissue cells, serves to delimit
the process and thereby aid in the prevention of widespread dis-
semination of the organisms. In superficial inflammations the ex-
foliation of diseased cells, as in scarlatina, may aid in the elimination
of the infective virus. This does not mean, however, that such cells
retain an infective character after long periods of desiccation.

The physiological activity of cells in the body may be so excited
as to aid in the elimination of toxic products, as exemplified by the
early increase of activity in infectious disease. If the toxic mate-
rial be sufficiently virulent this period of hyperactivity may be sue-

i8 THE PRINCIPLES OF IMMUNOLOGY

ceeded by one of depression. The stimulation of cells in the produc-
tion of antitoxic and anti-bacterial substances will be discussed sub-
sequently.

Classification of Natural Immunity — Species Immunity. — As has
been indicated above, natural immunity may be found in species,
races, families, or individuals. It is profitable to emphasize again
that what we speak of as species immunity expresses a difference in sus-
ceptibility exhibited by certain species as contrasted with others.
Whereas man is susceptible to such diseases as syphilis, gonorrhea,
cholera, and diphtheria, numerous other species are resistant to these
diseases. It is possible to inoculate syphilis in higher apes, in the
rabbit, possibly in the guinea-pig and other animals, but even suc-
cessful inoculation shows a greater degree of resistance than is pos-
sessed by man. Conversely, man is not susceptible to hog-cholera,
chicken-cholera, rat-typhoid, and certain other diseases. Man is
susceptible to the bacillus of human tuberculosis, but less so to
that of bovine tuberculosis, still less to that of avian tuberculosis,
and not at all to that of fish tuberculosis. In fact, with the exception
of the rabbit, fish tuberculosis is not transferable to any of the warm-
blooded animals. Fish are not susceptible to human tuberculosis.
Practically all animals are susceptible to snake venoms except the
hog. Man is highly susceptible to pneumococcus and to bacillus
pestis, but fowl are resistant to both these organisms. Metchnikoff
showed that certain species of insects are susceptible to diphtheria
toxin whilst others are not. Man is susceptible to trypanosoma gam-
biense, but is resistant to trypanosoma naganae. In some instances
these variations in susceptibility and resistance depend upon the
environment. For example, frogs kept in low temperature are not
susceptible to anthrax, but if kept in a temperature of 35° C. they
succumb to the disease. Similarly it was found that if lizards are
kept at 16° C. they could not be infected with plague, but at a higher
temperature were susceptible. The work of Pasteur with anthrax
in fowl is a classical experiment. He found that if he kept fowl at
low temperatures they became susceptible to anthrax because of the
decrease of body temperature ; but if they were allowed to maintain
their normally high body temperature they were resistant. The
temperature of most of the lower mammalia is higher than that of
man, but the difference is not sufficiently great to explain all the varia-
tions in susceptibility and resistance.

Racial immunity probably exists but cannot be so conclusively
proven in man as is true of species immunity. It is generally be-
lieved that Caucasians are less susceptible to tuberculosis than
negroes. That this is an inherent character of the race appears to be
somewhat doubtful. Difference in hygienic conditions and in de-
gree of exposure to the disease may account for much that appears
to be racial susceptibility. It is possible that the superior hygienic
conditions of whites in northern latitudes explains this difference.
It is also possible that having been the victims of tuberculosis for

GENERAL PHENOMENA OF IMMUNITY 19

many centuries a certain degree of racial immunity has been estab-
lished by virtue of the elimination of more susceptible individuals
and the survival of the more resistant. It is apparently true that
when an infectious disease first attacks a race, it is more virulent
than in those races where it is commonly found. The native African
when brought into contact with tuberculosis appears to be attacked
violently. The decimation of the population of Iceland after the
introduction of measles was one of the horrors of improved com-
munications ; subsequent epidemics of the disease in the same people
have been considerably less fatal. The introduction of syphilis into
the American Indian showed a virulence unknown among the Cau-
casians. Smallpox materially aided the Spaniard in his conquest of
Mexico. The negro is supposed to be less susceptible to yellow
fever than is the Caucasian, but careful investigation would make
it appear that in infancy and childhood acquired immunity is estab-
lished by mild attacks of the disease. The recent work of Love and
Davenport shows that among 500,000 troops illness was 19 per cent,
more frequent among negro than among white troops. The negro
was apparently less resistant to pneumonia, tuberculosis, and small-
pox than the white. The negro was more resistant to skin diseases,
but contracted venereal disease readily and suffered more than the
whites from extension and complications of venereal disease. Borell
has reported that the Senegalese are very susceptible to pneumonia
even in their own country. On transportation to France during the
World War more than 5 per cent, succumbed to pneumonia before
they had become acclimated, but in those who had been in France
two or three years, the death-rate from pneumonia was much re-
duced ; only 2 in 7000 troops died of pneumonia. Whether this
reduction is due to acclimatization or the early elimination of the
more susceptible is an open question. An apparent racial immunity
to malaria may be explained by the persistence of this disease for
many years following a childhood infection. In Australia, New
Zealand, and Tasmania during the years 1906-1908 there were only
about half the deaths per thousand inhabitants as the result of tuber-
culosis than occurred in Ireland, Norway, and Japan, during the same
period ; whilst the rate decreased regularly in the former countries
it increased in the latter. This appears to favor the idea of racial
differences of susceptibility, but a careful analysis of all the condi-
tions may show that climate, mode of life, and hygienic conditions
have a considerable influence. In the lower animals racial differ-
ence may be more satisfactorily illustrated. Common sheep are sus-
ceptible to anthrax, whereas the Algerian sheep seem to be immune.
The culex mosquito rarely harbors the malarial parasite, whereas
the anopheles are commonly infected. The field mouse is highly
susceptible to glanders, whilst the white mouse is immune. The
gray mouse is more resistant to streptococcus infections than is the
white mouse. The common rat is more resistant to anthrax than
is the white rat.

20 THE PRINCIPLES OF IMMUNOLOGY

Family Immunity. — Members of certain families may through
generations appear to be especially susceptible to such diseases as
tuberculosis and rheumatism or the converse may be true. In the
case of tuberculosis this difference may be the result of conforma-
tion of the body. The physical character of flat, narrow chest and
thin skin apparently go hand in hand with susceptibility to tuber-
culosis, whereas the well-rounded chest appears to indicate resist-
ance. In a family with whose history we are familiar the blondes
have almost invariably succumbed to tuberculosis and the brunettes
living under the same conditions and in intimate association have been
resistant. This must be due to inherent constitutional characters and is
not to be considered as a difference due to complexion alone.

Individual Immunity. — Variations of individual resistance or im-
munity are seen frequently. It is true that the extremes of age
show a certain proneness to infection and that this varies somewhat
with individuals. Excellent examples of individual resistance are
seen in great epidemics where some of those exposed apparently in
the same manner and under the same hygienic conditions as others
show either complete resistance to the disease, or, if they are at-
tacked, develop only moderate or slight attacks. Infected water and
foods consumed by a population may lead to disease in only a small
portion of those exposed. Individual variations in animals are very
frequent and offer a considerable source of error in the interpreta-
tion of experimental results. If a series of guinea-pigs be injected
with the same dose of anthrax bacilli, all will die at practically the
same time, but if rabbits be treated in the same way some die
within two days, others die subsequently, and still others are com-
pletely resistant. On the other hand, rabbits are all susceptible to
chicken-cholera, whereas the guinea-pig shows great individual dif-
ference. Although a large number of children suffer from tonsillar
infections, yet the incidence of acute articular rheumatism or of
endocarditis is small and variable. Instances might be multiplied
indefinitely of individual variations in resistance, but the phenom-
enon is one of common knowledge.

Inherited Immunity. — The immunity transferred from parent to
offspring may be a natural immunity or an immunity acquired by
the parent. The transfer of natural immunity may be seen in racial,
species, and family manifestations, and is probably a true transfer
through the germ plasm. Congenital immunity may arise either in
the form of an active immunity developed in the fetus because of
the presence of antigens in the circulating blood of the mother, or
may be in the form of passive immunity transferred from the blood
of the mother to that of the fetus. It is conceivable that the fetus
may survive an attack of disease transmitted from the mother and
thereby become immune. It has been known since the time of
Pasteur that certain dogs are immune to rabies. Remlinger has
found that the guinea-pig may transfer rabies to the fetus and
puppies have been known to become rabid several months after

GENERAL PHENOMENA OF IMMUNITY 21

birth without any evidence of having been bitten, the disease there-
fore probably having been contracted in utero. Immunity in dogs
may be explained by direct transmission of immunity from the
mother, or by survival of the disease in uterine or early post-
uterine life.

Acquired Immunity— ^Naturally Acquired Immunity. — The acqui-
sition of immunity may be through so-called natural processes, such
as passing through and recovering from an infectious disease, or it
may be induced and artificially acquired by special methods of im-
munization to be described. In both these instances, although the
normal level of resistance cannot always be accurately determined,
yet there is no doubt that the acquired immunity represents a higher
level of resistance than is normally possessed. For example, the
fact that when a patient has survived an attack of such a disease as
scarlatina and then in spite of repeated and intimate exposure re-
sists infection, leaves no doubt that his acquired immunity repre-
sents a higher level of resistance than he possessed before the attack
of the disease. The diseases which confer a lasting immunity include
acute anterior poliomyelitis, chickenpox, cholera, epidemic cerebro-
spinal meningitis, measles, mumps, plague, scarlatina, smallpox,
typhoid fever, typhus fever, whooping-cough, and yellow fever.
The question as to whether or not syphilis confers a lasting im-
munity has been reopened by the discovery of the Wassermann test
and by the work of Warthin. The Wassermann test has shown that
many cases of apparently cured syphilis are really in a latent stage
of the disease. Warthin has found the treponema pallidum in
various organs at autopsy on syphilitics who clinically appeared to
be free from the disease. If Warthin's work can be confirmed in a
large number of cases it would appear that syphilitic infection re-
mains latent throughout the life of the individual in the vast ma-
jority of cases, even in spite of the fact that the Wassermann test is
negative and no clinical signs of the disease are demonstrable. If
syphilis be curable, the reported occurrence of second infections in a
small number of instances would make it appear that any immunity
which may develop is not permanent. The long duration of the
disease would account for the small number of reinfections reported.
Immunity in tuberculosis has been extensively studied, and as yet
no final and conclusive statements can be made. It seems probable
that tuberculosis is never completely eliminated from the body, and
although the patient exhibits no symptom nor sign, he still may
harbor the disease. The studies of Opie and others would make it
appear that the development of tuberculosis in adult life is traceable
directly to old lesions which occurred in childhood. The fact that a
very large number of individuals show at autopsy small lesions indi-
cates the prevalence of the disease. Subsequent active development,
following encapsulation of a lesion, appears to be due to certain fac-
tors which either reduce the protective properties of the body or
excite the organisms to renewed activity, or both.

22 THE PRINCIPLES OF IMMUNOLOGY

Artificially Acquired Immunity. — The artificial acquisition of im-
munity may be the result of active development of immune sub-
stances in the organism or it may be due to the transfer into the
organism of immune substances from an immune animal. Arti-
ficially acquired immunity differs from naturally acquired immunity
in that it is likely to be less durable. If acquired by active immuniza-
tion the duration is likely to be considerably greater than if acquired
by passive immunization. In the discussion of immunity it is well
to keep clearly in mind the definition of antigen and antibody. The
antigen is a substance which upon introduction into the body in
proper amounts and under suitable conditions induces the formation
of a special antagonistic substance, the antibody. Conversely the
antibody is the substance produced as a result of the introduction
of antigen. Experimentally the antigen is usually introduced by
parenteral routes, meaning routes other than by way of the ali-
mentary canal, such as intravenous, intraperitoneal, subcutaneous,
intrathecal, intraocular, and by other similar pathways. The nature
of antigens and antibodies will be discussed in the subsequent chap-
ters, but it may be said here that both are of protein nature. Every
soluble complete protein, with the exception of the racemized pro-
tein of Dakin, may serve in at least some degree as an antigen.
The proteins employed are for the most part native, but synthetic
proteins may also act as antigens. Wells states that " of the cleav-
age products of proteins it is certain that none of the ammo-acids
and simple polypeptids can act as antigens, and it is not yet fully
established that even such large complexes as the proteoses are
antigenic, although there is some evidence in favor of this view."
There have been numerous reports of the use of lipoids as antigens,
but this relation has not been definitely established. If lipoids are
obtained from animal tissues favorable results may be obtained, but
in none of these experiments is it proven that the lipoids are entirely
free from proteins. Ford has successfully employed a hemolytic
glucoside obtained from the poisonous mushroom amanita phalloides
as an antigen for the production of an anti-hemolysin, but this is
the only well-established exception to the general rule that antigens
are of protein nature.

Actively Acquired Immunity. — This may be produced by actual
infection of an individual during a period of good health by the
virus of the disease to which he is to be immunized. The classical
example of this form of immunization was the practice for many
centuries of inoculating smallpox into the healthy, so as to induce
a mild attack of the disease. The danger lies in the uncertainty
of action of the virus, since apparent health does not necessarily
presuppose resistance to any special disease. If the virus can be
measured in some way so that an extremely small amount can be
inoculated, the procedure is somewhat safer. Protection against Texas
fever in cattle has been practised by permitting nursing calves to be

GENERAL PHENOMENA OF IMMUNITY 23

bitten by a small number of infected ticks or by injecting intraven-
ously a small amount of blood from an infected animal.

Somewhat similar to the above examples is infection with attenu-
ated virus. Such attenuation may be obtained by prolonged cultiva-
tion on artificial media, by heat, by passage through animals, by
desiccation, by the use of chemical agents, and by pressure. If heat
be employed for attenuation, rather than for killing the organisms,
it must be properly adjusted. Toussaint employed this method in
his early experiments with anthrax in which he heated infected
blood to 55° C. for ten minutes. This method, however, is not re-
liable, probably because of variations in the resistance of individual
members of a culture of any given organism. Heat may be applied
also during the cultivation of organisms upon artificial media, a
method practised by Pasteur in producing anthrax vaccine. The
heat must be of such a degree as to permit growth of the organisms,
but at the same time reduce the virulence. As has been pointed
out before, the cultivation of organisms upon artificial media through
many generations leads to a reduction of virulence. This latter
method was employed by Pasteur in the development of the vaccine
for chicken-cholera. The attenuation of smallpox virus by passage
through the calf so reduces virulence that the virus may safely be
inoculated into man. Pasteur found that the virus of swine ery-
sipelas could be attenuated by passage through rabbits, and it is
well known that the passage of rabies virus through dogs and
through monkeys reduces its virulence. An excellent example of
attenuation by desiccation is found in the preparation of anti-rabic
vaccine. For this purpose the virus is raised by passage through
rabbits to a standard degree of virulence, the " virus fixe." The
spinal cord of a rabbit so infected is desiccated at 25° C. over KOH.
This method of attenuation is so delicate that there are distinct
variations in virulence between fragments dried for five, six, and
seven days, as well as virus dried for thirty-five, thirty-six, and
thirty-seven days or intervening periods. The longer the desicca-
tion the greater the reduction of virulence and the greater the safety
of the inoculation. Attenuation by the use of chemicals, such as
phenol, potassium bichromate, and sulphuric acid, has been prac-
tised. Chemical attenuation may also be applied to toxins, as in the
use of iodine terchloride and potassium iodide. A pressure of eight
atmospheres at a temperature of 28° to 39° C. has been employed
for the attenuation of anthrax cultures, but is probably not widely
applicable, is difficult, and possesses no superior advantages.

Immunization with Dead Bacteria. — In the study of immune
processes it was finally found that killed bacteria could be used for the
production of immunity. The organisms may be killed by heat or
by chemicals. In either case, it is necessary so to apply these agents
as to kill the organisms without destroying their proteins. The
use of heat sufficiently high to destroy spores leads to destruction
also of the proteins, and therefore the method does not apply to

24 THE PRINCIPLES OF IMMUNOLOGY

spore^bearing organisms. Those organisms which do not produce
spores can be killed by heat of 58° to 60° C. for thirty to sixty
minutes, and this degree of heat does not alter the character of the
proteins. 'The chemicals most frequently employed for killing bac-
Iteria so as not to alter the proteins are formaldehyde and phenol.

Immunization with Bacterial Products. — In addition to the use
of dead bacteria, as indicated above, it has been found possible to
produce immune reactions by the use of extracts of the organisms,
these extracts containing a considerable amount of bacterial pro-
tein. Immunization of this sort leads to the formation of anti-
bacterial sera which agglutinate the bacteria or precipitate bacterial
extracts. It is possible also that this method of immunization leads
to the formation of other immune substances. How far protein,
either in solution or in the bodies of bacteria, may be broken down
and still be capable of leading to the formation of immune bodies is a
iquestion that has been extensively studied. Certainly any change
that breaks up the protein into its fundamental amino-acids is likely
to destroy its antigenic properties. Simple fractionation by means
of salting still leaves sufficient native protein to serve to immunize.

Of bacterial products which have been employed for immuniza-
tion none is more important than those poisonous bodies called
toxins. In the classification of toxins we have referred to the true
toxins or exotoxins and to the endotoxins. There is little support
for the belief that endotoxins as such, except in rare instances, can
produce immune substances. On the other hand, the production of
a neutralizing antitoxin against the exotoxins has constituted one
of the most brilliant chapters in the study of immunology, and it
will be given discussion in the chapter on toxins and antitoxins.
The use of toxins as antigens involves the employment of these
substances in non-fatal doses, their attenuation by chemical and
physical means, or their primary neutralization by means of previ-
ously prepared antitoxins. In experimental work on animals the
first two methods are commonly employed and may be combined
with the third method. In man immunization by the use of toxins
is practised, mainly in connection with active immunization to diph-
theria. The combination between toxin and antitoxin is not in the
nature of a fixed and final reaction, and under certain circumstances
partial dissociation may occur. The active immunization of man
by the use of neutralized mixtures of toxin and antitoxin appears
to provide conditions whereby dissociation progresses gradually,
and the toxin is liberated in such small amounts that it does no
harm and yet induces in the body antitoxin formation. In the mean-
time the individual is protected by the antitoxin simultaneously
dissociated. Recent studies make it appear that several organisms
which formerly were supposed to produce only endotoxins elab-
orate in addition true toxins, and some of the earlier studies sup-
porting the assumption that antitoxins could be produced by these

GENERAL PHENOMENA OF IMMUNITY 25

endotoxins are probably fallacious, because of the mixture of un-
recognized exotoxins, the latter producing the immune reaction.

Active immunization may be produced not only by toxic sub-
stances elaborated by bacteria, but also by toxic substances produced
in animal life, such as snake venoms, spider poisons, and similar
substances. Higher plant poisons, such as ricin, abrin, crotin, etc.,
may produce specific neutralizing antibodies. The practical value
of the antitoxins prepared against bacterial toxins and against the
venoms produced by animals is such as to have added greatly to the
combating of poisoning by these substances.

Passive Immunization. — In active immunization the animal
manufactures within its own body immune substances which serve
to protect against and combat infection. Passive immunization,
however, utilizes these immune substances, through the transfer of
blood serum containing the products of active immunization. The
most common example of passive immunization is found in the
therapeutic use of diphtheria antitoxin. For practical purposes the
diphtheria antitoxin is manufactured in the body of the horse. The
injection of immune horse serum transfers to man the immunity
actively produced in the horse. Passive immunity of this sort serves
to protect against infection, and until the possibility of active im-
munization of man against diphtheria was demonstrated, the former
method was widely employed for protection of exposed individuals
against diphtheria. This method of protection has the great advan-
tage of quickly conferring immunity and is widely employed when
time does not permit the use of methods for developing active im-
munity. After the disease has developed the use of immune serum
to combat the infection has the utmost value. In the case of tetanus
antitoxin the protective value of prophylactic injections has been
amply demonstrated, but in this instance the great affinity between
nerve tissues and tetanus antitoxin is such that the therapeutic use
of tetanus antitoxin after the disease has developed has not given
such beautiful results as has been true of the serum treatment of
diphtheria. Much encouragement has recently been afforded by the
use of similarly prepared antitoxins against the toxin of the bacillus
of gas-gangrene, and there is little doubt that the methods may be
much more widely employed as it becomes possible to demonstrate
the formation of true exotoxins by other bacteria. Not only may
advantage be taken of substances produced by artifically acquired
immunity, but in certain instances it is feasible to use the blood
serum of individuals who have acquired immunity by survival of an
attack of certain diseases. In this field, however, the facts have not
been accumulated in sufficient number to justify unqualified ap-
proval of the method.

Passive immunity may be not only antitoxic in character, but
also anti-bacterial. Anti-bacterial immune sera have been prepared
against the streptococcus, the meningococcus, the pneumococcus,
and other organisms. The success with passive immunization by

26 THE PRINCIPLES OF IMMUNOLOGY

the use of these sera has not always been so clear cut as in the case
of antitoxic sera. Nevertheless the use of anti-meningococcus sera
has reduced the mortality of epidemic cerebrospinal meningitis from
75 or 80 per cent, down to 35 or 40 per cent, or lower. The results
with anti-streptococcus sera have been variable. Although the
early reports of the use of anti-pneumococcus sera were highly en-
couraging, later study has thrown some doubt upon the value of
this method of treatment. Much further study of the subject is re-
quired before a definite conclusion can be reached.

Theories of the Nature of Immunity. — In the early study of im-
munity numerous hypotheses were advanced as to the action and
development of immune bodies. It was known, for example, that
when bacteria are grown for a long time upon a culture medium
certain substances are produced which have a deleterious influence
upon the further growth of the organisms and may actually lead to
their death. It was easy to assume, therefore, that recovery from
an infectious disease might be due to the development of similar
antagonistic substances within the infected host. Another theory
was to the effect that bacteria growing in the body utilize and
exhaust the specific nutritive substances necessary for their growth
and then die. It was also thought that the death of bacteria in the
body was due to changes in reaction of the blood, and further that
altered osmotic conditions changed the permeability of cell mem-
branes so as to permit ready entrance of poisonous substances.
These theories, however, could not withstand the demonstration of
passive transfer of immunity, the production of immunity by the
use of killed organisms, or perhaps more important, the clear demonstra-
tion of immune reactions in vitro.

The Ehrlich Side-chain Theory. — As more and more facts were
added to the knowledge of the subject, Ehrlich propounded his side-
chain theory. This was based upon the law of Weigert, which
states that when animal cells are required to repair an injury
they not infrequently exceed the absolute necessity for repair and
produce tissue in excess. Ehrlich, therefore, hypothesized that the
injurious substances of infection demand of the cells the forma-
tion of protective bodies, and that the cells respond to this demand
in such excess that the protective bodies are formed in amounts not
only sufficient to meet the requirements, but in such excess as to free
circulating immune substances in the blood. This hypothesis in-
troduced an entirely new terminology into the subject. It was
supposed that cells normally possess certain specific receptors or
combining groups for the injurious substances much as a struc-
tural chemical formula exhibits free valencies on the part of certain
elements or groups. When all these combining groups of the cell
are utilized and uncombined poisonous material exists in the circula-
tion, the cell produces and liberates additional receptors even in
excess of demand. These free receptors constitute the circulating
immune substances. The study of immune substance demon-

GENERAL PHENOMENA OF IMMUNITY 27

strates somewhat variable activities. For example, it was found
that antitoxins operate differently from other immune substances;
that agglutinins and precipitins operate in a special fashion which
is practically identical for both substances ; and that cytolysins, includ-
ing the lytic bodies for bacteria as well as for animal cells, require
the presence of fresh serum containing the so-called complement or
alexin. The specific cytolysins were found to be similar to certain
other substances which are now referred to as complement-fixing
bodies. Finally the discovery of opsonins and tropins showed that
there is in all probability a fourth group or sub-group of these
immune substances.

The Ehrlich Classification. — Ehrlich, on the basis of the general
outline given above, divided the immune bodies into three groups,
depending upon demonstrable differences in their nature. He found
that the receptors in some instances are not immunologically simple
bodies, but that even in this sense they show varying degrees of
complexity. In the more complex forms the actual receptor or
combining groups constitute only a part of the immune substances,
and he therefore applied a more comprehensive term, the haptines.
•He included in the haptines of the first order the antitoxins, in the
haptines of the second order the agglutinins and precipitins, and in
the haptines of the third order the cytolysins and other amboceptors.
The early studies of antitoxins made it appear that the neutralizing
effect of these substances was similar to the neutralizing action of
alkalies and acids, but it was subsequently discovered that such
combinations may be, at least in part, dissociated. It was then
found that toxin may undergo a variety of changes as the result of
preservation. Subsequently it was learned that the antitoxin would
combine not only with the toxin, but with its degeneration products.
This has complicated the conception considerably, and we may say
in brief that according to the Ehrlich conception the antitoxin con-
stitutes a simple receptor or combining group capable of entering
into combination with a special combining group in the toxin called
the haptophore. In order to account for the combination of anti-
toxin with the degeneration products of toxins it was necessary to
assume that the toxin exhibits its essential combining property in
the haptophore group and that the toxin also possesses a toxophore
group, serving to give it its poisonous character and partly de-
stroyed during preservation or by certain degrees of heat. The
second category of Ehrlich includes the agglutinins and precipitins.
In the study of these substances it was found that the agglutinins
and precipitins may be deprived of the agglutinating and precipitat-
ing properties by preservation or by the application of certain de-
grees of heat. Ehrlich, therefore, conceived the idea that in this
instance we have to deal with a somewhat more complicated haptine
containing a combining group and a so-called zymophore group,
the latter leading to the special reaction. These constitute the re-
ceptors or haptines of the second order. This assumption is neces-

28 THE PRINCIPLES OF IMMUNOLOGY

sary, because even although the agglutinating and precipitating
properties are destroyed by heat or other means, nevertheless, there
remains a group capable of entering into combination with the anti-
genie substances, so that the addition of a complete agglutinin or
precipitin produces no effect. The third category of Ehrlich includes
the receptor or haptine which has been named by Ehrlich the ambo-
ceptor, and by Bordet the sensitizer. In this instance the receptor
produced by the cell is conceived as a body possessing two com-
bining groups, one serving to combine with the antigen and the
other serving to combine with complement. These two groups have
been called the cytophilic group and the complementophilic group.
The complement is a thermolabile substance which has little or no
capacity for combining with the antigen. Accepting Ehrlich's hy-
pothesis, this haptine of the third order constitutes an intermediary
body through the action of which the complement is brought into
contact with the cells, be they bacterial or animal, so as to lead to
solution. Bordet, however, believes that the immune body enters
directly into combination with the antigen, thereby " sensitizing "
it so that the complex is operated upon by the complement, or as he
calls it, the alexin. The discovery of the phenomenon of comple-
ment-fixation demonstrated that a similar substance may operate in
the presence of dissolved protein and complement, so as to engage
the complement in such a fashion that it is not available for other
reactions. In these reactions the participation of the complement is
an essential and necessary condition of the reaction. The original
Ehrlich theory could not consider the subsequently discovered
opsonin or tropin. This substance prepares bacteria and other
cells for phagocytosis. It was at first supposed to be a simple
immune substance, but as the study of its activity progressed it was
found that the presence of complement increases its activity, al-
though this latter body is not essential and necessary. We, there-
fore, propose to consider the opsonin as belonging essentially to the
haptines of the third order. The way in which this differs from the
original haptine of the third order is simply in the fact that com-
plement may or may not be utilized in the reaction. In order to
differentiate we suggest that the amboceptors of Ehrlich be looked
upon as " obligate " amboceptors and the opsonin be regarded as a
" facultative " amboceptor.

Recent Criticism of the Ehrlich Hypothesis. — Following the
earlier discoveries of immune phenomena numerous studies were
made of the chemistry of immune substances and immune reactions.
These will be discussed in the chapters on the special immune reac-
tions. It may be said at this time that many objections have been
raised to the Ehrlich hypothesis, particularly as the study of physi-
cal chemistry, more particularly that part which refers to colloids,
has advanced. As will be seen from the brief review of the Ehrlich
hypothesis given above, this investigator was much influenced by
the status of chemistry which prevailed when he announced his

GENERAL PHENOMENA OF IMMUNITY 29

views. The idea predominated that the immune reactions resemble
the more or less fixed changes which are seen in the chemical reac-
tions of crystalloids. As it was found that practically all immune
substances are colloidal in nature and either are proteins or are very
closely related to proteins the similarity of the immune reactions to
colloidal reactions became more and more strongly emphasized. In
fact, in a general way, practically all immune reactions parallel in
their general phases similar demonstrable reactions with colloids.
There is but one feature of immune reactions which has not yet
been explained on the basis of colloid chemistry, namely, their
specificity. This does not mean, however, that further investiga-
tion will not clear up this phase of the problem. It is but fair to say
that the Ehrlich hypothesis provides an excellent basis for the classi-
fication of immune phenomena, but as will be shown subsequently,
the conception underlying the Ehrlich hypothesis is not adapted to
the more modern views of the mechanism of immune reactions. The
combination of toxin and antitoxin shows numerous features not to
be explained by the simpler reactions of crystalloids. The same is
true of agglutination, precipitation, cytolysis, complement-fixation,
and anaphylaxis.

Specificity of Immune Reactions. — The antitoxin elaborated in
the response to injections of diphtheria toxin or to the presence of
the disease itself is a substance which reacts only with diphtheria
toxin. The agglutinins and precipitins produced by injection of
bacteria and of dissolved proteins act most powerfully upon the sub-
stances used for injection. In this case, however, these immune
bodies may also react less strongly with other closely related bac^
teria or proteins. Cytolysins induced by the injection of certain
cells react strongly with those cells, but also less strongly with
closely related cells. This phenomenon of reaction with closely re-
lated bodies is spoken of as the group phenomenon and may be
exhibited also in connection with complement fixation and ana-
phylaxis. Even where purified proteins are employed the same
phenomenon may be observed. In spite of the group reaction, how-
ever, the immune substances are most highly specific for their spe-
cial antigens. Specificity has been employed for the detection of
particular proteins of animal species, of bacterial species, and it
has lent support to the Darwinian theory of species relationship and
evolution. Much thought and study has been given to the resem-
blance between immune substances and enzymes, but in no sense
can enzymes be said to have the same specific character as immune
bodies. There is no satisfactory explanation of specificity. Why
the injection of red blood-corpuscles of the sheep should induce the
formation of a hemolysin capable of dissolving the red cells of the
sheep but not of other animals, except in minor degree of those
closely related to the sheep, cannot be explained. As can readily be
understood, specificity involves the use of special antigens and the
formation of more or less specific immune substances. The wide

30 THE PRINCIPLES OF IMMUNOLOGY

range of possibility in this connection is indicated in the building-
stone theory of Abderhalden. Bearing in mind that practically all
immune substances are protein in nature and that proteins are made
up of numerous amino-acids, Abderhalden calculated that twenty
amino-acids could be so combined as to form 2,432,902,008,176,640,000
different compounds. He illustrates this possibility by stating that
if three amino-acids are building stones which may be designated
A, B, and C, they can be grouped together so as to form six different
combinations, ABC, ACB, BCA, BAC, CAB, and CBA, and that
four building stones can form twenty-six such combinations and so
on until the enormous possibility of different combinations of
twenty amino-acids is reached, as illustrated in the figures given
above. Chemically no such enormous number of proteins is known,
but if immune specificity could be shown to depend upon slight
differences of molecular arrangement, Abderhalden's figures indi-
cate the number of immunologically specific proteins obtainable.
Taking for granted the phenomenon of specificity, that of the group
reactions can be more readily explained. In this case it is assumed
that in the proteins of closely related species there is some group of
molecules common to these species, and further that the formation
of immune substances in response to injections of this common
group leads to the production of a substance which may react with
the common group. In each species, however, there is in addition
to the common group special groups which determine the specificity
of the substance as an antigen as well as the production of an im-
mune substance with a higher degree of affinity for the combined
groups of the particular species than for the common group.

Non-specific Therapy of Infectious Disease. — As a result of the
extensive studies of infectious disease various modes of treatment
have been elaborated. It is well understood that the organism
offers resistance to these infections and that the support of circula-
tion and excretion by simpler pharmacological methods aids mate-
rially in the treatment. Not only is this true, but the investigation
of various drugs has determined the specific chemo-therapeutic treat-
ment of infections. Examples of this are seen in the use of quinine
in malaria, arsenic in trypanosomiasis and spirochetosis, and of
emetin in amebiasis. The treatment based more particularly upon
immunological methods has been largely specific, but more recent
studies have given encouragement in the use of certain non-specific
methods of treatment. It was found, for example, that the use of
typhoid vaccine is of value not only in the treatment of typhoid
fever, but in other diseases, and typhoid vaccines either in the form
of the usual killed organisms or organisms sensitized with specific
immune sera have produced beneficial results in such diseases as
acute articular rheumatism, sub-acute and chronic arthritis, and in
certain other infections. Similarly the use of blood serum, of pure
proteins, of leucocyte extracts, of fibrin derivatives, and of certain
other protein derivatives has appeared to be beneficial. It is not to

GENERAL PHENOMENA OF IMMUNITY 31

be assumed that this method of non-specific treatment is of con-
clusively proven value, but the effects observed in a certain per-
centage of cases offers the hope that the method may be so perfected
as to give improved results.

The general reaction following subcutaneous injection of these
substances may or may not be severe, but if they are administered
by the intravenous route the reaction is likely to be pronounced.
Frequently a chill appears and almost all cases develop fever which
may be very high. Sometimes there is a general feeling of discom-
fort associated with headache and nausea. In typhoid fever it is
reported that hemorrhages not infrequently occur as the result of
the therapeutic use of sensitized and of non-sensitized typhoid vac-
cine. This does not appear in other diseases, and although pro-
tein substances and their cleavage products, upon injection, tend to
decrease the coagulation time, yet the use of blood serum in the treat-
ment of hemophilia often has a favorable effect in preventing hemor-
rhage. In addition to the possibility of hemorrhage in typhoid fever
there are definite contraindications to this form of therapy in preg-
nancy, in patients with organic heart disease, and in those with high
blood-pressure. The influence of this non-specific method of treat-
ment is not clearly understood. The question as to whether or not
the known forms of antibodies are liberated or stimulated has been
studied by numerous workers with contradictory results. Some
have found an increase of agglutinins and precipitins for the specific
organisms concerned in the disease, following non-specific protein
injections, but this is contradicted by other workers. Regardless of
the question of stimulation of special immune bodies it is important
to know what other protective influences may be set at work.

Fever is a common incident of the injection of proteins or pro-
tein products, especially when they are given intravenously. This
is sometimes accompanied by leucocytosis, but neither leucocytosis,
marked acceleration of pulse-rate, nor the other clinical accompani-
ments of fever necessarily appear. It has been demonstrated that
increased temperature aids in the production of agglutinins and bac-
teriolytic substances. In most instances the degree of temperature
reached in fever has no deleterious effect directly upon the bacteria
concerned, except possibly in the case of infections with the
gonococcus and with the spirochete of relapsing fever. It has
been suggested that high body temperature may favor the com-
bination of the antigen and immune substances, but this has not
been conclusively demonstrated. The injection of proteins may lead
to an increase in the number of circulating leucocytes, although this
is not invariably the case. The influence of such a hyper-leucocytosis
in combating infection is at least partly because of the fact that
these cells ingest and destroy bacteria. Nevertheless, certain
infectious diseases, such as typhoid fever, may run their course
without exhibiting leucocytosis, and it is therefore not essential for
recovery that the leucocytes be increased in number. It must be

32 THE PRINCIPLES OF IMMUNOLOGY

pointed out, however, that phagocytosis is not the only way in
which an increase of leucocytes may operate beneficially. The
studies of Hiss and Zinsser indicate that extracts of leucocytes have
a beneficial effect on infections and others have confirmed these
results. Bail claims that a fresh emulsion of leucocytes will aid in
neutralization by the specific anti-serum of endotoxin obtained from
cholera vibrios. Jobling and Bull, however, demonstrated that leu-
coprotease " will destroy the toxic extracts of typhoid bacilli and
meningococci, and it is not improbable that a similar explanation
will apply to the results obtained by Bail."

There are other possible changes in the blood as the result of the
injection of protein. The work of Jobling and his collaborators has
thrown great light on the alterations of ferments and anti-ferments
in the blood under a wide variety of conditions. The injection of
various substances is almost invariably followed by a considerable
mobilization of the serum ferments, more particularly the protease,
and usually also the esterase. The value of the protease is probably
in the direction of breaking down toxic split-protein products, which
probably originate during the course of infectious disease, as the
result of the splitting of bacteria and perhaps also of the body pro-
teins. Protease does not act directly upon living bacteria, but it is
to be considered possible that the esterase may break up the lipoid
or lipoid-protein surface of the bacteria and therefore aid in their
destruction. If we concede that the toxic protein split products aid
in the virulence of bacteria it is possible that even although the
protease simply breaks down these products into simpler non-toxic
substances and does not directly attack the bacteria, yet the relief to
the body afforded by this detoxifying action may assist it more
permanently in combating disease. In certain states, such as preg-
nancy, in disease such as cancer, and in the course of vaccine treat-
ment the anti-ferment titer of the blood has been found to be high.
Jobling and Peterson found that the anti-ferment power of the blood
depends upon the amount of unsaturated lipoids present in highly
dispersed phase in the serum and Bogolemez suggests that lipoids
may serve to inhibit toxins, as is true in relation to the toxin of
bacillus botulinus. Anti-ferment is not increased following protein
injections and plays no part in the non-specific therapy of infectious
disease, but inasmuch as the change may be seen in immune states,
such as that following vaccination, it may be of importance in non-
specific resistance to infection. In addition to the changes in fer-
ments Jobling has found that the injection of non-specific proteins
may produce changes in the viscosity of the serum. It is known
that if precipitates are formed in serum by the action of a specific
precipitating serum, conditions favorable to protease activity are
produced and the changes in viscosity produced by protein injec-
tions may similarly aid proteolytic activity. These changes in
ferment content and physical character of the serum are of short dura-
tion and are probably contemporaneous with the chill and fever.

GENERAL PHENOMENA OF IMMUNITY 33

They do not directly account for permanent improvement seen in
many patients, but if they rid the body of toxic substances for a
short period of time the natural resistance may thereby become
more effective than would otherwise be the case.

The Site of Antibody Formation. — Aside from a few fairly well-
established facts the problem as to exactly where antibodies are
formed still remains obscure. In general it is assumed that anti-
bodies are not products of simple inversions 'of the foreign protein
substances parentally introduced or as particular functions of spe-
cial organs, but are the result of general cell reactions on the part
of the host. Much evidence points to the lymphatic organs, the
spleen, the liver, and the bone marrow as places where antibody
formation is most active. Metchnikoff thought that antitoxins and
bacteriolysins originate in the lymphatic organs and more particu-
larly in the spleen and the bone marrow. Bordet attempted to
show that bacteriolysins are derived from the leucocytes. Pfeiffer
and Mark injected dead cholera spirilla into animals, exsanguinated
these five days after the injection, and found the antibodies more
concentrated in the spleen than in the blood serum itself. These
authors also found that after a single injection of these organisms,
the spleen, the bone marrow, and the lymph-nodes contained the
specific antibodies before they could be detected in the blood, and
further that as time passed these tissues became less active in spite
of the fact that the bacteriolysins increased in the blood. Deutsch
corroborated these findings with bacillus typhosus and Castellani
with bacillus dysenterise. These authors agree, however, that the
spleen is not essential, since its removal but slightly inhibits the
formation of antibodies,. Hektoen's experiments demonstrated that
in dogs splenectomy just before and after the injection of alien
blood-corpuscles was followed by a much lower, but otherwise typi-
cal antibody curve, than is usually the case in dogs under normal
conditions. London also reported a decreased formation of
hemolysins after splenectomy, but this work has been contradicted
by Yakuschewitch. Karsner, Amiral, and Bock found that splenec-
tomy produces no change in hemopsonins of the circulating blood
that is clearly demonstrable by in vitro test, and that the blood from
the spleen is no richer in hemopsonins than is blood from other
organs. Carrel and Ingebrigsten have produced hemolysins in the
growing embryonic spleen. More recently Przygode succeeded in
producing precipitins in vitro by culture of splenic tissue, and Miiller
by transplanting splenic tissue from guinea-pigs, previously injected
with sheep corpuscles, into the peritoneal cavity of normal guinea-
pigs. It seems to us that since the spleen is an organ physiologically
designated for the destruction of erythrocytes and also of other
foreign substances through the activity of its hemophages, splenic
tissue on transplantation will carry with it much antigenic sub-
stance. Whether or not these hemophages participate in antibody
production is at present difficult to say.
3

34 THE PRINCIPLES OF IMMUNOLOGY

For rapid production of antibodies Violle injected organisms
directly into the gall-bladder. This fact is of interest because it
indicates a possible function of the liver in the production of immune
bodies. Miiller claims to have been able to stimulate the formation
of hemolysin in liver tissue suspended in Ringer's solution outside
the animal body. By perfusing the organ with solutions contain-
ing iodine (iodipin) the effect was augmented, and he believes that
in the normal animal the iodine of the thyroid may play a certain
role in stimulating this special activity of the liver. Gay and Rusk
found no evidence to uphold the supposed influence of iodipin.
Hektoen and Carlson believe that both the spleen and liver are equally
concerned in antibody formation, but Hektoen and Curtis found
that in rats removal of about one-half of the liver appears to have
no effect on the development of hemolysin for sheep corpuscles.
The liver, just as the spleen, possesses highly active phagocytic
endothelial cells which may play an important role in the produc-
tion of antibodies.

Numerous authors have shown that agglutinins appear in the
blood stream before they are present in the extracts of any organ.
The question, however, of whether or not the leucocytes are in-
volved in this generative process is a matter of considerable contro-
versy. Achard and Bensaud and others controvert the leucocytic or
local origin of agglutinins, whereas Cantacuz^ne and also Swerew
support this local origin in the formation of precipitins ; they noted
a hypoleucocytosis followed by a marked hyperleucocytosis, which
they think is responsible for the liberation of precipitins. Petit and
Carlson, Vaughan, Cumming, and McGlumphy found that sub-
stances like egg-white and serum disappear quickly from the cir-
culating blood ; in fact, within a few hours after the introduction of
these substances. Gay, however, has shown by means of comple-
ment-fixation that even in immune animals such antigens are dem-
onstrable after twenty-four hours, but not after forty-eight hours.
It was not possible to demonstrate the antigen by the fixation method
in organs like spleen, lymph-nodes, liver, kidney, and muscles,
either at the time antigen was present in the blood or twenty-four
hours thereafter. That the cells lining the blood-vessels may have
certain powers of antibody production may be shown by the fact
that a blood-vessel from an animal which has received several injec-
tions of sheep erythrocytes and which has been dissected out soon
after death of the animal and washed free from blood, has the power
to hemolyze a suspension of fresh, non-sensitized sheep cells (Van
Calcar). Kraus and Levaditi furthermore have shown that there
exists a certain relationship between precipitins and the number of
circulating leucocytes. Acute loss of blood profoundly affects anti-
body production. The earliest observations seem to have been those
of Roux and Vaillard. They found that in horses actively im-
munized against tetanus toxin, bleeding causes a drop in the anti-
toxin content in the blood, followed by a sharp rise in a short time.

GENERAL PHENOMENA OF IMMUNITY 35

By continuous daily bleedings Hahn and Langer recently succeeded
in increasing the agglutinin content 250,000 times its original value.
Similarly Madsen and Tallquist have shown that certain poisons
which destroy erythrocytes may increase the production of anti-
bodies possibly by the action of the same mechanism as that whereby
hemorrhage stimulates antibody formation. Rusk has found that
animals intoxicated with benzol produce hemolysins and precipitins
much less efficiently than normal animals. Since benzol affects
particularly the bone marrow and the lymphatic apparatus, this
evidence points in favor of the view that these tissues are largely
involved in the production of hemolysins and of precipitins.

According to Hektoen and Curtis, adrenalectomy in normal dogs
and in dogs at the height of the antibody curve after the injection
of rat corpuscles does not cause a decrease in the antibody content
of the blood serum. Gates was able to remove approximately three-
quarters to seven-eighths of the adrenal tissue of guinea-pigs with-
out causing symptoms of adrenal insufficiency. Guinea-pigs thus
treated were injected with bacillus typhosus and with hen cor-
puscles, and the results demonstrated that adrenalectomy had no
influence upon the rise or persistence of antibodies in the blood,
and therefore the adrenals appear to play no essential part in the
mechanism of antibody production.

The results of Tjeldstad had shown that thyroidectomy failed to
influence antibody production. Similar observations were recorded
by Hektoen and Curtis, and others, but Frouin was more conserva-
tive in his conclusions, and Garibaldi has recently renewed an in-
terest in this matter by reporting that the hemolytic titer of the
serum of thyroidectomized rabbits is much higher than that of his
control animals, therefore concluding that thyroidectomy definitely
favors antibody production.

We know from the experiments of Was,sermann and Takaki that
brain substance neutralizes tetanus toxin, but this fact does not
indicate this organ to be of much importance in the production of
antibodies. In fact, we have learned from the experiments of Loewi
and Meyer that injection of toxin into the nervous system produces an
increased susceptibility of the animal rather than increase of resistance.

Production of Antibodies at Site of Injection. — Certain experi-
ments indicate that antibodies may also be produced at the place of
introduction of the antigen. Romer and also von Dungern have
shown that immunization by way of the conjunctiva or anterior
chamber of the eye results in the formation of antibodies in the
aqueous humor before they can be demonstrated in the blood. These
experiments also demonstrated that the opposite eye produces no
antibody. Wassermann and Citron ligated a rabbit's ear at its base
after a subcutaneous injection of bacteria. The ligation was left
for several hours, and after nine days the bactericidal titer of the
blood serum determined and the ear amputated. An immediate and
rapid drop of antibody in the blood which occurred after the ampu-

36 THE PRINCIPLES OF IMMUNOLOGY

tation indicates that the main source of antibody formation was
removed or the absorption of the ahtigenic substances entirely
stopped. Forsmann and Lundstrom studied the curve of pro-
duction of botulinus antitoxin following single intravenous or
subcutaneous injection of the toxin. The curve following the
subcutaneous injection reached its highest level on the fifteenth day,
while that following the intravenous injection attained the maximum
on the tenth day. It must be inferred that the subcutaneous method
of injection introduces the factor of slow absorption, but it is also
possible that some local factor may enter into the phenomenon.
Immunization of horses with diphtheria toxin results in a greater
yield of antitoxin when the horses are injected subcutaneously, but
this does not necessarily prove a local production of antitoxin at the
site of inoculation. Local cellular participation in immune reactions
will be discussed further in the chapter on hypersusceptibility.
There is little doubt that local reactions are of significance and that
absorption may be influenced by local changes. The production of
circulating antibodies in any considerable amounts undoubtedly re-
quires more extensive cellular activity than that about the site of
local inoculations.

CHAPTER IV
TOXINS AND ANTITOXINS

GENERAL NATURE OF TOXINS.
THE BACTERIAL TOXINS.

CLASSIFICATION.
PATHOLOGICAL EFFECTS.
FORMATION OF ANTITOXINS.

TECHNIC OF PRODUCING DIPHTHERIA TOXIN.
THE NATURE OF ANTITOXINS.

STANDARDIZATION OF DIPHTHERIA ANTITOXIN.
THE MINIMUM LETHAL DOSE (M.L.D.).
THE L0 DOSE (LIMES NULL).
THE L+ DOSE (LIMES DEATH).
TITRATION OF DIPHTHERIA ANTITOXIN.
THE TOXIN ANTITOXIN UNION.
THEORIES OF UNION.
EHRLICH THEORY.
LAW OF MASS ACTION.

THE DANYSZ EFFECT.

THERAPEUTIC USE OF DIPHTHERIA ANTITOXIN.
VALUE.

MODES OF ADMINISTRATION.
NATURAL IMMUNITY TO DIPHTHERIA.

THE SCHICK TEST.
ACTIVE IMMUNIZATION IN MAN.
TETANUS TOXIN AND ANTITOXIN.
TETANOTOXIN.
TETANOSPASMIN.
ROUTE OF ABSORPTION OF TOXIN.
THERAPEUTIC USE OF TETANUS ANTITOXIN.
PROPHYLAXIS.

TREATMENT OF THE DISEASE,
DYSENTERY TOXIN.

THERAPEUTIC USE OF DYSENTERY ANTISERA.
BOTULINUS TOXIN.

THE USE OF IMMUNE SERA IN BOTULISM.
GAS BACILLUS TOXINS.

THE USE OF IMMUNE SERA IN GAS GANGRENE.
TREATMENT OF THE DISEASE.
PROPHYLAXIS.
BACTERIAL HEMOTOXINS.

STAPHYLOLYSIN AND ANTILYSIN.
THE PHYTOTOXINS.
RICIN.
ABRIN.
CROTIN.
CURCIN.
PHASIN.
THE ZOOTOXINS.

SNAKE VENOMS.

SCORPION AND SPIDER POISON.

CENTIPEDE POISON.

BEES, WASPS, AND HORNETS.

TOADS, FROGS, AND SALAMANDERS.

POISONOUS FISH.

EEL SERUM.

PARASITIC PROTOZOA.

MAMMALIA.

ANIMAL SERA.

37

38 THE PRINCIPLES OF IMMUNOLOGY

General Nature of Toxins. — Toxins are soluble poisonous prod-
ucts of life processes, which on injection into animals lead to the
formation of antitoxins. A corollary of this definition sometimes in-
sisted upon is that the injurious effect of these toxic bodies must be
preceded by an incubation period, but in certain instances this in-
cubation time is a matter of minutes or hours, as is the case with
certain snake poisons. The toxins are divided according to their
origin into phytotoxins, produced by vegetable life, and zootoxins,
produced by animal life. The most important of the phytotoxins
are the bacterial toxins, but the group includes also ricin, abrin,
crotin, robin, and curcin. Certain of the higher plant poisons which pro-
duce the varieties of " hay fever " in susceptible individuals were
formerly considered as toxins, but this view has now been discarded.
The poison of rhus toxicodendron (poison ivy) and of rhus diversiloba
(poison oak) might be considered a phytotoxin, but is chemically a glu-
coside and does not produce antitoxin. The poisoning of non-edible
mushrooms is due, in the case of amanita muscaris and helvella escul-
enta, to definite chemical compounds, muscarine and helvellic acid,
which do not produce antibodies. In the case of amanita phalloides
there are two substances of toxic nature, a thermolabile hemolytic
glucoside capable of producing an anti-hemolysin, and a thermo-
stabile toxin of unknown composition and incapable of producing a
definite immune body. The most important of the zootoxins are the
snake venoms, but this group also includes the poisons of spiders,
scorpions, centipedes, bees, wasps, hornets, dermal glands of toads
and salamanders, various sera, notably that of the eel, and certain
poisonous fish.

Nicolle, Cesari, and Jouan divide the toxins into those that ap-
pear in the form of definite secretions, as snake venoms, those which
are determined by logical inference, as the toxins in microbial fil-
trates, and those which are obtained by simple maceration, expres-
sion, grinding, or autolysis, the endotoxins. Experiments with the
endotoxins are performed in large part with the microbial bodies,
and therefore these workers refer to the endotoxins as solid toxins.

The bacterial toxins are synthetic products of the life of the organ-
isms themselves. It was thought for years that the bacteria could
synthesize the protein toxin from simple nitrogen-containing com-
pounds. More modern studies oppose this view and state that more
complex substances, such as proteoses and polypeptids, are essen-
tial. It seems certain that nothing less complex than the amino-
acids can be synthesized, and recent studies indicate that diphtheria
toxin is not a synthetic product, but rather a catabolic substance
elaborated by the bacteria only in the presence of amino-acids and
certain additional substances, probably of the nature of vitamines.
They are thus to be distinguished from the ptomains, which al-
though products of bacterial growth, are in reality formed from the
culture medium and vary according to the medium rather than
according to the organism. The chemical nature of the bacterial

TOXINS AND ANTITOXINS 39

toxins is uncertain, but they appear to be more closely related to the
proteins than to any other known substance. They diffuse through
membranes less slowly than do proteins, and therefore are pre-
sumed to have a smaller molecular size. On the other hand, they
are digested less readily than proteins. Like proteins they are
electro-positive colloids, and are precipitated by protein precipitat-
ing agents, such as ammonium sulphate. As against this is the
statement that toxins may be so far purified that they do not give
the protein reactions. They resemble enzymes in that both are
colloids, both thermolabile, dialyze with difficulty, lose strength in
passing through porcelain filters, resist drying and dry heat, resist
low temperatures, both produce antibodies, both deteriorate after
standing in solution with loss of zymophore group, but without loss
of haptophore or combining groups. The difficulty of establishing
the toxins as enzymes lies in the fact that neither toxin nor enzymes
have been isolated in the pure state. Furthermore, they do not act
according to the same chemical laws, the enzyme operating re-
peatedly to produce a large effect in the course of time and the toxin
acting in almost direct proportion to its quantity. In summary we
may quote Oppenheimer as saying of toxins that, " we must be
contented to assume that they are large molecular complexes, probably
related to the proteins, corresponding to them in certain properties,
but standing even nearer to the equally mysterious enzymes with
whose properties they show the most extended analogieG, both in
their reactions and in their activities."

Toxins may be injured in a variety of ways. They may be de-
stroyed, with certain exceptions, by moist heat at about 80° C., and
resist dry heat to over 100° C. Light operates in a general way
according to its intensity and penetrating power and the action is
intensified by the presence of oxygen. Diffuse daylight operates
slowly, but direct sunlight, X-ray, and ultra-violet rays more rapidly.
They are destroyed by fluorescent substances. Oxygen and oxi-
dizing substances injure and destroy toxins both in vivo and in vitro.
Certain chemicals are injurious, as the salts of bivalent and trivalent
metals, but not of monovalent metals. Certain toxins, particularly
dysentery and diphtheria, may be rendered non-toxic by acids and
restored to toxicity by alkali. They may be bound by fats and
lipoids, as illustrated in part, at least, by the neutralization of
tetanus toxin by brain substances. Enzymes, such as pepsin and
pancreatic juice, as well as bile, destroy certain toxins, so that they
produce no symptoms following ingestion, the striking exception
being botulinus toxin. The action of digestive ferments upon toxins
has recently been studied in detail by Loewi. He finds that diph-
theria toxin is destroyed by pepsin and ptyalin, that tetanus toxin
is destroyed by trypsin and ptyalin, but not by pepsin ; and that
dysentery toxin is destroyed by the action of the duodenal mucosa
of rabbits, but resists digestion with trypsin, ptyalin, pepsin,
and papayotin.

40 THE PRINCIPLES OF IMMUNOLOGY

Classification of Bacterial Toxins. — The bacterial toxins are
classified as exotoxins and endotoxins, the former appearing in the
culture medium as soluble substances and the latter appearing within
the bacterial bodies. These intracellular toxins can be liberated by
digestion, autolysis, freezing and fine grinding, and by expression
with a Buchner press. They cause the symptoms of their special
diseases, and in the natural course of the disease are probably liber-
ated either by autolysis or by the action of the enzymes of the cells
or fluids of the host. The tendency to-day, however, is to accept
the view that the so-called endotoxins are not produced as such, but
are produced from the bacteria during the process of hydrolytic
cleavage of the bacterial proteins by ferments provided by the host.
Certain bacteria, such as diphtheria and tetanus, produce only exo-
toxins, whereas the typhoid group and certain other organisms were
supposed formerly to produce only endotoxins. However, Bull has
shown that certain strains of the gas bacillus of Welch produce an
active exotoxin, and Ecker has shown that certain strains of bacillus
paratyphosus B produce exotoxins, and this has been confirmed by
Aronowitch. Kraus has shown a similar relationship in bacillus
dysenteriae. Studies of Admont Clark and Felton indicate that the
strepto'coccus hemolyticus produces a filterable toxic product
answering all the requirements of a true toxin. The production of
exotoxins is important for practical purposes because the endo-
toxins do not lead to antitoxin formation with the same degree of
facility as do exotoxins. Recent studies by Olitsky and Kligler have
shown that the dysentery bacillus (Shiga) produces a thermolabile
exotoxin and a thermostable endotoxin, the latter not being neutral-
ized by anti-exotoxic serum. A potent antiserum for both toxins
can be developed in the horse. The exotoxin appears to have spe-
cial affinity for the nervous system of the rabbit and the endotoxin
operates particularly upon the intestine.

Organisms which produce exotoxins show a considerable variation of
this property, but, on the whole, such toxins are more virulent and more
highly antigenic than the exotoxins of those organisms which are essen-
tially endotoxin producers. Being more highly antigenic the antitoxins
produced by exotoxins are the more powerful, as is well known in the
case of diphtheria and tetanus antitoxins. Nicolle, Cesari, and Jouan
maintain, on the basis of certain work with the bacillus of Nocard, that
exotoxins and endotoxins are identical in the case of a given organ-
ism, but the more recent studies of Olitsky and Kligler quoted above
would indicate that this is not true of dysentery bacillus (Shiga),
and therefore not a general law.

The exotoxins include diphtheria toxin, tetanus toxin, botulinus
toxin, dysentery toxin, paratyphoid toxin, and bacillus aerogenes
capsulatus (perfringens) toxin. Toxins are produced also by
bacillus edematiens (Weinberg), vibrion septique, bacillus of symp-
tomatic anthrax, bacillus pyocyaneus, streptococcus, and bacillus in-
fluenzse. In addition it is claimed by Kolmer and his co-workers

TOXINS AND ANTITOXINS 41

that they have demonstrated in pneumonic exudates a pneumococcus
toxin. A number of other organisms produce lytic bodies for red
blood-cells or hemotoxins, such as staphylolysin and megath-
eriolysin, capable of inducing the formation of antilysins. A lytic
body for leucocytes is also produced by staphylococcus aureus.

The pathological effects of toxins are fundamentally seen in the
production of cloudy swelling and even fatty degeneration of the
parenchymatous viscera, heart, vascular muscle, liver, kidney, and
secreting glands. Local inflammation at the site of injection, some-
times leading to necrosis, is a frequent rinding. Diphtheria toxin
may show, in cases of paralysis, myelin sheath degeneration or in-
flammation of nerves, and in guinea-pigs usually shows marked
congestion or hemorrhage in the adrenals. Botulinus toxin leads to
meningeal and even cerebral thrombosis and small hemorrhages.
Both botulinus toxin and tetanus toxin have a marked affinity for
the nervous system, but the effects are seen in the form of func-
tional disturbance rather than morphologically demonstrable change,
except for the vascular changes produced by botulinus toxin.

Formation of Antitoxins. — Antitoxins are produced by the re-
peated parenteral injection of the toxin. Parenteral injection signi-
fies introduction into the body by routes other than absorption
through the alimentary canal. The selection of the species of
animal to be used depends in part on its demonstrated ability to
produce antitoxin and in part in commercial establishments on the
possibility of obtaining large volumes of immune serum. It is often
found desirable to select a species which has natural immunity to
certain toxins and by inoculation raise that immunity to a higher
degree. This may be accomplished with less difficulty than if a
susceptible species were used. This is true of the use of the horse
in producing gas bacillus antitoxin. The same principle is employed by
Kyes in using fowl for the production of an anti-pneumococcus
serum, although in this case it is not clear that the serum is an anti-
toxic serum. Kyes states that the antiserum is antibacterial, i.e.,
agglutinating and bacteriolytic. Especially in the case of suscep-
tible animals and also in relatively immune animals it may be neces-
sary either to dilute the toxin to a very high degree or to attenuate
it by other means, and thus consume considerable time in develop-
ing a high degree of immunity. For immunizing guinea-pigs against
diphtheria toxin Behring and Kitasato used iodine terchloride,
and Roux and Martin, Lugol's solution. Frankel heated the
toxin to 60 degrees. Behring in the case of tetanus toxin used
a neutralized mixture of toxin and antitoxin, gradually reduc-
ing the amount of antitoxin, and finally using unmodified toxin. In
a sense this latter method has been employed by Behring and by
Park for producing active immunity to diphtheria in children, al-
though here it has been found unnecessary to use pure toxin with-
out antitoxin to attain the desired result. It is believed that after
injection there is a dissociation of the mixture, which permits the

42 THE PRINCIPLES OF IMMUNOLOGY

toxin to induce active antitoxin production by the patient's own
body. In the work with animals the injections are given at inter-
vals of a few days, sometimes interspersed with rest periods of
about a week, until testing of small amounts of serum shows that a
satisfactory result has been attained.

Technic of Producing Diphtheria Toxin, and Antitoxin. — Some details of
the technic of producing diphtheria antitoxin may well serve as an example of the
general phases of the method. It was early noted that different strains of the
diphtheria bacillus produced toxins of variable strength and also that the same
strain showed slight variation. Finally the strain isolated by Park and Williams,
now well known as Park No. 8, a strong toxin producer, was selected as a standard
and is so used throughout the world. In order to obtain the best aerobic conditions,
a wide-bottom flask is employed so as to expose a large surface of the medium,
"bob" veal broth being selected as most desirable. The culture is planted super-
ficially and allowed to grow for seven or eight days. It is wise to use a culture that
has been grown for several generations on the surface of broth tubes, the tubes
so inclined as to expose a large broth surface. This accustoms the organisms to
freely aerobic conditions. After the period of growth in the flask the organisms
are killed by formaldehyde or by phenol, or even by heat, and the broth filtered
either through paper or through a porcelain filter, preserved with toluol and per-
mitted to " ripen." This ripening is made desirable because of progressive deteriora-
tion of the toxin, a phenomenon which will be more profitably taken up in the
discussion of the toxin-antitoxin combination.

The following table shows a scheme as actually practised for producing
diphtheria antitoxin. All the injections are subcutaneous.

INJECTION SCHEME FOR PRODUCTION OF DIPHTHERIA ANTITOXIN
July 22 6,000 units antitoxin

25 400 minimum lethal doses of toxin (see page 45)
27 800 minimum lethal doses of toxin

29 1,200 minimum lethal doses of toxin

Aug. i i, 600 minimum lethal doses of toxin

3 2,000 minimum lethal doses of toxin

5 2,500 minimum lethal doses of toxin

8 3,000 minimum lethal doses of toxin

10 3,600 minimum lethal doses of toxin

12 4,400 minimum lethal doses of toxin

15 5,200 minimum lethal doses of toxin

17 6,200 minimum lethal doses of toxin

19 7,200 minimum lethal doses of toxin

22 8,500 minimum lethal doses of toxin

24 10,000 minimum lethal doses of toxin

26 13,000 minimum lethal doses of toxin
29 16,000 minimum lethal doses of toxin
31 20,000 minimum lethal doses of toxin

Sept. 2 24,000

5 28,000

7 32,000

9 36,000

12 40,000

Lengthen intervals by 24 hours if made
necessary by severe reaction.

Trial bleeding separation of and testing of serum, September 21. After that
if further immunization is necessary the dose is raised 5000 M.L.D. each injection.

The Nature of Antitoxins. — The serum thus obtained contains
the antitoxin. The exact nature of the antitoxin is unknown, but
chemical examination and other studies have thrown a certain
amount of light upon its properties. If we can accept the division
of the serum protein into fibrinogen, euglobulin and pseudo-globulin
by precipitation with magnesium sulphate or ammonium sulphate,
the antitoxin is found in that water-soluble fraction known as

TOXINS AND ANTITOXINS

43

pseudo-globulin, which constitutes about 78 per cent, of the serum
protein. This fact is taken advantage of in the so-called concentra-
tion of antitoxin, in which the pseudo-globulin is thrown down by
the addition of ammonium sulphate. Heinemann states that pseudo-
globulins may be broken into fractions, one of which contains the
antitoxin in highly concentrated
form, thus making the bulk even
smaller than by the use of pseudo-
globulin. The precipitate is col-
lected, dialyzed free of salt, and
taken up in water, the final volume
being considerably less than the
original amount of serum, there-
fore containing a greater number
of antitoxic units per c.c. than the
whole serum. This does not mean
that the antitoxin is necessarily a
globulin, for it resists trypsin diges-
tion in greater degree than does
globulin. It is, however, an elec-
tro-positive colloid. Antitoxin is
not thrown down in indifferent
precipitates, and in this respect dif-
fers from the enzymes, nor does it
operate in the same quantitative re-
lations as enzymes. The large size
of the antitoxin molecule is indi-
cated by the famous Martin and
Cherry experiment, which showed
that if toxin and antitoxin are
mixed and passed through gelatin

filters the toxin appears first. The PIG. i.— Apparatus for filtration through porce-
Same point Was brought OUt by ^in of small quantities of material. ]

Arrhenius and M a d s e n , who
showed that toxin diffuses ten times
more rapidly than antitoxin. Anti-
toxin is injured by moist heat of 60° to 70° C, destroyed by moist heat
of 100° C., and by dry heat of 140° C.

The influence of temperature on antitoxin is of the utmost prac-
tical importance in regard to its preservation for therapeusis.
Anderson has estimated the yearly deterioration at different tem-
peratures as follows :

rubber tube and the suction apparatus must be
inserted a small trap to prevent entrance into the
flask of water when suction is released. The trap
is a salt neck bottle with an inlet and outlet tube
through a rubber stopper.

Temperature

5°C.

Yearly deterioration
20 per cent.
10 per cent.
6 per cent.

44 THE PRINCIPLES OF IMMUNOLOGY

McConkey has given the following rates of deterioration :

Temperature Deterioration in 6 months

36° C. 37 per cent.

6°-i6° C. 14 per cent.

Ice chest 7 per cent.

The second figures in McConkey's table indicate room temper-
ature in winter and summer. Although the two series of investi-
gations differ in actual figures, they serve to show that the only
temperatures for satisfactory preservation are those of the ice chest.
Antitoxin is destroyed by putrefaction of the serum, by acids and
alkalies, by ultra-violet rays and deteriorates in solution, by expo-
sure to light and air. Ingestion into the alimentary tract destroys
antitoxin. Nevertheless, it is stated that suckling infants can absorb
antitoxin from the mother's milk. Toxin disappears from the blood
in the neighborhood of from seven to eleven days after injection, it
being in part destroyed, in part bound by the tissues, and in very
small part excreted in the urine. Antitoxin appears in man very
early in life, as determined by the Schick test (see page 53). It has
not been proven why the antitoxin develops, that is, whether it is
natural or the result of slight attacks of the disease. As indicated
above, it may possibly be transferred in mother's milk. Sherman
states that lysins and complement are inappreciable in the youngest
swine embryos, but that after the ninth week of gestation they can
be demonstrated in varying amount. Whether they are autoch-
thonous or transmitted from the mother has not been determined.
Wells states that, " taken together, the evidence indicates a closer
resemblance of antitoxins to proteins than has been shown for the
toxins, and all attempts to separate antitoxins from proteins have
so far failed."

The manner in which antitoxin neutralizes toxin is the subject
of much discussion, experiment, and hypothesis. Before discussing
the matter from a theoretical point of view, it is advisable to explain
some of the technical operations in the standardization or titration of
the antitoxin. From the practical point of view this is now rela-
tively simple, although requiring an extremely precise method, but
the earlier investigators were beset with many difficulties.

Standardization of Diphtheria Antitoxin. — In diseases such as
diphtheria and tetanus, where the symptoms are the results of the
action of the toxin, it is necessary to determine the amount of anti-
toxin required to protect an animal against the effect of a given
amount of toxin. The earlier investigators attempted to determine
the amount of antitoxic serum necessary to protect against inocula-
tions with living organisms, but the variability in biological proper-
ties of growth and toxin production, infection, and resistance, soon
showed the unreliability of this method. Behring then took up the
determination of antitoxin against toxin, but found it difficult to
standardize such a method over a wide geographic area because of

TOXINS AND ANTITOXINS 45

differences in bacterial strains and variations in the same strains
growing under even slightly different conditions. Ehrlich sought to
reduce the factor of error by determining the antitoxic " unit " as
the amount of antitoxic serum necessary to protect against ten times
the minimum lethal dose. Even this was unsatisfactory, and Behring
and Ehrlich in collaboration settled upon an arbitrary method of
determining a " normal " toxin and a " normal " therapeutic serum.
The "normal" toxin contained in i.o c.c. one hundred times the
minimum lethal dose for a guinea-pig of 250 grams. The " normal "
therapeutic serum was tested and diluted so that o.i c.c. contained
sufficient antitoxin to neutralize i.o c.c. of the "normal" toxin or,
in other words, i.o c.c. antitoxic serum, as a unit, was capable of
neutralizing 100 minimum lethal doses of toxin. The fundamental
error, however, had not been overcome by this method, and it was
found that no method which had as its basis a toxin, could be ap-
plied over a large area and the method was finally abandoned.
The toxin deteriorates rapidly on standing, and even though
after a time it becomes fairly stable, it still is insufficiently so
to justify its use for purposes of standardization. On the other
hand, the antitoxic serum resists drying for an indefinite period, and
if used as a standard can be shipped great distances. The standard
in this country has been established by the United States Public
Health Service. The unit of antitoxin as now used has no direct
relation to the unit of " normal " therapeutic serum as defined by
Behring and Ehrlich, but by interchange between nations it is
practically constant throughout the world. Hence, if a laboratory
wishes to prepare an antitoxin the standard unit of antitoxin can be
obtained from the Public Health Service. With the standard anti-
toxin on hand, the antitoxic content of a newly prepared antiserum
may be determined. This must be done through the medium of a
toxin whose strength is titrated against the standard antitoxin ; the
toxin is thereby standardized, so that the strength of the new anti-
toxic serum can be measured. The toxin must be one which has
been ripened, so that any deterioration during the few days' time
necessary for titration against the standard immune serum and then
against the new serum, is reduced to a negligible minimum. In
order to make the titration against the standard antitoxic unit some-
what easier it is well to know the minimum lethal dose of toxin.
There are then to be determined :

1. The minimum lethal dose of toxin (M.L.D.).

2. The L0 dose of antitoxin.

3. The L+ dose of antitoxin.

The minimum lethal dose of toxin is determined by injecting subcuta-
neously, varying doses of toxin into a series of guinea-pigs 250 grams in
weight. Healthy pigs of this weight are usually young and less expensive
than fully-grown animals. The M.L.D. is the smallest dose that kills a
pig in from four to five days. Less than four days means too great strength,

46 THE PRINCIPLES OF IMMUNOLOGY

more than five days too little strength. It can be seen that the selection of
the weight of the pigs and the length of time are arbitrary but universal
standards. A strong toxin might give results as follows:

Guinea-pig Toxin dose Result

1 0.0036 c.c. Lives

2 0.0038 c.c. Dead 6 days

3 0.0040 c.c. Dead 4 days 8 hours

4 0.0042 c.c. Dead 3 days 20 hours

5 0.0044 c.c. Dead 2 days

Guinea-pig No. 3 died at the right time interval and 0.004 is the M.L.D.
of this toxin. Experiments with a preliminary series using more widely vary-
ing doses of toxin would be necessary before the final experiment could
be set up.

The Lo dose (Limes null) is that amount of toxin which is so thoroughly satu-
rated with one unit of antitoxin that neither local nor general symptoms
appear following the injection of the mixture. An experiment follows:

Standard
Guinea-pig antitoxin Toxin Result

1 i unit 0.36 c.c. No reaction

2 i unit 0.38 c.c. No reaction

3 i unit 0.40 c.c. Barely visible congestion

4 i unit 0.42 c.c. Moderate inflammation

5 i unit 0.44 c.c. Distinct inflammation

In this experiment the dose of toxin, 0.40 c.c., given pig No. 3, is the L0
dose. The note as to reaction refers to the shaven site of injection.

The L+ dose (Limes death) indicates the smallest amount of toxin which
after mixture with one unit of antitoxin will produce death in four-five days.
The plus sign is the mark used in English texts to correspond to the cross
mark used in German literature to signify death. An experiment follows :

Standard
Guinea-pig antitoxin Toxin Result

unit 0.44 c.c. Lives

unit 0.46 c.c. Dead 6 days

unit 0.48 c.c. Dead 4 days

unit 0.50 c.c. Dead 3 days

unit 0.52 c.c. Dead 2 days

In this experiment 0.48 c.c. given guinea-pig No. 3 is the L+ dose of toxin.

Titration of Diphtheria Antitoxin.— In the actual titration of an
antitoxin as practised to-day there must be at hand a standard anti-
toxin of known strength as well as a toxin, whose M.L.D. has been
at least approximately determined. The antitoxin has been dried
in a vacuum and preserved in sealed U-shaped ampoules which con-
tain the antitoxin in one arm and P2O5 or some other hygroscopic
substance in the other arm, in order to maintain the dryness of the
antitoxin. The ampoule is best kept in a light-proof box in the re-
frigerator. Against this antitoxin the L+ dose of a toxin is deter-
mined, and against this toxin the new antitoxin is titrated. The
amount of antitoxin which protects against the L+ dose for four
days is the antitoxin unit of the new serum. In preliminary experi-
ments the antitoxin is roughly titrated in dilutions of i : 100, i : 200,
1:300, and so on. In each case the antitoxin is used in i.o c.c.
amounts and the toxin so diluted that 2.0 c.c. contain the M.L.D.,
the two being mixed and allowed to stand at room temperature for

TOXINS AND ANTITOXINS

47

one hour. The Rosenau glass syringe for this purpose has an
oblique side arm for salt solution, so that after the toxin-antitoxin
mixture is injected, the side arm is swung around, emptying the
saline into the main body of the syringe. The salt solution is then
injected, thus washing out the remnants of the toxin-antitoxin mix-
ture that may remain in the lower part of the syringe and needle.

The following experiment will serve to illustrate, granting that the pre-
liminary titration showed a strength of antitoxin between 1—200 and 1-400.

Antitoxin

Toxin

Guinea-pig i c.c. of each
dilution

2.0 C.C

= M.L.D.

I

-200

2.O C.C.

2

-220

2.O C.C.

3

-240

2.0 C.C.

4

-260

2.O C.C.

-280

2.O C.C.

6

-300

2.O C.C.

7

-320

2.O C.C.

8

-340

2.O C.C.

9

-360

2.0 C.C.

10

-380

2.0 C.C.

Dead
Dead
Dead
Dead
Dead
Dead
Dead
Dead
Dead
Dead

Result

5 days
5 days
4 days
4 days
3 days
3 days
3 days
3 days
2 days
2 days

Thus doses i and 2 were more than sufficient to protect four days, and
doses 5-10 were insufficient. Doses 3 and 4 protected for four days, and in
order to be safe dose No. 3 of 1-240 would be selected. If the antitoxic unit is
1/240 of i.o c.c., each c.c. of serum contains 240 units of antitoxin. In com-
mercial work, the practice is to be absolutely on the safe side, and the next
larger dose of antitoxin would be employed as the unit, and the serum
marketed as containing 220 units per c.c.

In the therapeutic use of such a serum the unit
content of the serum is simply a guide to its use,
the dose employed being rather on an empirical
basis than otherwise, because of the uncertainty of
the amount of toxin present in the body of the
patient. It is generally assumed that the larger
the extent of the exudate, the greater the amount
of toxin produced and the larger the absorbing sur-
face, but it can readily be seen from the theoretical
standpoint that variations may be produced by dif-
ferences in toxin production by the different strains
of bacillus diphtheriae which may be encountered
in patients. It is unwise to stress this latter possi-
bility and preferable to regulate the dosage on the
former basis. More will be said later regarding
therapeusis.

The Toxin-antitoxin Union. — T he E hrlic h
Theory. — With the foregoing practical considera-
tion of antitoxin titration in mind, the theoretical
problems of the nature of the toxin-antitoxin com-
bination will be taken up. The simplest conception
is that antitoxin neutralizes toxin in the same way
that a strong acid neutralizes a strong base. As has been seen,
the neutralization is quantitative and follows in a general way

48 THE PRINCIPLES OF IMMUNOLOGY

the law of multiple proportions. If this were true, how-
ever, the L+ dose which in combination with the antitoxin unit
kills a pig in four days should contain one unit more of toxin
(i M.L.D.) than the L0 dose which just fails to produce symptoms.
Reference to the experiments offered to illustrate the determination
of M.L.D. , L0 dose, and L+ dose show that the M.L.D. of the toxin
was 0.004 c-c-» tne L0 dose of toxin was 0.40 c.c. or one hundred times
the M.L.D., and the L+ dose 0.48 c.c. or one hundred and twenty
times the M.L.D. The difference between L0 and L+ doses is, there-
fore, twenty times the M.L.D. instead of exactly equal to it. This
has been interpreted to indicate that some body or bodies, other than
the toxin, has combined with the antitoxin, thus limiting its ability
to combine with toxin. Ehrlich, after numerous experiments and
hypotheses, reached the assumption that the toxic broth contains
two bodies other than toxin, which he named toxon and toxoid. The
toxon is a body with a smaller degree of affinity for the antitoxin
than has the toxin. In a determination of the L0 dose the antitoxin
neutralizes both toxin and toxon, so that no symptoms appear, but
if more toxin be added to the mixture it combines with antitoxin,
displacing the more loosely combined toxon. Finally, after suffi-
cient addition of toxin the antitoxin is fully saturated, and any addi-
tional toxin will be free, and if in sufficient quantity (i M.L.D.)
will lead to the death of the experimental animal. In more detail
the 20 M.L.D.'s necessary to make the difference between the L0 and
L+ doses were so used that 19 M.L.D.'s were employed to displace a
proportionate amount of toxon and toxoid from combination with
the antitoxin unit, and the remaining i M.L.D. sufficed to kill the
pig in four days. If more than i M.L.D. were present in excess
death would ensue after a shorter period, and if less than i M.L.D.
were present death would occur later than four days or not at all.
It is believed, on the basis both of experimental and clinical obser-
vation, that toxon is responsible for the late paralyses of diphtheria.
The conception of the toxoid is based on the Ehrlich assumption
that the toxin molecule has a toxic fraction or " toxophore group "
and a combining fraction or " haptophore group." A toxin will
retain its binding power for antitoxin for a considerable length of
time with little change in the L+ dose, but with marked deterioration
of toxic power and corresponding reduction of the M.L.D. This is
interpreted as meaning that the toxic fraction is labile and the com-
bining fraction much more stable. The toxoid, then, is the toxin
molecule so altered that its toxic part is reduced and the combining
part practically intact. As can readily be seen, this can account
also in part for the discrepancy between L0 and L+ dose. The
discrepancy between L0 and L+ dose in fresh toxic broth is be-
lieved to be due to the presence of toxon rather than toxoid, be-
cause too short a time has elapsed to account for toxoid formation.
As the toxic broth becomes older the discrepancy becomes greater,
even after a relative equilibrium has been established, and the differ-

TOXINS AND ANTITOXINS 49

ence is believed to be due to the progressive formation of toxoid
from toxin and perhaps also from toxon. Ehrlich and also Madsen
found that the combination of one antitoxic unit with toxin in the
determination of the L0 dose was in multiples of 100 M.L.D/s.
These multiples were rarely less than 100 and never more than 200.
This would indicate that the multiple is not less than 100, but even
though values of 200 are not obtainable, the failure may be ex-
plained by the fact that pure toxin is not procurable. By means of
the phenomenon of " partial absorption " Ehrlich established a
formula for the antitoxin-toxin combination which he expressed as
"toxin200 antitoxin." This has been illustrated by means of a toxin-
antitoxin " spectrum " based on a total valency of 200, the total valency
including toxin, protoxoid, and toxon. In spite of the great academic
interest of this discussion, its immediate practical value is not apparent
and the reader is referred to larger works for complete discussion.

Objections to the Ehrlich Theory. — As indicated previously, the
Ehrlich hypothesis is based on the assumption that the toxin-anti-
toxin reaction follows in a fairly close manner the chemical reaction
between a strong acid and a strong base. Certain features of the
process of combination support the idea of chemical union, as, for
example, the fact that warmth accelerates the reaction, dilution
slows it. Furthermore, there is a liberation of heat in the reac-
tion, that is to say, about half as much heat per gram molecule as
would be liberated by the reaction between a strong acid and a
strong base. It is well known, however, that the union of toxin and
antitoxin is loose and within certain limits reversible. The Martin
and Cherry experiment referred to earlier in this chapter is of great
importance in this connection. They mixed snake venom and anti-
toxin to a point of neutralization and filtered through gelatin filters,
with the result that the toxin came through the filter first. This
they interpreted as being due to the smaller size of the toxin mole-
cule. It also shows the looseness of combination and the reversi-
bility of the reaction. They further showed that the longer the
mixture stands, the smaller the amount of toxin that comes
through the filter. Zinsser states that the " chief value of these
experiments lies in their proof of the element of time as an
important factor in the toxin-antitoxin union." Calmette had previ-
ously shown that venoms of certain snakes would remain virulent
after heating even to 100 degrees, and that the antitoxins were
thermolabile. He demonstrated that if the two were mixed so as to
be non-toxic, subsequent heating would liberate the toxin probably
through thermic destruction of the antitoxin. If the union were a
fixed one, this should not have been true. Martin and Cherry failed
to confirm this with the venom of an Australian snake, but this can-
not be regarded as a refutation of Calmette's work, especially as the
principle was found to apply to other toxins and antitoxins.
Morgenroth showed that acidulation with HC1 of a venom lysin-
antilysin mixture produced an acid-toxin molecule that resisted
4

50 THE PRINCIPLES OF IMMUNOLOGY

heat and could by heat be dissociated from the thermolabile anti-
lysin. The subsequent chemical neutralization of the toxin (or
lysin) resulted in the restoration of its toxicity. In this laboratory
Wahl has shown that titration of diphtheria toxin using normal
guinea-pigs in one series and guinea-pigs with only one kidney in
another, gives materially different results. These experiments were
carefully controlled and may be offered as a further indication of the
loose combination and its corollary the reversibility of the reaction,
on the probable assumption that the toxin is more readily excreted
by the animals with two kidneys than by those with one. The
recitation of these few experiments to which others might be added
is sufficient to illustrate the objection to the Ehrlich theory of fixed
combination, and two other important hypotheses are offered for
consideration : (i) The conception that the combination follows the
law of mass action and (2) the theory of colloidal reaction.

The Law of Mass Action. — The application of the law of mass
action has been worked on by Arrhenius and Madsen principally.
This law is usually illustrated in the chemical laboratories by the
reaction between one gram molecule ethyl alcohol and one gram
molecule acetic acid which yields ethyl acetate and water, the reac-
tion, however, stopping at a point of equilibrium where there is
found in the mixture ^3 gram molecule alcohol, l/$ gram molecule
acetic acid, 2/z gram molecule ethyl acetate, and 2/z gram molecule
water. The same end-result is obtained if instead of mixing ethyl
alcohol and acetic acid, we mix ethyl acetate and water, thus indicat-
ing the reversibility of the reaction as stated in the formula :

C,H6OH + CH3COOH ^± CH8COOC2H5 + H2O

Arrhenius and Madsen compared the reaction between tetanoly-
sin and its antitoxin and the reaction between boric acid and am-
monia. This was of advantage because ammonia is hemolytic and
boric acid is not. Thus a reversible reaction is found in which the
addition of boric acid reduces the hemolytic activity of the ammonia.
As with the alcohol-acetic acid experiment, however, a point of
equilibrium is established whereby there .always remains a small
amount of free ammonia in spite of the addition of boric acid to a
point of saturation. The same general proposition holds in regard
to tetanolysin and antilysin, and these authors were able to con-
struct similar curves of neutralization for both of these reactions.
With this idea as a basis, the late paralyses of diphtheria, either ex-
perimental or clinical, would depend not upon the toxon of Ehrlich,
but rather upon a small non-fatal amount of toxin that is never com-
pletely neutralized in the reaction.

The Danysz Effect. — It will readily be seen, however, that the
reversible reactions illustrating the law of mass action deal with
crystalloids, while it is probable both toxins and antitoxins are of
colloidal character. Certain colloids are known as " reversible col-

TOXINS AND ANTITOXINS 51

loids," but as yet there is little definite proof that reversible reac-
tions between two colloids take place. According to certain inter-
pretations, the most important observation in support of the
colloidal theory is the Danysz effect. If the toxin is added to the
antitoxin in fractions with an interval of time elapsing between, less
toxin is needed to saturate the antitoxin than if the toxin were added
in one volume. In other words, if i.o c.c. toxin were saturated
in the usual way with o.i c.c. antitoxin and if in another test-tube
the toxin is added to the same amount of antitoxin, not in a single
dose, but in successive doses of 0.2 c.c., until i.o c.c. is present, this
latter mixture instead of being neutral would be toxic. Wells, how-
ever, states that this " indicates that the toxin antitoxin union is
physical rather than chemical, for it seems to be quite analogous to
such a phenomenon as the taking up of more dye by several pieces
of blotting paper added in series to a dye solution, than by the same
amount of paper added in one piece." Of somewhat similar import
is the absorption theory of Bordet and of Landsteiner, which states
that when toxin is added to antitoxin in smaller quantities than
saturation, let us say five molecules of antitoxin to ten of toxin, this
does not result in complete molecular combination with five mole-
cules of toxin, but rather in half saturation of the entire ten mole-
cules of toxin. This results in attenuation of the toxin, so that
instead of there being five free molecules of toxin there are ten units of
partly detoxified toxin. It is not to be expected that this follows in exact
arithmetical progression, but Biltz has made comparisons with absorp-
tion phenomena in general and finds fairly consistent results.

In summary it may be said that in explaining the union of toxin and
antitoxin the Ehrlich hypothesis does not withstand critical examination
and that the reaction is in all likelihood of an intricate physico-chemical
nature referable, in part at least, to the probable colloidal nature of the
reacting bodies, but not as yet satisfactorily explained.

Therapeutic Use of Diphtheria Antitoxin. — In 1892 von Behring
and Wernicke found that the serum of animals immunized against
diphtheria toxin protects other animals of the same and different
species against the action of the toxin. In 1894 Roux demonstrated
the value of the treatment of diphtheria in man by means of anti-
toxic serum. This method of treatment rapidly attained widespread
use and has markedly reduced the mortality from the disease.
Numerous statistical studies have been made since that time, and
it is safe to say that the introduction of antitoxin treatment has
reduced mortality from approximately 40 per cent, to approximately
7 per cent. In interpreting the figures it has been found necessary
to take account of two important factors; namely, the cases of
laryngeal diphtheria and the time at which treatment is instituted.
Laryngeal diphtheria presents not only the element of toxic absorp-
tion, but in addition mechanical obstruction to respiration and
the possibility of extension downward, so as to produce pneumonia.
Furthermore, the operative procedures for relieving the respiratory

THE PRINCIPLES OF IMMUNOLOGY

obstruction are such as to introduce an additional minor element of
danger. The accidents following tracheotomy are distinctly more
numerous than those following intubation, but neither operation can
be regarded as absolutely without risk. It is now well established
that the earlier in the course of disease the antitoxin is administered
the more favorable is the prognosis. In order to present this
graphically, however, we insert the following table taken in large
part from Dieudonne and Weichardt :

Total
number
of cases

Mortal-
ity per-
centage

ISt

day

2nd
day

3rd
day

4th
day

5th
day

6th

day

After
6th
day

Welch

I48Q

14.2

2.^

8.1

13.5

19.0

29.3

34.1

33.7

Hilbert

**rwif

2428

T^

18.3

•o
2.2

7.6

I7.I

23.8

33.9

34.1

38.2

American Pediatric

^T^

fcV^»O

Society

S794

12.3

4-99

7.4

8.8

2O.7

35.3

Brook Hospital,

\} / "T^

London

8007

Q.5

o.o

A. 3

II. 12

17.24

18.72

Germany

71

7*C/

*T*O

* / "-^T-

Kais-Gesundh. Amb-

tes (Berlin) (Sam-

melforschung) ....

958i

15-5

6.6

8-3

12.9

17.0

23.2

26.9

Russia

(Rauchfusz) (Sam-

melforschung)

44,631

14.6

3-7

8.2

16.2

25.9

Austria

Sammelforschung

(Sanitatswesen) . . .

1103

12.6

8.0

6.6

9.8

25.5

28.8

30.7

2I.O

The therapeutic efficiency of the antitoxin also varies accord-
ing to the method of administration. According to Berghaus,
intravenous injections are five hundred times more effective and
intraperitoneal are eighty to ninety times more effective than sub-
cutaneous injections. In the earlier days of antitoxin treatment the
method was almost entirely subcutaneous injection, but subse-
quently the intravenous method was employed in severely toxic
cases. Injections were given at various intervals, usually a day
apart, until the disease showed marked improvement. The studies
of Park and his collaborators have modified the treatment consid-
erably. Park was able to show that a single dose of antitoxin in
sufficient quantity is more effective in neutralizing the circulating
toxin than the multiple small doses, largely because of the fact that
with subcutaneous and intramuscular injections the absorption is
continuous, whereas during the period usually occupied by giving
several doses, the absorption occurs for only a short time after each
injection. Were it possible to determine for clinical purposes the exact
amount of toxin absorption during the disease the dosage of antitoxin
could be accurately regulated. Unfortunately, however, different strains
of the bacilli vary in capacity for production of toxin and the depth
and extent of the local lesion, as well as the nature of the underlying
tissues, have some influence upon the rate and amount of absorption.

TOXINS AND ANTITOXINS 53

Therefore, the actual dose employed is to a large extent upon an empiri-
cal basis. Park recommends the following table of doses and methods
of administration :

DOSAGE OF UNITS OF ANTITOXIN IN DIPHTHERIA. SINGLE DOSE ONLY.

Infant, ten to thirty pounds (under two years of age).

Mild Moderate Severe Malignant

2,000 3,000 5,000

3,000 5,000 10,000 10,000

Child, thirty to ninety pounds (under fifteen years of age).
3,000 4,000 10,000 10,000

4,000 10,000 15,000 20,000

Adults, ninety pounds and over.

3,000 5,ooo 10,000 15,000

5,000 10,000 20,000 40,000

METHOD OF ADMINISTRATION.

Mild Moderate Severe Malignant

Subcutaneous or Intramuscular or Intramuscular or ^ intravenous and
intramuscular subcutaneous ^ intravenous y2 intramuscular

and y2 intramus- or subcutaneous
cular or subcu-
taneous

McCombie recommends the following dosages :
Mild: 4000 to 8000 units in one dose.

Moderate: 12,000 to 16,000 units in one dose or two doses.
Severe : 20,000 to 50,000 units or more in two or three doses.
Laryngeal: 16,000 to 24,000 as initial dose, and repeat once or twice according
to persistence of symptoms.

Improvement following the administration of antitoxin is strik-
ing when given early and exhibits itself in fall of temperature, reduc-
tion of leucocytosis, reduction of inflammation, and separation of
the fibrinous membrane. This improvement varies somewhat with
the method of administration, the intravenous method effecting im-
provement in somewhat less time than the intramuscular, and the
latter in somewhat less time than the subcutaneous. In a series of
cases studied in the City Hospital in Cleveland by Ruh the intra-
venous form of administration was followed in 83 per cent, of the
cases by a severe general reaction with chills and prostration com-
ing on a few minutes after the administration and lasting for about
twenty minutes. At the present time it is impossible to state the
exact cause of this reaction. Such reaction does not follow subcu-
taneous and intramuscular injections. It is not due to the preserva-
tive, nor as far as can be determined, to the age of the serum. The
most reasonable explanation appears to us to be that the reaction is
due to foreignness of the horse protein. None of Ruh's cases showed
prolonged or fatal reactions, and it is not probable that these reac-
tions represent individual hypersusceptibility, because if this were
true fatalities would be likely to occur (see page 230).

Natural Immunity to Diphtheria — The Schick Test. — It has long
been known that many individuals, even as many as 80 per cent, of
adults and 50 per cent, of children, are immune to diphtheria, as indi-

54 THE PRINCIPLES OF IMMUNOLOGY

cated by demonstrating antitoxin in the blood. The methods of
demonstration were not easily applicable until the development
of the Schick test. This test is performed by injecting intra-
cutaneously one-fiftieth of the minimum lethal dose of a specially
prepared toxin contained in 0.2 c.c. of salt solution. Injection is pref-
erably on the flexor surface of the arm or forearm. Six-day broth
cultures of the organism are killed with phenol, sedimented in the
ice-box for two or three days, the supernatant fluid filtered through
a Berkefeld candle, and the clear filtrate accurately standardized. It
is well to keep this filtrate for several months or a year, so that its
rate of deterioration is reduced to a minimum. A control injection
is given with the same quantity of toxic broth heated to 75° C, so
as to destroy the toxin.

The reactions to injections may be as follows:

A. Positive reaction. This indicates that no antitoxin is present in the
body, thereby permitting the toxin to act upon the unprotected cells. Slight
reaction appears in from twelve to twenty-four hours in the form of redness,
which becomes more distinct in from twenty-four to forty-eight hours, reach-
ing its maximum on the third or fourth day, then gradually disappearing and
leaving an area of scaling and brown pigmentation. The area attains a
diameter of 10 to 20 mm., and varies in intensity, depending on the sensitive-
ness of the individual.

B. Negative reaction. If no distinct reaction appears any more than is
seen in the control area the failure to react indicates that an amount of anti-
toxin is present in the body sufficient to neutralize the introduced toxin. Such
a reaction in a child of about three years of age probably indicates perma-
nent immunity. By varying the quantity of toxin injected, the amount of
antitoxin can be titrated.

C. The pseudo-reaction. This is usually urticaria] in nature, appearing some-
times immediately and sometimes in from six to eighteen hours, reaching its
maximum on the third or fourth day. It fails to leave pigmentation after it
subsides. This is a reaction of hypersusceptibility to the protein substances
present in the toxic broth as the result of the autolysis of the diphtheria
bacilli, and is in nature the same as other reactions of hypersusceptibility
described subsequently (see page 236). Such a pseudo-reaction may intensify
the true reaction and represent a summation of the protein reaction and a
reaction to the toxin. This must be taken to indicate that an individual may
be hypersensitive to the proteins of diphtheria bacilli but at the same time
not possessing in his circulating blood any antitoxin. The differentiation
depends upon the difference between the reaction at the site of the test
injection and at the site of the control injection.

Zingher divides the positive reactions as follows: -f -f indicates a strong
positive reaction with marked local redness, infiltration, and occasionally super-
ficial vesiculation ; -|- indicates positive reaction with redness but little or no
infiltration; ± indicates moderately positive reaction with moderate degree of
redness and no local infiltration ; i indicates a faintly positive reaction with only
slight redness and no local infiltration.

The test has been found to be of great value in determining the
immunity of groups of individuals, particularly in institutions
where there has been exposure to diphtheria. It has also given con-
siderable information as to the incidence and duration of this variety
of active immunity. Immunity to diphtheria may be derived from
the mother and lasts for about six months after birth. The largest
number of positives is found from the ages of six to eighteen months.
This gradually decreases throughout life.

Another method for determining the presence of antitoxin in the
blood is that of Romer. This depends upon the well-known fact

PLATE I.

POSITIVE SCHICK REACTION

Reaction of moderate severity seventy-two hours after the

intracutaneous injection of one-fortieth the minimal lethal

dose of diphtheria toxin. Patient's blood serum was found to

contain no antitoxin (International Clinics).

TOXINS AND ANTITOXINS 55

that the intracutaneous injection of toxin into guinea-pigs leads to
localized necrosis in the course of forty-eight hours. The minimum
amount of toxin sufficient to produce necrosis can be determined,
the protective power of antitoxin determined, and subsequently
with a standard antitoxin any new toxin may be titrated after the
same general principles as described previously for antitoxin titra-
tion. By this method the presence of toxin in human blood may be
determined, inasmuch as normal human serum does not produce
necrosis upon intracutaneous injection in the guinea-pig.
Harriehausen and Wirth found that the serum from patients suffer-
ing with diphtheria produced necrosis owing to the presence of
toxin, and this was demonstrated in five cases for as long as thirty-
five days after the onset of the disease. By the use of a titrated
toxin the method may also be employed for determining the pres-
ence and amount of antitoxin in human blood.

Active Immunization Against Diphtheria. — For many years im-
munization to this disease was entirely in the form of passive im-
munization, practised by giving protective doses of antitoxin. The
antitoxin was given in doses of 500 to 1000 units and served to pro-
tect for a period of about three to six weeks. This was of special
importance in institutions and families exposed to the disease. The
disadvantages are the short period of immunity and the fact that
the patient may thereby become hypersensitive to any subsequent
injections of horse serum. Active immunity had been observed by
Park in guinea-pigs which had been used for the titration of anti-
toxin, and Park, in 1905, reported that horses treated with neutral-
ized mixtures of toxin and antitoxin had produced immune sera as
strong as 400 units per c.c. Theobald Smith suggested a similar
method of immunization in man, and in 1913 von Behring reported
the successful immunization of children and adults. The method
of immunization with toxin and antitoxin mixtures has now attained
a widespread use and is employed even as early as the fourth day of
life. Active immunity of this sort is demonstrable by the Schick
test in about ten days after treatment, and increases so that in the
eighth week about 80 per cent, of the treated individuals are immune,
by the twelfth week 96 per cent, are immune, and at the end of
four months 98 per cent, are immune. According to Park, the re-
maining 2 per cent, become immune if reinjected. The method of
immunization is to give three injections subcutaneously one
week apart.

For the preparation of the mixture a ripe toxin is used and so diluted
that i. c.c. will contain 200 minimum lethal doses as tested against guinea-
pigs. This is slightly over-neutralized with antitoxin and the mixture should
cause no symptoms in guinea-pigs even when given in very large doses. As
indicated above, antitoxin may deteriorate in the moist state, and this must
beQavoided in the toxin-antitoxin mixtures. If the mixtures are kept at about
21 C. the mixture remains good for at least one year, although it is prefer-
able to keep it at a lower temperature. The injection is given subcutaneously
in the arm at the insertion of the deltoid muscle. The immunity developed
following injection of this sort is against toxin, but vaccination against diph-

56 THE PRINCIPLES OF IMMUNOLOGY

theria bacilli themselves may also be practised at the same time by adding
1000 millions killed organisms. The value of this latter procedure has not
been demonstrated as yet and it is not widely practised.

The advantage of the use of toxin-antitoxin mixtures is that a last-
ing active immunity is established. It has been suggested, however,
that the use of the antitoxin in the mixture may lead to the de-
velopment of anaphylaxis. It has been maintained, on the other
hand, that the mixture of the toxin probably avoids the sensitizing
effect of the horse serum, but this is not borne out experimentally
or in human medicine. Nevertheless, the amount of horse serum
given is relatively small and the development of hypersusceptibility
in man following these small amounts is not very common. Park
has found that even with the therapeutic dose of diphtheria antitoxin
the danger of anaphylaxis is extremely small, and states that
among 330,000 cases on record there were only five deaths. As we
will point out subsequently (see page 230), the reports of deaths
following antitoxin administration would indicate that, in a certain
percentage at least, factors other than anaphylaxis are operative.
If sensitiveness to horse serum is known it is suggested that anti-
toxin prepared in some other animal, such as the goat, may be em-
ployed, but sera of this sort are not easily obtainable in the market.

Tetanus Toxin and Antitoxin. — In the foregoing consideration
much stress has been laid on diphtheria toxin and antitoxin, with
the idea that the problem might thus be presented as simply as pos-
sible. Tetanus toxin and antitoxin have been studied almost if not
quite as intensively as diphtheria and deserve, both practically and
theoretically, more than passing mention. The important facts may
be given briefly. The toxic broth produced by growth of bacillus
tetani contains a body which is actively hemolytic, tetanolysin, and
more important, a body which produces the symptoms of tetanus,
tetanospasmin. These are capable of specific absorption, so that
one or the other remains free. In other words, the tetanolysin may
be removed from the mixture by saturation with red blood-cor-
puscles, leaving in the broth the tetanospasmin which may be in-
jected with the production of symptoms and death. An antitoxin
may be produced specifically for the tetanospasmin, but in practice
the antitoxin is made without separation of the two toxins.

The toxin is produced in anaerobic broth cultures. It is readily
injured by heat and light, and is best preserved in the dried state.
The white mouse is extremely susceptible, the guinea-pig less so
and the horse somewhat less, whilst pigeons and fowl are highly
resistant. The antitoxin is produced for commercial purposes in the
horse, the earlier doses being with an attenuated toxin following a
previous injection of antitoxin. The serum is standardized by the
use of white mice or by guinea-pigs, the procedure being practically
the same as for standardization of diphtheria antitoxin. In the
United States the toxin is used as a standard. It is precipitated by
ammonium sulphate and dried. The minimum lethal dose is that

TOXINS AND ANTITOXINS

57

which kills a pig of 350 grams in four to five days. The antitoxic
unit is ten times the amount of antitoxin necessary to protect
against 100 minimum lethal doses.

In man the symptoms appear, as a rule, first in trismus of the jaw
muscles, but in experimental animals the first spasms are near the
injection site when the toxin has been given subcutaneously or
intramuscularly. For demonstration purposes the dried toxin is
freshly dissolved and five minimum lethal doses injected into the
thigh muscles of one hind leg of a guinea-pig. In the course of
about two days the leg is found stiff and extended, the animal show-
ing excitable reflexes. In the course of another day a sudden noise
or other stimulus will excite convulsions, and later the animal will
be found in tonic spasm and dies with all four extremities in exten-
sion. If the toxin is given intravenously or intraperitoneally, the
first symptoms are excitable reflexes, then general clonic and finally
tonic spasm. If given intracerebrally, the onset is by epileptiform
convulsions. Rabbits are much more resistant to the toxin, and
an intravenous injection will lead to gradual wasting and a cachectic
death, which has been called " tetanus sine tetano." The suscep-
tibility of animals varies with the temperature of the body. Cold-
blooded and hibernating animals are resistant at cold winter
temperatures, but become susceptible at summer temperatures.

Tetanolysin. The tetanolysin is easily demonstrable in a toxin. The best
red blood-cells for use are those of the goat, sheep and horse. The fol-
lowing protocol will show the method of titrating the tetanolysin. The toxin
is dissolved so as to make a I per cent, solution in saline, and is further diluted
for the experiment 1-2, 1-5, i-io, 1-20. The blood-cells are washed three
times and suspended in salt solution so as to make a 5 per cent, solution. For
method of washing red blood-cells see page 118.

Hemolysis

Complete

Complete

Partial

Slight

None

The mixtures are incubated in a water bath at 37° for one hour.

The minimum lytic dose in the above instance is I c.c. of a 1-5 dilution of
the i per cent, toxin solution. This is used as the unit to determine the
antitetanolysin in an antitetanic horse serum as in the following protocol:

Tube

Toxin

S % sheep cells

I

1-2

(i

o

c.

c.)

.0

c.c.

2

i-5

(i

0

c.

c.)

.0

c.c.

3

I-IO

(i

0

c.

c.)

.0

c.c.

4

i -20

(i

0

c.

c.)

.0

c.c.

5

Saline

(i

.0

c

c.)

.0

c.c.

Tube Toxin

Immune serum 5 % sheep cells*

Hemolysis

I

.0 C.C.

-5

dil.

I-I,OOO (l.O C.C.)

.0 c.c.

None.

2

.0 c.c.

-5

dil.

1-2,000 (i.o c.c.)

.0 c.c.

None

3

.0 C.C.

-5

dil.

I-IO,OOO (l.O C.C.)

.0 c.c.

Slight

4

.0 C.C.

-5

dil.

1-20,000 (i.o c.c.)

.0 c.c.

Partial

5

.0 C.C.

-5

dil.

1-50,000 (i.o c.c.) ]

.0 c.c.

Complete

Normal horse serum

6

.0 C.C.

-5

dil.

I-IOO (l.O C.C.)

.0 c.c.

Complete

7

.0 C.C.

-5

dil.

1-1,000 (l.O C.C.)

.0 c.c.

Complete

8

.0 c.c.

-5

dil.

None

.0 C.C.

Complete

9 None

I-IOO (l.O C.C.)

.0 C.C.

None

*Add the sheep cells after the mixture of toxin and serum has been incubated for one-half
hour and then incubate one hour. For method of diluting serum so as to obtain required
strengths see page 84.

58 THE PRINCIPLES OF IMMUNOLOGY

Tubes 6-9 are controls to show that normal horse serum is not antilytic,
that the laking dose still operates after the preliminary half-hour incubation,
and that horse serum itself has no lytic effect.

Tetanospasmin. For the demonstration of the neutralization of tetano-
spasmin by antitoxin and by brain substance, the following experiments are
of value. Five guinea-pigs of about 250 grams are needed.

Pig No. i. Inject five minimum lethal doses of toxin into the thigh muscles.

Pig No. 2. Mix ten minimum lethal doses of toxin with one unit of
antitoxin. Allow to stand at room temperature for about twenty minutes
and inject as in pig No. I.

Pig No. 3. In a sterile mortar grind one-half the fresh cerebrum of a
guinea-pig with five minimum lethal doses of toxin, adding salt solution in
the smallest amount necessary. Allow to stand two hours, centrifuge and
inject the supernatant fluid as in pig No. I.

Pig No. 4. The other half of a guinea-pig brain is boiled for twenty
minutes in water, then ground up with five minimum lethal doses of toxin,
allowed to stand two hours, centrifuged, and the supernatant fluid injected
as in pig No. i.

Pig No. 5 serves as a control.

The guinea-pig injected with toxin will show typical symptoms as de-
scribed above, beginning with 'extension of the leg injected, then showing excita-
ble reflexes followed by convulsions, tetanic spasm and death. The antitoxin
and fresh brain substance will protect the animals, but the boiled brain will
not. The normal animal serves best as a control for the elicitation of
excitable reflexes and slight convulsions.

Route of Absorption of Toxin. — It is of interest to note that in
man, horse, and guinea-pig the central nervous system alone has
the power of neutralizing tetanus toxin, but in the case of the
rabbit, liver and spleen in addition have this power. It is main-
tained that the gray substance of the nervous system possesses
this special affinity, and the white matter does not. Most authorities
believe that the toxin is carried along nerve tracks, but Zupnik
maintains that it travels through the blood stream and is found not
only in the nervous system, but also in the muscles. Studies of
Meyer and Ransom and of Marie and Teale indicate that both
routes are followed. Depending on the size of the dose, the site of
inoculation, and perhaps certain other factors, one or the other
route may be followed predominantly, but never to the exclusion of
the other. According to Teale and Embleton, the mode of transit
along nerve trunks is by way of the axis cylinders and the peri-
neural lymphatic vessels. These authors, however, maintain that
toxin cannot pass from the choroidal plexis into the cerebrospinal
fluid, nor from the capillaries of the central nervous system to the
nerve tissues. The special affinity of tetanospasmin for nerve sub-
stance is not peculiar and is also exhibited by the neurotoxins of
snake venom and by the toxin of bacillus botulinus. Teale and
Embleton believe that tetanus antitoxin does not enter the sub-
stance of the central nervous system following either intravenous or
intrathecal injection, but simply acts by neutralization of the toxin
at the site of formation. Clinical experience is not entirely in
agreement with the experimental work of these authors, since cases
have been improved by the use of serum after tetanic spasms
have appeared.

Therapeutic Use of Tetanus Antitoxin. — As is well known, tetanus

TOXINS AND ANTITOXINS 59

follows the introduction of the bacilli or their spores into wounds in
such a fashion that anaerobic growth is permitted. The incubation
period is usually considered to be eight days, but there are many
variations from this standard period, including cases that have an
incubation period of over sixty days. The mortality from the dis-
ease is extremely high, the average ranging between 78 and 90 per
cent. Its incidence in civil life is not very great, but in time of war
it is likely to occur with considerable frequency because of the con-
tamination of war wounds by soil containing the organism or its
spores. In the American Civil War the disease occurred in 2.5 per
cent, of the wounded; in the Franco-Prussian War in 3.5 per cent.;
and in the World War 6.5 per cent. In the earlier wars the mortality
ranged between 80 and 90 per cent., but in the World War, owing
in all probability to prophylaxis and treatment, the mortality was
50 per cent. In carefully studied statistics it is found that the longer
the incubation period the lower is the rate of mortality. This general
statement held true before the use of anti-tetanic serum was instituted
and still holds true. The difference between the mortality rate of
80 to 90 per cent, in the earlier wars and 50 per cent, in the World
War gives an excellent illustration of the decrease in mortality that
has followed the introduction of serum prophylaxis and treatment
as well as rational surgery. Knowing that the organism is anaerobic
in growth, surgery demands that contaminated wounds be kept open
for the access of air.

Prophylactic Use of Serum. — The use of tetanus antitoxin is
directed toward prophylaxis and toward cure. As can readily be
understood from the experiments outlined above, the toxin of this
disease is very firmly bound to nerve tissues; therefore, treatment
established after the disease has appeared is not likely to be so
effective as in the use of some other antitoxins. Nevertheless, not-
able success has been attained in some cases where the disease has
become well advanced before serum treatment has been instituted.
Prophylactic treatment with serum is given as early after the
wound as possible, and in both military and civil life all wounds
contaminated with soil should receive protective doses of tetanus
antitoxin. This is given subcutaneously in doses of 500 to 1000
units. Wolff reported that in the German army prior to December,
1914, prophylactic injections were not regularly given and the inci-
dence of tetanus amounted to 1.4 per cent, of the wounded. During
the following seven months prophylactic injections were given in
the field to all those wounded by grenades and shrapnel, but not
those wounded by rifle bullets, and the incidence of the disease was
reduced to 0.16 per cent. Protection was equally as successful in
the Allied armies, and instructions were given to administer serum
as soon after injury as possible, either in the first-aid station or in
the field hospitals. Experiences in the British army demonstrated
that cases might develop a considerable time after the wound was
inflicted, and for this reason subsequent orders directed the use of

6o THE PRINCIPLES OF IMMUNOLOGY

500 units of tetanus antitoxin every ten days for four doses. Ic was
further recommended that the serum be given subcutaneously not
more than seven days and not less than two days before any oper-
ative procedure upon an old wound. If haste is necessary the serum
may be given intramuscularly twelve hours before operation. Ob-
viously this suggests the possibility that organisms may remain
dormant in wounds, to become active at a later period ; it is further be-
lieved that the antitoxin is probably eliminated in about ten days,
and the later doses of immune serum are given in order to neutral-
ize any toxin that might be produced subsequently.

Golla tabulated the following cases not receiving prophylactic
doses of antitoxin :

Incubation period Cases Mortality

i- 7 days 17 (32.7 per cent.) 82.5 per cent.

8-14 days 24 (46.2 per cent.) 79.0 per cent.

15-21 days 6 (11.5 per cent.) 54.0 per cent.

Over 21 days 5 ( 9.6 per cent.)

This shows that the commonest incubation period is eight to
fourteen days, and also illustrates the fact that the shorter the incu-
bation period the more serious is the disease. In another series of
patients who had received prophylactic treatment with serum, the
following data were collected ;

Incubation period Cases Mortality

i- 7 days 6l (22.6 per cent.) 75.5 per cent
8-14 days 93 (34.6 per cent.) 70.0 per cent

15-21 days 33 12.2 per cent.

21-30 days 19 7.05 per cent.

30-40 days 14 5.2 per cent.

40-50 days 9 3.3 per cent.

50-60 days 18 6.7 per cent

Over 60 days 22 ( 8.2 per cent.)

/ w.w J-/V-.1 WMt

60.8 per cent.

62.8 per cent.

57.0 per cent.

33.4 per cent.

27.7 per cent.

40.8 per cent.

In the Franco-Prussian War only 5.7 per cent, exhibited an in-
cubation period of more than twenty-one days, whereas, according
to Golla, in the last war 30.54 per cent, showed an incubation period
of more than twenty-one days. In summary it may be stated that
the introduction of prophylactic injections of the tetanus antitoxin
not only reduces the incidence of the disease, but also lengthens
the incubation period, and therefore reduces the mortality. The
delay in incubation usually leads to more moderate symptoms as
well as reduces mortality, and oftentimes the cases exhibit tetanic
spasms in only one extremity.

Treatment of Tetanus with Serum. — When the disease has de-
veloped, treatment must be prosecuted vigorously. In the earlier
years of its employment subcutaneous, intramuscular, and intra-
venous administration was practised, but arguing from the nature of
the disease it was soon suggested that intrathecal injections be given.
This suggestion was followed by experimental and clinical investi-
gation, and in the hands of the majority of workers the method has
been found to have great value. Park and Nicoll injected twice the

TOXINS AND ANTITOXINS 61

fatal dose of toxin into the hind legs of guinea-pigs and seventeen
to twenty-four hours subsequently injected antitoxin by various
routes. Six animals receiving the immune serum subcutaneously
died ; fifteen received it intracardially and two survived, whereas
sixteen animals received it intrathecally and thirteen survived. It
was found that the dose necessary for intrathecal injection was con-
siderably smaller than the dose necessary for injection into the cir-
culation. Sherrington conducted a similar series of experiments
upon monkeys with essentially the same results. He used twenty-
five monkeys for his series of injections and the following table
gives the results ;

Time between

Route of injection giving of toxin Recoveries Deaths

and antitoxin

Lumbar intrathecal 47-78 hours 14 n

Bulbar intrathecal 47-78 hours 13 12

Intravenous 47-7$ hours 7 18

Intramuscular 47~78 hours 3 22

Subcutaneous 47-78 hours 2 23

Cerebral subdural, ten cases 47~78 hours o 10

Clinically Irons was not able to demonstrate such a marked dif-
ference in results, and Leishman and Smallman came to the conclu-
sion that the intramuscular route is the best. The work of Andrew
and Golla demonstrated the clinical value of the intrathecal method.
Experimental work also shows that although antitoxin can take up
toxin after fixation with nerve tissue, such a release of toxin is re-
stricted by long contact with the nerve tissue. This explains the
necessity for early administration of antitoxin. For example, the
experiments of Doenitz show that the amount of antitoxin neces-
sary for protection increases tremendously with a lapse of time.
He injected twelve times the fatal doses of toxin and found that
after the lapse of four minutes a slight excess of antitoxin was suffi-
cient to protect the animal, but after eight minutes six times this
dose of antitoxin was required ; after sixteen minutes twelve times
the dose, after 1 hour twenty-four times the dose; in four to six
hours six hundred times the original dose, and after six hours he
was unable to save the animals. As a result of long experience with
treatment it has finally been determined that a combination of
modes of injection is desirable in order to procure complete and
lasting saturation of the body with antitoxin. When giving intra-
thecal injections it is well to draw off the spinal fluid and then
immediately inject 3000 to 5000 units of toxin, diluting the serum
to a volume of 10 to 15 c.c. with sterile salt solution. Where no
fluid can be withdrawn from the spinal canal the antitoxin is intro-
duced very slowly by gravity. The intrathecal injection is further
supplemented by 10,000 to 15,000 units intravenously, and three to
four days later a similar injection subcutaneously. It is often ad-
visable to repeat the intrathecal injections each day for three or
four days. The following outline taken from the Memorandum

62 THE PRINCIPLES OF IMMUNOLOGY

on Tetanus published by the British War Office gives a plan for
combined injections in a case of acute tetanus;

Day Subcutaneous Intramuscular Intrathecal

First 8,000 units 16,000 units

Second 8,000 units 16,000 units

Third 4,000 units 8,000 units

Fourth 4,000 units 8,000 units

Fifth 2,000 units

Seventh 2,000 units

Ninth 2,000 units

This outline is offered as a suggestion for treatment and has
been applied successfully. The doses are arranged in multiples of
8000 because that was the size phial issued in the British army.
Doses may be varied, but it is strongly advised to administer a
total of 75,000 to 100,000 units. In those cases with long incuba-
tion period the dose may be smaller, and if the case is one of spasm
in one extremity, without evidence of involvement of higher centers,
such as spasm of jaw muscles (trismus), the serum may be given
by intramuscular and subcutaneous routes in amounts of 3000 to
6000 units. The patient should be placed so that he lies with the
feet considerably higher than the head, so as to allow drainage to-
ward the head. It has also been suggested that the antitoxin be
applied near or in the wound. Calmette recommended that pow-
dered antitoxic serum be applied locally. Suter recommended rub-
bing the fluid serum into the wound. Bockenheimer recommended
that it be introduced in the form of ointment, and Robertson satur-
ated pads of cotton with antitoxin, dried these for twenty-four hours
at 40° to 45° C, and applied them locally. As will be seen, these
latter measures are probably more in the nature of prophylaxis than
treatment, and no definite information has accrued as to their value.
The disadvantages of serum treatment are essentially the same as
those in the use of diphtheria antitoxin, but in addition we have to
deal with the factor of introduction of foreign serum into the spinal
canal. This frequently leads to the development of a sterile menin-
gitis with a formation of purulent fluid. As far as can be learned,
this inflammation does no damage. A few reports of nerve and
cord lesions following the use of antitetanus serum intrathecally
have been reported, but they are extremely small in number com-
pared to the number of cases treated, and it would appear that the
high percentage of mortality in this disease justifies the intrathecal
treatment in spite of the minor element of danger.

Dysentery Toxin and Antitoxin. — Dysentery toxin may be pro-
duced in broth by the growth of the Shiga bacillus. It is probable
that the Flexner and Hiss-Russell types produce only an endotoxin.
This is consistent with the greater clinical and pathological severity
of the Shiga type of dysentery. The broth must be definitely alka-
line, the optimum stated by Doerr being reached where 0.3 per cent.
soda is added to a broth neutral to litmus. Rabbits are very sus-
ceptible and the intravenous injection of a filtered toxin broth in

TOXINS AND ANTITOXINS 63

proper doses will produce marked, often bloody, diarrhea, wasting,
paresis, or even paralysis of extremities, and death. The autopsy
shows marked inflammation, often hemorrhagic, particularly severe
in the cecum, but also involving the large intestine and lower
ileum. Monkeys, cats, and dogs are also susceptible, but fowl, pigeons,
and guinea-pigs are resistant. Antitoxin can be produced in horses
and goats. There is considerable difficulty in standardizing such a
serum, owing to the variation in individual susceptibility of ani-
mals. Kraus and Doerr have shown that the immune serum first
shows a capacity to neutralize toxin in vitro, then in vivo (simul-
taneous injection of toxin and antitoxin into opposite ear veins),
and finally it attains a definite curative value as demonstrated by
primary injections of toxin followed after certain time intervals by
antitoxin. A serum must have a high curative value before it is
acceptable and is used in doses of cubic centimeters rather
than units.

Therapeutic Use of Anti-dysentery Sera. — After the discovery of
the dysentery bacillus by Shiga in 1898 it was found that the sepa-
rate types of this organism vary greatly in their power to produce
toxic substances. The most toxic varieties are those of Shiga and
Kruse, and their toxins are not only endotoxic but also exotoxic in
nature, a fact clearly established by the work of Todd, Liidke,
Kraus, Doerr, and Rosenthal. Shiga was the first to immunize
horses with killed cultures of his organism and produced highly
protective sera capable of saving guinea-pigs injected with six
times the lethal dose of living bacilli. This specific anti-bacterial
serum was used by Shiga with encouraging results during a dysen-
tery epidemic in Japan, the mortality among cases treated with
Shiga's serum being one-third of that among cases treated by the
usual routine procedures. Not only was the mortality greatly re-
duced, but the total period of illness decreased from forty to twenty-
five days. A similar serum was prepared by Kruse and its use
reduced the mortality among Kruse's cases from n to 5 per cent.
Kraus and Doerr also obtained favorable results from the use of
their serum, which was mainly an antitoxic serum produced by the
injection of filtrates of young cultures into horses. Vaillard and
Dopter treated a large number of cases with a serum prepared by
themselves and possessing both antibacterial and antitoxic prop-
erties and reported highly encouraging results with a mortality of
2 per cent., while the mortality otherwise would have been between
ii and 25 per cent. More prompt effects were obtained when the
serum was given at the earliest moment in the course of the disease.
Vaillard and Dopter used 20 to 30 c.c. in moderate cases and from
40 to 80 c.c. in grave cases. In late cases serum injections were
often of value. Graham more recently has added a valuable contri-
bution to the serum therapy in bacillary dysentery, his studies being
made during the campaign in Macedonia. Graham used a serum
prepared at the Lister Institute and gave intravenous injections of

64 THE PRINCIPLES OF IMMUNOLOGY

60 to 80 c.c. once or twice daily during the first three days of treat-
ment. Three injections were followed by 150 to 300 c.c. of saline
daily for the first two days and once for the next two days, the
saline injections being made to prevent dehydration of the tissues.
In mild cases no saline injections were necessary. Most of the cases
arrived after the third day, so that they were not placed under
treatment at the earliest possible moment. On entering the hos-
pital all cases received 20 c.c. of the serum subcutaneously. Klein
also maintains that anti-dysenteric serum given early and in large
doses intravenously (60 to 100 c.c.) is efficacious. He found that the
use of the serum produced the best results when given during the
first five or six days. When the disease has entered into the inter-
mediate stage, from the sixth to the tenth day, the outcome of the
disease is irrespective of serum treatment. In the third stage —
tenth day — the use of serum is practically without value. Waller
treated 140 cases with the Lister Institute serum and found that
the early use of the serum resulted in shortening the duration of the
disease. He gave three subcutaneous injections of 140 c.c. at eight-
hour intervals during the first twenty-four hours to fairly severe
cases. Rosenthal, in a series of serum-treated cases, found a mor-
tality of 0.6 per cent. In other units the mortality was 10 to 15 per
cent. Sixty c.c. of sera were given by Rosenthal on the first day,
followed by 40 to 60 c.c. on the second, and if no improvement was
observed subsequent doses of 40 c.c. were given up to a
total of 400 c.c. Usually the stools were free of blood in forty-eight
hours, and their number reduced from 60 to 15 or 10 per day.
Lantin also thinks that the use of serum constitutes an efficient
specific method of treatment. He gave the serum by rectum in doses
from 30 to 50 c.c. Neumann used human convalescent serum in 400
cases. Intestinal irrigations with silver solutions were also em-
ployed by this author. Only six of his cases ended fatally. Jacob,
on the other hand, and with him also Nolf, failed to obtain success
with serum therapy in this disease. Jacob treated ninety cases,
using polyvalent sera and injecting subcutaneously or intravenously
doses ranging from 20 to 400 c.c. during the first or second week of
the disease. According to the British Medical Research Com-
mittee, serum treatment of bacillary dysentery is not satisfactory.
Nevertheless, numerous investigators showed that this method of
treatment has a well-established clinical value as expressed in the
view of Schittenhelm, who states that it should be employed in all
cases of more than three or four days* duration, and in all cases
showing toxemia and severe symptoms, and in cases where the
number of stools are more than twelve in the course of twenty-four
hours. It should be given early in the disease and in massive doses.
If possible the type of the infecting organism should be known prior
to the administration of these massive doses. This can readily be
done in twenty-four hours in a well-equipped laboratory. The
serum used should be polyvalent, because there are a number of

TOXINS AND ANTITOXINS 65

serologically distinct types of dysentery bacilli. Schmitz, for in-
stance, found in a dysentery outbreak among prisoners of war in
Roumania strains which resembled the Shiga bacillus but were
serologically entirely distinct types. Pribram also found that an
antitoxin specific for the Shiga-Kruse toxin is inactive toward the
toxin of a strain D118H (Hallmann). Furthermore, the curative
action of anti-dysentery serum is due first to its content in antitoxin,
and second to its anti-bacterial properties.

The serum can further be employed for prophylactic injections in
doses from 10 to 30 c.c., but the immunity thus produced will be only
of a short duration. Recently Boehneke and Elkeles have inocu-
lated over 100,000 persons with a polyvalent bacillary toxin-anti-
toxin preparation called dys-bakta, but complete protection was not
secured. It was noted, however, that infections occurring in the
inoculated individuals were usually of slight severity, and death a
very unusual occurrence. The reaction following inoculations was
no more severe than that following typhoid inoculations. Immunity
thus produced lasted for at least three months.

Botulinus Toxin and Antitoxin. — Bacillus botulinus produces a
toxic body leading to symptoms often called " ptomaine poisoning."
The toxin, however, is apparently independent of the medium used,
is destroyed by moist heat of 58° C. for three hours, and of 80° C.
for one-half hour, and is capable of inducing the formation of an
antitoxin. The symptoms produced by the toxin are marked in-
crease or decrease of saliva flow, vomiting, sometimes diarrhea,
but more often constipation, often retention of urine, paralysis of
eye muscles, aphoria, rarely fever or disturbance of sensitivity.
Death frequently ensues following the appearance of symptoms of
bulbar paralysis with disturbances of respiration and heart action.
The necropsy shows marked general passive congestion and throm-
bosis of the meningeal vessels sometimes with slight hemorrhage.
Unlike other toxins, that of botulism resists the digestive juices and
is absorbed by way of the alimentary canal. It can be neutralized
by brain substance and by the lipoids, lecithin, cholesterol, and by
fats, such as butter and oil. It is toxic for man, monkey, cat, rabbit,
and guinea-pig.

The Use of Immune Sera in Botulism. — Van Ermengem in 1895
discovered the cause of botulism poisoning to be an exotoxin pro-
duced by a strictly anaerobic Gram-positive bacillus which he iso-
lated from portions of a ham that had caused fifty cases of poisoning
at Ellezelles, Belgium. The disease has an exceptionally high mor-
tality of almost loo per cent., and up to the present time the per-
centage of fatal cases has been as great as it was fifty years
ago. The reason for this lies in the fact that the early symptoms of
the disease are not recognized until the toxemia is well established.
In the year 1897 Kempner showed that susceptible animals may be
successfully immunized to the toxins of this organism and obtained
a potent antitoxin from goats, I c.c. of the serum protecting against
5

66 THE PRINCIPLES OF IMMUNOLOGY

100,000 minimal lethal doses. Forssman and Lundstrom were also
successful in their immunization attempts, using attenuated toxins.
Wassermann immunized horses and produced sera of undeniable
value in animal experiments. In this country sera were prepared
by Graham, Brueckner and Pontius, Buckley, Hart, Meyer, Hurwitz
and Taussig, Burke, Dickson, and Howitt mainly for experimental
purposes, using rabbits, sheep, goat, cattle, and dogs for immuniza-
tion. According to Dickson and Howitt, laboratory experiments
show that the antitoxin may protect against the action of the toxin
for at least twenty-four hours after the administration of one test
dose of toxin, but the effectiveness is, to a certain extent at least,
dependent upon the amount of toxin injected. Like tetanus anti-
toxin, botulinus antitoxin should be given early if it is to be effec-
tive, and even in well-established cases it is strongly advisable to
give antitoxin in massive doses, because Kob has demonstrated that
this toxin may persist in the blood nine days after the poisoning.
If symptoms of botulism, such as hypersecretion of mucus from
mouth and nose, visual disturbances, aphonia, dysphagia, and
paralysis of the intestinal tract appear, antitoxin should be admin-
istered as soon as possible, and should be given in large doses
intravenously. Dickson also advises the use of antitoxin to all
persons who have eaten fowl that have suffered from limberneck.
Of importance is the use of polyvalent sera because of the discovery
of Leuchs that two strains, the one of Van Ermengem and a
Darmstadt strain, were distinct, that the toxin of one was not
affected by the specific antitoxin of the other, and vice versa. As
for the effect of botulinus antitoxin in man, little is known, as it
has been used only in isolated instances. Dickson and Howitt, in
1918, gave 85 c.c. of immune goat serum (i c.c. equivalent to 3000
M.L.D. for a guinea-pig) to each of two patients at Madera, Cali-
fornia. Both patients recovered, but as the antitoxin was given very
late, in fact, after all the more seriously poisoned patients had
succumbed, there is no definite evidence that the course of the ill-
ness was favorably influenced by the antitoxin, although it was
later shown that the toxin of the strain recovered from the food was
Type A. McCaskey used small doses of antitoxin in three patients
(5 to 10 c.c.). One died and two recovered and this author there-
fore thinks the serum to be of some aid. Nonnenbruch obtained
rapid improvement in his case after the use of antitoxin. His pa-
tient became poisoned after eating sausage. Jennings, Haas and
Jennings in the recent Detroit outbreak used Graham's serum in a
dose of 42 c.c. intravenously in one case without apparent effect, and
20 c.c. in two injections to another patient, who recovered, and state
that the latter case was not of mild type. Dickson and Howitt
found that of all the outbreaks in which the serum had been used,
with the exception of the cases of McCaskey, the toxin was that of
Type A, and consequently when Type B serum was used it could
not be expected to give any satisfactory results. As it is impos-

TOXINS AND ANTITOXINS 67

sible to determine quickly the type of toxin in a particular outbreak,
it is of the greatest importance to use polyvalent sera.

Gas Bacillus Toxin. — The frequent occurrence of gas gangrene
in the Great War has given especial interest to the preparation of
antitoxins for the organisms causing the disease. Klose, in 1916,
and Bull and Pritchett, in 1917, were able to prepare a soluble toxin
of the bacillus Welchii or as it is often named bacillus perfringens.
Bull and Pritchett drew especial attention to the necessity for select-
ing a strain which is capable of producing toxin in fairly large
amounts. The British Medical Research Committee reports that
the toxin of vibrion septique has very little effect following
subcutaneous injection. Upon intravenous injection, however, it
produces convulsions and usually death in a few minutes. An
antitoxin may be produced, but it is not effective after the toxin has
been injected. The toxin of bacillus edematiens produces massive
edema about the site of inoculation. The toxin of bacillus aerogenes
capsulatus was found to have a necrotic action upon the tissues ; it is
generally toxic in large doses and animals may be protected by
antitoxic serum.

The Use of Immune Sera in Gas Gangrene — Treatment of the
Disease. — Leclainche and Vallee, Sacquepee, Weinberg and Seguin,
Bull and Pritchett were the first to apply serum therapy in wounds
infected with the gas bacilli. Leclainche and Vallee's and Weinberg
and Seguin's serums were polyvalent and also antibacterial, while
Bull and Pritchett's serum was antitoxic. In 1917 Bull and
Pritchett produced an exotoxin from twenty-four-hour cultures of
bacillus aerogenes capsulatus, which when injected into pigeons
or guinea-pigs caused local edema, necrosis, and hemolysis of red
cells, and was capable of stimulating the formation of an antitoxin.
Bull's claim for the potency of his antitoxic serum was based on ex-
periments in which he used pure cultures of bacillus aerogenes cap-
sulatus and made no attempt to discriminate between the different
types of the organism, such as have been found to exist by Henry,
or to consider the fact that in war wounds the bacillus aerogenes
capsulatus is not the only causal factor of gas gangrene. From
Nevin's work it would appear that neither anti-perfringens serum
(bacillus aerogenes capsulatus anti-microbial serum) nor Bull's anti-
toxin afford any protection when other pathogenic anaerobes inci-
dent to war wounds are present, together with bacillus aerogenes
capsulatus, whereas when the vibrion septique and bacillus
edematiens are present in mixed infections without bacillus aero-
genes capsulatus, the prophylactic use of the specific sera, even
when diluted by another serum, is effective. Weinberg and Seguin,
who have contributed extensively to the serum therapy of gas
gangrene, found treatment by serum alone limited because of rapid
absorption of toxin in this disease. The association of rational
surgery and of serum therapy gives the best results. In a series of
sixty-six cases reported by these authors in which sixty did not

68 THE PRINCIPLES OF IMMUNOLOGY

receive serum treatment, three received non-specific treatment and
three suffered complications, thirty-five deaths were recorded, while
in a series of twenty-four specifically treated cases only five deaths
occurred, thus reducing the mortality from more than 55 per cent,
to less than 21 per cent. The serum used in these cases was poly-
valent, produced against bacillus aerogenes capsulatus, the vibrion
septique, and bacillus edematiens. Duval and Vaucher, in 1917,
reported fifty cases in which a combination anti-perfringens, anti-
edematiens, and anti-vibrion septique serum prepared by Weinberg
and Seguin was injected prophylactically. In none of these patients
did gas gangrene develop, although all were of the most severely
wounded type. Twenty-five died as a result of severe multiple
wounds without any signs or symptoms of gas gangrene.

Prophylactic Use of Sera. — A year later these same authors re-
ported a series of 281 cases in which severely wounded patients
were injected with polyvalent serum prepared at the Pasteur Insti-
tute. Eighteen developed gas gangrene (6.4 per cent.), and of these
ten died, resulting in a mortality of 3.5 per cent., the usual mortality
from gas gangrene in severely wounded being 16 per cent. Mairesse
and Regnier found among 1016 wounded men examined bacteriologi-
cally 297 gas bacillus infections. They received prophylactic injec-
tions of anti-serum depending on the type of organism present. In
thirty instances, or 10 per cent, of the cases, the disease developed.
Ivens also used Weinberg and Seguin's serum in 222 cases for
prophylactic injections. Among these no deaths occurred, and
fourteen amputations were performed without fatal results. With
Leclainche and Vallee's serum (154 cases) four died, and in fifty-
seven other cases treated with both sera two deaths occurred.
Further favorable reports were made by Quenu, Bazy and Routier,
Vincent and Stodel, Marquis, Dufour and Samelaigne. Curative
injections were given by Duval and Vaucher with 20.7 mortality.
Rouvillois, Guillaume, Louis, Pedeprade, and Thibierge treated
twenty-five cases, five of whom died. Of these three were moribund
on entrance to the hospital. Mairesse and Regnier's thirty treated
cases had a mortality of 16.6 per cent.

Van Beuren, who reports a personal communication from Lieut.-
Col. W. Elser, states that prophylactic doses were given to 15,000
soldiers and controlled by 15,000 others. According to this finding
there was not sufficient difference in the incidence rate to warrant
any definite declaration as to the protective value of the sera used.
Apparently these investigators were favorably impressed; for they
laid the failure to secure better results to the weakness of the
serum then available. Elser advises the following routine for the
serum treatment :

1. A prophylactic dose of polyvalent serum, combined with
tetanus antitoxin, given as early as possible after the receipt of
the wound.

2. Bacteriologic examination of the wound and establishment of

TOXINS AND ANTITOXINS 69

the presence of gas bacillus infection and determination of the
variety of the bacteria.

3. Administration of specific serum, either single or polyvalent
or " pooled," according as there are one or more gas formers found,
and also the administration of anti-streptococcus serum, since the
latter organism is very commonly found in association with the
other organisms.

From the general reports obtained during the Great War it is
considered that intravenous injection is to be preferred, in combina-
tion with deep muscular injections in the vicinity of the wound.
From these reports it seems, then, that the use of a polyvalent anti-
bacterial and antitoxic serum is advisable, but much work on the
subject must yet be done. From all the observations at hand it is
safe to state that the best results are to be obtained from
preventive injections.

Bacterial Hemotoxins. — As an example of the hemotoxins pro-
duced by bacteria certain details of staphylolysin may be consid-
ered. The hemotoxin is produced by twelve to thirteen days'
growth of staphylococcus pyogenes aureus or albus in broth. The
organisms are killed and the broth filtered through a porcelain
filter. The filtrate can be preserved by the addition of 5 per cent, of
a solution made up of 10 parts phenol, 20 parts glycerol, and 70 parts
water. Doses of 0.025 to 0.05 c.c. should completely hemolyze one
drop of rabbit blood after two hours at 37° C. Antilysin may be
produced by immunization of animals and is found normally to a
slight extent in normal human blood and in that of certain lower
animals. The victims of staphylococcus infections frequently show
an increased antilysin content of the serum. This fact has been em-
ployed by Bruck, Michaelis, and Schultze to diagnose staphylococcus
infections, some cases showing increases of ten to one hundred times
over the normal antilysin. The simplicity of bacteriological exami-
nation, however, makes this method of diagnosis by comparison
rather cumbersome and time consuming. Whether or not antilytic
sera would be of value in the treatment of those cases that resist or
are unsuitable for vaccine treatment has not been determined so far
as we have been able to learn.

PHYTOTOXINS

Introduction. — Although literally the phytotoxins include all the
toxins of vegetable origin the term usually is restricted to include
those originating in forms of vegetable life higher than the bacteria.
With this definition thought would be first directed to the poison-
ous fungi, but as has already been shown, only one of the poisons so
far isolated is capable of inducing antibody formation. The poison-
ous elements of poison ivy and poison oak produce no antibodies.
The poisonous elements of those plants that produce " hay fever "
require separate discussion, because the toxic factor operates only
on individuals who show a peculiar susceptibility or " hypersus-

70 THE PRINCIPLES OF IMMUNOLOGY

ceptibility." The element of hypersusceptibility in this connection
will be deferred until after the presentation of the fundamental
material on anaphylaxis and hypersusceptibility. The following
paragraphs will present briefly the essentials concerning ricin, abrin,
robin, crotin, curcin, and phasin. This brevity is justified by the rela-
tively small practical importance of these substances.

Ricin is the toxic principle of the castor-oil bean, ricinus com-
munis. It was isolated by Gibson in 1887 and named ricin by
Stillmark in 1888. Gushing made very strong toxic preparations and
Field states that ricin will kill rabbits in doses of o.oooi mg. per
kilo ; guinea-pigs, 0.0008 mg. ; dogs, 0.0006 mg. ; cats, 0.0002 mg. ; and
goats, 0.003 mg. Following injection there is an incubation period
succeeded by diarrhea, somnolence, weakness of extremities, and
death. At the necropsy are found reddening and swelling of Peyer's
patches, mesenteric and retinal hemorrhages, ulcers of stomach,
nephritis, general lymphatic swelling, and softening and degenera-
tion of the pyramidal cells of the cerebral cortex. Beauvisage re-
ported 150 cases of ricin poisoning in man of which nine were fatal.
Many of these were children who ate the seeds, but there were also
soap makers who handled the beans in soap manufactories. Ricin
and the other toxins in the group may be precipitated with the pro-
teins by ammonium sulphate ; they are precipitated by alcohol and are
gradually destroyed by proteolytic enzymes. Jacoby, however,
claims to have produced ricin and abrin which failed to give pro-
tein reactions. Osborne, Mendel, and Harris maintain that ricin is
inseparably associated with protein, and that Jacoby's error was
due in all probability to the fact that he obtained a product so toxic
that the small amounts necessary for toxic action were too small to
give the protein reactions. The most striking character of ricin in
vitro is its capacity to agglutinate the red blood-corpuscles of prac-
tically all warm-blooded animals. It may agglutinate other body
cells, precipitates protein, and is adsorbed by casein, fibrin, coagu-
lated serum albumin, and by silk. Jacoby concludes that ricin is a
mixture of agglutinin and toxin, the two having certain molecular
groups in common. Ehrlich believes that these may undergo altera-
tion into agglutinoid and toxoid. The mechanism of the agglutina-
tion is not clear and many hypotheses, none quite satisfactory, have
been advanced. Ehrlich produced an antiricin by giving increasing
doses to animals by mouth, and then changing to subcutaneous in-
jections. This antiricin was used by Ehrlich in the development of
much of his hypothesis of the toxin antitoxin union because of the
ease of manipulation as compared with the time-consuming and
expensive method of working with animal injections of toxin anti-
toxin mixtures. In addition to the antitoxin there are present in the
serum a closely related antiagglutinin (with which Ehrlich worked)
and a precipitin for ricin solutions.

Abrin is obtained from paternoster or jequirity bean, abrus pre-
catorius, and was described by Warden and Waddell in 1884. It is

TOXINS AND ANTITOXINS 71

much less toxic than ricin, producing gastro-enteritis, hemorrhages,
and swelling of lymph-nodes. Local applications led to an acute
conjunctivitis and in hairy regions to transitory loss of hair, both
of which may be protected against by immunization. Robert states
that in India and Ceylon cattle were immunized (by feeding beans)
against the effects of wounds by abrin-coated projectiles. Roemer
found that by repeated application to the conjunctival sac of one
eye, he could produce an immunity which first protected that eye
and, after further immunization, served to protect the opposite
untreated eye, in this later stage becoming a general immunity with
antiabrin in the serum. Abrin is also a hemagglutinating agent,
and can be distinguished from ricin by immunological experiment.
In many respects abrin and its immunity resemble ricin very closely.

Crotin is derived from croton seed, croton tiglium, and is less
toxic than either ricin or abrin. According to Elfstrand, it agglu-
tinates the red blood-corpuscles of beef, sheep, swine, and frog; it
hemolyzes the cells of rabbit, cat, and crow, and has no effect on the
erythrocytes of man, dog, guinea-pig, rat, hen, goose, and pigeon.
Immune sera can be produced by the usual methods. Jacoby found
in Grubler's pepsin a body which he called pseudo-anticrotin, cap-
able of neutralizing the action of crotin on erythrocytes in vitro but
not in vivo, and he found the same substance in gastric and
intestinal mucosa.

Curcin is produced from the seeds of jatropha curcus, and robin
from the leaves and bark of robinia pseudacacia. Immune sera can
be produced against both of these.

Phasin is a name given by Landsteiner and Raubitschek to a
hemagglutinating substance found in the seeds of the bean, pea,
lentil, and vetsch. Antiagglutinins are found in normal serum and
may be increased experimentally, but this substance or group of
substances can hardly be regarded as belonging to the class of
toxins because of little or no toxic symptoms following injection.

Pollen Proteins or Pollen Toxin. — The modern studies of hay
fever and of asthma place this subject so clearly in the group of
anaphylactic phenomena that its consideration is deferred (see
page 233).

ZOOTOXINS

Introduction. — The zootoxins include the poisonous elements
produced in animal life. They may be, and most frequently are, in
the form of excretions of special poison glands or are found in secre-
tions of other glands, in blood and in tissues. The most important
are the snake poisons, but there are also included the poisons of
spiders, scorpions, bees, centiped.es, tarantula, toads, poisonous fish,
duck-bill platypus, and the sera of various animals.

The snake venoms differ somewhat in their action according to
family, the colubridae, including the cobra, Australian black snake,
and others ; the viperidae, including the European viper and Ameri-

72 THE PRINCIPLES OF IMMUNOLOGY

can rattlesnake ; and the hydrophinse or poisonous sea snakes. The
venoms secreted by special glands are injected during the bite
through fine canals in the fangs (not the forked tongue), and are
all hemolytic. The fact that the blood of snakes contains poisons
similar to those of the venom indicates that the poison glands
secrete with little alteration the poison of the blood. Never-
theless, snake bites may be poisonous for snakes of other species,
and also for other members of the same species. Geoffroi
and Hunauld, in 1737, and Fontana, in 1781, noted the anticoagu-
lant action of venom, but the work of Weir Mitchell in 1860,
and of Weir Mitchell and E. T. Reichert in 1886, served as the great-
est stimulus to modern investigation. Mitchell and Reichert
showed that the venom of the rattlesnake produces rapid coagula-
tion of the blood and death, but that if the animal survives the blood
is reduced in coagulability. C. J. Martin confirmed this in regard to
Australian snakes and showed that the phenomenon could be con-
trolled by dosage of the venom. In addition to hemolysis and
alteration of coagulation, other properties are present, and Flexner
and Noguchi showed in venom the presence of hemotoxins, in-
cluding hemolysins and hemagglutinins, leucocytolysins, and an
endotheliotoxin which they named hemorrhagin. Pearce showed
that hemorrhagin produced lysis of endothelium leading to hemor-
rhage. In addition, venoms contain proteolytic enzymes, invertase,
lipase, and probably certain ferments dealing with coagulation.
Martin found fibrin ferments which probably aid in thrombus for-
mation. Lamb found that even citrated blood could be clotted by
venoms. Negrete found the anti-coagulating element closely asso-
ciated with the proteins of the venom. Morowitz claims the pres-
ence of an antithrombokinase. Modern studies by Houssay
Sordelli and Negrete with the venoms of fourteen snakes, Indian,
American, and Australian, show that clotting time does not parallel
closely the dose of venom, that venoms clot whole blood, plasma,
and fibrinogen solutions, and that mammalian blood is more sus-
ceptible than that of birds, batrachians, and snakes. The addition
of citrate, oxalate, magnesium sulphate, hirudin, and peptone delay
the action of the venom, the oxalate acting the most intensely. It
seems likely that large doses of venom bring to bear a sufficient
amount of fibrin ferment to produce clotting and that the later
effects are due to the anti-coagulating power of the venom after the
fibrin ferment is exhausted. Inasmuch as the hemolysis of venom
is somewhat closely related in mechanism to hemolysis in general,
it will be discussed in the chapter on Hemolysis (see page 141).
Venom toxins resemble other toxins in that they are precipitated
with proteoses, whilst the factor which produces local irritation
comes down with globulin, although Faust maintains that the active
principles of venoms are glucosides. Venom toxins are destroyed
by heat, the cobra poisons as a class by 100° C., and the viper
poisons by 85° C. They do not dialyze and deteriorate under the

TOXINS AND ANTITOXINS

73

influence of light, radium, and oxiding agents. There is an incuba-
tion period and the venoms are definitely and specifically antigenic.

Venoms act in extremely small amounts. The fatal dose of
cobra venom for man is probably o.oi to 0.03 gm., rattlesnake venom
0.15 to 0.3 gm., and poisonous sea snakes o.ooi to 0.003 Sm-> or ten
times as toxic as cobra venom. The bite of the cobras produces
little pain and local reaction, probably due to its small content (2
per cent.) of globulin, which contains the local irritant property of
the venom. A feeling of stiffness spreads from the region of the
wound, followed by vertigo and weakness of muscles of locomotion,
tongue, jaw, esophagus, and preservation of senses, resembling a
very acute bulbar palsy with death in a few hours. Gushing, how-
ever, finds that the action of the poison is particularly upon motor
nerve termini. The venom of the vipers produces a marked local reac-
tion, probably due to its large (25 per cent.) globulin content, with
pain, swelling, local bleeding, blood in the serous membranes and
hematuria. Nausea and vomiting, excited reflexes, and even con-
vulsions are followed by prostration, paraplegia of lower extremities
which extends upward and resembles an acute ascending spinal
paralysis with death in one to three days. Langmann states that,
" if the patient recovers from the paralysis, a septic fever may de-
velop ; not rarely there remain suppurating gangrenous wounds
which heal poorly." The suppuration of snake bites (viperidae)
has been the subject of considerable study; Welch and Ewing
ascribed this to loss of bactericidal property of the blood after venom
poisoning. Flexner and Noguchi demonstrated a loss of the com-
plement of the blood, an element necessary to its full bactericidal
power. They believed that the complement was used up by the
venom whose amboceptors require complement for their action,
therefore leaving little or none free for the bactericidal amboceptors.
Morgenroth and Kaya claim that the complement is actually de-
stroyed by the venom. Of considerable importance in favoring infec-
tions must be the local necrosis of tissue caused by the venom and the
associated hemorrhage, aided by the customary radical surgery of
the wound.

The production of antisera was placed on a practical basis by
Calmette in 1894 and Frazer in 1895. Calmette attenuated cobra
venom for the first four injections by the addition of equal volumes
of i per cent, gold chloride solution, and then gave small doses of
the native toxin, gradually increasing until a powerful antivenin
was developed. Phisalix and Bertrand attenuate viper venom by
heating the first dose to 75° C. and then after two days giving one-
half the minimum lethal dose of toxin. It was at first thought that
the antivenin produced by cobra venom would protect against all
venoms, but it was soon shown that the sera were specific for the
venom employed. Antivenin also neutralizes that element of venom
which induces loss of bactericidal power of the blood. Noguchi has
shown that the antivenin of rattlesnake venom neutralizes the

74 THE PRINCIPLES OF IMMUNOLOGY

hemorrhagin. Such sera also contain precipitins for the proteins of
the special venoms employed and for the serum proteins of the
same species of snake. These are highly but not absolutely specific.
The mechanism of venom-antivenin union is probably very closely
similar to that of toxin-antitoxin unions of other varieties, although
Kyes holds that the former is distinctly in the nature of the chemi-
cal reaction between a strong acid and a strong base.

Scorpion poison is secreted by special glands in the abdomen. In
human adults the symptoms are rarely severe, except for marked
local reaction, but it is stated that the bite of an African scorpion
may kill children. As a rule, the most serious effects are from the
subsequent infection of the wounds. Todd was able to prepare a
specific immune serum for the poison of scorpions. According to
Houssay, scorpion venom acts pharmacologically much as veratrin ;
it is a smooth muscle stimulant. He states that serum therapy is
useful and specific.

Spider poison is secreted by glands in the thorax. The common
spiders are not venomous, except the " cross spider " whose venom,
much weaker in the saliva than in the ovaries, closely resembles
snake venoms in chemical properties and agglutinin, and probably
contains a neurotoxin. Sachs prepared an antivenin against this
venom. Some of the larger spiders are extremely poisonous, par-
ticularly the Malmigatte of southern Russia and related species in
South America and Africa. Large numbers of cattle have been
poisoned with as high as 12 per cent, mortality, but the bite is
rarely fatal for man.

The tarantula produces a poison which operates almost entirely
locally, and it is stated that an antitoxin can be produced against the
Russian tarantula.

Centipedes. — Certain centipedes secrete a poison in special glands
that discharge through the claws, capable of producing considerable
local reaction. But one case of fatal poisoning has been reported
from Texas, that of a child four years old.

Bees, wasps, and hornets secrete a poison closely similar for all
three. Bee poison contains formic acid and in addition a poison
which does not give the usual protein reactions, but is destroyed
by proteolytic enzymes ; it resists heat to 100° C., weak acids, and
alkalies. The poison contains a hemolysin which operates in much
the same manner as does cobra hemolysin. The bite produces
marked local reaction, but only in cases of extreme hypersuscep-
tibility are there general effects or death. Part of the lack of
severity of bee poison is due to the small dose injected, for if col-
lected in large amounts and injected intravenously into dogs it can
produce death. The resistance of professional bee keepers to the
bites is probably due to the fact that repeated bites lead to de-
velopment of immunity, although it is possible that the doctrine of
the survival of the fittest may play its part. Ants probably pro-

TOXINS AND ANTITOXINS 75

duce a somewhat similar poison in addition to formic acid. The
" black flies " of the woods produce a poison not as yet identified,
but no poison has as yet been isolated from the mosquito. Numer-
ous other insects appear to have poisonous secretions, but as yet no
studies have been made in detail as to their isolation and identification.

Toads, frogs, and salamanders produce dermal secretions which
are poisonous, several of which operate like digitalis and some like
epinephrin. These poisons are interesting from a pharmacological
point of view, but as they are not capable of producing immune re-
actions in animals they deserve no extensive discussion here.

Poisonous fish comprise several groups. One group secrete
poisons in special glands, the poison being discharged through
spines. Such poisons contain a hemolysin which requires an
activator, as in the case of cobra- venom hemolysins. These poisons
act as powerful local irritants and as cardiac depressants and may
cause death. Only one variety of fish produces poisoning by its
bite, the poison being secreted in the gums. Other fish are poison-
ous when eaten even when quite fresh, the poison being found
especially in the ova and ovaries. The symptoms may be of a
severe choleriform type frequently fatal, or of a less severe gastro-
intestinal type, not commonly leading to death. Certain fish, par-
ticularly in the tropics, rapidly decompose with the formation of
poisonous products or ptomains. The bites of crabs may produce
peculiar erysipelas-like lesions or " erysipeloid," but the origin and
nature of the poison are not known. Many individuals develop
toxic symptoms after eating shell-fish and other sea food, in some
cases due to the decomposition of the food, but in most instances
due to a peculiar hypersusceptibility which will be discussed under
Hypersusceptibility (see page 230).

Eel serum deserves special consideration because of the fact that
immune bodies can be produced. It is not poisonous when ingested,
but is highly so if given intravenously and it produces conjunctivitis
when instilled into the sac. Relatively large doses lead to rapid
death and small doses may produce cachexia and death after sev-
eral days. The toxic 'element is in the albumin fraction of the
serum and is destroyed by 58° C. for fifteen minutes. It contains
a hemolysin and probably also a neurotoxin. The hemolysin does
not act as an amboceptor, reactivation by fresh serum being im-
possible after the eel serum has been heated. Immune sera can be
produced which neutralize the hemolysin in vitro and also protect
animals from death by the eel serum. The serum of lampreys and
rays is similarly toxic.

The parasitic protozoa and other animal parasites are strikingly
free from substances which induce immunity. The protozoa show
few exceptions to this rule. Cytolysins can be produced experi-
mentally for amebse, but no such reaction takes place in human
patients. Active immunity to trypanosome infection can be pro-

76 THE PRINCIPLES OF IMMUNOLOGY

duced, and it is claimed that immunity can be conferred passively.
The trypanosomes, however, can become immune to trypanocides.
Malarial parasites produce among other things a hemolysin, but
there is no indisputable evidence that immunity occurs in malaria,
nor have immune reactions been developed. Sarcosporidia of sheep
produce a toxin fatal for rabbits in doses of o.oooi gm., against which
an antitoxin may be produced in rabbits. Complement-fixation re-
action is positive in infested sheep. Man may be infested by the
cyst of one tape worm, the tenia echinococcus, the cyst contents
being definitely toxic, as shown when a cyst ruptures into a body
cavity, e.g., the peritoneum. Serum of infested patients contains a
precipitin for the cyst proteins and also a complement-fixing body,
Zapelloni reporting 93 per cent, positive complement-fixations in 500
cases examined. Of the adult tape worms which infest man the
dibothriocephalus latus is the most important from the immunologi-
cal standpoint, although this parasite is rare in America. The
proglottids contain a thermostabile hemolytic lipoid liberated on
the death of the segments by auto-digestion. There is also a ther-
molabile hemagglutinin. It is probable that the hemolysin is
either associated with other cytolysins or that a species cytolysin is
present which also acts as a hemolysin. This is responsible for the
primary type of anemia seen in dibothriocephalus latus patients.
The serum of these patients contains a precipitin for the fluid ob-
tained by antolytic digestion of the segments. Of the nematodes
the ascaris, the trichinella spiralis, the hook worm, and certain forms
of filaria have been investigated. Certain ascarids produce poison-
ous substances without immunological relations. In regard to
trichinosis Salzer has found that the serum of recovered patients
has distinct therapeutic value in infested patients and protects ani-
mals against experimental infestation. Complement-fixation has
been found to be of value in the diagnosis of trichinosis. It has been
claimed that the anemia of hook-worm infestation is due to a
hemolytic poison, but there is doubt that this is as important as
the small repeated hemorrhages produced by the bite of these para-
sites. There is little of immunological significance in the studies of
the filariae. The guinea-worm (filaria medinensis) contains in its
body a violent irritant which may be discharged by rupture of the
worm during forcible attempts at its removal, and leads to severe
local inflammation and even to gangrene.

Mammalia do not produce poisons except in the somewhat ques-
tionable case of the male duck-bill platypus of Australia, a survivor
of the very earliest forms of mammalian life. Special glands are
said to secrete a poison like that of Australian snakes, which is dis-
charged through a hollow movable spur on the hind foot. There is
serious question as to the toxic properties of this secretion, certain
authorities believing that the sequences of such wounds are due to
infection and that the secretion is of importance only as a secondary
sex character. The serum of certain mammals is toxic on injection,

TOXINS AND ANTITOXINS 77

as, for example, beef serum, which in doses of 0.5 c.c. will kill a
guinea-pig in a few minutes. Dog serum is also toxic for guinea-
pigs in somewhat larger doses (i.o to 2.0 c.c.). Horse serum is toxic
for cats in doses of i.o c.c. per kilo, and for guinea-pigs in doses of
20.0 c.c. per kilo., but man is practically insusceptible, except in
those cases of hypersusceptibility in which small doses of serum
produce serious symptoms and even death. Such toxic sera con-
tain hemolysins and agglutinins in small amounts and reduce the
coagulability of the blood, but death is probably due to other factors.
Except for cases of natural or artificial hypersusceptibility, the toxic
element is destroyed by heat of 56° C. and is removed by
animal charcoal.

CHAPTER V
AGGLUTININS AND PRECIPITINS

GENERAL INTRODUCTION.
AGGLUTININS.

BACTERIAL AGGLUTINATION.
INTRODUCTION.

PRODUCTION OF IMMUNE AGGLUTININS.
PRELIMINARY TITRATION.

BLEEDING THE IMMUNE RABBIT.

METHODS OF TITRATION.
MACROSCOPIC.

MICROSCOPIC (THE WIDAL TEST).
SPECIFICITY OF AGGLUTININS.

GROUP REACTIONS.

ABSORPTION OF AGGLUTININS.
INHIBITION ZONES.
INFLUENCE OF HEAT.
INFLUENCE OF ELECTROLYTES.
INFLUENCE OF HYDROGEN ION CONCENTRATION.
THE MECHANISM OF AGGLUTINATION.

ALTERATIONS OF CELL AGGLUTINABILITY.

THE NATURE OF AGGLUTININS.

PHYSICAL BASIS OF AGGLUTINATION.

THE DREYER TEST.
HEM AGGLUTININS.

HETERO-HEMAGGLUTININS.
ISO-HEMAGGLUTININS.

CLASSIFICATION OF HUMAN ISO-HEMAGGLUTININS.
CHARACTERS.
MECHANISM.

ISO-HEMAGGLUTININS IN OTHER ANIMALS.
BLOOD TRANSFUSION.

METHODS FOR TESTING HUMAN BLOOD.
REACTIONS.

CHEMICAL AGGLUTINATION OF ERYTHROCYTES.
CONGLUTI NATION.
PRECIPITINS.

INTRODUCTION.

NATURE OF REACTION.

EXPERIMENTAL DEMONSTRATION.

DELICACY OF REACTION.

INFLUENCE OF HEAT AND OTHER AGENTS.

PRACTICAL APPLICATION.

THE FORENSIC BLOOD TEST.
BIOLOGICAL RELATIONSHIPS.

ORGAN SPECIFICITY.
DETECTION OF FOOD ADULTERATION.
FUNCTION IN IMMUNITY.

General Introduction. — Jf a clear albuminous urine be boiled the
invisible protein aggregates clump together, become visible as
flocculi, and sink to the bottom of the test-tube. If to a colloidal
suspension of mastic be added a proper concentration of common salt
a similar flocculation of the mastic occurs. Red blood-corpuscles or
bacteria shaken in physiologic salt solution form a cloudy suspen-
sion of particles invisible to the naked eye. They may be clumped
together by a variety of methods in similar flocculi which become
78

AGGLUTININS AND PRECIPITINS

79

clearly visible as small particles and sink to the bottom of the tube
more quickly than would the individual cells in the original suspen-
sion. The first example is one of precipitation and the last of
agglutination. In the immunological sense, precipitation implies
flocculation of a protein solution by means of specific antibodies, so
that large aggregates are formed and thrown out of solution. Simi-
larly the term agglutination signifies clumping together by means
of specific antisera of cells originally in smooth emulsion, so that
the clumps are visible microscopically or grossly, and sink rapidly
to the bottom of the containing vessel. Animals may be immunized
to a protein in solution, as, for example, blood serum or egg white,
so that the animal's serum contains a body, the precipitin, capable
of precipitating the protein used for immunization. Similarly bac-
teria, red blood-corpuscles, or even other cells may be injected re-

FlG. 3. — Wooden box for holding rabbits during injections into or bleeding from the ear vein.

0

peatedly into animals leading to the formation within the animal of
a body, the agglutinin, appearing in the blood serum and capable of
clumping the type of cell injected. These phenomena, although
closely related and probably fundamentally identical in nature, will,
for eminently practical reasons, be discussed separately.

AGGLUTININS

Bacterial Agglutination. — Although others had observed the
phenomenon of agglutination, Gruber and Durham, in 1896, were
the first to study it intensively in the course of work on the colon
bacillus and the cholera vibrio. They pointed out the specificity of
the reaction and the fact that it differed in certain essentials from
previously studied serum reactions. These points will be discussed

So

THE PRINCIPLES OF IMMUNOLOGY

in some detail, but it must be pointed out at once that the specificity
is not absolute. It was soon found that blood-cells and later 'other
body cells could be agglutinated by specific sera. It was also
found that agglutinins of various kinds exist normally in certain
sera, these being called normal agglutinins as opposed to the arti-
ficially produced or immune agglutinins. It was found that the
agglutinins resist heat of 56° C, a degree sufficient to destroy com-
plement, and that after being rendered inactive by heat cannot be
reactivated by fresh normal serum. It was soon observed that in
the course of infectious disease due to a specific organism agglu-
tinins^are likely to develop, and this led to the discovery in Widal's

FIG. 4. — Method of obtaining blood from the posterior auricular vein of the rabbit's ear. The

vein has been incised by means of a small hypodermic needle. The same position of the animal

serves for intravenous injections which are given into the posterior auricular vein.

clinic in Paris, a few months after Gruber and Durham's publica-
tion, of the now widely used Widal reaction for typhoid fever. Con-
versely with a serum of known type, the antigenic bacteria may be
identified. The demonstration of agglutination may be by the
microscopic method or by the macroscopic method. In our pre-
sentation of the subject it is considered desirable to illustrate the
points by actual experiment, and for this reason we proceed to take
up the method of producing immune agglutinins in the laboratory
and subsequently present the factors which qualify and modify
the process of agglutination.

Production of Immune Agglutinins. — Injections for producing
agglutinins may be subcutaneous intraperitoneal, intravenous, or a

AGGLUTININS AND PRECIPITINS

81

combination of these, using first the subcutaneous or intraperitoneal
routes followed later by intravenous injections. Bacteria are usu-
ally killed by heat or chemicals before injection, although after im-
munization is well under way living organisms may be employed.
The use of living organisms is often of service in the development
of a serum of high titer.

POUPACTd U a.

PIG. 5. — Method of complete bleeding from the femoral vessels of the rabbit (see
text page 83).

The following will serve as a fairly typical example of the process of
immunization for the production of an anti-typhoid agglutinin. The cultures
used are twenty-four hour agar slants inoculated by zig-zagging the loop
back and forth over the surface so as to have the surface well covered by
growth. A measured amount, 5.0 c.c. or 10.0 c.c. of sterile salt solution is
added, the tube allowed to stand ten or fifteen minutes and then vigorously
rotated between the palms of the hands. This procedure gives a much
smoother emulsion than washing off by sucking in and blowing out from a
pipette or by scraping off with a platinum loop and is less susceptible to
6

82 THE PRINCIPLES OF IMMUNOLOGY

contamination. The suspension is pipetted into a sterile tube and the growth
killed by placing in a water bath of not less than 56° C. or more than 60° C.
for two hours. Rabbits are desirable animals because of the ease of intra-
venous injection. For ease in handling, the animal is placed in a box as shown
in Figs. 3 and 4. The ear is shaved along the course of the posterior auricular
vein situated near the posterior margin of the ear on its upper surface, is cleansed
with soap and water and sponged over with alcohol. Usually the alcohol makes the
vein stand out prominently, but if it does not, the ear may be pinched near its root
so as to distend the vein, or, if necessary, brushed over lightly with a sponge dipped
in xylol. Xylol should be used very sparingly, because of the danger of an
inflammation, which may make subsequent injections difficult. Bleeding from
the puncture may be stopped by pinching the ear for a few moments at the
site of injection. Usually one ear is used for injections and the other for

PIG. 6. — A flask placed upright after blood has clotted

with oblique surface. The serum drains to the bottom

of the flask and is easily withdrawn.

test bleeding. The earlier injections or bleedings are near the tip of the ear,
the later ones approaching the base. The following protocol illustrates
an immunization:

Day Killed typhoid emulsion

I 0.05 agar slant *

6 o.i agar slant

ii 0.2 agar slant

16 0.2 agar slant

21 0.2 agar slant

* If 10 c.c. saline had been added to
the culture, 0.5 c.c. suspension would
contain 0.05 agar slant.

Preliminary Titration. — One week after the last injection 0.5-1.0 c.c. blood
is withdrawn from an ear vein and the serum separated and titrated for the
agglutinin. (See Fig. 4.) If the titer is not satisfactory, the immunization
may be continued with living bacilli as follows :

Day Living typhoid emulsion
I 0.05 agar slant

4 o.i agar slant

8 0.2 agar slant

AGGLUTININS AND PRECIPITINS

After 7-10 days a further titration is made, and if still unsatisfactory the
animal is discarded. As a rule, three animals are employed, and from these
at least one will produce an agglutinin which will titrate 1-5000 or higher.
The titer may be maintained by subsequent injections at longer intervals, but
it is usually found desirable to kill the animal by bleeding and to preserve the
serum in ampoules in the refrigerator.

Bleeding the Immune Rabbit. — The rabbit may be " bled out " by strapping
it on a flat board, lightly anesthetizing and plucking the hair from the groin
on one side. The skin is scrubbed
with soap and water and then
alcohol, and a long incision made
in the line of the groin groove.
(See Fig. 5.) This goes through
the f ascias and exposes the femoral
vessels. The neck of a sterile
150 c.c, flask is placed over the
vessels just below Poupart's liga-
ment and the vessels cut with the
knife, the blood being caught as
it spurts. As the bleeding con-
tinues the head end of the board
is raised and the animal's body
squeezed until the flow ceases.
Before disposing of the body,
death should be assured by a
blow fracturing the cervical spine.
The flask is placed in an oblique
position until the blood is firmly
clotted, then placed upright in
the refrigerator. (See Fig. 6.)
This leaves an oblique surface of
clot from which the serum flows
out in the bottom of the flask.
The blood may also be obtained
from the carotid artery, but this
requires more careful dissection,
longer anesthesia and may require
the insertion of a cannula. Practi-
cally it gives no better results
either in quantity of blood with-
drawn or sterility of the process.
Usually 15-20 c.c. serum are
obtained after twenty-four hours
in the ice-chest, and only occasion-
ally is it necessary to centrifuge in
order to obtain a clear serum. The
serum is withdrawn as shown in
Fig. 7 and placed in small sterile
ampoules of dark glass, sealed and
kept in the refrigerator.

Macroscopic Titration. — Titra-
tion is usually by the macroscopic
method, but an alternative is the
microscopic method. For the ti-
tration by the macroscopic method
it is necessary to have the growth
from two or three agar slants,
adding about 10.0 c.c. sterile salt

solution to each tube and making an emulsion as described for immunization.
The suspension is placed in a flask and killed either by heat (56° C.-6o° C. for
two hours), or by phenol i.o per cent, or formalin (40 per cent.) i.o per cent.
The killing of the organisms is not necessary, but is desirable because of the
added safety and because such killed emulsions may be preserved in the refrigera-
tor for several days or a few weeks. Broth cultures may be used, but the
hydrogen ion concentration of the broth may add a small factor of error not
present in the saline suspensions.

PIG. 7. — Method of drawing up measured volumes into
graduated pipette. The rubber tube enables the

worker to observe the ascent of fluid in the pipette

and the position of the tip of the pipette. Fluid is

withdrawn from flasks in the same fashion.

84 THE PRINCIPLES OF IMMUNOLOGY

Dreyer, who has given much attention to agglutination in the diagnosis
of typhoid and paratyphoid fevers in individuals who have been vaccinated
against these diseases, maintains that heat and chemicals other than formal-
dehyde are inferior to the latter in killing and preserving the bacterial suspension.
He has given great attention to standardization of the reaction, an important
but not infallible precaution, where a patient, as in the army, is likely
to be examined in different laboratories during the course of the disease.
On this basis Dreyer has shown that saline emulsions from agar cultures are
inferior to broth cultures. Scheimann maintains in addition that the broth
cultures furnish a more permanent standard. Laboratories in which such
standards are prepared determine the optimum density of the agglutinable
cultures and also keep the emulsions until the early deterioration of agglu-
tinability produced by the formalin has reached a stationary point, after
which the standards remain practically unchanged for ten months and
probably longer.

The primary test is carried out in small test tubes, with each dilution
one-half that of the preceding one. This simplifies making the dilutions,
especially if only one serum is to be tested. A row of twelve tubes is placed
in a rack and each tube receives 0.5 c.c. salt solution. To the first is added
0.5 c.c. immune serum, the mixture blown in and out of the pipette three times
and 0.5 c.c. transferred to the next tube, the processes repeated and 0.5 c.c.
transferred to the next tube, and so until the last tube is reached. In order
to preserve the constant volume in each tube, 0.5 c.c. is discarded from the
last tube. Thus there are dilutions 1-2, 1-4, 1-16, 1-32, 1-64, 1-128, 1-256, 1-512,
1-1024, 1-2048, 1-4096. To each tube is added 0.5 c.c.^bacillus emulsion, thus
doubling each of the dilutions, so that instead of ranging from 1-2 to 1-4096,
they range from 1-4 to 1-8192. In the twelfth tube are placed 0.5 c.c. salt
solution and 0.5 c.c. bacterial emulsion to serve as a control of the emulsion
and prevent error due to spontaneous clumping of the organisms. The tubes
are placed in a water bath at 37° C. for one hour and then in the refrigerator
over night. The clumping is observed with the naked eye, the clumps being
visible and settling more rapidly than the bacterial emulsion. Should 1-512
of the final dilution show agglutination and 1-1024 fail to^ show it, the titer
lies between these two, and it is advisable to set up a series of tubes 1-500,
1-600, 1-800, 1-900, i-iooo, and repeat. The same, of course, is true of the
weaker dilutions, although beyond i-iooo the scale is more easily placed in
grades of 200 rather than 100. The preparation of such dilutions is illustrated
as follows:

0.5 c.c. serum + 4.5 c.c. saline = i-io dilution

0.5 c.c. No. i + 12.0 c.c. saline = 1-25 dilution

0.5 c.c. No. i + 4.5 c.c. saline = i-ioo dilution

0.5 c.c. No. 2 -f 4.5 c.c. saline = 1-200 dilution

4 0.5 c.c. No. 2 -r 4.5 c.c. saline -

5 0.5 c.c. No. 3 + 1.0 c.c. saline =

6 0.5 c.c. No. 2 + 5-5 c.c. saline ==

7 0.5 c.c. No. 3 + 1.5 c.c. saline —

8 0.5 c.c. No. 2 + 8.5 c.c. saline —

9 0.5 c.c. No. 4 + 8.5 c.c. saline =

-300 dilution
-350 dilution
-400 dilution
-450 dilution
-500 dilution

Should we wish to determine a titer between 1-200 and 1-500, dilutions
4-9 are placed, 0.5 c.c. in each of six tubes, 0.5 c.c. emulsion added to each,
and in a seventh tube 0.5 c.c. saline and 0.5 c.c. emulsion as a control. The
tubes are placed in the water bath and incubated as before. Similar protocols
may be made if higher dilutions are required for the final test. Some workers
prefer to set up primary dilutions of i-io, 1-50, i-ioo, 1-200, 1-500, i-iooo,
1-2000, 1-4000, but this has no particular advantages as compared with the
primary titration outlined above.

Microscopic Titration. — The microscopic method may be employed with
the same method of dilution and mixing, simply removing a drop for obser-
vation in a hanging drop preparation at the end of the period of incubation
and examining with a 4-mm. lens. Another somewhat less accurate method is
to place one loopful of each dilution on a coverslip and mix with a loopful of
bacterial suspension, inverting the slip on a hollow ground slide, sealing with
vaseline, incubating and reading the result. A still less accurate method is
to place on coverslips or slides a row of loopfuls of salt solution, adding a
loopful of serum to the first drop, mixing, transferring a loopful to the second

FIG. 10. — Microscopic drawing showing the agglutina-
tion of a suspension of bacillus typhosus by blood serum
from a human case of typhoid fever, as seen in the
Widal test.

AGGLUTININS AND PRECIPITINS

drop and so on until the series of dilutions has been made, discarding a loop-
ful of the last mixture and leaving one loopful of salt solution as a control,
then adding to each drop a loopful of bacterial suspension. The slips or
slides are inverted, sealed, incubated and read. In using slides, the trouble
of sealing may be avoided by incubating in a moist chamber. The micro-
scopic method is usually employed in the Widal test, the dilutions of patients'
blood or serum being made by the same drop method, 1-20, 1-40, 1-80. Some-
times a drop of dried blood is used, this being laked and dissolved by a drop
of water and then made up to the first dilution of 1-20 by the addition of
nineteen drops of saline. Frequently twenty-four-hour broth cultures of the

FlG. 8. — The Wright tube for obtaining small
quantities of blood serum.

FIG. p. — Coiled pipette for taking up small quantities of fluids. Bubbling in the
coil gives warning of the filling and prevents suction into the mouth. The tube may
be made straight and plugged with C9tton. Either may be used for withdrawing
serum from the Wright pipette. Suction may be applied by the mouth directly or
with a rubber tube or by means of a small nipple.

typhoid bacillus are employed as the emulsion. Clearer results, however, are
obtained by collecting blood in Wright tubes (Fig. 8) and allowing the
serum to separate for dilution, and then employing a salt solution suspension
of a twenty-four-hour agar slant culture.

Specificity of Agglutinins — Group Reactions. — >The specificity
of the reaction may be shown by setting up dilutions of the anti-
typhoid serum obtained from the immunized rabbit against suspen-
sions of bacillus typhosus, bacillus paratyphosus (A or B), and
bacillus coli communis. An illustrative protocol follows :

Typhoid immune serum

B. typhosus

B. paratyphosus A.

B. Coli

1-4

1-8

+

+

+

-16

-f

+

+

-32
-64
-128

|

+

-

-512

+

—

—

-1024
-2048
-4096

Salt solution

1

—

—

This protocol illustrates two points, first, that the serum agglu-
tinates its homologous bacteria in high dilutions, and second, that
in strong concentrations it also agglutinates other organisms of the
same group. Thus the specificity is not absolute throughout, but
there is a " zone of absolute specificity," in this case between the

86 THE PRINCIPLES OF IMMUNOLOGY

dilutions of 1-128 to 1-4096. The fact that the other two organisms
are agglutinated is due to the phenomenon of " group reactions."
In the same way, if an animal were immunized to bacillus coli the
serum would agglutinate coli in high dilutions and typhosus in
lower dilutions. The principle is also well shown in a table taken
from Citron :

Typhoid Cholera

Agglutination of immune immune

serum serum

Against B. typhosus , , . 1-2,000 i-io

Against B. paratyphosus i-ioo i-io

Against B. coli 1-25 i-io

Against V. cholerae i-io 1-3,000

Absorption of Agglutinins. — If specific sera for paratyphosus and
coli were interposed in the above diagram it would be seen that these
sera would clump the homologous bacteria in high dilution, and the
others of the group in only low dilutions. This indicates that in
each serum we may assume there are several agglutinins, one for
the homologous organism, a major agglutinin or main agglutinin,
and one for each of the other organisms of the group, minor agglu-
tinins or partial agglutinins. This statement may be accepted for
the present, although the conception will be somewhat altered in the
theoretical discussion. Castellani has shown that if the major
agglutinin is absorbed by its homologous organism the minor agglu-
tinins disappear also, but that if one or several of the minor agglu-
tinins be absorbed by other members of the group of organisms the
major agglutinin remains. In order to make this clear we shall first
illustrate the process of absorption and then apply it to the group
reaction. It is well known that an animal may be simultaneously
immunized to two or more types of organisms ; for example, bacillus
typhosus and bacillus coli. The resulting serum may agglutinate
typhosus in dilution of 1—4000 and coli in dilution of i— 1000. The
absorption of the agglutinins may be shown as follows :

Prepare thick suspensions of bacillus typhosus and of bacillus coli com-
munis by suspending the twenty-four-hour surface growth of three slant agar
cultures in about 5 c.c. saline. This is done by placing 5 c.c. in the first tube,
making the suspension, then transferring to the second tube, suspending that
culture and repeating in the third tube. The typhoid emulsion is killed by
heat of 56° C. for one hour and the colon by heat at 60° C. for one hour.
Add to 1.5 c.c. serum an equal volume of thick suspension of dead bacillus
typhosus and in another tube place 1.5 c.c. serum with an equal volume of
thick suspension of dead colon bacilli. The tubes are marked A and B. After
mixing the emulsion of bacilli and serum the tubes are incubated at 37° C
and placed in the ice-chest for twelve hours. The tubes are centrifuged and
the supernatant fluid pipetted off. The bacteria are resuspended and the sus-
pensions diluted with salt solution about 1-20 or more, in order that agglu-
tination may be easily observed. The supernatant fluid represents a 1-2
dilution of the original serum. Place 0.5 c.c. each into test tubes and add 4.5 c.c.
saline, making a dilution of 1-20, well under the titer of the serum. Of the
diluted fluid A which has been absorbed by typhosus place 0.5 c.c. in a series
of two tubes and add 0.5 c.c. thin emulsion of colon. After incubation the first
tube will show no agglutination, and the second tube containing colon, whose
agglutinin has not been absorbed, will show agglutination. Conversely place
0.5 c.c. diluted fluid B in a series of two tubes, and add in order 0.5 c.c. thin

AGGLUTININS AND PRECIPITINS

emulsion of typhosus and 0.5 c.c. thin emulsion colon. After incubation only
tube i shows agglutination, because the colon agglutinins have been ab-
sorbed. The protocol of this experiment with the controls follows:

SERIES A (ABSORBED BY TYPHOSUS)

1. Fluid A 0.5 c.c. 4- 0.5 c.c. typhosus = no agglutination.

2. Fluid A 0.5 c.c. T 0.5 c.c. colon = agglutination.

SERIES B (ABSORBED BY COLON)

3. Fluid B 0.5 c.c. + 0.5 c.c. typhosus = agglutination.

4. Fluid B 0.5 c.c. + 0.5 c.c. colon = no agglutination.

CONTROLS

5. Saline 0.5 c.c. + 0.5 c.c. typhosus — no agglutination.

6. Saline 0.5 c.c. -f- 0.5 c.c. color = no agglutination.

This experiment shows only the essentials of the specific absorption. It
may be further elaborated by making a series of dilutions of the treated
serum so as to show the fact that the titer is essentially unimpaired.

Result

Tube

Fluid A 0.5 c.c.

Typhosus emulsion

I

1-8

0.5 c.c.

2

1-16

0.5 c.c.

3

1-32

0.5 c.c.

4

1-64

0.5 c.c.

1-128

0.5 c.c.

6

1-256

0.5 c.c.

7

1-512

0.5 c.c.

8

1-1,024

0.5 c.c.

9

1-2,048

0.5 c.c.

10

1-4,096

0.5 c.c.

ii

1-8

Colon emulsion

12

1-16

0.5 c.c.

13

1-32

0.5 c.c.

14

1-64

0.5 c.c.

15

Saline

0.5 c.c.

16

Saline

Typhosus 0.5 c.c.

At the same

time set up tubes

as follows:

Tube

Fluid B 0.5 c.c.

Colon emulsion

i

1-8

0.5 c.c.

2

1-16

0.5 c.c.

3

1-32

0.5 c.c.

4

1-64

0.5 c.c.

1-128

0.5 c.c.

6

1-256

0.5 c.c.

7

1-512

0.5 c.c.

8

1-1,024

0.5 c.c.

9

1-8

Typhosus emulsion

10

1-16

0.5 c.c.

ii

1-32

0.5 c.c.

12

1-64

0.5 c.c.

13

Saline 0.5 c.c.

0.5 c.c.

14

Saline 0.5 c.c.

Colon emulsion 0.5 c.c.

Result

This experiment shows that the process of absorption removes
only the specific agglutinin and leaves the other agglutinin un-
changed. As a matter of practical fact, the typhoid agglutinin re-
mains unchanged, but the colon agglutinin may be somewhat reduced
in titer, perhaps to 1-800 or even as low as 1-300. In a combined
serum of this sort with the typhoid agglutinin of high titer, part of
the agglutinin for colon is the result of a typhoid minor agglutinin
which is removed by absorption with typhosus, thus reducing the

88

THE PRINCIPLES OF IMMUNOLOGY

colon titer. The primary colon titer of 1000 would have a very low
content of minor agglutinin for typhosus, the removal of which
would leave the primary titer for typhosus practically unchanged
after absorption with colon bacilli.

The differences of absorption of major and minor agglutinins may be
illustrated by the use of a typhosus immune serum. We may use, for illustra-
tion, as closely related organisms bacillus typhosus and bacillus paratyphosus B.
Preliminary titration of the serum is carried out as usual against bacillus
typhosus and bacillus paratyphosus B. Let us suppose that the serum shows
a titer of 1-4096 for typhosus and 1-512 for paratyphosus B. Thick emulsions
of typhosus and para B are made as described in the previous experiment,
killed by 56° C. for one hour and mixed in equal volume with 1.5 c.c. serum,
incubated for one hour and refrigerated for twelve hours, then centrifuged
and the fluid pipetted off. The experiment with the results may be illus-
trated in the following protocol:

Tube

I
2

3
4

Fluid A 0.5 c.c.

(absorbed
by typhosus)

-16

-128

-256

-512

-1,024

-2,048

Typhosus emulsion

0.5 c.c.

0.5 c.c.

0.5 c.c.

0.5 c.c.

0.5 c.c.

0.5 c.c.

0.5 c.c.

0.5 c.c.

Fluid A 0.5 c.c.

Tube

(absorbed by

Para B emulsion

typhosus)

10

1-16

0-5

II

1-32

0-5

12

1-64

0-5

13

1-128

0.5

14

1-256

'0-5

15

16

Saline 0.5 c.c.
Saline 0.5 c.c.

Typhosus 0.5 c.c.
Para B. 0.5 c.c.

Fluid B 0.5 c.c.

Tube

(absorbed by

Typhosus emulsion

Para B)

11

-16

-32

0.5 c.c.
0.5 c.c.

19

-64

0.5 c.c.

2O

-128

0.5 c.c.

21

-256

0.5 c.c.

22

-512

0.5 c.c.

23

-1,024

0.5 c.c.

24

-2,048

0.5 c.c.

25

-4,096

0.5 c.c.

Para B emulsion

26

-16

0.5 c.c.

27

-32

0.5 c.c.

28

-64

0.5 c.c.

29

-128

0.5 c.c.

30

-256

0.5 c.c.

3i

Saline 0.5 c.c.

0.5 c.c.

32

Saline 0.5 c.c.

Typhosus 0.5 c.c.

Untreated serum

33

1-2,048

Typhosus 0.5 c.c.

34

1-256

Para B. 0.5 c.c.

Result

Result

Result

AGGLUTININS AND PRECIPITINS 89

It will be seen from these protocols that absorption by the major agglu-
tinogen, bacillus typhosus, removes both the major and minor agglutinins,
and that absorption by the minor agglutinogen removes only the minor agglu-
tinin, although it is true that even though the titer of the major agglutinin is
not reduced it may agglutinate in smaller clumps.

Inhibition Zones. — It is sometimes found that in powerful agglu-
tinins there is an " inhibition zone " in the more concentrated dilu-
tions. Thus a serum may agglutinate as follows :

Tube Serum dilution Result

^ I I-IO

2 I-IOO +

3 1-1,000 +-H-

4 1-2,000 -f-H-

5 1-4,000 -}— f-

6 1-6,000 +

7 1-8,000

This phenomenon is somewhat more frequently observed in sera
that have been preserved for a considerable time in the moist state.
If a serum with a titer of i-iooo, which originally showed agglu-
tination in all dilutions up to 1000, is preserved and after several
months titrated again, it may fail to agglutinate in i-io, may
agglutinate only weakly in i-ioo, and completely in 1-500. If the
tube containing i-io dilution is centrifuged, the supernatant fluid
drawn off, the bacteria again suspended and placed with the serum
in dilution of 1-500, there is no agglutination. The same is true if
these treated organisms are placed in contact with a fresh agglu-
tinating serum. The same phenomenon is obtained if the serum
first used is a fresh one of high titer with an inhibition zone,
and the bacteria are removed from the low dilutions in which they
have failed to agglutinate. The bacteria have become inagglutin-
able by treatment with the serum in these concentrations. Simi-
larly, heating an agglutinating serum to 65° to 70° C. destroys its
agglutinating properties, but if it is added to bacteria they become
inagglutinable when treated with fresh active serum. This phe-
nomenon is strictly specific and operates only in the presence of the
homologous organism. This peculiar character of agglutinins has
been closely linked with the Ehrlich conception of immune bodies
and is explained as due to the presence in sufficient concentration of
" agglutinoids." The term agglutinoid is applied to that part of the
agglutinin which has a specific binding affinity for the cell, but has
been deprived of the thermolabile and more easily destructible frac-
tion which has the power of producing clumping. This explanation
will be discussed more in detail in the general discussion
of agglutinins.

The influence of heat on agglutination has been studied exten-
sively. As has been indicated, heat will destroy agglutinins, but
certain agglutinins are destroyed by degrees of heat which fail to
destroy others. Most agglutinating sera are rendered inactive at
60° to 65° C., but anti-plague agglutinin is destroyed at 56° C,

90 THE PRINCIPLES OF IMMUNOLOGY f>

whereas others do not disappear until 80° C. has been reached.
Wells states that " purified typhoid agglutinin may resist 80 to 90
degrees." Agglutinins cannot be reactivated by the addition of
fresh serum, even though the temperature may have been adjusted
so that the agglutinoid remains.

A simple experiment for the demonstration of the influence of heat on
agglutinins is as follows: The typhoid immune serum, the production of
which has been described above, and also the killed typhoid suspension may
be used. In each of three tubes place 0.5 c.c. serum diluted i-io, and into a
fourth tube 0.5 c.c. salt solution. Tube I is heated in a water bath at 56° C.
for one-half hour, tube 2 heated at 70° to 75° C. for one-half hour, and
tubes 3 and 4 kept at room temperature. After cooling tubes i and 2, add
0.5 c.c. bacterial emulsion to each tube and incubate for one hour at 37° C.
Agglutination will not occur in tube 2, the serum having been heated to 70° to
75° C., nor in the control tube with saline. The unheated serum and the
serum heated to 56° C. will agglutinate powerfully. It will be found also that
the addition of o.i c.c. fresh guinea-pig serum (complement) to tube 2, and
subsequent incubation will fail to produce agglutination.

It is of interest to note that the degree of concentration of serum
has some influence on the degree of heat necessary for destruction.
For example, Koeckert in this laboratory found that normal un-
diluted iso-hemagglutinins are destroyed at 65° to 66° C. for
thirty minutes, but that in high dilutions they are destroyed at 62° C.
for thirty minutes.

The influence of electrolytes on the phenomenon of agglutination
is of considerable importance from the theoretical point of view
because of the resemblance to flocculation of colloidal suspensions.
Bordet, who discovered this fact, compared the reaction to the
throwing down of the alluvial matter in rivers as the fresh water
meets the salt water of the ocean. By previously dialyzing the salts
out of the bacterial suspension and the specific serum he showed
that agglutination would not occur, but that if the mixture was
salted in proper concentration the reaction would take place. It is
possible, however, to agglutinate bacteria by certain concentration
of salts, particularly of the heavy metals, but such concentration is
always much stronger than is necessary for salting, as described in
the Bordet experiment.

The demonstration of the influence of salts may be seen in the following
experiment, taken from Zinsser, Hopkins and Ottenberg. For this experi-
ment the killed typhoid suspension and the anti-typhoid serum as employed
in previous experiments may be used. " Place in each of two centrifuge tubes
with pointed tip 2.0 c.c. of the suspension. To tube A add 2.0 c.c. of agglu-
tinating serum diluted 1-50. To tube B add 2.0 c.c. distilled water. Allow
the tubes to stand at 37° C. for thirty minutes. Centrifugalize the tubes at
high speed until the supernatant fluid is clear." Pipette off the fluid and " to
the washed sediments add 2.0 c.c. distilled water and draw the mixture re-
peatedly in and out of the capillary pipette in order to break up the clumps and
obtain an even suspension. Set up the following tests in agglutination tubes :

1 Sediment A 0.5 c.c Distilled water 0.5 c.c.

2 Sediment A 0.5 c.c. 10 per cent NaCl 0.09 c.c. Distilled water 0.5 c.c.

• 3 Sediment A 0.5 c.c. 0.8 per cent. CuSO4 0.02 c.c. Distilled water 0.5 c.c.
4 Sediment B 0.5 c.c. 0.8 per cent. CuSO4 0.02 c.c. Distilled water 0.5 c.c.

• 5 Sediment B 0.5 c.c. 0.8 per cent. CuSO4 o.i c.c. Distilled water 0.5 c.c.
6 Sediment B 0.5 c.c Distilled water 0.5 c.c.

AGGLUTININS AND PRECIPITINS 91

"The tubes are placed in the water bath at 37° C for one hour and then
observed. Tubes 2, 3 and 5 should show agglutination." In tube A the
bacteria have been ' sensitized ' with the immune serum, and after the clumps
have been broken up are ready again for clumping under proper conditions.
In tube i the addition of distilled water does not provide the essential con-
ditions, but in tubes 2 and 3 the addition of electrolytes favors the reaction.
In tube B the bacteria have not been sensitized, but of the tubes 4, 5 and 6,
the concentration of the copper sulphate is such as to induce clumping in
itself, a phenomenon frequently seen in certain concentrations of salts of
the heavy metals, such as zinc, lead and mercury.

Influence of Hydrogen Ion Concentration. — It has been shown
by Michaelis and others that bacteria may be agglutinated by pro-
viding a proper hydrogen ion concentration, and it was hoped that
this might provide a means of rapid identification of organisms.
Proteins, for example, have a specific and constant optimum con-
centration of H ions for their precipitation. In the case of bacteria
it was shown, for example, that bacillus typhosus was agglutinated
by a hydrogen ion concentration of 4 to 8 X io~5, whereas para-
typhosus requires 16 to 32 X icr5, colon bacilli not being agglutin-
able by this method. It has been shown, however, that this
differentiation is not so sharp as was at first supposed, that differ-
ent strains show considerable irregularity, and that there is over-
lapping of one species with another. A combination of serum and
acid agglutination has shown that bacteria sensitized by serum can
be more readily agglutinated than are non-sensitized bacteria. The
specific characters of bacterial proteins are probably due to such a
slight variation in the arrangement of the molecular structure that
a satisfactory differentiation by changes in hydrogen ion concentra-
tion is not at present feasible. Eisenberg has recently studied the
problem with 584 races of bacteria, of which 537 were of the colon-
typhoid group, and found no differential diagnosis possible with the
acid agglutination method. He also found flocculation with salts
of the heavy metals extremely variable.

The Mechanism of Agglutination. — The data given in the pre-
ceding paragraphs outline the most important phases of the phe-
nomenon of agglutination, and any discussion of the mechanism of
the process must be based on these fundamentals. The chemical
nature of the agglutinogen is, of course, closely combined, if not
identical, with the protein of the cells, but is in no sense dependent
for its activity on the existence of life within the cell. Agglutinogens
are not destroyed by mild concentrations of formalin, phenol, heat,
or ultra-violet rays which are sufficient to destroy the life of the cell
itself. They pass through dialyzing membranes more rapidly than
do the agglutinins, and therefore are probably made up of smaller
molecules. That they pass through collodion sacs can be shown by
implanting such sacs, filled with killed typhoid organisms, in the
peritoneal cavity of rabbits and observing the development of agglu-
tinins in their blood; an observation which has been confirmed by
Reimann in this laboratory. Old broth cultures contain in the fluid
agglutinogens which may neutralize agglutinins and which may

92 THE PRINCIPLES OF IMMUNOLOGY

serve also to produce agglutinins upon injection. Thus it would
appear that agglutinogens are bodies of small molecular size capable
of slow diffusion and almost certainly protein, although Stuber
maintains that they are of fatty nature. The influence of heat on
agglutinogens has been carefully studied by Joos, who concluded
that the agglutinogen consists of relatively thermolabile and ther-
mostable constituents (the dividing line being 60° to 62° C.) which
induce the formation of separate agglutinins. The thermostable
fraction resists heat up to 165° C., is soluble in alcohol, and does
not give protein reactions, whilst the thermolabile fraction gives all
the protein reactions. This work is more fully discussed subsequently.

Alterations of Agglutinability. — Of considerable interest in con-
nection with agglutinogens is the alteration of agglutinability of the
cell. This probably is more closely associated with the cell as such
than with the agglutinogen. If bacteria are heated above 65° C.
they are not agglutinable by specific immune sera, but can absorb
agglutinin from the sera. Organisms freshly isolated from cases of
infectious disease often show similar reductions of agglutinability,
but recover it after prolonged growth on artificial media. This is
likely to be true in the case of " carriers," and Welch has referred to
it as a quasi-immunity which the bacteria themselves have acquired
by acting against the immune bodies of the host, an immunity, how-
ever, which the organisms lose on living in the environment of the
artificial culture media. Such inagglutinability may be produced
artificially by growing the bacteria on media containing a specific
immune serum, heated to destroy any bacteriolytic influence. In a
personal communication to us M. Cooper has stated that the pres-
ence of capsules about bacteria serves to establish a quasi-immunity
for the organisms against antibodies, and that such capsules appear
after cultivation in immune sera. This peculiar phenomenon is ex-
plained on the Ehrlich theory by assuming that the bacteria are
practically exhausted of receptors. Nevertheless, such inagglu-
tinable bacteria upon injection into animals lead to the production
of agglutinins for agglutinable strains, but not for inagglutinable
strains. It has also been assumed that they are saturated with
agglutinoid, but in America, at least, the Welch theory has been
given wide acceptance as an important philosophical conception.
Not only may agglutinability be altered, but different strains of an
organism show natural differences in agglutinability. For example,
Cole has shown that against a specific agglutinating serum five
strains of pneumococcus showed titers of 1-4000, 1-4500 (2), 1-7000,
and 1-8000. These are not " types " of a species but strains, and show
no specific agglutinability for sera produced by the strain in question.

The Nature of Agglutinins. — The chemical study of the agglu-
tinins shows that, like antitoxins, they are precipitated out of the
serum in the globulin fraction, and so far they have not been fur-
ther purified. They pass through filters less readily than their
antigens, and therefore have a larger molecular structure. Pepsin

AGGLUTININS AND PRECIPITINS 93

digestion destroys the agglutinins fairly readily, but trypsin acts
more slowly. Alkalies even when dilute are destructive, but acids
operate less actively. They are absorbed by charcoal. They are
not thrown down in the precipitate formed by specific precipitating
sera. The influence of heat on agglutinins has been the subject of
much study. The work of Joos was conducted with both agglu-
tinogen and agglutinin. As mentioned above, he demonstrated the
presence in the bacterial antigen of a thermolabile A agglutinogen
and a thermostable B agglutinogen, the dividing line being 60° to
62° C. The injection of heated antigen (B agglutinogen) gives rise
to the formation of B agglutinin, which in contrast to the antigen is
destroyed by heat of 60° C., but reacts with both A and B agglu-
tinogens. The injection of the unheated bacilli containing both A
and B agglutinogen leads to formation of both agglutinins, but the
B agglutinin can be removed by heat leaving the thermostable A
agglutinin, which reacts only with the A agglutinogen. The essen-
tials of this work have been confirmed, although Scheller working
with bacillus typhosus found that the B agglutinin is reduced in titer
but not completely destroyed at 60° to 62° C. Scheller showed
further that the heated bacteria (B agglutinogen) absorb agglu-
tinins from the sera more readily than do unheated bacteria, and that
they give the highest titers with the serum.

According to the Ehrlich scheme, agglutinins have a haptophore or
combining group and a zymophore group which causes the agglutination.
This zymophore is killed by heat and deteriorates on long standing to form
the agglutinoid (or agglutinin free from zymophore), which has combin-
ing but not agglutinating power. Thus in the side-chain theory the
agglutinins (and precipitins) differ from the theoretical simplicity of
the antitoxins and constitute the receptors of the second order.

The Physical Basis of Agglutination. — The mechanism of agglu-
tination is such that the reaction takes place in constant proportions,
thus likening it to a simple chemical reaction. The reaction is re-
versible, however, in that simple shaking, the use of organic and
inorganic acids and acid salts, as well as alkalies and heat of 70° to
75° C., can break the clumps into cell units; but after this separa-
tion fresh agglutinating serum cannot operate again. It has been
shown further that agglutinins can be separated from bacteria-
agglutinin combinations by the electric current; therefore, the agglu-
tinins are not destroyed by the union with the bacteria. Many of
the older workers believed that the reaction occurred because of
changes in the outer layers or ectoplasmic substance of the cells.
Gruber at first maintained that a substance, glabrificin, was taken
from the serum by the cejls which made their outer surfaces sticky
and caused adhesions when their motility brought the bacteria in
contact with one another. Malvoz and others held that the reaction
depended upon the entanglement of the flagella of the bacteria.
Neither of these ideas is consistent with the fact that non-motile

94 THE PRINCIPLES OF IMMUNOLOGY

bacteria and other cells are subject to agglutination, but no definite
proof is at hand to show that the ectoplasmic substance is not of
considerable importance. The influence of salts on agglutination
lends much support to the conception that agglutination is a col-
loidal phenomenon. As has been indicated above, the presence of
electrolytes is essential to the reaction, but salts, acids, and salts of heavy
metals, if present in sufficient concentration, may of themselves produce
agglutination. On the other hand, salts in strong concentration
serve to prevent the action of agglutinin. When bacteria have ab-
sorbed agglutinin, very small amounts of salt serve to bring about
agglutination. If a suspension of bacteria and an agglutinating
serum are each dialyzed free of salt and the two mixed, the bacteria
absorb agglutinin. This is shown by the fact that the supernatant
fluid after centrifugalization is free of agglutinin, but agglutination
occurs on addition of salt. Bordet interpreted the phenomenon of
agglutination as having two phases, first that of sensitization of the
bacteria by the agglutinin, and second, that of agglutination of these
agglutinin-bacteria by the salt. It may be stated in other terms
that the bacteria are primarily suspensions of protected colloids
which are so altered by the agglutinin that they become unprotected
and precipitable by salts, or that they become more permeable for
electrolytes. In fact, it has been shown that sensitized bacteria
take up salts more readily than unsensitized. The similarity of
bacteria to protected colloids is also borne out by Porges, who
showed that while encapsulated organisms are inagglutinable, the
solution of their capsules by heating in weak acid renders the bac-
teria agglutinable. Bacteria carry electro-negative charges and move
toward the anode, whereas agglutinins are electro-positive. The
sensitized bacteria are agglutinated by the current between the poles,
although the sensitized bacteria move slowly toward the anode.
The small amount of salt necessary for agglutination further sup-
ports the influence of electrical charge and thus furnishes further
analogy with colloidal precipitation. Neisser and Friedemann have
studied the similarities of agglutination and colloidal precipita-
tion and offer much in support of such analogy. Two protocols
may serve to show the importance of their work, one dealing
with the so-called sensitization and the other with inhibition zones.
Just as salt influences agglutinin and agglutinogen, so may it
influence mastic and gelatin solutions, as may be seen in the follow-
ing experiment :

i.o c.c. mastic i.o c.c. mastic + o.oooi c.c.
(i-io original emulsion) 2% gelatin sol. and

10% NaCl Sol. diluted to 3.0 c.c. diluted to 3.0 c.c.

I.O C.C. + +

0.5 c.c. + .

0.25 c.c.

0.125 c.c. +

0.05 c-c.

0.025 c.c. — —

AGGLUTININS AND PRECIPITINS 95

Furthermore, they offer a protocol showing the similarity be-
tween the reaction of colloidal iron hydroxide upon mastic emul-
sions and the agglutination phenomenon in reference to inhibition
zones. It will be seen that stronger concentrations of the iron
hydroxide fail to precipitate, thus simulating the action of strong
concentrations of an agglutinating serum of high titer or of an old
serum. The protocol follows :

Colloidal iron hydroxide Mastic emulsion Result

I.O .0 C.C.

0.5 .o c.c.

0.25 .o c.c.

O.I .O C.C.

0.5 .o c.c.

O.O25 2.O C.C.

O.OI .0 C.C.

0.005 .0 c.c.

0.0025 .o c.c.

O.OOI .O C.C.

This latter protocol is of significance not only in relation to
agglutination, but is of importance also in connection with the
Neisser-Wechsberg phenomenon of complement-deviation (not
fixation) discussed in connection with bacteriolysis. As Zinsser says,
" it seems to be a universal fact governing the union of colloidal
substances, that definite quantitative proportions must be main-
tained in order to lead to reaction, this being, possibly, explicable on
the basis that actual union can take place only after disturbance of
the electrical balance which keeps the particles apart." The assump-
tion that agglutinoids have an important bearing on the presence of
inhibition zones is not necessary if we accept the colloidal nature of
agglutination. This does not entirely controvert the existence of
altered agglutinin with a binding power for agglutinogen.

Not only may salt-free bacteria-agglutinin combinations be
agglutinated by salts but, as Friedberger has shown, certain organic
substances, such as dextrose and asparagin, serve also to produce
agglutination in such salt-free mixtures. These substances do not
dissociate in solution as do salts, and therefore produce no electric
phenomena. This fact presents a certain objection to the final
acceptance of the colloidal theory of agglutination, but it is possible
that the mechanism in this instance is of a nature different from
that of the immunological process, and certainly the great mass of
evidence is in favor of the reaction of agglutination being of
colloidal nature.

Nothing has been definitely brought forward in the physico-
chemical examination of agglutination to explain specificity, except
the fact previously indicated, that variations of hydrogen ion con-
centration have a relatively specific action on bacteria. As is
known, the definite identification of bacteria by this method has
not been satisfactory. The specificity of immune serum agglutina-

96

THE PRINCIPLES OF IMMUNOLOGY

FIG. ii.— The nip-
ple pipette for
making mixtures
of fluids and bac-
teria] suspension.
The pencil mark is
seen a short dis-
tance above the
tip.

Tube

I
2

3

4
5

tion is also a relative matter, as is shown in the group
reactions, and if electric phenomena play a part in spe-
cificity they are more delicate than can be demon-
strated by present chemical or electrical methods.
Bordet, who laid no emphasis on the electrical reac-
tions, thought that the process of sensitization of bac-
teria by agglutinins is in essence a denaturing of the
bacterial proteins, and that the specificity of the process
depends on the degree of denaturation.

The Dreyer Test.— The Widal test has been described
(page 85). This test has been of the greatest service in
the diagnosis of typhoid and paratyphoid fevers but the
introduction of vaccination on a large scale has reduced the
value of the test as a diagnostic sign of actual disease, be-
cause vaccinated individuals give a positive test. Dreyer
studied the course of agglutination in typhoid and paratyphoid
fevers, and found that the agglutinative titer of the blood
follows, during the course of the disease, a fairly regular curve,
increasing to the third week and then declining. Although
the titer may be higher at the beginning of the disease in
vaccinated individuals than in others, the titer follows the
same general curve. Of more importance is the differentia-
tion between typhoid and other infections in the vaccinated.
This has been of the utmost importance in the World War
in distinguishing between febrile disease, such as trench fever
or malaria, and typhoid or paratyphoid. The test is made by
the macroscopic method for agglutination, and must be re-
peated at weekly intervals in order to determine the curve
of agglutinins. Not infrequently the first test may show a
titer so much higher than occurs after vaccination that a
presumptive diagnosis is justifiable. Under war conditions
the transfer of patients often made it necessary to perform
the tests in several different laboratories, and to provide for
this the Oxford Standards Laboratory prepared emul-
sions of the bacilli for distribution. For this purpose
the organisms were grown for twenty-four hours in pep-
ton veal broth, then shaken well and o.i per cent, for-
malin (40 per cent, formaldehyde) added. The culture was
stored at 2° C. and shaken frequently during four or
five days. At the end of this time it was usually sterile.
It was then diluted to standard opacity by means of salt
solution, to which was previously added o.i per cent,
formalin. It was further standardized as to ^agglutina-
bility and labeled with a factor so as to provide means
whereby tests in different laboratories could be estimated
on the same basis.

The blood for the test can be obtained in a Wright
tube, but it is preferably taken from the cubital vein
into a centrifuge tube, so as to provide a fairly large
amount of serum. In order to make the method appli-
cable in laboratories where graduated pipettes are ^ not
available, Dreyer made all the dilutions with a nipple
pipette of drawn-out glass tubing similar to that illus-
trated in Fig. n, except that the drawn-out part is wider
and shorter. Three rows of 7 x 75 mm. test tubes are then
set up and further dilutions made according to the follow-
ing scheme;

Water Serum Bacterial suspension Dilution equals

.o drop 10 drops 15 drops 1-25

5 drops 5 drops 15 drops 1-50

8 drops 2 drops 15 drops 1-125

9 drops i drop 15 drops 1-250
10 drops o drop 15 drops control

AGGLUTININS AND PRECIPITINS

97

The three rows of tubes are set up so as to use suspensions in each row
of bacillus typhosus, paratyphosus A, and paratyphosus B. The dilutions
may be carried further if necessary. The tubes are incubated in a water bath
at 55° C. for two hours, are read immediately, and, if desired, again after
twenty-four hours in the refrigerator. The standard method of Dreyer may
be adapted to other methods of dilution and incubation, but must be the
same in the study of every case.

In unvaccinated individuals agglutination in a dilution of 1-25
against bacillus typhosus justifies suspicion, and if marked in dilu-
tion of 1—50 is nearly always diagnostic. Browning offers the fol-
lowing table as indicating positive reactions in each of the
diseases indicated.

Organism

B. typhosus

B. paratyphosus A.

B. paratyphosus B.

Serum dilution
I-IOO

1-50 (or even lower 1-20)
1-200

These criteria are not applicable to vaccinated persons or those
who have previously had typhoid or paratyphoid fever. Martin and
Upjohn examined seventy-five persons from; seven to fourteen
months after typhoid vaccination and found that the serum of two-
thirds agglutinated bacillus typhosus in serum dilutions of 1-200,
and that of one-tenth agglutinated in dilutions of 1-800. These are
higher levels than are usually reached by unvaccinated persons dur-
ing the course of the disease. Vaccination with typhoid vaccine
produces minor agglutinins for para A and B, but in very low con-
centration. Triple vaccines produce agglutinins for para A and B,
but rarely in dilutions exceeding 1-50 or i-ioo. The following
chart, taken from Mackie and Wiltshire, as quoted by Browning,
illustrates the change in titer of blood serum in the course of infec-
tion with bacillus paratyphosus A.

Serum dilutions

1—50

I— 100

1—200

I—SOO

I— 1000

1—2000

Fourth or fifth day of illness:
B. typhosus

+
+ + +

+ + +
+ + + +
+ +

+

+ +
+ + + +

+
+ + + +

+ + + +

+++

+ +

B. paratyphosus A

B. paratyphosus B
Thirteenth day of illness:
B. typhosus . ....

B. paratyphosus A
B. paratyphosus B

The first test in this patient was strongly suggestive, since it is
rare in a vaccinated individual to find the titer for either para A or B
to exceed that of typhosus. In our experience typhoid in the vac-
cinated is likely to show titers in the first week of 1-500 for typhosus,
i-ioo for para A, and 1-50 for para B ; toward the end of the
second week they are likely to be, respectively, 1-2500, 1-750,
1-250; and in the third week 1-3000 or higher for typhosus with
slight increases for para A and B. The titers then subside. It will
be noted that infection increases not only the major but also the
minor agglutinins.
7

98 THE PRINCIPLES OF IMMUNOLOGY

Space does not permit a complete discussion of the results of the
test, but it may be said that a positive Dreyer test indicates the
presence of some form of enteric fever. If, however, the isolation
of organisms from the stools indicates the nature of the disease the
test may sometimes mislead. For example, we have found para-
typhosus B in the stools of a patient whose serum titer curve indi-
cated the presence of a para A infection. The test should go hand
in hand with careful clinical study and bacteriological examination
of the blood, feces, and urine.

Hemagglutinins. — The agglutination of blood-cells and other
body cells follows the same general principles laid down for bacterial
agglutinins. In the case of agglutinins for red blood-corpuscles the
name hemagglutinins has been adopted. These may be divided into
auto-hemagglutinins, iso-hemagglutinins, and hetero-hemagglu-
tinins. The auto-hemagglutinin is contained in the same blood as
the cells it agglutinates, but certain factors operate to prevent agglu-
tination in the living body. For example, Rous and Robertson have
shown the presence in rabbits, which had received repeated small
blood transfusions, of an auto-hemagglutinin which operates at
temperatures lower than that of the animal, but on raising the tem-
perature to 38° to 40° C. the clumps break up and a homogeneous emul-
sion results. The same workers also demonstrated the presence of
auto-agglutinins in rabbits subjected to repeated withdrawal of
small quantities of blood. It has been stated that this phenomenon
may also occur in acquired hemolytic jaundice (Hayem-Widal
type), pernicious anemia, malaria, and other diseases, but more
recent studies tend to contradict this statement. Hornby states
that auto-hemagglutinins have been demonstrated frequently in
animals infected with trypanosomes. Hetero-agglutinins were dis-
covered by Creite and Landois, who noted that the serum of certain
animals produced agglutination when brought in contact with the
cells of certain other species; for example, the serum of the goat
and the erythrocytes of rabbit, man, or pigeon. Bordet discovered
in the course of his studies on hemolysins that if an animal is im-
munized with the erythrocytes of another species, the blood serum
will contain not only hemolysin, but also hemagglutinin for the
cells used in immunization. Thus we have to consider normal
hetero-hemagglutinins and immune hetero-hemagglutinins. Such
normal antibodies are present in low titer, but immune agglutinins
of this sort may be induced up to titers of several thousand. The
methods employed for the production of such agglutinins are the
same as those for producing hemolysis and will be considered under
that subject. The determination of the titer of hemagglutinative
sera is by essentially the same methods as for bacterial agglutinins,
save that the cells are washed as for experiments in hemolysis,, and
usually a fixed percentage emulsion of cells is employed. The influ-
ence of heat and other physical agents, as well as chemicals, is much
the same as for hemolysins (see page 115).

AGGLUTININS AND PRECIPITINS

99

Iso-hemagglutinms, Classification. — Iso-hemagglutinins are
those which exist in certain members of a species for cells of cer-
tain other members of the same species. Although iso-hemagglu-
tinins may somewhat rarely occur in lower animals, they appear
with great regularity in human blood. They were discovered in
1906 by Landsteiner and Shattock, working independently.
Landsteiner, by a study of the interaction of sera and corpuscles,
classified all human bloods in three groups and determined that the
property of iso-agglutinination is normal to man and does not vary
under pathological conditions. Hektoen noted in 1907 that the three
groups do not include all individuals, and in the same year Jansky
published the classification in four groups. This was confirmed by
Hektoen and subsequently adopted by Ottenberg. Moss, in 1910,
without knowledge of Jansky's work, also found that it is necessary
to divide bloods into four groups in order to include all individuals,
but unfortunately employed a system of numbering the groups the
opposite of that of Jansky. Because of the priority of Jansky's sys-
tem and its important support by Hektoen and by Ottenberg and
others, we prefer to use it rather than that of Moss. Groups I and
IV are transposed in the two systems but Groups II and III re-
main the same, hence, groups are transposable from one basis to the
other. The groups are not present at birth, but become established at
about the end of the first year of life and remain constant thereafter ; they
are heritable according to the Mendelian law. Disease does not change
the group of an individual, although, according to some of our experi-
ments, it seems possible that the agglutinin titer may be somewhat
reduced by prolonged disease. Jansky included in Group I those
bloods whose sera agglutinate cells of all^^ier groups and whose
cells are not agglutinated by any sera; C^l^tV is the reciprocal
of Group I in that the sera agglutinate « K, but the cells are
agglutinated by sera of all the other groups^^ffoups II and III are
reciprocals of each other and occupy intermediate positions between
Groups I and IV. This may be rendered clearer by the following table :

Group I. Serum agglutinates cells II, III and IV.

Cells agglutinated by no sera.
Group II. Serum agglutinates cells III and IV.

Cells agglutinated by sera I and III.
Group III. Serum agglutinates cells II and IV.

Cells agglutinated by sera I and II.
Group IV. Serum agglutinates no cells.

Cells agglutinated by sera I, II and III.

The following chart presents the classification graphically; the
+ sign indicates agglutination :

JANSKY CLASSIFICATION

Sera

I. II. III. IV.
j

5 II. + — + —

3 in. + -

IV. -f -

100

THE PRINCIPLES OF IMMUNOLOGY

Inasmuch as the Moss classification has been widely adopted we
include the chart of that system so as to show the relation of the
two systems of grouping :

Moss CLASSIFICATION

Sera
ii.

I.

II.

III.

IV.

III.

IV.

+
+

It is of the utmost importance that when the groups are deter-
mined in any individual the method of classification should be
clearly stated.

The incidence of the groups varies somewhat, according to the
figures of different investigators, and there is probably a factor of
error due to " random sampling," in spite of the large number of
individuals examined. Selected figures follow, according to the

Jansky classification :

Groups

II.

III.

IV.

per cent.

47-

per cent.

ii.

per cent.

6.

per cent.

per cent.

40.

per cent.

7-

per cent.

10.

per cent.

per cent.

39-

per cent.

13.

per cent.

2.

per cent.

per cent.

42.4

per cent.

8.3

per cent.

3-i

per cent.

per cent.

38.5

per cent.

12.5

per cent.

6.

per cent.

I.

Von Dungern
Hirschfeld . 36.

Moss 43.

Olmstead 46.

Karsner 46.2

Koeckert 43.

Average 42.84 per cent. 41.38 per cent. 10.36 per cent. 5.42 per cent.

The table shows that about four-fifths of all individuals fall in
Groups I and II, about equally divided between the two groups, the
next most frequent being Group III, and the least frequent being

Characters of /• magglutinins. — The iso-hemagglutinins are
neither filterable noi^B^zable, and are destroyed by heat of 62° to
66° C. for thirty minutes, depending on concentration, i.e., the agglu-
tinins in high dilutions (1-32, 1-64) disappear at 62° C., and in the
undiluted sera at 65° to 66° C. They are present in transudates and
exudates as well as in the plasma and serum, the serum showing a
greater concentration than the plasma. In serum the titer is usu-
ally between 1-16 and 1-32, although it may be as low at 1-2, and
has been reported as high as 1-320, irrespective of group. There is
variation of agglutinin content and probably of agglutinability of
cells at different times in the same individual.

The fact that a blood contains an iso-agglutinin does not nec-
essarily mean that it will similarly dissolve corpuscles, but the
converse is true ; namely, that if a serum shows iso-hemolytic prop-
erties it is also iso-hemagglutinative ; the group relationship prevails
in both agglutination and hemolysis. In fact, agglutination always
precedes hemolysis. In spite of this generally accepted view,
Kolmer claims recently to have demonstrated the presence of iso-
hemolysins independent of iso-agglutinins.

AGGLUTININS AND PRECIPITOUS A L \\

The Mechanism of Iso-hemagglutination. — Numerous theories
have been offered, of which we present that of Landsteiner. It has
recently received support in this laboratory by the painstaking spe-
cific absorption experiments of Koeckert. Landsteiner considers
that the division into four groups depends upon the presence, dif-
ferently distributed in bloods, of two agglutinins, a and b, and two
agglutinogens, A and B. The distribution of these may be tabulated
as follows (Jansky classification) :

Group Agglutinins (serum) Agglutinogens (cells)

I. a b

II. — b A —

III. a — — B

IV. A B

Aside from the support offered by Koeckert, in his demonstration
that specific absorption experiments prove the presence of these
bodies, further confirmatory evidence is found in the fact that the
agglutinogenic character of cells is demonstrable in the early months
of life, whereas the agglutinins do not appear until near the end of
the first year. It is also stated that transfusion with a certain group
may lead to the development in the recipient of specific iso-agglu-
tinins for the group injected. Kolmer's work, however, shows that
immunization of animals with the blood of the various groups pro-
duces a hemagglutinative and hemolytic serum without group char-
acters. Karsner and Koeckert have shown that desiccation leads to
a loss of specificity of the sera, and that at a certain period in the
desiccation a common agglutinin is found which clumps the cells of
all groups, including Group I. This is probably in part due to
alterations in physical character of the redissolved sera, and to
alterations in hydrogen ion concentration, as shown by Karsner
and Collins. Therefore, although the Landsteiner hypothesis offers
an excellent working basis, it seems probable that an intricate
physico-chemical mechanism is largely concerned in the phenom-
enon of iso-hemagglutination.

Iso-hemagglutinins in Lower Animals. — The presence of iso-
hemagglutinins in animals other than man is extremely irregular
and infrequent. Certainly no classification into definite groups has
so far been demonstrated. In our own experience the examination
of from ten to twenty members each of dog, rabbit, cat, and guinea-
pig species has failed to show iso-hemagglutinins, but others who
have examined larger numbers have found an occasional instance
of iso-hemagglutination.

Relation of Iso-hemagglutinins to Blood Transfusion. — The
principal importance of iso-hemagglutinins and the related iso-hemo-
lysins in human medicine relates to the transfusion of blood, a thera-
peutic measure which civil and military practice have shown to be
of the utmost value in combating secondary anemia following
hemorrhage. It is also recommended for prolonged sepsis with or
without severe anemia, for primary anemias, and for certain other

PRINCIPLES OF IMMUNOLOGY

diseases, but results are not so brilliantly successful as in secondary
anemias, particularly those resulting from acute hemorrhage. Ill
effects following transfusion are spoken of as reactions and include
fever, chills, cyanosis, hemoglobinuria, and even death. Cases com-
ing to autopsy show parenchymatous degenerations of solid organs,
marked congestion of all viscera, acute splenic hyperplasia, hemo-
globin staining, and sometimes multiple small emboli of agglutin-
ated erythrocytes. Blood studied in life has shown phagocytosis of
erythrocytes by the recipient's white corpuscles. The reactions de-
pend in large part on intravascular agglutination and hemolysis,
but probably certain other factors play a part. The prevention of
these other factors awaits the determination of their nature, but the
avoidance of agglutination and hemolysis can easily be accomplished
by use of the very simple tests for the determination of the presence
of conflicting iso-agglutinins. The simplest of these tests is the deter-
mination of the groups to which recipient and prospective donors
belong. The most desirable means of selection, in our opinion, is
that whereby the donor is chosen from the same group as the patient.
Lee and others have maintained that it is equally safe to use members
of Group I as donors for recipients of any group. The argument in
favor of this procedure is based on the statement that the real danger
in transfusion is the use of a donor whose cells are agglutinated by the
recipient's plasma and that the converse has little or no significance.
The cells of Group I are not agglutinated by any sera and are, there-
fore, safe to use. In our own experience we have seen occasional
reactions following this procedure and prefer to use a donor in the same
group as the recipient. Reactions following the general use of Group I
donors do not necessarily mean that the trouble is the result of agglu-
tination or hemolysis, for, as has been indicated above, other factors
may be concerned. Nevertheless, it holds true that thousands of trans-
fusions have been done with Group I as the " universal " donor and
without reaction. The explanation of the fact that a donor may thus
be used, whose plasma or serum is capable of agglutinating the recipi-
ent's erythrocytes in vitro, is not settled, but certain theories have
been offered. It must be remembered that in transfusion a small bulk
of blood is introduced, as compared with the total bulk in the recipi-
ent's body. Therefore, agglutinins introduced in this way are much
diluted, and as they ordinarily occur in low titer they may be sufficiently
diluted to be ineffective. Another possibility is that the agglutinins
are absorbed equally by an extremely large number of cells, each cell,
therefore, taking up too small an amount to be subjected to agglutina-
tion. A third possibility is that an excess of non-agglutinable cells
and the presence of the patient's own plasma permits of the formation
of only small clumps of cells, so small that they are of no significance
in the circulation. Our own work has failed to demonstrate anti-
agglutinins in a large number of tests, and it seems improbable that a
mechanism of this type operates to protect the recipient. It is conceiv-
able, however, that these possible factors of safety may not operate and

AGGLUTININS AND PRECIPITINS 103

reaction follow this type of transfusion. We cannot enter here into
a discussion of methods of transfusion.

Methods for Testing Human Blood. — The simplest method depends upon
the preservation in the laboratory of known Group II and Group III sera. These
should be selected so that they have a relatively high titer, and should not be
employed if they titrate less than i to 16. The method to be described is essen-
tially that of Lee and Minot. The apparatus includes a few 7x75 mm. test-tubes,
a platinum loop, microscope slides with at least one built up on the ends with
pieces of glass rod or match sticks glued on by means of balsam so that another
slide may be inverted upon it with hanging drops. A microscope is useful but
not essential, since a hand lens of 10 diameters magnification is satisfactory. A
small moist chamber is desirable but not essential. In well equipped labora-
tories the serum may be kept in the ice chest in sterile ampoules or small bottles
and drops removed as required. Somewhat more satisfactory is preservation
in sections of drawn out glass tube similar to that used for vaccine virus. Each
small tube contains serum for one test and the serum may be blown out exactly
as is done with vaccine virus. Phenol 0.5 per cent, may be used as a pre-
servative. One-half cubic centimeter of physiological salt solution is placed in a
test-tube, and to this are added one or two drops of blood, obtained by ear or
ringer puncture, sufficient to make a slightly opaque emulsion. Clotting of the
mixture is not harmful since subsequent shaking of the tube will produce a
homogeneous suspension. Upon a microscope slide are placed one drop eacli of
the sera of Groups II and III. With the platinum loop a drop of blood suspension
is mixed, by gentle rubbing, in each of the serum drops and the slide immedi-
ately inverted upon the prepared slide or a small rack so as to make hanging
drops. At the end of five or ten minutes the reaction occurs and may be seen
with the naked eye ; in order to avoid mistakes owing to slight agglutination it
is important to observe with the 16 mm. lens of the microscope or a hand
lens. If a small number of specimens is examined it is well to have controls
with known I, II or III cells. If the reaction is delayed the slide should be kept
in a moist chamber for one-half hour and then observed. The group to which
the cells belong is determined by the following section from the chart of
inter-agglutination :

STANDARD SERA

II. III.

2 I. _

3 n- - +

CJ III. + -

IV. + +

Thus if the cells are agglutinated by both sera they belong to Group IV; if
not agglutinated at all and the control cells show that the sera agglutinate prop-
erly the cells belong to Group I; if agglutinated by only III serum they belong to
Group II, and if agglutinated by only II serum they belong to Group III.

Hanging drops are not essential, but serve to make the reaction somewhat
clearer. The reaction occurs with the slides upright. In this case cover slips
may be used. Many employ undiluted blood and cover with cover slips, but
rouleaux formation sometimes offers a confusing picture.

It has been suggested that since the important point of determination is as
to whether or not the donor's corpuscles are agglutinated by the patient's serum,
the latter may be separated and placed on a slide with the donor's corpuscles.
The separation of the serum requires more time than a complete test as given
above, and is subject to serious error if the patient's serum happens to be of low
agglutinin titer.

If standard sera are not available they may be prepared if a known II or
III blood can be obtained. The interaction of the cells and serum with fifteen or
twenty other bloods can be worked out on the basis of the chart on inter-
agglutination. Space does not permit of giving the details, but Brem's method
gives them accurately. If this cannot be done the method of Rous and Turner
is probably the best of the methods for use where standard sera are not to be
had, since this method determines the activity of both the cells and serum of
the donor and recipient. The method with slight omissions is taken directly
from the article of Rous and Turner in volume 64 of the Journal of the Amer-
ican Medical Association.

104 THE PRINCIPLES OF IMMUNOLOGY

" Collection of the Blood. — The blood is taken from the patient and the pros-
pective donors in a I— 10 mixing pipette, such as is used in counting leucocytes.
The pipette is rinsed beforehand with 10 per cent, sodium citrate in water; the
citrate solution is drawn up to the mark i ; the pipette is rapidly filled with blood
from a puncture of the ear or finger ; and without pause the mixture is expelled
into a small, narrow test-tube. There is thus obtained a citrated blood containing
slightly less than I per cent, of citrate. The pipettes which we have employed hold
only 0.25 c.c. of fluid. This much blood is easily obtained from a single puncture.
There is no objection to increasing the flow" by pressure. Should it cease before
the pipette is full, the blood must be at once expelled into a test-tube, in order
that it may mix with the citrate and clotting be avoided. The mixture is then
taken up again, a new puncture made, and the pipette completely filled. After
each blood is obtained, the pipette is rinsed with citrate, then with distilled water,
then with fresh citrate, and it is ready for another blood. If several donors are
to be tested, two pipettefuls of Citrated blood should be obtained from the patient.
It is best to take them from different puncture wounds, in order to avoid a. pos-
sible clotting in the pipette.

" Mixing. — The mixing is done in pipettes with a capillary end — the so-called
Wright pipettes obtained by drawing out glass tubing in the flame. (Fig. u.)
The citrated bloods are used as such, and two combinations are made of the
patient's blood with that of each prospective donor, a mixture containing nine
parts of the patient's blood to one of the donor's, and a mixture of equal parts
of the two. The proportions used need be only approximate. In case of
emergency the first of the mixtures will suffice, since by its use the most
dangerous possibility, namely, that the blood of the recipient might destroy that
of the donor, can be ruled out. Following the technic usual with Wright pipettes,
the capillary tube is marked, blood is drawn to the mark, and each column of
the blood is separated by an air bubble from the next that is drawn up. To
insure proper mingling, each mixture should be expelled on a slide, or Widal
plate, and then drawn high in the pipette, which may be sealed off in the flame
in case the examination is not to be made for some time.

" Incubation. — No incubation in the ordinary sense is necessary. The pipettes
are kept at room temperature, and readings are begun after two minutes if
there is need to hurry. Readings are for agglutination, and even within two
minutes this is plainly evident, except when the agglutinating forces are notably
weak. In the final choice of a donor it is safest to rely on results obtained after
the mixtures have stood for fifteen minutes. But the ruling out of individuals
with unfit blood may be begun practically at once.

" Readings. — The capillary end of each pipette is broken, a small drop of the
blood expressed on a slide, a large drop of normal salt solution superimposed
without mixing, a coverslip put on, and the preparation examined for agglutin-
ation under the microscope. Fresh preparations can be made at intervals if de-
sired. The salt solution is not absolutely necessary ; but very clear pictures are
obtained as the blood spreads in it. When agglutination has occurred, the red
cells show a characteristic clumping, sometimes in small masses, often in large
ones that are very evident microscopically.

" If there is no clumping in the preparations made after the mixtures have
stood fifteen minutes, the assumption is warranted that the bloods do not agglu-
tinate or hemolyze each other. But if clumping is present in the 9-1 mixture
and to a less degree or not at all in the i-i mixture, it is certain that the blood
of the patient agglutinates that of the donor, and may perhaps hemolyze ^it.
Transfusions in such cases are dangerous. Clumping in the i-i mixture with
little or none in the 9-1 indicates that the plasma of the prospective donor
agglutinates the cells of the prospective recipient. For practical purposes these
findings suffice. But if there is a desire to know whether both bloods contain
agglutinins, a 1-9 mixture should be made. If this and the 9-1 mixture show
large clumps, whereas the clumps are smaller when the bloods are mixed in equal
parts, two agglutinins must be present. Should there be only one agglutinin,
little clumping or none will be observed when the blood containing the agglutinin
is diluted with nine parts of the other blood."

By the use of the technic indicated in the last paragraph, it is
possible to overcome error due to weak agglutinin content of the re-
cipient's blood. This we believe is of especial importance if the
patient has been ill for a long time.

AGGLUTININS AND PRECIPITINS 105

Reactions to Transfusion. — The effects on the body of introducing
high titer hemagglutinins have been studied experimentally in normal
animals and in those which have been splenectomized. In normal animals
there is found agglutination of red blood-corpuscles with embolism in
liver, lungs and other viscera. The liver is often found to show hyaline
necrosis in connection with the emboli. The spleen is large and dif-
fluent, and there are small areas of necrosis as well as phagocytosis of
erythrocytes by endothelial cells. Necrosis is also found in the fol-
licles of lymph-nodes. Multiple hemorrhages may also be noted. After
splenectomy the phagocytic function of the spleen is taken over by the
lymph-nodes. The incident hemolysis in either case leads to hemo-
globinemia and hemoglobinuria, but in splenectomized animals the
threshold of excretion of hemoglobin is somewhat higher than in
normal animals.

In severe and fatal reactions in man the phenomena are not likely
to be so marked. Phagocytosis of erythrocytes by the recipient's leuco-
cytes has been observed. We have performed autopsies on twelve
cases in which transfusion was practised shortly before death, in three
of which the death was at least in part due to the use of unsuitable
blood. In all of these the spleen was considerably enlarged (170,
390, 400 grams), and in one there were multiple small hemorrhagic
infarcts. One case showed enlarged soft white lymph-nodes. The
bone marrow was normal in all. All showed marked cloudy swelling
of the kidneys. Two showed hemoglobinuria, and in one of these
there was post-mortem staining by hemoglobin. In the case with
multiple infarcts of the spleen 50 c.c. Group II blood had been given
to a Group III recipient, and there was neither hemoglobinemia nor
hemoglobinuria. In the case with hemoglobinemia and hemoglobin-
uria about 700 c.c. Group III blood was given a Group I recipient.
Unfortunate accidents led to these errors, and the groups were dis-
covered subsequent to the operations. In the nine cases where the
transfusions were satisfactory the spleen was either normal or if
enlarged was accompanied by septicemia.

Chemical Agglutination of Erythrocytes. — Blood-corpuscles are
agglutinated not only by various sera but also by certain chemical sub-
stances. Gay has examined the function of the tonicity of the sur-
rounding medium in determining iso-hemagglutination and maintains
that the bloods of that group whose cells are non-agglutinable (Group
I) are constantly of higher total molecular concentration than the
other bloods. He further states that a " simple hypertonic solution
of CaCl2, but more particularly solutions hypertonic both in respect to
NaCl and CaCl2, produces a cohesion of any human blood after sev-
eral hours resembling iso-agglutination." Studies of hypotonic solu-
tions and of variations , of any considerable degree in hydrogen ion
concentration have been rendered difficult because hemolysis is likely
to occur under these conditions and render conclusions difficult. Land-
steiner and Jagic in 1904 were the first to call attention to the fact that
a well-defined colloid, namely silicic acid, agglutinates erythrocytes.

io6 THE PRINCIPLES OF IMMUNOLOGY

Gengou reported agglutination and hemo lysis by means of such chemi-
cal precipitates as calcium fluoride and barium sulphate, but in these
instances serum served to prevent agglutination. This appears to be
another example of protective colloidal action. According to Girard,
Mangin and Henri, the red cells carry electro-negative charges, but
agglutination has been produced by colloids regardless of the electrical
charge they carry. We, in collaboration with Hanzlik, have exam-
ined a wide variety of colloids and have determined that many of
those which produce thrombosis upon intravenous injection into animals
also produce agglutination in the test tube.

Conglutination. — Bordet and Gay, as well as Muir and Browning,
independently described in 1908 the phenomenon of conglutination — an
agglomeration of corpuscles in the presence of two normal sera. The
result of this reaction is the agglutination of corpuscles, but what is
known of its mechanism makes it advisable to consider the phenomenon
after the discussion of hemolysis (see page 126).

PRECIPITATION

Introduction. — The discovery of agglutination led to the discovery
by R. Kraus in 1897 of the precipitin reaction. His problem was to
determine whether or not agglutinating sera would act in any way on
extracts of bacteria, and in his work with typhoid bacilli and cholera
vibrios he found that the addition of the specific antisera to the bac-
terial extracts led to the formation of a precipitate and that this
reaction is specific. This was confirmed by Nicolle. Previously Widal,
Levy and Bruns had shown the converse, namely, that filtrates of
typhoid and cholera cultures upon injection led to the formation of
agglutinins. In 1899 Tchistovichs published the results of his work
with horse serum and eel serum, demonstrating the formation of
specific precipitates when the serum of rabbits previously inoculated
with these sera was added to the antigenic sera. Bordet confirmed
this with chicken serum and later showed that cow's milk upon injec-
tion induces the formation of a specific precipitating serum for the
casein of the milk. Kraus states that previous to the publications of
Tchistovitchs and of Bordet he had also, in collaboration with Winter-
berg and E. P. Pick, experimented with proteins of animal origin.
Fish demonstrated the specificity of various milk antisera for their
respective antigens. The reaction was enlarged in scope for various
other animal proteins. Kowarski showed that the reaction is specific
for higher vegetable proteins as well as for those of bacteria. Certain
authors have claimed that peptones, globulins, albumoses and other
protein products are antigenic in a similar manner, but the weight of
evidence is that the whole protein molecule is necessary. A recent
review of the literature on this subject by Fink has shown that state-
ments in regard to the proportion of the entire protein molecule neces-
sary to take part in the reaction are confusing and obscure. Frequently,
instead of testing against the decomposition product itself, the serum
obtained by its use has been tested against the entire protein molecule.

AGGLUTININS AND PRECIPITINS 107

Fink worked with the precipitates obtained by salting protein solutions
and found that rabbits inoculated with one-fourth, one-third, one-half,
and two-thirds saturation products produced no precipitins nor com-
plement-fixing bodies. In guinea-pigs, however, the three-fourths
saturated and completely saturated products possess slight sensitizing
and intoxicating properties, the latter being apparently the more
active. Nevertheless, three-fourths saturated and completely saturated
products of egg-white were sufficient to produce definite formation of
precipitin and complement-binding antibodies but not in as high a titer
as entire protein.

Nature of the Reaction. — In analogy with the terms used in the
phenomenon of agglutination Kraus named the antigen, precipitinogen
and the immune body precipitin. The reaction is similar to agglutina-
tion in all respects save that here we have to deal with proteins in
solution. Aging or heating leads to the formation of precipitoids,
group reactions as well as inhibition zones appear, heat has much the
same influence in all respects as in agglutination, salts play an im-
portant part in the reaction and specific absorption can be demonstrated.
It is known, however, that some protein molecules are largely built
up of alkaline amino-acids and that others are built up largely of the
acid amino-acids. Salmine, for example, a product of the spermatozoa
of certain fish, consists almost entirely of strongly alkaline amino-acids.
Gliadine of wheat is chiefly built up of dibasic amino-acids, glutaminic
acid. The fermentation end product of salmine is alkaline and of gli-
adine acid in nature. An antigliadine serum gives with a homologous
precipitinogen, a beautiful precipitate, while a mixture of salmine and
antisalmine-serum gives no visible precipitate. This would indicate
that the alkaline salts are of importance in the actual formation of the
precipitins, and we know by simple titration that during the precipitin
reaction there occurs a reduction of acidity. Nevertheless, it is also
asserted that when the acidity is due to an organic acid or acid salt
the reaction appears to be promoted. The precipitin is precipitated in
the euglobulin fraction of the serum, is destroyed slowly by trypsin
and rapidly by pepsin. The immune serum contains the precipitin
which constitutes the bulk of the precipitate, the latter thus represent-
ing, according to Wells, " the insoluble modification of the previously
dissolved precipitin and originates chiefly or entirely in the proteins
of the immune serum." Welch and Chapman obtained, with a precipi-
tinogen containing only i gram of protein, a precipitate containing 21.1
grams of protein. Pick employed a precipitinogen which did not give
the biuret reaction and with this obtained a voluminous albuminous
precipitate. It must not be understood that precipitins are always the
result of immunization, for Vaughan states that goat serum contains
a normal precipitin for rabbit and for dog sera. Such normal pre-
cipitins are not of very high titer and are not so sharply specific as
the immune precipitin. Puppies, kittens and rabbits up to ten days
old may absorb native protein from the milk of the mother which
apparently stimulates the formation of precipitins. Sera of human

io8 THE PRINCIPLES OF IMMUNOLOGY

infants have been observed to precipitate the protein of cow milk. It
appears possible, then, that from absorption through the intestinal tract
early in life the protein may appear in the circulating fluid in native
form and thus stimulate the formation of precipitins. These, of
course, are not normal precipitins in the sense indicated above for goat
serum but similarly are always precipitins of low titer and not
highly specific.

Experimental Demonstration. — For practical demonstration of the reaction
the serum proteins are the simplest to use. For immunization of animals the
intravenous route is by far the best, injecting 2.0 c.c. serum at five-day intervals
and bleeding ten days after the last dose. Three doses are usually sufficient, but
five doses frequently give a precipitin of very high titer. In order to get clear
serum it is necessary to fast the animal for twenty-four hours before bleeding,
thus eliminating fat from the serum. Rabbits are the animals usually selected
for this purpose because of their availability in the laboratory and because of the
relative ease of intravenous injection. Hektoen has shown, however, that the
domestic fowl is a prompt, reliable and liberal producer of precipitins, even more
so than the rabbit. A single intraperitoneal injection of 20 c.c. of defibrinated
blood or serum in most cases yields at the end of ten or twelve days a precipitating
serum of sufficient strength and specificity for practical purposes; but on ac-
count of an unwelcome tendency to give non-specific reaction, great care must
be exercised in all the tests with fowl antiserum, and it is necessary to use
salt solution 1.8 per cent, in strength. Man is also a good producer of precipi-
tins, as has been shown by investigation of human serum after the individual
has been given doses of horse serum. For performing the test, narrow tubes,
not more than 5 mm. in diameter are most suitable in order to save reagents
and get clear-cut results. Instead of diluting the antiserum, it is customary
here to dilute the antigenic serum. Nevertheless the titer thus obtained is
referred to the immune precipitin. Two methods are in use, the original
method of actual precipitation, and the Fornet ring test. In either case dilutions
of the antigenic serum are made i-io, i-ioo, 1-1,000, 1-10,000, 1-100,000, and
1-1,000,000, with provision for a salt solution control. After such a preliminary
test the serum may be more accurately titrated with intermediate dilutions. For
determining precipitation i.o c.c. of each dilution is run into tubes with a nipple
pipette, and to each is added o.i c.c. immune serum, the latter settling into the
dilutions, without shaking. Immediate observations are made and then the
mixtures incubated for one hour at 37° C, followed by subsequent observation,
and if desirable further observation after twenty-four hours in the ice chest.
The Fornet ring test is more clear-cut and is more commonly used. Here o.i c.c.
immune serum is placed in the tubes and the dilutions of antigen added with
nipple pipettes, so as to form a contact ring as in the Heller test for albuminuria.
A white ring gradually spreading both up and down indicates a positive re-
action. A good immune serum titrates 1-10,000 or more, although titers of
1-100,000 are obtainable.

The production of bacterial precipitins is somewhat more difficult
and requires longer immunization, the precipitins appearing, as a rule,
somewhat later than the agglutinins. Zinsser recommends the use of
salt solution emulsions of agar cultures killed at 6o°-7o° C., rather
than extracts or filtrates of broth cultures. The intravenous route is
best unless the bacteria are extremely toxic, when the subcutaneous
or intraperitoneal method may serve. Intravenous injections should
be given four or five times at five- or six-day intervals, the animal
(rabbit) being bled eight or nine days after the last injection. Ex-
tracts of bacteria for similar purposes are obtained by growth for
three weeks to three months in slightly alkaline broth, filtration through
Berkefeld filters and injection of the filtrate. Salt solution suspen-
sions of agar cultures may be shaken in a machine for twenty-four

AGGLUTININS AND PRECIPITINS 109

hours, filtered through a Berkefeld filter and the filtrate used. Kraus,
in his original studies, used broth filtrates and also juice expressed
from the bacteria. Kraus points out that the broth filtrates of toxin-
producing organisms such as bacillus diphtheriae do not precipitate
when mixed with antitoxic serum. That this is a general rule, how-
ever, is not true, since Jacoby has shown that it is possible to obtain a
precipitate by mixing ricin and antiricin serum, and others have ob-
served similar reaction with the use of abrin and antiabrin serum as
well as crotin and anticrotin serum.

The delicacy of the precipitin reaction is great and only exceeded,
in certain respects, by complement fixation and the anaphylaxis reac-
tion. It is of interest to note that whereas the Biuret and the Millon
test for protein will hardly exceed dilutions of i-iooo, the precipitin
reaction will detect not only the presence of protein but the species
from which it originates, commonly in dilutions of 1-10,000 or 1-20,000
and even 1-100,000.

Physical Basis of Precipitation. — The influence of heat on pre-
cipitation and also the group reactions are of considerable importance in
the practical application of the phenomenon and will be dealt with more
fully as this side of the question is considered. The comparisons offered
between agglutination and certain colloidal phenomena (see page 94)
are equally applicable to precipitation and require no extensive dis-
cussion here. It must be borne in mind, however, that the colloidal
interpretation of these phenomena is not proven. Essentially the same
arguments are available against the conception of precipitoids as
against that of agglutinoids, but none of these explains satisfactorily
the specific absorptive capacities of these hypothetical bodies. As ag-
glutinogen and agglutinin may exist in the blood of a living animal,
so may precipitinogen and precipitin coexist. This is compared by
Zinsser to the fact that if gum arabic is added to a mixture of thin
gelatin and arsenic trisulphide the precipitation which ordinarily occurs
will be prevented. The gum arabic in this instance is a protective
colloid. It is assumed that such a protective colloidal action operates
to prevent precipitation when precipitinogen and precipitin coexist in
the blood of a living animal. After the blood is withdrawn and
allowed to stand, this protective action disappears and precipita-
tion occurs.

The fact that precipitin and precipitinogen can coexist in circulating
blood and that experiments on the attempted production of iso-
agglutinins with their conflicting results has led to the question of
whether or not it is possible to produce precipitins in an animal by
the injection of proteins of a closely related species. Uhlenhuth and
Weidanz claim to have produced precipitins for human serum by
injecting human serum into monkeys, the resulting precipitin acting
on human but not on monkey serum. Berkeley and later Sutherland
were unable to confirm this experiment and we are forced to the con-
clusion that precipitin formation in closely related species is by no
means a constant phenomenon. Such precipitins would be practically

no THE PRINCIPLES OF IMMUNOLOGY

iso-precipitins, and, as we have seen, their existence is irregular
and questionable.

Practical Application. — Wladimiroff first applied precipitation
practically in the diagnosis of glanders in horses, using the serum of
suspected horses against a filtrate from cultures of glanders bacilli.
Kraus employed the reaction to identify closely-related bacteria. At
the present time, however, agglutination is employed for the detection
of glanders and also for identification of bacteria rather than precipita-
tion, because the latter procedure introduces the more cumbersome
technic of obtaining filtrates.

The Forensic Blood Test. — Uhlenhuth and Beumer published their first re-
sults on the use of the precipitin reaction in legal medicine in 1903. Other
studies were rapidly contributed, and to-day the method has an established place
in the identification of stains by blood and other fluids such as seminal fluid.
If spots on clothing or other material are suspected of being blood, this must
be proven by chemical, microscopic or spectroscopic examination. Subsequently
the precipitin test is used to determine the species from which the blood orig-
inated. Before proceeding to this test it is necessary to have immune pre-
cipitating serum against the suspected species, usually man, an additional
immune precipitating serum against some other species and a normal rabbit serum.
The immune sera are prepared according to the method outlined on page 108.
The suspected material must be carefully guarded against possible substitution
or contamination until the immune sera are prepared. It is then dissolved in
physiological salt solution and a perfectly clear filtrate used. If the material is
on cloth the latter should be teased so as to permit of solution ; if on some solid
material, such as a knife blade, it should be scraped off, ground in a mortar and
a small amount of salt solution added. Cloth should be placed in a test-tube or
bottle, and it is well to have a control with unstained cloth. The time for
extraction depends to a certain extent on the freshness of the material, but it
is wise to allow it to extract in the refrigerator over night, adding a few drops
of chloroform to prevent bacterial growth. If extraction does not proceed well
in salt solution it may be necessary to extract with I per cent, potassium cyanide
solution, correcting the alkalinity after extraction, by means of tartaric acid.
To prove that the solution contains protein a small amount may be boiled and
treated with acetic or nitric acid as in the ordinary test for albuminuria. A
final solution of the suspected material in a dilution of i-iooo is usually emr
ployed, and this dilution may be approximately determined by the foam test.
For this purpose make a i-iooo solution of any convenient serum, blow air
through it in a test-tube and note the persistence of bubbles above the fluid.
Dilute the extract gradually and blow air through it, repeating until that dilution
is obtained which will produce a foam of about the same viscosity as that in
the control tube. If the solution is not perfectly clear it may be centrifuged or
passed through a filter of washed asbestos or cotton. On the assumption that
the spot is suspected of being human blood the test is set up as f ollows :

Tube Material Result

1 Suspected extract -f- anti-human serum +

2 Suspected extract 4- normal rabbit serum

3 Control extract 4- anti-human serum

4 NaCl solution -{- anti-human serum

5 Human serum (i-iooo)-f- anti-human serum -f

6 Beef serum (i-iooo) -f- anti-human serum

7 Sheep serum (i-iooo) -f- anti-human serum

The amounts throughout are o.i c.c. of each reagent, and the test is made
by the Fornet ring method. Inasmuch as acceptable antisera should titrate
1-10,000, the reaction in i-iooo dilution occurs within a few minutes, and the
above result would be interpreted as a clear-cut positive for human blood pro-
vided the preceding chemical and other tests for blood had been positive. It
should be remembered that the precipitin reaction in this instance simply iden-
tifies the material as human protein, and a similar result might be obtained
from human seminal fluid, albuminous urine, purulent sputum, exudates and
transudates, unless the preliminary tests had been carried out.

AGGLUTININS AND PRECIPITINS in

Biological Relationships. — The question of the specificity of this
reaction has been somewhat confused by quotation from the famous
studies of Nuttall in regard to interrelationship of species. Nuttall's
book, published in 1904, wasi of the utmost importance to biology in
general, because it demonstrated anew by the use of the precipitin
reaction the interrelationship of animal species. He showed, for ex-
ample, the close biological relationships between man and the higher ape,
also similar relationships in the lower animals, as between the goat
and the sheep, the horse and the ass. Reference to the tables which
he published would seem to indicate, however, that the relationship
between man and the higher ape was so close as to be indistinguishable
by the precipitin reaction. An example in point is the statement which
he makes that whereas human blood will respond to antihuman precipi-
tating serum to the extent of 100 per cent., the blood of the chimpanzee
responds to the extent of 130 per cent. These figures, however, refer
to the bulk of the precipitate thrown down in relation to standard
dilution of the various bloods employed. He used relatively low dilu-
tions, allowed the sera to remain in contact for several hours and then
measured the amount of precipitate. This, as can readily be seen, is
different from the method which is employed at the present time in
determining the titer of the sera against the immune serum. The
latter method is distinctly more delicate in determining the specificity
of the reaction. For that reason it is the method employed in the for-
ensic test of to-day, as well as in ordinary laboratory procedures. Fur-
thermore, we find at the present time that the test demonstrates its
specificity particularly in the presence of strong sera by reading very
shortly after the contact has been made. Hektoen offers an excellent
example of this in the following table (antihuman serum) :

Blood

Fish

Chicken . .
Rabbit ...
Guinea-pig

Rat

Cat

Dog

Swine

Sheep

Beef

Horse

Goat .

Monkey (Macacus rhesus)
Human

-10

-10

-10

-IO
-IO
-IO
-10
-10
-IO
-IO
-10
-TOO

-5000

It will be noticed by reference to the above table that the titer of
the serum used in this particular test was only 1-5000, and we would
expect an even greater difference between the titer with the different
animal sera if the antihuman serum had been of higher titer. Con-
cerning the group reactions in the precipitin test an interesting instance
is given by Hamburger in regard to the action of the serum of a
rabbit inoculated simultaneously with sheep serum, goat serum and ox
serum, all of which are fairly closely related to each other biologically.

ii2 THE PRINCIPLES OF IMMUNOLOGY

The serum of the rabbit when mixed separately with each of these
three antigenic sera gave the most voluminous precipitates in the
presence of the sheep serum, less in the presence of the goat serum
and least in the presence of the ox serum. This observation has been
confirmed by Arrhenius. Just why the sheep antiserum should be
the most powerful is difficult to say, but it might be assumed that the
sera of closely-related species may augment the antigenic power of
the strongest of the three species used. Of further interest, Hektoen
has shown that in rabbits previously injected with foreign serum the
subsequent injection of a different serum may reawaken the production
of precipitin for the antigen previously injected. The practical value
of this fact is that rabbits which have once been used for the production
of precipitin should not be used again for the same purpose with
another protein because of possible decrease in specificity of the
second antiserum.

Organ Specificity. — The question of organ specificity is of con-
siderable importance in the discussion of the specificity of the precipitin
test. Numerous experiments have been made by various immunological
methods to determine whether or not it is possible to identify the
protein of a given organ within the same species. It may be stated
very briefly that these experiments have not met with any great degree
of success. However, in regard to the protein of the crystalline lens
of the eye and the protein of the testicle, certain interesting facts have
been discovered. Immunization with protein extracts of the crystalline
lens will produce precipitating sera which operate not only on the
lens protein of the same species but on the lens protein of all animals
as low in order as fish. In this example the species specificity has been
entirely replaced by a curious organ specificity. The organ specificity
in this case is so strict that the immune serum will not react with other
tissue extract even of the same species. Lens protein may, indeed, be
injected into the same species from which the lens was taken and give
rise to specific precipitins. By the use of the complement-fixation test
it has further been shown that in adult human beings it is possible to
detect the presence of an antibody for lens protein which is not detect-
able in children. This phenomenon will be mentioned later in con-
nection with the autocytotoxins (see page 142). Zinsser comments to
the effect that biologically these phenomena probably signify that al-
though there are fundamental species differences between the general
body proteins of various animals, there are still in certain highly spe-
cialized organs varieties of protein which possibly because of functional
exigencies have developed similar chemical characteristics. In regard
to the testicle and the placenta, it might be supposed that the germ char-
acter of these tissues is retained as distinctive from the somatic char-
acter of the other body tissues. This would not apply to crystalline
lens, since it is not of germ character. On the other hand, although
the lens can be regarded as a highly-specialized organ in both morpho-
logical and physiological senses, the testicle and the placenta can
hardly be so considered. Such discussions are likely to be fruitless

AGGLUTININS AND PRECIPITINS 113

until it is possible to isolate the protein of other body organs without
contamination by the animal's blood. Up to the present time this
seems to be impossible. Studies by Bell, for example, with perfusion
of various organs has demonstrated the impossibility of removing the
blood completely.

Detection of Food Adulteration. — The precipitin reaction is ap-
plied not only to detect blood as indicated above but also various other
.body proteins; for example, it may be used to detect the nature of
bone fragments or other tissue scraps. Of great significance is the
fact that the precipitin test is employed for the detection of adultera-
tion of food products. It has been utilized, for example, in detecting
adulteration of sausages by the use of horse and other meats. In the
preparation of such food products, heat is often employed, and there-
fore it is necessary to know the influence of heat on the precipitin
reaction. The relation of heat to the agglutinin reaction has already
been discussed (see page 93), and it is found that similar conditions
exist in regard to the precipitin test. Obermeier and Pick studied this
problem experimentally and found that an antiserum, even of high titer,
produced by an unheated antigen, failed to precipitate when brought
into contact with heated serum. If, however, animals are immunized
with serum boiled for a short time, the resulting immune serum forms
a precipitate when brought in contact with either heated serum or
unheated serum. Therefore, the precipitin produced by the latter
method is regarded as more comprehensive in its precipitating activity,
but nevertheless its species specificity remains unimpaired. By em-
ploying a lower degree of heat, namely 70° C, Schmidt found that
this marked difference was not so apparent and that an immune serum
prepared by injecting unheated serum would produce precipitation
with unheated serum and with the moderately-heated serum. How-
ever, the titer of antiserum prepared by the use of moderately-heated
antigen was not as high as with the use of unheated antigen. Schmidt
ifurther found that he could produce an even more comprehensive
immune serum by boiling the antigen until a coagulum was formed,
namely, for three hours. The coagulum was washed with salt solution,
dried, powdered and then taken up with a normal NaOH solution.
Zinsser and Ottenberg found that the use of a boiled antigen led to
the production of a comprehensive precipitin, but nevertheless they
determined that this resulted in some loss of specificity of the precipitin.

This outline of the influence of heat will serve to show that in the
detection of the adulteration of food products extreme care must be
taken in the selection of material. Wherever possible, fresh material
should be obtained, and the material for testing should always be taken
from near the middle of the specimen. This precaution prevents con-
tamination with other meat, and in the case of sausage yields material
likely to be less influenced by heat or smoke. The meat is cut into fine
pieces and allowed to extract in salt solution. Clarke used 30 grams
meat and 50 c.c. physiological saline, extracting in the ice chest for
twenty-four hours, and further diluting 1-300 for the test. Such
8

ii4 THE PRINCIPLES OF IMMUNOLOGY

extracts must be proven as to protein content by the nitric acid test and
the foam test. Violent shaking is to be avoided, because it liberates
fats and lipoids which cloud the extract. If precipitation occurs by the
use of this extract with an immune serum prepared by injecting un-
heated protein, the test can be regarded as highly specific. If, however,
it is necessary to use serum which is prepared by injecting heated pro-
tein, the specificity cannot be regarded as being so high. In practice
it is the rule to use serum prepared by injecting unheated protein rather
than otherwise, unless the special indications of the case indicate the
use of an immune serum prepared from heated antigen. The technic
in case of food adulteration is. essentially the same as for the detection
of blood. Inasmuch as the specificity of this reaction is a species speci-
ficity, it is satisfactory to utilize the animal's serum for immunization
rather than extracts of the flesh under suspicion.

In mixtures of meat such as one finds in sausages, the mixture in
itself sometimes interferes with the delicacy of the test. In these
cases it has been found that the complement-fixation test is likely to
give more satisfactory results.

The precipitin test is also applied in the enforcement of game
laws. For example, cases arise in which the unlawful possession
of venison is suspected, and the identity of the meat may be estab-
lished by the precipitin reaction.

Numerous suggestions have been made regarding1 the identification
of racial strains within species, but we agree with Hektoen in saying
that "suggestion to the contrary notwithstanding, it is not possible
to distinguish between different human races, and far less between
individuals, by means of the precipitin test."

Function of Precipitation in Immunity. — The function of pre-
cipitation in the protection against infection is not clear, and, indeed,
according to certain theories, it may play a part in hypersusceptibility.
Friedberger has shown that the addition of complement to a precipitin-
precipitinogen mixture leads to the formation of a toxic body, but there
is no convincing evidence that this actually takes place in the living
animal (see page 218) . It is to be considered possible, on the other hand,
that a certain amount of protection against foreign proteins may depend
on precipitation, the precipitate being less harmful and more suscep-
tible to the destructive action of ferments.

CHAPTER VI
CYTOLYSINS

INTRODUCTION.
HEMOLYSINS.

IMMUNE HETERO-HEMOLYSINS.
HEMOLYTIC AMBOCEPTORS.

PREPARATION OF IMMUNE HEMOLYSINS.
OBTAINING ANTIGENIC BLOOD.

PREPARATION AND COLLECTION OF IMMUNE SERA.
TITRATION OF IMMUNE SERA.
TITRATION OF COMPLEMENT.

QUANTITATIVE RELATIONS OF AMBOCEPTOR AND COMPLEMENT.
QUANTITATIVE RELATIONS OF AMBOCEPTOR AND ANTIGEN.
RELATIVE AFFINITIES OF AMBOCEPTOR AND COMPLEMENT.

SELECTIVE ABSORPTION OF AMBOCEPTOR.

INFLUENCE OF AMOUNT OF COMPLEMENT.

RATE OF ABSORPTION OF AMBOCEPTOR.

DISSOCIATION OF AMBOCEPTOR ANTIGEN UNION.

SPECIFICITY OF AMBOCEPTORS.

GROUP REACTIONS.
NATURE OF THE ANTIGEN.
NATURE OF THE AMBOCEPTOR.

MECHANISM OF OPERATION OF AMBOCEPTOR.

CONGLUTININS.
COMPLEMENT.

DISTRIBUTION.

ALTERATIONS OF AMOUNT.

METHOD OF OBTAINING COMPLEMENT.
ORIGIN OF COMPLEMENT.
NATURE OF COMPLEMENT.

PRESERVATION.

VARIABILITY OF COMPLEMENT.

MULTIPLICITY OF COMPLEMENTS.

COMPLEMENTOIDS.

COMPLEMENT FRACTIONS.

NORMAL HETERO-HEMOLYSINS.

PROPORTIONS OF AMBOCEPTOR AND COMPLEMENT.
NORMAL ISO-HEMOLYSINS.
A NTI-A M BOCEPTORS .
ANTI-COMPLEMENTS.
PHYSICAL HEMOLYSIS.

FRAGILITY OF ERYTHROCYTES.
CHEMICAL HEMOLYSIS.
BACTERIAL HEMOLYSINS.

OTHER VEGETABLE HEMOLYSINS.
' VENOM HEMOLYSINS.
CYTOTOXINS.
SPECIFICITY.
LENS CYTOTOXIN.
BACTERIOLYSINS.

THE PFEIFFER PHENOMENON.
BACTERIOLYSIS IN VITRO.
WRIGHT'S METHODS.
NEISSER-WECHSBERG PHENOMENON.
BUXTON'S METHOD.
BIOSCOPIC METHOD.

SUMMARY.

n6 THE PRINCIPLES OF IMMUNOLOGY

Introduction. — In the study of resistance to disease it was learned
very early in the course of the investigations that the blood serum
possesses the property of destroying bacteria. Later it was found that
blood serum may possess similar power in regard to other cells, in-
cluding various animal cells, particularly the erythrocytes. Rather
than consider the subject of cytolysis in a historical fashion, we believe
that it may be much more clearly discussed by first presenting the
established facts which have been learned concerning the power of
blood serum to destroy red blood-corpuscles. Many substances other
than blood serum may destroy erythrocytes and immunology has
profited from the study of hemolysis resulting from chemical and
physical agents, but the greatest advance has been made by the investi-
gation of the hemolytic properties of the blood.

Hemolysins. — Hemolysins are classified, in the same manner as
hemagglutinins, into autohemolysins, iso-hemolysins and hetero-
hemolysins. These may be present normally or may be produced as a
result of immunization. Landois in 1875 studied those normal hetero-
hemolysins which for years have made blood transfusion a dangerous
operation and showed that fresh sera from various species possess the
power of dissolving or laking the erythrocytes of certain foreign species.
In 1898 Belfanti and Carbone noticed that the serum of a horse which
had received numerous injections of rabbit blood was, upon injection,
specifically toxic for rabbits, but they did not determine the cause of
the toxicity. In the same year Bordet published his discovery of the
fact that several injections of defibrinated rabbit blood into the peri-
toneal cavity of guinea-pigs led to the production of an immune body in
the guinea-pig serum capable of rapidly laking rabbit erythrocytes,
whereas normal guinea-pig serum possesses the same property in only
slight degree or not at all. Shortly afterward von Dungern and Land-
steiner independently published similar results. The immune body
in the serum has been named hemolysin and also hemotoxin, but the
former term has received much wider usage, because the constitution
of this body is not that of true toxins, because the effect is seen on the
blood-corpuscles rather than the whole blood, and because the hemo-
globin is liberated for solution in the surrounding medium without
actual destruction of the stroma. Bordet in his study of the subject
showed that heating the serum to 55° C. for thirty minutes so altered
it that it no longer produced hemolysis; in other words, it became
" inactive." It could, however, be reactivated by the addition of a
small amount of fresh normal serum. This indicated that two sub-
stances are concerned in the hemolytic activity of blood serum, a
thermostable substance present in the immune blood and a thermolabile
substance present in normal blood as well as in immune blood. Bordet
named the immune thermostable body " substance sensibilisatrice " and
Buchner named the thermolabile body " alexine." Ehrlich and Morgen-
roth, whose studies have been of fundamental importance, named the
thermostable body " amboceptor " and the thermolabile body " com-
plement." Others have given other names, but these two forms of

CYTOLYSINS 117

nomenclature are most frequent in the literature. We have elected to
use the terms amboceptor and complement because of our belief that
these terms have attained the more widespread usage. Complement
appears in the blood of many species, but may be very small in amount
or absent from certain species. Certain complements may operate
with the amboceptors of only a few species, whereas others may act
with amboceptors of a large number of species. Within a given species
different individuals may possess complement in variable quantity, and
it may vary at different times in the same individual. The complement
of guinea-pig blood is usually large in amount and applicable to the
amboceptors of a considerable number of other species. Complement
does not appreciably change in amount by the ordinary processes
of immunization.

Immune Hetero-hemolysins. — Hemolytic amboceptors may be
natural to a blood or may be developed by immunization. Autolysins
and isolysins may be produced but with great difficulty and variability.
Isolysins may be present normally, notably in man. Heterolysins may
be found normally and can be readily produced by artificial immuniza-
tion. Bordet produced heterolysins by intraperitoneal injection of ery-
throcytes. They may also be induced by the subcutaneous and by the
intravenous routes of inoculation. Two important conceptions of the
mode of action of the amboceptor have been proposed. Bordet, Metch-
nikoff and the French school consider the action to be in the nature of a
sensitization or fixation of the antigenic cells, so that they are more
readily acted on by the complement, in somewhat the same fashion
that a mordant prepares a cell so that it will stain more readily. Ehr-
lich and Morgenroth and the German school consider the amboceptor
as a link which brings together antigen and complement ; in other words,
in their conception the amboceptor possesses two binding groups, a
cytophilic and a complementophilic group, each capable of acting as a
specific receptor. In order to discuss the various theoretical con-
siderations more clearly, it is essential that the well-established facts in
regard to hemolysis be presented as they are ordinarily demonstrated
in a practical way.

Preparation of Immune Hemolysins. The Blood Antigen. — In immuniza-
tion for the production of a hemolysin it is necessary to select the animals to
be used. The rabbit is usually chosen as the animal to be immunized because
of the fact that it is easily available, relatively inexpensive, and yields a fairly
large amount of blood. In selecting the animal whose blood-corpuscles are to
be used for the production of hemolysin, convenience again plays a part. The
sheep is the animal most commonly employed, although the goat is equally use-
ful. Reasonably large amounts of blood can be secured from such animals at
short intervals of time, without deleterious effects. Dog blood is unsatisfactory
because the corpuscles do not resist standing for any length of time. The cat is
undesirable because of its relatively small size. In order to secure blood from
a goat or sheep the animal is either strapped on a board, or may be held by a
skilled attendant. The neck is shaved over the jugular vein, the area washed
with soap and water, cleansed with alcohol, and the vein distended by pressure
over the jugular bulb at the base of the neck. The blood is collected through a
fairly large needle into a sterile flask containing glass beads or fragments of
glass tubing. Rotation of the flask or shaking during the collection and con-
tinued shaking for five or ten minutes after the collection completely defibrinates

ii8 THE PRINCIPLES OF IMMUNOLOGY

the blood. The blood may be injected as defibrinated blood for purposes of im-
munization, but as a rule in order to avoid any influences the serum may have,
the blood is washed so that only corpuscles are injected. For purposes of washing
50 c.c. centrifuge tubes are desirable, but if these are not obtainable, the 15 c.c.
size may be employed. The blood is measured into the tube with a pipette,
usually to the amount of 5.0 c.c. The amount of blood is marked with a
grease pencil and the tube filled with physiologic salt solution. The tube is
centrifuged until the blood is thrown down. The supernatant fluid is poured off
and the tubes again filled with salt solution. The sedimented corpuscles are
shaken up into the salt solution and again centrifuged. This operation is re-
peated again, and the blood is said to have been washed three times. After
the last centrifugation the supernatant fluid is poured off and the sedimented
blood-corpuscles restored to original volume by addition of salt solution. In
order to make a five per cent, suspension, this is washed into a 100 c.c. cylinder
and made up to 100 c.c. volume with salt solution. Any other percentage
desirable may be made by appropriate additions of salt solution to the original
blood mass.

Preparation and Collection of Immune Sera.— The injection into the rabbit
may be by subcutaneous, intraperitoneal, or intravenous routes. The intravenous
route produces immune bodies most rapidly, as has been shown by Bullock,
and as a rule produces an immune serum of higher titer than is obtainable by
other methods. An excellent way to produce hemolysin rapidly is to inject in-
travenously into the rabbit three doses 4.0 c.c. each of 50 per cent, suspension
of washed sheep or goat erythrocytes at intervals of five days. The method of
intravenous injection has previously been described in connection with the pro-
duction of agglutinins (see page 82). A test bleeding may be made from the
posterior ear vein five to seven days after the last injection, and if the titer of
the serum is not sufficiently high, one or two more injections may be given. When
sufficiently high the rabbit is bled from the femoral artery as previously
described (see page 83). The blood is collected in a flask, the flask inclined
at an angle of about 45° until the blood is firmly clotted. The flask is then placed
in an upright position in the refrigerator for about twenty-four hours, after
which the collected serum is pipetted into a sterile container. Melick, in a study
of the influence of colloidal suspensions on the production of hemolysis, finds
that if he gives preliminary intravenous injection of aleurpnat suspension and
subsequently immunizes with blood-corpuscles, the hemolytic sera are of con-
siderably higher titer than in animals not so treated.

Titration of Immune Sera. — For titration of the hemolysin in the rabbit
serum, it is necessary to have a 5 per cent, suspension of the antigenic corpuscles,
the serum to be tested and in addition fresh guinea-pig serum which serves as
complement. In such a titration the 5 per cent, suspension of corpuscles and the
complement are regarded as standards and employed in the same doses through-
out the series of tubes. If 0.5 c.c. of serum and 0.5 c.c. of corpuscle suspension
are employed, 0.05 c.c. of complement is usually sufficient, Before attempting
the titration the rabbit immune serum should be inactivated by heating in a
water bath at 56° C. for one-half hour. Dilutions of the inactivated immune
amboceptor are made as a rule i-io, i-ioo, 1-500, i-iooo, 1-1500, 1-2000, 1-2500,
1-3000, 1-4000. The guinea-pig serum (complement) is diluted i-io. The follow-
ing protocol will show how the series of tubes is set up :

Amboceptor Complement i-io Result

0.5 c.c. -ioo 0.5 c.c. 0.5 c.c. CH

0.5 c.c. -500 0.5 c.c. 0.5 c.c. CH

0.5 c.c. -1000 0.5 c.c. . 0.5 c.c. CH

0.5 c.c. -1500 0.5 c.c. 0.5 c.c. CH

0.5 c.c. -2000 0.5 c.c. 0.5 c.c. CH

0.5 c.c. -2500 0.5 c.c. 0.5 c.c. CH

0.5 c.c. -3000 0.5 c.c. 0.5 c.c. PH

0.5 c.c. -4000 0.5 c.c. 0.5 c.c. N

0.5 c.c. ...... 0.5 c.c. 0.5 c.c. N

0.5 c.c. i-ioo 0.5 c.c. ...... N

The last two tubes are controls and should be made up to volume by addi-
tion of 0.5 c.c. saline in place of amboceptor in the one and of complement in the

other. The letters CH indicate complete hemolysis, PH partial hemolysis, and

a

FIG. 12. — a shows a saline suspension of blood-corpuscles before

hemolysis; b the same after hemolysis. a' and b' present the

appearance of a and b after sedimentation of corpuscles. From

Noguchi, Serum Diagnosis of Syphilis.

Green -» Complement
Purple <= Amboceptor
Red =• Haemolysis

/unit of Amboceptor
used in esc/7 w/mvar/ous
fractions or 3 comp/ement
unit

FIG. 13. — Diagrams showing relative proportions of reagents in hemolysis. The
group of four columns shows the reduction possible in amounts of_ complement with
increasing amounts of hemolytic amboceptor, in the production of complete hemol-
ysis. The second and third parts of the figure show the curve of reduction in
percentage, or degree, of hemolysis following reduction in amount of comple-
ment in the presence of constant quantities of amboceptor. From Noguchi, Serum
Diagnosis of Syphilis.

CYTOLYSINS 119

N no hemolysis, the reading being made after a period of incubation in the
water bath at 37° C. This period may be thirty minutes, one hour or two hours,
but subsequent experiments with the same system of amboceptor, complement
and corpuscles must be made with the same period of incubation as practised in
the original titration. In this laboratory one hour is the standard time for incu-
bation. In order to make results somewhat more clear-cut, the rack of ' test
tubes may be placed in the refrigerator over night and the results read the fol-
lowing morning. The lapse of twelve or eighteen hours time permits the cor-
puscles to settle to the bottom' of the test-tube ; therefore any red coloring of
the supernatant fluid may be interpreted as a partial or complete hemolysis, de-
pending on the depth of color and the amount of sediment remaining on the
bottom of the tube. The controls which are used in this experiment demon-
trate that neither complement nor inactivated amboceptor will produce hemoly-
sis. The result given in the above experiment indicates that at some point
between the dilutions 1-2500 and 1-3000 the exact end point of titration is to be
found. In order to determine the exact end point it is well to set up an addi-
tional series with dilutions of 1-2500, 1-2600, 1-2700, 1-2800, 1-2000, and 1-3000
with the necessary controls. If it is found that complete hemolysis takes place
in a dilution 1-2700 and not in the dilution 1-2800 the dilution 1,2700 is taken as
the end point or titer. The unit of amboceptor therefore is 1-2700 of 0.5 c.c. or
1-5400 of i c.c. In the experiment outlined above, the unit of amboceptor would
be designated as 0.5 c.c. of a 1-2700 dilution of the immune serum.

Titration of Complement. — As has been indicated previously, the amount
of complement in guinea-pig serum varies in different animals. Therefore sub-
sequent experiments with this amboceptor must be controlled by titrating the
complement. This may be done by setting up a series of tubes as follows, the
control tubes being made up to volume with salt solution :

Erythrocytes Amboceptor Complement PPC,,H

suspension 1-2700 i-io

0.5 c.c. 0.5 c.c. 0.5 c.c. CH

0.5 c.c. 0.5 c.c. 0.4 c.c. CH

0.5 c-c. 0.5 c.c. 0.3 c.c. PH

0.5 c-c. 0.5 c.c. 0.2 c.c. N

0.5 c-c. 0.5 c.c. o.i c.c. N

0.5 c.c. ... 0.5 c.c. N

0.5 c.c. 0.5 c.c. ... N

0.5 c.c. ... ... N

In this experiment it is found that 0.4 c.c. of the new complement is sufficient
for activating the unit of amboceptor. Therefore, whereas in the first experiment
0.5 c.c. i-io complement dilution was the unit of complement, in the second experi-
ment 0.4 c.c. i-io dilution complement is the unit. If it is found that in none of
these tubes complete hemolysis takes place because of weak complement, it will
then be necessary to set up an additional series with complement diluted 1-5
instead of i-io.

Quantitative Relations of Amboceptor and Complement. The

quantitative relationship between the amount of complement and ambo-
ceptor used has been very extensively studied. It is now known that
a larger amount of complement will require a smaller amount of ambo-
ceptor for the production of complete hemolysis in the standard blood-
corpuscle suspension and conversely a smaller amount of complement
requires a larger amount of amboceptor. Thus, if we use two units of
complement, hemolysis will occur in the presence of less than one unit
of amboceptor. If we use two units of amboceptor, it will require
less than one unit complement to produce complete hemolysis. This
relationship, however, is not in definite proportion. For example, if
four units of amboceptor are employed, one-third unit of complement
is necessary. This relationship is beautifully illustrated in the dia-
gram (Fig. 13) taken from Noguchi.

120 THE PRINCIPLES OF IMMUNOLOGY

Quantitative Relations of Amboceptor and Antigen. — By subse-
quent studies of different mixtures, it was found that the unit of
standard corpuscle suspension can take up considerably more than one
unit of amboceptor. This amount varies with the total quantity of im-
mune body present. For example, Muir found that on addition of
twelve doses of amboceptor one dose remained free, on addition of six-
teen doses of amboceptor two doses remained free, on addition of
twenty doses three doses remained free and on the addition of twenty-
three doses of amboceptor, four doses remained free. When, how-
ever, the mixture of complement, amboceptor and red blood-
corpuscles is properly adjusted, the reaction completely uses up the
amboceptor, complement and, by hemolysis, all the red blood-corpuscles.
Correspondingly, if two units of complement are employed in the
presence of one unit of amboceptor, it does not follow that after
the reaction one unit of complement will remain free. As a matter
of fact, practically the entire two units of complement will be utilized
in the reaction. Nevertheless, increasing the amounts of complement
will leave more and more complement free in the supernatant fluid.
These points will be made somewhat clearer after subsequent experi-
ments have been outlined.

Relative Affinities of Amboceptor and Complement. — In the in-
troductory paragraph it was pointed out that the amboceptor has a
special affinity for the antigenic red blood-corpuscles, but that the com-
plement has no such affinity. This is illustrated by the fact that when
red blood-corpuscles are set up against amboceptor they will absorb
the amboceptor, but if they are set up in the presence of complement
they will not absorb complement.

The following experiment illustrates this point: Two centrifuge tubes
are marked A and B. In tube A are placed i.o c.c. standard erythrocyte sus-
pension (5 per cent, suspension) and i.o c.c. inactivated immune serum so
diluted as to contain two units amboceptor. This tube is incubated at 37° C.
for thirty minutes and then centrifuged. The supernatant fluid is pipetted
into a tube marked A 2. The erythrocyte sediment in tube A is washed in
salt solution, again centrifuged and the supernatant fluid discarded. The
sediment in tube A is resuspended in i.o c.c. salt solution and two units of
complement, i.e., i.o c.c. i-io dilution are added. To tube A 2 are added two
units complement (i.o c.c. i-io dilution) and i.o c.c. 5 per cent, 'erythrocyte
suspension. These tubes are incubated for one hour at 37° C. Tube A will
show hemolysis because the sedimented corpuscles have absorbed ambo-
ceptor and the addition of complement is sufficient to complete the reaction.
Tube A 2 will not show hemolysis because the amboceptor is not in the
supernatant fluid and the complement is not sufficient to lake the added
corpuscles. At the same time the converse of the foregoing experiment may
be conducted. In tube B are placed i.o c.c. standard erythrocyte suspension
(5 per cent, suspension) and i.o c.c. fresh guinea-pig serum diluted i-io.
This is incubated thirty minutes at 37° C, centrifuged and the sediment
washed. The supernatant fluid is placed in tube B 2. The sediment in tube B
is resuspended in i.o c.c. salt solution and i.o c.c. immune serum so diluted
as to contain two units amboceptor is added. To the supernatant fluid in
tube B 2 are added i.o c.c. immune serum (two units amboceptor) and i.o c.c.
erythrocyte suspension. These tubes are incubated for one hour at 37° C.
Tube B 2 will show hemolysis because the supernatant fluid after the centrifu-
gation still contains complement, so that the addition of amboceptor and
erytnrocytes permits of completion of the reaction. Tube B will not show

CYTOLYSINS 121

hemolysis because the corpuscles in the sediment have not taken up any
complement, and the addition of amboceptor is not sufficient for the
reaction to occur.

Selective Absorption of Amboceptor and Complement. — Not only
is it possible to show, as has been done in the preceding experiment,
that red blood-corpuscles will combine with amboceptor and not with
complement, but if conditions are so arranged that hemolysis is pre-
vented, it is possible to demonstrate that red blood-corpuscles will
selectively absorb amboceptor from a mixture of amboceptor and
complement. In order to prevent hemolysis, it is necessary to permit
the absorption to take place at o° C. Not only must this precaution
be observed, but the tubes must be cooled, the various reagents in the
mixture must be cooled in advance and the centrifuge carrier must
also be cooled.

The various reagents are placed in test tubes and all the tubes placed in
a mixture of salt and ice. Into a cold centrifuge tube are placed i.o c.c.
5 per cent, erythrocyte suspension, i.o c.c. inactive immune serum so diluted
as to contain two units amboceptor and i.o c.c. guinea-pig serum, i-io dilu-
tion. This tube remains in the salt-ice mixture for thirty minutes and is
then centrifuged. The supernatant fluid is poured off and divided so that
one-half the amount is placed in each of two tubes. The sediment is washed
in cold salt solution and resuspended in 4.0 c.c. cold salt solution. These
4.0 c.c. are divided between two tubes. The four tubes so prepared are set
up as follows:

TUBE i

Supernatant fluid 1.5 c.c.

Fresh guinea-pig serum, i-io 0.5 c.c.

5 per cent, erythrocyte suspension 0.5 c.c.

TUBE 2.

Supernatant fluid 1.5 c.c.

Immune rabbit serum 0.5 c.c.

5 per cent, erythrocyte suspension 0.5 c.c.

TUBE 3.

Sediment 2.0 c.c.

Fresh guinea-pig serum, i-io 0.5 c.c.

TUBE 4.

Sediment 2.0 c.c.

Immune rabbit serum 0.5 c.c.

These tubes are incubated for one hour at 37° C. Inasmuch as the
supernatant fluid no longer contains amboceptor, tube i will fail to show
hemolysis, but in the case of tube 2 the amboceptor is added, and since the
supernatant fluid contains complement which has not been absorbed by the
corpuscles, hemolysis will result. The sediment has absorbed amboceptor
from the mixture ; therefore, in the case of tube 3, the addition of fresh guinea-
pig serum will serve to produce hemolysis. The sediment has not, however,
taken up any complement, the addition of the immune serum in tube 4 will
not serve to complete the reaction, and hemolysis will not occur. By the
use of sera containing other hemolytic amboceptors, it is possible to show
not only that absorption of amboceptor may occur from a complement
amboceptor mixture, but that this absorption is specific for the particular
amboceptor concerned.

Influence of Amount of Complement. — Although, as will be shown
subsequently, the concentration of complement plays a part in the com-

122 THE PRINCIPLES OF IMMUNOLOGY

pletion of hemolysis it can be demonstrated that the absolute amount
rather than the degree of concentration is of importance in regard to
the amboceptor.

This may be shown by placing in each of four tubes 0.5 c.c. 5 per cent,
suspension of corpuscles and adding to the second, third and fourth tubes,
respectively, four, nine and fourteen volumes of salt solution. To each of the
four tubes is added one unit amboceptor, and the mixture incubated at 37° C
for one-half hour to permit absorption of amboceptor. The tubes are cen-
trifuged and the supernatant fluid is discarded. To each tube is added i.o c.c.
complement so diluted as to contain one unit, and the mixtures again incu-
bated at 37° C. for one hour. Hemolysis will occur equally in all tubes
showing that the complete absorption of amboceptor by the cells occurred
in spite of marked dilution in some of the tubes.

Rate of Absorption of Amboceptor. — The absorption of ambo-
ceptor varies in rapidity under different conditions. For example,
absorption takes place more readily at 37° C. than at 20° C., and more
readily at 20° C. than at o°. An exception to this rule appears in
cases of paroxysmal hemoglobinuria. Some of these cases possess in
the blood an autohemolysin which does not enter into combination with
erythrocytes at body temperature. If the blood is withdrawn and
placed at a temperature of o° to 10° C. for an hour the cells absorb the
amboceptor and subsequent incubation at 37° C. permits the inter-
action of complement so that hemolysis results. With this and
possibly some other exceptions the general rule holds true that tem-
peratures approaching 37° C. favor the union of amboceptor and antigen.
Certain physical conditions also play a part in rapidity of absorption
as may be shown by the following experiment in which the mixture of
corpuscles and amboceptor is made under different conditions.

Two wide test tubes or small beakers are marked A and B. In A are
placed six units of cell suspension; namely, 3.0 c.c. 5 per cent, suspension.
To this are added drop by drop 3.0 c.c. amboceptor, so diluted that it con-
tains six units, the tube being shaken constantly during the addition. In
tube B the process is reversed, the amboceptor being placed in the tube and
the cell suspension added drop by drop. These mixtures may be titrated
against varying amounts of complement in a series of tubes, or six units of
complement may be added to tube A and tube B. An hour's incubation at
37° C. will show less active hemolysis in tube B than in A. The probable
explanation is that the first cells added to the amboceptor in tube B absorb
all or nearly all the amboceptor, and the subsequently added cells are only
partly saturated or take up no amboceptor at all.

This experiment illustrates the very rapid absorption of amboceptor
by cells and also the fact that cells may absorb considerably more than
one unit of amboceptor.

Dissociation of Amboceptor-Antigen Union. — Whereas tempera-
tures up to 37° C. appear to favor absorption of amboceptors, Bail,
Tsuda and others have shown that a temperature of 42° C. results in
a partial dissociation of amboceptor. That dissociation of amboceptor
and cells could occur was shown independently by Muir and by Mor-
genroth. Muir mixed i.o c.c. 5 per cent, corpuscle suspension with
ten units amboceptor and allowed the mixture to stand at room tem-
perature for one hour. The tube was then centrif uged, the corpuscles

CYTOLYSINS 123

washed three times and resuspended in salt solution to a volume of i.o
c.c. To this was added i .o c.c. untreated corpuscle suspension, the tube
shaken and placed at 37° C. for one hour. At the end of this time
four units of complement were added, the tube incubated again for
one hour and complete hemolysis was found. Thus it was found that
the original cells yielded at least one unit of amboceptor for the new
cells. Although in the report cited on page 120, twelve units gave one
free unit, Muir states that usually one unit of amboceptor can be
obtained from corpuscles containing six units. In this experiment the
dissociation was at 37° C., but dissociation takes place at room tem-
perature, although more slowly, and at o° C. it is practically nil. By
working with sensitized cells and with supernatant fluids, it is possible
to titrate the latter so as to determine the exact quantities of ambo-
ceptor dissociated. Kosakai, in working with so-called pure hemolysins,
has recently shown that the antigen and amboceptor union is reversible to
a greater extent than has previously been supposed. He main-
tains that the reversibility under these circumstances is almost or
quite complete.

Specificity of Amboceptors. Group Reactions. — The hemolytic
amboceptors are highly specific, but show, as do other immune bodies,
group reactions. Ehrlich and Morgenroth showed that immune sera
prepared against ox blood are hemolytic also for goat and sheep blood
and that a hemolysin prepared against goat blood also dissolves ox
blood. Marshall showed that an antihuman hemolysin acts on monkey
blood and vice versa. In any case the hemolysin is most active in the
presence of the antigenic corpuscles. Treatment of a hemolytic im-
mune serum with heterologous corpuscles removes more of the specific
immune body than is the case in other group reactions. For example,
Muir developed an anti-ox-blood serum which, in a dose of 0.0005 c.c.
dissolved i.o c.c. 5 per cent, suspension ox corpuscles and, in a dose
of 0.0012 c.c. dissolved a similar suspension of sheep corpuscles. Ab-
sorption by sheep corpuscles in excess reduced the titer against ox cor-
puscles so that the serum dissolved the latter in doses of 0.0012 c.c. ;
in other words, the titer of the serum was reduced to about half its
original strength, Ehrlich and Morgenroth showed that if the quantity
of sheep corpuscles is carefully adjusted so as exactly to equal the
hemolytic power for such corpuscles, the fraction of amboceptor lytic
for sheep corpuscles may be absorbed without reducing the titer against
ox cells. If, however, the amboceptor for ox blood is removed by
absorption with ox corpuscles the hemolytic power for sheep corpuscles
is entirely destroyed. Thus it is seen that there is close similarity with
the group reactions of agglutinins and other immune bodies. Ehrlich
and Morgenroth explain the phenomenon by assuming that each ambo-
ceptor contains numerous " partial amboceptors " formed in the im-
mune animals in response to relatively undifferentiated receptors of the
antigenic cells. In other words, ox-blood corpuscles are supposed to
contain a certain number of receptors specific to those cells, and in
addition other receptors that are closely* similar to or identical with

124 THE PRINCIPLES OF IMMUNOLOGY

certain receptors of sheep cells and goat cells. Therefore, the injec-
tion of ox cells leads to the production of an amboceptor containing
partial amboceptors specific for ox blood and partial amboceptors
specific for the common receptors of ox, sheep and goat cells. The
removal of the partial amboceptors common to all three cell receptors
will not remove that specific for ox cells, but ox cells will remove both
the specific and common fractions. This explanation has been the
subject of much experiment, particularly with anti-hemolysins,, and
modern views are not entirely in accord with the original views of
Ehrlich. The subject will be referred to again in connection with a
discussion of anti-amboceptors and anti-complements. In the same
place will be found a discussion of the interpretation of the ambo-
ceptor as made up of a cytophilic and complementophilic group.

Nature of the Antigen. — In ordinary practice the entire erythrocyte
is employed for immunization, but attempts have been made to deter-
mine what fraction of the cell is truly antigenic. Ford and Halsey
have shown that the use of either stroma or the laked hemoglobin may
serve to produce hemolysins, but they obtained only questionable results
following the use of pure hemoglobin. Stewart obtained essentially
the same results. Nucleo-proteins obtained from dog blood are capable
of producing specific hemolysins. Pearce and his co-workers have
shown that nucleo-proteins from washed organs also lead to. the forma-
tion of hemolysins specific for the homologous species. Organ and
cell extracts free from blood also serve as hemolysinogens ; the best
example is an extract of spermatozoa, for in this instance there is
no question of blood contamination of the extract. Of further interest
is the fact that ether extracts of erythrocytes, alcohol-ether extracts,
and extracts in 1.5 per cent, sodium bicarbonate induce the formation
of weak hemolysins without the coincident formation of hemagglu-
tinins. This indicates that the hemagglutinin and hemolytic ambo-
ceptor are probably separate and distinct antibodies.

Nature of the Amboceptor. — The amboceptor, although it resists
heat of 56° for one hour or more, is injured by heat of 60° C. for twenty
minutes, is almost completely destroyed by 70° C. for one hour and is
completely destroyed by boiling. Like antitoxin, it does not dialyze,
is electro-positive and is resistant to ultra-violet rays. It is carried
down in the euglobulin fraction of the serum protein, but by various
methods of purification may be obtained in an almost protein-free
state. The method of purification described by Kosakai is of im-
portance from various points of view and deserves some description
at this point. He requires a hemolytic serum which titrates I— 10,000.
This is diluted to 100 times its volume with salt solution and 5 c.c. of
the diluted serum are poured into 4 c.c. blood-cell suspension. The
union of amboceptor and red cells is accomplished by exposure at room
temperature for fifteen to twenty minutes, after which the cells are
freed from serum by repeated washing. To the antigen-amboceptor
combination is added isotonic or slightly hypertonic aqueous solution
of a sugar such as saccharose, glucose or lactose, and the mixture incu-

CYTOLYSINS 125

bated at 55° C. for fifteen to twenty minutes, during which period it
is shaken several times. The mixture is centrif uged and the supernatant
fluid placed in a separatory funnel with five to ten volumes of ether
and shaken for one or two hours until the solution becomes quite
colorless. The saccharose solution is separated from the ether and
dialyzed in running water in order to free it from sugar and salt.
After dialyzation the solution is concentrated in a vacuum until it
reaches the original volume of blood serum employed. Strong salt
solution may prevent amboceptor from entering into combination with
complement, but it does not interfere with the amboceptor cell union.
Alkalis may prevent either form of union and may serve partly to
dissociate amboceptor cell combinations.

Mechanism of Operation of the Amboceptor. — As has been
pointed out previously, the action of amboceptor is differently inter-
preted by the Ehrlich and the Bordet schools. If the Ehrlich view of
the two-fold binding group is to be adhered to, it should be possible to
show on the one hand a combination with antigen, and on the other
a combination with complement. Of these possibilities there is no
doubt that combination with cells is possible, but as yet no conclusive
evidence has been produced to show a combination between comple-
ment and an amboceptor not united to its antigen. The discovery of
the Neisser-Wechsberg phenomenon (see page 147) was regarded as
demonstrating a combination between free amboceptor and comple-
ment. This explanation, however, does not take into account the
possible relationship to certain colloidal reactions such as have been
described in connection with the inhibition zone of strong agglutinins
and is therefore not to be regarded as settled. Ehrlich and Morgen-
roth stated that if amboceptor is repeatedly injected into animals an
anti-amboceptor is produced which serves to combine with the cytophilic
group of amboceptor, but Bordet found that a normal serum, free from
hemolytic amboceptor could be used to produce the same immune body,
and argued therefrom that this antibody could not be regarded as a
specific receptor. Ehrlich and Sachs admitted the fact of Bordet's
experiments and came to the conclusion that the substance is anti-
complementophile, rather than anti-cytophile. As will readily be seen
this argument presupposes the correctness of the Ehrlich conception
of amboceptor, and is therefore not to be accepted as conclusive. With-
out the actual demonstration of the union of free amboceptor and
complement, the union of antigenic cells and amboceptor is of quite as
much value in support of the Bordet view of sensitization as in support
of the Ehrlich hypothesis. Nevertheless, Ehrlich and Sachs have
reported what they believe to be a crucial experiment in that it appears
to show that at least in some instances free amboceptor and comple-
ment may combine. Horse serum is slightly hemolytic for guinea-pig
erythrocytes and ox serum is somewhat more so. If inactivated ox
serum and fresh horse serum are added to guinea-pig cells, hemolysis
occurs, the ox serum acting presumably as an amboceptor, the horse
serum as complement. If the guinea-pig cells are treated with inac-

126 THE PRINCIPLES OF IMMUNOLOGY

tivated ox serum for a time ordinarily sufficient for amboceptor ab-
sorption, washed free of serum and then treated with fresh horse
serum as a complement, no hemolysis occurs. Furthermore, under
these conditions no hemolytic immune body has been absorbed from
the ox serum. Hemolysis only occurs when fresh horse serum and
inactivated ox serum are added as a mixture. The interpretation is
that in this particular hemolytic system the amboceptor must be com-
bined with complement before the amboceptor combines with the cells,
or, in other words, that the complementophilic group of a free ambo-
ceptor has united with complement independently of the cyto-
philic group.

Conglutinin. — Bordet and Gay have studied the phenomenon de-
scribed in the preceding paragraph and have come to a different
conclusion as to interpretation because of their discovery of a
so-called " bovine colloid " in the ox serum. They attribute the
hemolysis in the Ehrlich-Sachs phenomenon almost entirely to the
amboceptor and complement of the horse serum. The complex
of guinea-pig cells and the two bodies in the horse serum serves
to attract the bovine colloid which augments the complementary action
of the horse serum so as to produce complete hemolysis and at the same
time produces marked agglutination of the cells. This colloid is
thermostable, is probably of protein nature, unites with a complex of
cells, amboceptor and complement, but does not act upon either normal
cells or cells saturated with amboceptor. Bordet and Streng in a later
study named the colloid " conglutinin." Streng found that the same
phenonemon could be demonstrated in regard to bacteriolysis and that
conglutinin is present in the sera of the ox, goat, sheep and certain
other herbivora but not in the sera of the cat, dog, guinea-pig, or bird.
Sachs and Bauer have not offered a better explanation of the phe-
nomenon unless the German theory of amboceptor is unqualifiedly
accepted. In our opinion both sides of this controversy deserve the
most careful consideration and much light may be thrown by further
study. The more modern views of immunological processes, influenced
as they are by the great advances in colloidal chemistry, tend toward
acceptance of the Bordet hypothesis of sensitization of antigen by the
thermostable constituent of cytolytic sera, at least until and unless
more conclusive contradictory evidence can be produced.

Complement. Distribution. — Complement is that thermolabile ele-
ment of normal blood which in the presence of amboceptor and antigen
completes the cytolytic reaction. As regards hemolysis, complement
in the presence of hemolytic amboceptor causes solution of the red
blood-corpuscles and thus renders the reaction visible. Complement
is found in the blood and in lesser amount in nearly all the other body
fluids except the aqueous humor of the eye. It is also found in inflam-
matory exudates and sometimes in transudates, but it is not present
in the urine, nasal secretion or the secretion of other glands except
that of the breast (milk). The amount in the blood is fairly constant
for any given individual, but during the first twenty-four hours after

CYTOLYSINS 127

birth the complement content of the blood has been found to be rather
small; Gay has found it to be somewhat less in women than in men.
Moro has found it to be less in bottle-fed than breast-fed babies.
Although individual variation may be great, there is a certain uni-
formity in different members of the same species. This is true through-
out a large number of species, except the horse, in which species it is
found to vary markedly. Different species as such contain different
amounts of complement. The guinea-pig contains, as a rule, more
complement per cubic centimeter than other species. Man and rabbit
contain less than the guinea-pig, and in the case of the mouse it is
very difficult to demonstrate any complement at all. It has recently
been found that insects and mollusks contain practically no complement.
Alterations of Amount of Complement. — The amount of comple-
ment in a given blood may be made to vary by artificial means. For
example, the injection of indifferent materials, such as foreign blood
plasma, bouillon, aleuronat, pepton, yeast, nuclein, physiological salt
solution, produces an increase in the amount of complement, but this
increase is not permanent. Similarly complement may be increased for
a short time following the injection of pilocarpin, phlorizin, staphylo-
cocci, oil of turpentine and thyreoidin ; exposing an animal to high
temperatures may also increase complement. Although it is generally
true that complement is not increased by immunization, nevertheless
Cantacuzene has recently shown that by injecting red blood-corpuscles
into certain marine invertebrates he is able to increase the amount of
complement in their blood. Complement may be reduced temporarily
by the injection of sodium taurocholate, potassium picrate, toluylendi-
amin and more permanently by experimental phosphorus poisoning,
experimental chronic suppuration, starvation and by alcohol poisoning.
If sensitized blood-cells, i.e., blood-cells saturated with amboceptor,
are injected into an animal, it can be demonstrated that the amount of
complement is reduced by the hemolysis which takes place in vivo.
Shaw has found that in the case of recently acquired syphilis, although
the blood before treatment shows no alteration of complementary ac-
tivity, yet the administration of salvarsan may reduce this activity
to a considerable degree. The experimental investigations of the effect
of disease in man on the complement content of his blood are very
unsatisfactory because human blood normally contains only a small
amount of complement and the detection of any variation is susceptible
to a wide margin of experimental error.

Method of Obtaining Complement. — Complement is usually obtained
from the guinea-pig-, although under special circumstances it may be obtained
from other animals. The blood may be withdrawn in any manner adapted to
such a procedure. In the case of the guinea-pig the method employed in this
laboratory is to anesthetize the animal very slightly, pull the hair from the
neck, make a longitudinal slit in the mid-line of the neck, place a 15 c.c. centri-
fuge tube toward the upper end of the slit with its lip firmly pressed into the
opening, then with a scissors snip the carotid artery, carefully avoiding the
trachea. The animal is then held head downward while the blood drains into
the tube. The blood is allowed to clot in the tube and the clot separated from
the side of the tube with a long sterile or clean needle, as the necessity of the
case indicates. The clot separates best at room temperature, but if centrifuga-

128

THE PRINCIPLES OF IMMUNOLOGY

tion cannot be done immediately the clot may be allowed to separate in the
ice chest. As soon as the serum has separated out of the clot, the tube is
centrifuged and the serum collected by means of a pipette. If guinea-pigs are
large, the blood may be collected in smaller quantities by heart puncture or by
bleeding from an ear vein, thus obviating the necessity for killing the animal.

FlG. 14. — Method of obtaining blood from guinea-pig (See text).

Origin of Complement. — Considerable controversy has been waged
concerning the origin of complement since the time that Hankin and

CYTOLYSINS 129

subsequently Metchnikoff expressed the belief that complement origin-
ates in the leucocytes of the body and is only liberated upon the»death
of these cells. Metchnikoff used the term cytase to indicate what we
now call complement and believed that the microphages gave rise to
a microcytase capable of dissolving blood and other body cells. Pfeiffer
and certain other German workers take a diametrically opposed posi-
tion and maintain that the leucocytes furnish none of the complement in
the blood. A. von Wassermann and also Landsteiner believe that the
leucocytes may constitute one source of origin for the complement,
and it seems practically certain from modern investigations that several
organs play a part in the formation of complement. Before the bac-
tericidal action of blood was thoroughly understood as due to the inter-
action of amboceptor and complement, certain studies seemed to indicate
that exudates rich in leucocytes were active as bactericidal agents, but
it is now understood that other constituents of the exudate take part
in this phenomenon and more recent experiments show that extracts
of leucocytes do not yield a complement. It has been shown further
that variations in the total leucocyte count in an animal produce no
corresponding variations of complement content. Neuf eld and certain
others take the view that even inside the living leucocytes there is no
complement because they have found that destruction of red blood-
corpuscles within living leucocytes takes place at a distinctly slower
rate of speed than is the case in ordinary hemolysis. Furthermore,
they point out that the method of destruction is quite different, in that
ordinary hemolysis shows simply liberation of hemoglobin without
destruction of the stroma. Metchnikoff 's belief that the death -of the
leucocytes yields complement was supported by an experiment which
apparently showed that complement is present in serum after clotting,
but not in plasma. A considerable amount of experimental evidence
has been adduced, since this statement of Metchnikoff, to show that
plasma contains complement in the same amount as does serum. Some
of these experiments appeared to be invalid on the ground that im-
munological work with a plasma is likely to lead to coagulation, thus
producing a serum for the actual experiments. After these objections
had been presented, further experiments were performed which over-
came such objection, and it now seems perfectly clear that plasma con-
tains complement. This fact has been firmly established by the
recent work of Watanabe.

Nature of Complement. — Complement is probably of protein
nature, inasmuch as it is destroyed in coagulation of the serum by heat
and is digested by trypsin. Noguchi and his co-workers were of the
opinion that complement is a combination of soap and a protein, but
numerous other workers failed to confirm these studies. This state-
ment of Noguchi, as well as the work of Kyes, with cobra venom led
to the hope that it might be possible to prepare an artificial complement.
Landsteiner and Jagic have investigated the question and have shown
that whereas it is possible to substitute for amboceptor a colloidal
solution of silicic acid, which nevertheless shows none of the specific
9

130 THE PRINCIPLES OF IMMUNOLOGY

characters of amboceptor, it is absolutely impossible up to the present
time to offer any substitute for complement. Complement resembles
an enzyme in that it is thermolabile, disintegrates cells, does not pass
through Berkefeld filters, is adsorbed by kaolin and destroyed by
shaking. Furthermore, it activates amboceptor much in the same
manner as entero-kinase activates trypsinogen. As against the idea that
complement is an enzyme is the fact that in the reaction of hemolysis,
hemoglobin is liberated without destruction of the stroma of the cells
and the further fact that complement acts quantitatively, following in
a general way the law of multiple proportions. As is well known, heat
at 56° to 60° C. for one-half hour destroys the complementary activity
of a serum. It has recently been shown, however, that if heat of
56° C. is applied for only a short period, i.e., from seven to te.n minutes,
the complementary action is restored after several hours have elapsed
(the phenomenon of Gramenitski). This is interpreted as due to an
agglomeration or aggregation of protein particles resembling heat
coagulation of protein. The restoration of activity after standing is
ascribed to a dispersion of the protein aggregates so that they can
act nearly or quite as they did originally. Ultra-violet rays destroy
complement, but it is stated that X-rays do not. Recent work in this
laboratory by Ecker has shown that the visible spectrum also serves to
reduce complementary activity. Experimental conditions in this in-
stance made it possible to work with three divisions of the spectrum,
namely, a division near the violet end, a division in the middle of the
spectrum and a division near the red end. It was found that those rays
toward the violet end of the spectrum were more active than the
rays in the middle of the spectrum and the latter were more active
than the rays at the red end of the spectrum. That this is a function of
the wave-length of the ray is not absolutely certain but seems probable
in view of the work of Bovie, Brooks and others, which shows that the
presence of cells in the serum reduces the activity of the ultra-violet
rays. That the destruction, however, is a function of the penetrability
of the rays is not borne out by the statement that X-rays fail to
destroy complement. We have also been able to show in this lab-
oratory that drying of complement produces some deterioration. Other
workers have stated, however, that if the complement is mixed with
a proper concentration of salt, preferably about 8 per cent, -and then
dried, the salting nullifies the destructive action of desiccation and
the dried serum under these circumstances may be preserved for a
considerable period of time. Complement may be inhibited by the
presence of hydroxyl ions but is restored to activity by the addition of
hydrogen ions. Complement can be made to combine with magnesium,
calcium, barium, strontium and sulphate ions and can be separated by
simple chemical precipitation. Acids and alkalis in sufficient concen-
tration also serve to destroy complement.

Preservation of Complement. — Owing to the extreme lability of
complement, the question of prolonged preservation assumes consid-
erable importance. The fresh serum may be desiccated in air, in

CYTOLYSINS 131

vacuum, in vacuum after freezing, or on filter paper. In the hands of
certain workers various methods of this sort have proven more or
less successful but do not seem to be widely applicable. It is of im-
portance to keep in mind that under such conditions the desiccation
of serum does not remove the possibility of the destructive action of
light. Other methods of preservation include salting with sodium
chloride and also with sodium acetate. The former has been fairly
successful, but the latter has been completely abandoned. Another
method is salting and then freezing, but this has been found to be in
no way superior to freezing without salting. According to Bigger,
it is of extreme importance that the serum should be sterile to ensure
the success of any method of preservation. Browning and Mackie have
found that frozen serum kept at a temperature of —15° C. retains its
complementary power three months without appreciable loss. Noguchi
and Bronfenbrenner found that at 10° C. the serum loses one-half
its original strength at the end of twenty-four hours. If it is kept at
37° C. it loses two-fifths of its strength at the end of six hours; at
45° C. one-half hour exposure reduces it to one-third to one-half its
original strength ; at 50° C. 50 per cent, is lost in five minutes. They
have examined the rate of destruction at 55° and find that this goes
on quite irregularly and is not in proportion to the length of time.
The irregularity, however, presents a certain rhythm, i.e., a period of
greater destruction alternating with a period of less active destruction.
Reudiger has studied the preservation of frozen complement and finds
that at the expiration of one week whether the complement is made
up of serum of a single guinea-pig or the pooled serum of several
guinea-pigs the activity in the Wassermann is somewhat stronger than
with fresh serum. At the end of two weeks the frozen complement
gives results that are practically identical with the results obtained
with fresh complement, but after two weeks the frozen complement
gradually loses strength apparently more rapidly in mild weather than
in very cold weather.

Variability of Complement. — Complementary activity varies con-
siderably in different sera ; in the same serum it may operate differently
with amboceptors from several different species. The explanation of
this difference of activity has led to a difference of opinion as to the
exact nature of the complementary activity. Ehrlich and Morgenroth
and the German school take the position that a given serum contains a
considerable number of complements, whereas Bordet and his school
take the point of view that the complement in any given serum is a unit,
although they admit that complements in different sera may represent
a somewhat different constitution.

Multiplicity of Complements. — Ehrlich and Morgenroth were able
to show that the complementary activity of a serum could be divided
by means of filtration in the following respect. They showed that
complement for sensitized guinea-pig cells passes through the filter,
whereas complement for sensitized rabbit cells remains in the filter.
It has also been shown by thermal and chemical differentiation that

132 THE PRINCIPLES OF IMMUNOLOGY

some complements are destroyed and others remain in the same serum/
Weak acids and weak alkalis may differentiate complement similarly;
it is stated that digestion by papain also serves so to differentiate. By
injection of a complementary serum into foreign species, a so-called
anti-complement is obtained which is said to act upon one of the
complements of a serum and not upon others, irrespective of whether
the complement of the antigenic serum or of some other serum be em-
ployed in the subsequent test. Practically all these experiments have
been performed in such a way that the complement acts with normal
amboceptors and the question at once arises as to whether or not the
same phenomena would be observed with immune amboceptors. Even
in the case of normal amboceptors, there is experimental contradiction
of the original supposition of Ehrlich and Morgenroth. Neisser stated
that anthrax bacilli deprive fresh rabbit serum of its bactericidal com-
plement, but not of its hemolytic complement. Wilde showed that if
a sufficient mass of anthrax bacilli were added to the fresh rabbit serum
all the complement is used, so that further bactericidal action does not
occur and no hemolytic action can be demonstrated. Similarly Bordet
found that unsensitized red blood-corpuscles deprive a serum of only
part of its complement but that cells strongly sensitized with hemolysin
use up all the complement both bactericidal and hemolytic. He believes
that the reason normal amboceptors do not utilize all the available
complement is due to the fact that such normal amboceptors do not suf-
ficiently sensitize the antigenic cells. Therefore, the complete sensitiza-
tion of the cells will result in a complete utilization of complement.
After the publication of these experiments, Ehrlich, who confirmed the
results, explained the phenomenon as being due to a multiplicity of
complements in the serum. In order to do so, he was obliged to alter
the original conception of the amboceptor, so that instead of having
a single cytophilic group it must contain several cytophilic groups.
Therefore, the term was altered to polyceptor instead of amboceptor.
The polyceptor was supposed to have one group with an especial affinity
for a dominant complement and other receptors with affinities for the
secondary complements. If the dominant complement is absorbed by
the polyceptor, the secondary complements are also involved, but, on
the other hand, if, as has been claimed, it is possible to obtain a serum
with only secondary complements present, these may be absorbed
without action upon the receptor for dominant complement. This expla-
nation, however, rests entirely upon the Ehrlich conception of the ambo-
ceptor, and, inasmuch as this conception is not conclusively proven, it
is not necessary to accept the idea that complements are multiple. This
question, however, is not settled at the present time, and reference
will be made to it again in connection with the phenomenon of com-
plement fixation.

Complementoids. — The similarity in action and nature of comple-
ment and toxin was early recognized, and it was therefore attempted to
determine whether or not complement could be broken up in the same
way as toxin so as to form complementoids. If such were the case, it

CYTOLYSINS 133

should be possible to break up a complement so as to demonstrate a
haptophore group and a zymophore or zymotoxic group. Thus, ex-
posure to increased temperature might be so arranged as to destroy
the zymophore group and leave the haptophore group intact. If this
were true, the haptophore group or complementoid might be added to
an antigen-amboceptor mixture and thus prevent any further action
by a fresh whole complement. Experimentally, however, it was found
that this, in the majority of instances, does' not occur. Nevertheless,
Ehrlich and Sachs found that if they mixed inactivated guinea-pig
serum, normal inactivated dog serum, and guinea-pig cells, hemolysis
did not occur, even after the addition of fresh guinea-pig serum. They
believed this to be the result of a blocking or plugging of the com-
plementophile group of the dog amboceptor by the complementoid of
the inactivated guinea-pig serum, thereby preventing the union when
fresh active complement was added. Fuhrmann supported this state-
ment and maintained that allowing the complement to stand for a
period of three weeks was even more adapted to separation of the hapto-
phore and zymophore group. Following this work, Muir and Brown-
ing conducted an extensive series of complicated experiments, which
in the main tend to support the view of Ehrlich that complementoids
actually exist. If they do exist, however, they are not uniformly demon-
strable, and it may very well be that this is due to the difference in
destructibility of the two groups being so slight that our methods of
differentiating by means of heat and standing are not sufficiently exact.
Complement Fractions. — Further light was thrown on the possi-
bility of fractioning complement by the experiments of Ferrata, who
found that dialyzation of the serum resulted in the destruction of com-
plementary activity. Dialyzation precipitates the so-called globulin
fraction of the serum as contrasted with the albumin fraction which
remains in solution. The precipitate may be dissolved in physiological
saline and the portion in solution may be restored to its original salt
concentration, thereby forming isotonic solutions of the two protein frac-
tions. Ferrata found that neither of these components in the presence of
an amboceptor was capable of producing hemolysis, but that if both were
added, sufficiently soon after dialyzation, hemolysis would take place.
Brand studied this phenomenon further and found that both fractions
are equally thermolabile and because of activities which he discovered,
named the fraction contained in the globulin precipitate " mid-piece " and
that in the albumin " end-piece." If the amboceptor- cell mixture is
treated first with mid-piece, no hemolysis occurs, but if end-piece is then
added, hemolysis occurs as it would have in the original amount of com-
plement. If the end-piece is first added and later the mid-piece, hemo-
lysis will occur, but in very much smaller degree than if the entire
complement had been used. Zinsser found, however, that when mid-
piece and end-piece are mixed and then added to the sensitized cells,
the hemolytic effect is reduced and is considerably less than if mid-piece
is added before end-piece. It has been found that the mid-piece may
enter into combination with the sensitized cells at o° C, but the end-

I34 THE PRINCIPLES OF IMMUNOLOGY

piece combines only at considerably higher temperatures. It has also
been found that the mid-piece of one animal species may be activated
by the end-piece of another animal of the same or different species.
Nevertheless, Ritz and Sachs have shown that the serum of an animal
may possess a mid-piece for certain sensitized erythrocytes, but does
not necessarily possess a corresponding end-piece. Marks has studied
the quantitative relations of mid-piece and end-piece and has found that
a ratio of i-i is not necessarily the optimum for hemolysis and that
very often it is necessary to change the ratio; this change must be by
increase of mid-piece, never by increase of end-piece. If the two
are mixed before addition to the amboceptor-cell mixture, an excess
of mid-piece inhibits hemolysis, but if the excess of mid-piece is added
first followed by end-piece, hemolysis is complete. Brand and later
Hecker found that if the globulin precipitate is preserved dry or in
solution in distilled water it will retain activity for several days, but in
physiological salt solution it loses its activity in three to four hours.
This, however, does not indicate destruction of mid-piece in salt solu-
tion since it will combine with sensitized cells if added before end-
piece. Marks holds that this phenomenon is due to the inhibition of
hemolysis by excess of mid-piece and does not occur if proper pro-
portions are maintained in the mixture. Swirski maintains that the
complement fixation of the Wassermann test binds mid-piece but not
end-piece. This has been investigated by Bronf enbrenner and Noguchi,
who believe that the free end-piece in Wassermann tests differs from all
other end-pieces in that it activates the complex which includes sheep
cells but has no effect upon the cells of other animals. Bessemans has
recently investigated again the question of thermostability of end-piece
and mid-piece. He finds that there are important differences in certain
of the sera he has examined, so that a general statement in regard to the
heat resistance of these fractions is not justified.

Browning and Mackie have found that by various methods of f rac-
tioning the serum it is possible to divide complement into four frac-
tions and that certain combinations consisting, as a rule, of at least three
of these reproduce quantitatively the full hemolytic effect of the whole
complement. This presents numerous intricate possibilities of experi-
ment, but the important point is that such a demonstration makes the
use of the terms mid-piece and end-piece no longer desirable.

Normal Hetero-hemolysins. — The preceding discussion has been
concerned chiefly with complement and immune amboceptor. Histori-
cally much study had been directed toward the normal cytotoxic powers
of blood serum before the immune amboceptors were recognized;
Fodor, Nuttall, Nissen and Buchner had investigated the action of nor-
mal sera in dissolving bacteria. Buchner in 1893 pointed out a similar
capacity of blood serum for dissolving animal cells, particularly ery-
throcytes. Ehrlich and Morgenroth took up the question as to whether
or not the globulicidal activity of normal serum is due to an interaction
of two substances similar to that in immune sera. They showed that
normal dog serum is capable of dissolving guinea-pig erythrocytes, but

CYTOLYSINS 135

that it is inactivated by heating to 55° C. Reactivation by fresh dog
serum was undesirable because of the normal amboceptor present.
Therefore, they employed fresh guinea-pig serum in fairly large doses
and reactivated the heated dog serum so that complete hemolysis oc-
curred. Thus they demonstrated the double nature of the normal
hemolysins and also that a complement may serve to hemolyze cells
of the same species from which the complement is obtained. Other
experiments have shown, however, that a complement operates less
actively against homologous cells than against heterologous cells. Ehr-
lich and Morgenroth showed similar relationships by employing as the
amboceptor normal calf serum and normal sheep serum, as well as
similar hemolytic complexes with sheep and goat blood. They also
showed that in a number of instances . the amboceptor could be dif-
ferentially absorbed by cells at o° C., leaving complement free in the
serum. Such absorption could not be accomplished with all normal
hemolytic sera; in some the cells absorbed both amboceptor and com-
plement, whereas in others no absorption whatever occurred. They
interpreted the absorption of both bodies as due to the possession on the
part of the amboceptor of equal avidity of both the cytophile and com-
plementophile group, whereas failure of absorption was supposed to be
due to a stronger affinity of complement for amboceptor than of cells
for amboceptor. We record the fact without accepting the explanation,
but it is important that in some instances normal hemolytic sera may
fail to exhibit a separability of complement and amboceptor by means
of differential absorption.

A normal serum may be hemolytic for cells of more than one species ;
this is true of goat serum, which is hemolytic for both guinea-pig and
rabbit cells. In such cases it is possible to absorb one amboceptor, leave
the other active in the serum and thus demonstrate multiplicity of
specific amboceptors in a serum. Ehrlich and Morgenroth also main-
tained that in the case of goat blood there is a different complement for
the two types of cells, but as has been indicated in discussing comple-
ment this possibility seems unlikely.

Proportions of Amboceptor and Complement in Normal
Hemolysins. — Further difference between a normal and immune
hemolytic serum lies in the different proportion of amboceptor and
complement. In a normal hemolytic serum the amount of amboceptor
present is small and the complement is usually present in at least suf-
ficient quantity to saturate the amboceptor ; it may be present in excess.
In immune sera the amboceptor is increased enormously, whereas the
complement is not altered in quantity. Therefore, such an immune
serum may contain amboceptor in greater quantities than can be sat-
urated by complement and for its full activity requires more com-
plement than can be furnished in the immune animal's serum. This
increase in amboceptor is out of all proportion to the amount of antigenic
cells injected. Muir, for example, calculated that in immunizing a
rabbit the total amount of ox blood injected was 30 c.c., and hemolytic
amboceptor was produced sufficient to dissolve the erythrocytes in

136 THE PRINCIPLES OF IMMUNOLOGY

6000 c.c. of ox blood. The practical bearing of the disproportion of com-
plement to immune amboceptor lies in the use of bactericidal sera. It
is easily conceivable that injections of such sera may meet in the injected
animals' blood with an insufficient amount of complement for complete
activation. Therefore, it may be advisable in such experiments or in
therapeutic use of sera of this type to activate with a sufficient quantity
of fresh complementary serum.

Normal Iso-hemolysins. — Attention has been called (page 99) to
the phenomenon of iso-hemagglutination. Similarly iso-hemolysins
can be demonstrated. For such a purpose the serum must be fresh
and the corpuscles exposed to it at incubator temperature. It is prob-
able that these hemolysins resemble other normal hemolysins. The
groups correspond to the groups of iso-hemagglutinins. As in other
experiments with hemolysins, agglutination appears and is followed
by hemolysis. Agglutination inhibits hemolysis to a certain degree, as
has been shown by Handel and by Karsner and Pearce. Kolmer, Trist
and Flick have maintained in a recent study that there are two varieties
of natural hemolysin and hemagglutinin in human sera. They find a
thermolabile variety of these antibodies which is destroyed at 56° C.
for thirty minutes and a less thermolabile or thermostable body which
is destroyed at 62° C. for thirty minutes. Exposure at 56° C. removes
the various iso-hemolysins but does not destroy the iso-hemagglutinins.
Sands and West have found that if the immune sera are filtered (more
particularly in I— 10 dilution) through perfectly clean Kitasato or Cham-
berland filters a large amount of the hemagglutinin is removed, with
slight or no reduction of hemolytic activity. In fact, the hemolytic
activity may be increased by the filtration and this may be explained
as due to the removal of whatever inhibiting power on hemolysis
hemagglutination may display.

Anti-amboceptors. — Inasmuch as the bodies which take part in
hemolysis, the amboceptor and complement, are of protein nature, it
is presumable that they might serve as antigenic substances. It should
be possible to prepare anti-amboceptor and anti-complement. As pre-
viously mentioned, experiments have been reported which have been
interpreted to indicate that it is possible to produce such immune anti-
complements, but the evidence offered has not withstood criticism ; at
the present time it is extremely unlikely that true anti-complements
have been demonstrated. Anti-amboceptors were first produced by Ehr-
lich and Morgenroth who injected as the antigen a hemolytic immune
serum. If a hemolytic immune serum is injected in fairly large amounts
into an animal for whose red cells the serum is specific, death results.
By carefully-graded injections, however, it is possible so to immunize
the animal that it becomes immune to the toxic effect of the serum.
When so immunized the serum of this animal when added to a cell
amboceptor mixture and incubated will prevent subsequent hemolysis
on the addition of complement. Similarly anti -amboceptor s may be
produced by injecting serum containing amboceptors into other animals
than those for which the serum is hemolytic. Ehrlich and Morgenroth

CYTOLYSINS 137

were of the opinion that such anti-amboceptors represented an excess
of cell receptors formed during the course of immunization and were
thus free in the serum to combine with the cytophilic group of the
amboceptor. Bordet found, however, that it was not necessary to use
a hemolytic immune serum as an antigen and demonstrated an anti-
amboceptor by injecting normal rabbit serum into guinea-pigs. The
anti-serum formed in the guinea-pig not only neutralized hemolytic
amboceptor of rabbit serum but other amboceptors of rabbit serum as
well, and therefore it could not be regarded as combining with such
a specific receptor as the cytophilic group of the amboceptor. This
work was confirmed by several investigators, including Ehrlich and
Sachs, who agreed with Bordet that the anti-amboceptor does not com-
bine with the cytophilic group but offered the new assumption that the
combination is with the complementophilic group. Muir and
his co-workers have studied anti-amboceptors extensively and find
no good ground for accepting the later interpretation of Ehrlich
and Morgenroth.

Muir offers an experiment as follows to illustrate the simple action
of anti-amboceptor. Two tubes are marked A and B. Into each are
placed one unit of cell suspension and three units hemolytic amboceptor
(contained in rabbit serum), the mixture incubated and then washed
and resuspended. To tube A is added 0.3 c.c. anti-amboceptor (pre-
pared by injecting rabbit serum into guinea-pig), and to tube B is
added 0.3 c.c. normal inactivated guinea-pig serum. The mixtures are
again incubated and washed; to each tube is added one unit comple-
ment and the tubes are again incubated. Hemolysis is complete in
tube B but is absent or much inhibited in tube A, thus demonstrating
the inhibiting effect of the anti-amboceptor. Such an anti-amboceptor
as is here illustrated will operate only against amboceptors contained
in rabbit serum. Similar amboceptors contained in goat serum would
not be affected by the anti-amboceptor prepared by injecting rabbit
serum into guinea-pigs. If in the preceding experiment the supernatant
fluid were examined after the first incubation it would be found that
the amboceptor had been absorbed; a fact also illustrated by the
hemolysis in tube B. Even were anti-amboceptor and amboceptor
mixed and then added to cells the amboceptor would not be prevented
from absorption. If the supernatant fluid were taken after the last
incubation complement would be found free in tube A but not in the
full original amount, as can be shown by careful titration. The anti-
amboceptor keeps a certain amount but not all the complement from
being utilized. The converse, however, cannot be demonstrated, that
is to say, complement cannot be shown to inhibit in any way the union
of amboceptor and anti-amboceptor. Intricate experiments demon-
strate, however, that the union of amboceptor and anti-amboceptor is
loose and a certain amount of dissociation may occur upon the addi-
tion of a normal serum homologous with the serum which contains
the amboceptor.

Anti-complements. — As has been indicated above, it is improbable

138 THE PRINCIPLES OF IMMUNOLOGY

that any so-called anti-complements operate differently from these anti-
amboceptors. Numerous substances and physical conditions are anti-
complementary in that they destroy or inhibit the action of complement.
These have been pointed out in discussing the nature of complement
and must be considered in all experiments which utilize complement.
It has been suggested that in the interaction between amboceptor and anti-
amboceptor a precipitate is formed which fixes complement and that
if such were the case the complement should not be recoverable. Muir
has shown that it is possible to recover complement, as we have pointed
out above. Nevertheless, even such a form of fixation may permit of
dissociation, and, as we shall show in discussing complement fixation,
there is much evidence in favor of the view that the action of these
antilysins is dependent upon the fixing properties of precipitates.

Physical Hemolysis. — Hemolysis is produced not only by the
serum components discussed in the preceding paragraphs but also by
chemical and physical agents, by bacterial products, by certain vegetable
poisons and by venoms. Studies of these forms of hemolysis are of
interest not only because of their intrinsic value but also because they
serve to throw some light on serum hemolysis.

The necessity for using an isotonic salt solution for the preservation
of erythrocytes is well known and equally well known is the fact that
reduction of salt content of the menstruum beyond a certain point leads
to solution of hemoglobin, which in distilled water is seen as complete
hemolysis. This is not merely a question of solubility of hemoglobin
for this substance is soluble in physiological salt solution to the same
degree as in distilled water. For the same reason it is not a matter
of simple osmosis of the hemoglobin. Although distilled water pro-
duces swelling of the cells before the solution of hemoglobin the
rupture of the cell is of no especial importance for cells may be cut
into pieces in physiological salt solution without hemolysis appearing.
From experiments of Fischer it would appear that the hemoglobin is
held in combination with the stroma by adsorption and that the action
of the water causes a physical separation. By combining fibrin, a
hydrophyllic solid colloid, with carmine, a hydrophobic colloid dye,
Fischer was able to produce phenomena closely resembling hemolysis.

Fragility of Erythrocytes. — The corpuscles of different animals
differ in the point to which reduction in salt concentration of the sur-
rounding menstruum leads to hemolysis. This is spoken of as a differ-
ence in resistance to hypotonic salt solution or a difference in fragility.
There may be a very slight difference in fragility of the corpuscles of
different individuals of the same species and diseased conditions may
lead to well-marked alterations. In man a simple secondary anemia may
lead to a normal or reduced fragility, whereas pernicious anemia leads to
increased fragility. Obstructive jaundice is accompanied by decreased
fragility, whereas familial hemolytic jaundice shows increased fragility.
In the anemia of animals following removal of the spleen there is a
decrease of fragility or, in other words, an increase of resistance to
hypotonic salt solutions and also to other hemolytic agents.

CYTOLYSINS 139

Hemolysis may be caused by other physical agents, such as
freezing (particularly when followed by thawing), heat of 62° to 64° C.
in the case of mammalian corpuscles or slightly less in the case of
cold-blooded animals and, as Rous has shown, by shaking.

Chemical Hemolysis. — The influence of chemicals on hemolysis
appears to be a factor of their permeation of the stroma. Wells states
that there seem to be two types of permeating substances, one such as
urea, which does not act in isotonic solutions of sodium chloride, and
the other such as ammonium chloride, which acts either in isotonic or
non-isotonic solutions. Hamburger, as quoted by Wells, states that
erythrocytes in relation to organic substances are (a) impermeable
for sugars, including cane sugar, dextrose, lactose, arabinose and man-
nose; (b) permeable for alcohols in inverse proportion to the number
of hydroxyl groups they contain, also for aldehydes (except paralde-
hyde), ketones, ethers, esters, antipyrin, amides, urea, urethan, bile
acids and their salts; (c) slightly permeable for neutral amino-acids,
such as glycocoll and asparagin. In relation to inorganic substances, not
including the salts of fixed alkalis, the erythrocytes are (a) " imperme-
able for the cations Ca, Sr, Ba, Mg, and (b) permeable for NH4 ions,
for free acids and alkalis." It will be noted that certain of the organic
substances for which the cells are permeable are solvents of lipoids,
particularly those lipoids of the stroma, cholesterol and lecithin, a
phenomenon which will be referred to again in discussing venom
hemolysis. Other chemical hemolysins include veratrin, digitalin,
arseniuretted hydrogen (in the body but not in the test tube), nitro-
benzol (important in denatured alcohol poisoning), nitrites, guaiacol,
pyrogallol, aniline compounds, alcoholic extracts of tissues and products
of tissue autolysis. Salt solution extracts of various organs are
hemolytic and have been called organ hemolysins. These bodies
resist boiling, do not act as antigens, hemolyze at 37° but not at o° C.,
are not increased in activity by complement but are inhibited by the
presence of serum. Noguchi has studied alcoholic tissue extracts ex-
tensively and finds them also hemolytic. He has come to the con-
clusion that the active elements in organ hemolysis are soaps.

Bacterial Hemolysins. — Bacteria may by their growth lead to suf-
ficient acid or base formation in the media as to make the latter
hemolytic. Of equal importance is the fact that certain bacteria may
produce hemolytic bodies not of definitely acid or alkaline character,
called bacterial hemotoxins. These substances include for the most
part the products of pathogenic organisms, such as tetanolysin,
staphylolysin, streptolysin, typholysin, vibriolysin (El Tor strain of
cholera) , anthrax-lysin and certain other less important forms. Certain
saprophytes also are capable of producing lysins, as for example mega-
theriolysin, proteus-lysin and the lysin of bacillus Welchii and others
of the gas gangrene group. An important bacterial hemotoxin is that
of bacillus pyocyaneus. The exact nature of these substances is not
known, but Burckhardt has shown that staphylolysin is dialyzable,
thermolabile, ether soluble and does not give protein or biuret reactions.

I4o THE PRINCIPLES OF IMMUNOLOGY

The action on the cells is independent of complement, there is no com-
bination at o° C, but at 6° C. combination occurs, leading to hemolysis
only at higher temperatures. It, therefore, seems likely that these
bodies are similar to toxins with a special affinity for erythrocytes.
The most favorable medium for developing these hemotoxins is broth,
but individual organisms require special conditions in the broth for
maximal production of hemotoxin. The development of the toxin fol-
lows fairly definite curves for the different organisms. For example,
staphylolysin begins to appear on the third day, reaches a peak on the
fifth day, drops on the sixth day, rises again on the eighth day, drops
again and reaches a final maximum on the thirteenth day. Megatheri-
olysin reaches a maximum on the seventh day and almost disappears
by the fifteenth day. De Kruif has recently shown that streptolysin
reaches its maximum in a few hours and has almost disappeared by
the end of twenty-four hours. The action is variable for different
species of corpuscles; staphylolysin acts powerfully on horse, sheep
and other bloods but only slightly on human and goat blood, whereas
megatheriolysin acts powerfully on human blood but not at all on horse
blood. Nakayama has studied the streptolysin and finds that it is
filterable. He also passed the organisms through two species of
animals and finds that after animal passage the streptolysin is more
actively lytic for the corpuscles of the species which last harbored the
organisms. Streptolysin unites with the corpuscles in the course of
hemolysis, but the filtrate, after absorption of lysin, remains toxic for
mice. Many of the bacterial hemotoxins are thermolabile, being de-
stroyed at 60° to 65° C., but others are resistant to temperatures as
high as 100° C. The bacterial hemotoxins are active in vivo as well
as in vitro and are capable of producing severe anemias and even
death. An intravenous dose of 2.0 c.c. of a ten-day-old filtered broth
culture of staphylococcus produces in the rabbit marked reduction in
hemoglobin and the number of erythrocytes and may cause death in
six or seven days. It is probable that the secondary anemias which
often follow attacks of acute infectious disease may be dependent upon
bacterial hemotoxins. Ford, Lawrence and Williams have found that
cultivation of the bacillus Welchii in milk leads to the formation of
bacterial hemolysins, thus disproving the opinion previously held
that the hemolysis in gas bacillus infection was due to the formation
of lactic or butyric acid. The hemolysin described by Ford and Law-
rence is relatively stabile, not being destroyed until a temperature of
62° or 63° C. has been reached. It has other characters of true toxin
in that it is digested by pepsin and hydrochloric acid as well as by
pancreatin. It is precipitated by ethyl alcohol. It is antigenic, and
these investigators found that they could, by immunization, produce an
anti-hemolysin or anti-hemotoxin in titers of i-iooo, 1-1250.

Vegetable Hemolysins. — -Among the hemolytic substances of
vegetable origin are to be included those already discussed as phyto-
toxins, namely ricin, abrin, crotin, robin, phallin. Crotin and phallin
are more markedly hemolytic than the others, which are rather hemag-

CYTOLYSINS 141

glutinative than hemolytic. The phytotoxins resemble some of the
bacterial hemotoxins in that they may serve as antigens for the pro-
duction of antitoxins but differ in that, as a rule, they are thermostable.
Both groups act according to the law of multiple proportions. Of
considerable importance from the experimental point of view are the
saponins " a closely related group of glucosides found in at least
forty-six different families of plants" (Wells). They are thermo-
stable, do not act as antigens, have a fairly definite chemical composi-
tion and are in these particulars to be separated from true toxins. They
operate injuriously not only upon the erythrocytes but also on other
body cells, especially those of the central nervous system. Cholesterol
and lecithin both combine with saponin, the former in such a way as
to prevent hemolysis. Therefore, it is assumed that the hemolytic
action of saponin is dependent upon its action on the stroma lipoids.
Normal serum is anti-hemolytic for some of the saponins, a property
which may be slightly increased by careful immunization; Robert be-
lieves this increase to be due to an increase of blood cholesterol.

Venom Hemolysins. — The venom hemolysins or hemotoxins are
found in different amounts in all venoms, and the phenomenon of venom
lysis is of considerable importance not only because of its scientific
interest but also because of its employment in certain clinical tests.
The venoms possess not only lytic but also hemagglutinative properties,
the two usually being present in inverse ratio. Flexner and Noguchi
demonstrated that the lysin of venoms requires activation by some
substance which exercises a complementary power. They found that
cobra hemotoxin dissolves the red corpuscles of certain animals (ox,
sheep and goat) only in the presence of serum,, but that it may dis-
solve other erythrocytes (dog, guinea-pig, man, rabbit, horse) in the
absence of serum. This difference is probably due to a content of
activator in the latter cells, which activator must be furnished by serum
for the lysis of the former cells. Kyes found that he could extract
an activator from those cells which do not require serum for venom
lysis but was unable to do so in the case of those cells which require
serum activation. This activating substance was found to be ether
soluble. Kyes subsequently found that lecithin can activate venoms and
assumed that this lipoid constituted the bulk of the activating sub-
stance. The substance in serum is usually active only after the serum
has been heated, but with some sera heating is not necessary. Kyes
and Sachs believed this to be due to differences in the nature of the
lecithin union in the serum. Kyes mixed cobra poison with a chloro-
form solution of lecithin and obtained a substance which he named
cobra lecithid capable of activating cobra venom. Von Dungern and
Coca, upon investigating the subject, came to the conclusion that the
cobra venom contains a ferment capable of splitting the lecithin so
as to yield certain substances such as oleic acid and that the resulting
hemolysis is due to the activity of these secondary substances. Noguchi
holds that although lecithin exists in the stroma of red blood-cells it
is not present in a form available for venom activation and that the

142 THE PRINCIPLES OF IMMUNOLOGY

degree of susceptibility to hemolysis depends upon the amount of such
ether soluble activators as fatty acids (particularly oleic acid) and
their soluble soaps. He regards fatty acids, neutral fats and soluble
soaps as endocellular complement and assumes certain similarities with
serum complement. Certain soap serum mixtures were found to be
capable of completing an amboceptor cell mixture but numerous objec-
tions have been interposed against both the fact and the interpretation
so that at the present time there is no good ground for believing that
the activator of cobra lysin is a true complement or that Noguchi's soap
mixtures are comparable to serum complement. If the activator cannot
be regarded as complement, the venom lysin cannot be looked upon
as an amboceptor, for it shows no specificity and does not require
serum complement for activation. Kyes in a recent publication
gives what may be regarded as the modern view in regard to venom
lysis as follows :

" i. There is present in all venoms a hemolysin existing as one of
a number of distinct toxins.

" 2. This hemotoxin effects hemolysis only in conjunction with
a so-called complementing substance which, however, may be found
within the erythrocytes.

"3. The reaction between the hemotoxin and lecithin is essentially
a chemical reaction resulting in the formation of a complete lysin.

" 4. This complete lysin is a true toxin in that it stimulates the
production of a specific antibody."

Although the experiments of Zunz and Gyorgy are not to be re-
garded as indicating that they have found other activating substances
for cobra venom hemolysis, nevertheless, they have determined that
hemolytic activity of cobra venom is increased by certain compounds of
protein destruction, including certain albumoses and amino-acids.

The hemolytic property of cobra venom has served as a basis for
proposed clinical tests. Calmette noted that in tuberculosis the blood
contains more than the usual amount of lecithin and that small amounts
of serum of such patients served to activate cobra venom lysin. The
test is not positive in more than 78 per cent, of tuberculous patients and,
furthermore, is by no means specific. Similar increases of lipoid con-
tent of serum have been found in certain diseases of the central nervous
system, a fact leading to the Much and Holzmann psycho-reaction,
which also is not specific. Weil has maintained that in syphilis the
corpuscles are more resistant to venom hemolysis than is normal,
except in the earlier stages where the corpuscles are said to be hyper-
sensitive. As time has passed the suggested clinical tests have not
come into general use largely because of lack of specificity. Un-
doubtedly the blood exhibits alterations in lipoid content at different
times in various diseases, and in all probability there is a parallel altera-
tion in its power to activate venom lysin, but no one disease shows this
change exclusively or even constantly.

Cytotoxins. Specificity. — As erythrocytes may act as antigenic
substance, so may other body cells. The antibodies produced by injec-

CYTOLYSINS 143

tion of these latter cells were called cytotoxins by Metchnikoff, and
the name has been retained in spite of the fact that the immune bodies
are not toxins in the strict sense of the word but are amboceptors
similar to the hemolytic amboceptors. It was thought that cytotoxins
might be strictly specific for the antigenic cells of different organs
within the same species, but more thorough investigation has shown
that such " organ specificity " is not demonstrable. Hemolysins, for
example, are lytic for other body cells, such as liver and kidney, pro-
vided these cells are of the same species. Hepatolysins and nephrolysins
are also active as hemolysins within the species. In other words,
these antibodies are species specific but not organ specific. It may be
true that a cytotoxin acts- more especially on its antigenic organ cells
than upon other cells, as is maintained by Pearce for the nephrolysins,
but the action is not exclusively upon the antigenic cells. In an ex-
tensive study Pearce, Karsner and Eisenbrey were unable to demon-
strate any strict " organ specificity " by means of cytolysis, precipitation,
agglutination or the anaphylaxis reaction. In this work the organs were
washed by perfusion with large amounts of salt solution and thus pre-
pared for injection. Bell has pointed out that even the most careful
perfusion will not entirely remove the blood from organs and therefore
a certain amount of blood must be injected with the other cellular
antigen. Nevertheless, the early work of Landsteiner, Metchnikoff and
of Moxter showed that spermatozoa which can be obtained free from
blood may lead to a spermatolysin which also acts as a hemolysin. The
amount of blood injected with carefully- washed organs is so small
that it can have but little antigenic power, too little to be consistent with
the well-marked hemolytic power of the cy to toxic sera. Recently,
however, Wilson and Oliver have absorbed the hemolysin from cyto-
toxic sera by means of erythrocytes and maintain that there is a very
definite organ specific cytotoxin contained in nephrolytic serum pre-
pared by immunizing with kidney substance. This specific nephrolysin
can be removed by absorption with kidney substance but not with other
organs. If these studies are extended and confirmed, much new light
may be thrown on the subject of organ specificity.

In spite of the apparent lack of strict organ specificity, the cytotoxins
of certain types of cells deserve mention, namely those resulting from
the injection of leucocytes and of crystalline lens. Following a brief
communication by Delezenne concerning leucotoxins, Metchnikoff
studied the matter by injecting guinea-pigs with material from the
mesenteric lymph-nodes and from the bone marrow of rabbits. The
resulting immune serum was highly toxic for guinea-pigs, but if given
in sufficiently small doses produced first a marked leucopenia, fol-
lowed in several days by a leucocytosis. This was confirmed by others
who used for injection also leucocyte emulsion, and although species
specificity was strict, the cellular specificity was not. Lucatello and
Malon were able to obtain a serum by the use of leucocytes from cases
of leucemia and treated a series of cases with this serum. The leuco-
cytes were reduced in number and the spleen diminished in size, but

144 THE PRINCIPLES OF IMMUNOLOGY

there was no permanent improvement. The lack of cellular specificity
in such sera is an a priori argument against their use.

Lens Cyiotoxin. — The injection of .crystalline lens leads to the
formation of a cytolysin which is organ specific but not species specific,
similar to the production of precipitins by lens protein. Such a cyto-
toxin prepared by the use of the crystalline lens of the dog is specific
for all mammals, birds and fish and will not act upon other cells from
these animals. The fact that the injection of lens into animals of the
same species or even into the same individual leads to the production
of isocytotoxins and autocytotoxins led Romer to build up a theory-
concerning the origin of cataract. He suggested that the constant
absorption of lens protein from the normal process of tissue wear and
tear leads to the development of an isocytotoxin which in later life
produces the degeneration of the Jens seen in cataract. If such were
the case cataract should be a much more frequent complication of age
than it is and the lens should be a soft pulpy organ as the result of cyto-
lysis. Furthermore, complement is not available in^the fluids of the eye
and the cytolytic amboceptor is not to be completed in that position.
Other theories as to the etiology of cataract are so much more logical
that Romer 's hypothesis has been practically abandoned.

Aside from the foregoing example of isocytotoxin and autocytotoxin
formation, there are no well-determined illustrations of this phenomenon
except for the demonstration by Metchnikoff of isospermatotoxins.

Bacteriolysins. — The death and solution of bacteria in the processes
of resistance to disease may be accomplished by the activity of phago-
cytic body cells or by virtue of properties of the blood and body fluids
similar in every way to those properties which lead to hemolysis. In-
deed, the discovery of bacteriolysis antedated that of hemolysis even to
the point of understanding the essentials of its mechanism. Nuttall
in 1888 demonstrated that fresh normal defibrinated blood has the
power of killing bacteria. He set up a series of tubes, each containing
the same amount (0.5 to i.o c.c. defibrinated blood) and added to each
a small platinum loopful of material from the spleen of a mouse previ-
ously inoculated with anthrax. These tubes were incubated and at differ-
ent time intervals gelatin plates were made from the tubes and a control
made from the splenic material. This showed that as incubation pro-
ceeded the bactericidal activity of the blood became apparent. Buch-
ner confirmed this fact in 1889 with a slightly different method,
whereby a larger amount of blood and bacteria were mixed in one
container and incubated and small standard amounts withdrawn by
pipette and plated. It was found that if the blood were heated or
allowed to stand, its bactericidal power was lost and Buchner named
the thermolabile element alexin. He believed it to be of the nature
of a ferment, suggested that it might originate in body cells, possibly
leucocytes, and recognized the fact that it is not specific.

The Pfeiffer Phenomenon. — The next important advance appeared
in the studies of Pfeiffer and his co-workers, who, in 1893, 1894 and
subsequently, published the details of what we now speak of as the

FlG. 15. — Stages in lysis of cholera vibrios,
showing the reduction to large coccoid
forms before final solution. (Modified
from Pfeiffer and Friedberger, Lehrbuch
der Mikrobiologie. Jena, 1919).

• ,

'V7

CYTOLYSINS 145

Pfeiffer phenomenon. These discoveries were incident to the investi-
gation of immunity to cholera spirilla. The method is essentially that
of studying the changes taking place in the spirilla following intraperi-
toneal injection in guinea-pigs. If the guinea-pig had survived preceding
inoculations and had thereby developed immunity the injection of or-
ganisms was followed by loss of their motility, transformation into
oval translucent granules and finally disappearance of the bacteria with
complete recovery of the animal. If the spirilla were of only low
degree of virulence the same phenomenon could be observed in a
normal animal, but if the animal were highly immune it could survive
doses of virulent organisms much greater than those fatal for normal
guinea-pigs. It was found that the simultaneous intraperitoneal injec-
tion of serum from an immune pig and of spirilla into a normal pig
served to protect the animal and that this protection could be conferred
as well by heated as by non-heated immune serum. The mechanism in
all cases was the same and not dependent upon phagocytic activity.
Furthermore, the protection was found to be specific. Pfeiffer was
unable to demonstrate the phenomenon in vitro (hanging drop prepara-
tion) and therefore assumed that some substance provided by the peri-
toneal endothelium served to activate the bacteriolytic process.

In the demonstration of the Pfeiffer phenomenon it is necessary to have
a series of fairly young guinea-pigs of about 200 grams in weight and a- culture
of cholera spirilla whose virulence is well established, because the virulence of
the organisms plays quite as important a part as their number. The immune
serum may be produced in the rabbit, goat or other animal by repeated inocula-
tion with the organisms. The organisms may be injected in measured volumes
of broth cultures or of saline suspensions of agar cultures ; they may also be
measured by weight by the use of a standard platinum loop which takes up
approximately 0.002 gm. organisms. The immune serum is diluted as indicated
in the following protocol and the bacteria and serum are injected simultaneously.
Peritoneal fluid is withdrawn at intervals of 10, 20, 30, 45, 60 minutes, the
intervals being altered as circumstances indicate. The withdrawal is by means
of drawn out capillary pipettes introduced into the belly cavity through a
small incision in the skin. The material may be examined in a hanging drop or
may be spread and stained by the ordinary bacterial dyes. A protocol from
Pf eiffer's own work follows :

PFEIFFER PHENOMENON

Weight of 0986 of Dose of

guinea-pig spirilla immune Examination of

in grams in grams serum in c.c. Result peritoneal fluid

320 o 002 0.05 Lives After 15 minutes, only gran-

ules present.

240 0.002 0.02 Lives After 20 minutes, only gran-

ules present.

200 0.002 0.006 Lives Sterile after 35 minutes.

220 0.002 0.003 Lives After 25 minutes, numerous

granules, isolated, non-
motile spirilla. After I
hour practically sterile.

220 0.002 o.ooi Died during After 25 and 50 minutes,

night numerous granules but

also numerous active spir-
illa. After loo minutes,
only active spirilla.

10

146 THE PRINCIPLES OF IMMUNOLOGY

PFEIFFER PHENOMENON— (Continued)

Weight of Dose of Dose of

guinea-pig spirilla immune Examination of

in grams in grams serum in c.c. Result peritoneal fluid

230 0.002 0.0005 Died during After 25 minutes, a few

night granules, numerous active

spirilla. Progressive in-
crease of spirilla.

200 0.002 0.2 c.c. Died during After 25 minutes, few gran-

normal night ules, numerous active

guinea-pig spirilla. Autopsy after

serum as several hours snowed pus

control on the liver, numerous

spirilla mostly free in exu-
date, with granules both
free and within cells.

In the foregoing experiment it is seen that 0.003 c.c. immune serum
serves to protect a guinea-pig of about 200 grams from an otherwise
fatal dose of cholera spirilla. Pfeiffer used this method to titrate bac-
teriolytic sera and in this case would have indicated the serum as
containing in each cubic centimeter 333 immune units.

Bacteriolysis in Vitro. — Further study of the phenomenon more
particularly by Metchnikoff and by Bordet led to the discovery that the
process may be demonstrated in vitro, in spite of Pf eiffer's failure to do
so. Metchnikoff was able to produce lysis of spirilla in hanging drop
preparations by adding to the mixture of spirilla and immune serum an
extract of leucocytes, thus offering evidence in favor of the influence
of leucocytes in destruction of bacteria. Bordet demonstrated that
although the activity of the immune serum is destroyed by heat of
50° C. to 60° C., the serum may be rendered active again by the addi-
tion of a small amount of fresh serum, an amount of fresh serum in
itself incapable of producing bacteriolysis. He found that the speci-
ficity of the immune serum resides in a substance which he later named
the sensitizer (the Ehrlich amboceptor). The alexin of Buchner
(complement) was found to exhibit no specificity and was not increased
by immunization. In the course of these studies Bordet found that
the corpuscles in the fresh normal guinea-pig serum were agglutinated
by the immune goat serum and that the spirilla were often likewise
agglutinated. Suspecting that if both blood-cells and bacteria are
agglutinable, the blood-cells might be the subjects of lysis as well as
are bacteria, Bordet was led to the discovery of the- phenomenon of
hemolysis. The studies of Toitsu, Matsunami and Kolmer would indi-
cate that all bacteriolysis is not necessarily dependent upon the activity
of complement, for they found that anti-meningitis sera which were
freed from complement possessed bactericidal properties. Ecker has
made similar observations in regard to a serum specifically bacteriolytic
for the diphtheria bacillus. Nevertheless, Ecker found that the addi-
tion of complement increases the bacteriolytic action of this serum.

The Pfeiffer phenomenon was found applicable to bacteria other
than the cholera spirilla, including particularly the typhoid bacillus,
paratyphoid, dysentery and colon bacillus. With these organisms the
phenomenon proceeds more slowly than with cholera spirilla. Were

CYTOLYSINS 147

no simpler means available, the Pfeiffer phenomenon might well serve
as a laboratory method of identifying cultures of the bacteria.

Wright's Method for Bacteriolysis. — In the course of subsequent
studies, other methods of investigation of bacteriolysis have been de-
vised, those of Wright, of Neisser and Wechsberg and of Buxton de-
serving especial mention. Wright exercised his usual ingenuity in
attacking this problem and devised two methods, one by dilution of
serum and the other by dilution of the culture of organisms. For the
collection of serum he used the Wright pipette such as is employed
for determining opsonic content of serum. The serum was diluted
with different amounts of bouillon. The culture was mixed with
melted gelatine and to measured amounts of this mixture was added
the proper amount O'f serum dilution. The final mixtures were incu-
bated in capillary pipettes for two to three days at 22° C, then placed
under low magnification of the microscope and the number of colonies
in the pipettes determined. In the second method the culture was
diluted in varying amounts of broth by means of a specially con-
structed capillary pipette and the suspension blown into a watch glass.
The culture dilutions were mixed with a standard amount of serum
and incubated in special pipettes. If the serum was insufficient to kill
all the organisms, there was bacterial growth, and the medium became
cloudy. Having, by previous plating, determined the number of organ-
isms in a given bulk of broth culture, it was possible to determine how
many organisms could be killed by the standard amount of serum.
The outlines of these methods are given because of the ingenuity dis-
played and the exact information gained, although at the present time
they are not extensively employed.

The Neisser- Wechsberg Phenomenon. — The Neisser and Wechs-
berg method was described almost contemporaneously with that of
Wright. They mixed inactivated serum dilutions in test tubes with
either broth cultures or salt solution suspensions of organisms, added
complement and incubated. Definite amounts of these mixtures were
added to melted solid culture media, such as agar, and plates poured.
After incubation of the plates, the colonies were counted and the bacte-
riolytic activity of the serum thus determined. A protocol taken from
the studies of Neisser and Wechsberg will serve to illustrate the method.

Tubes

I

NEISSER- WECHSBERG PHENOMENON

Amount Inactivated Fresh guinea-
of culture immune serum pig serum

1/5000 c.c. of a 24-

Number of colonies
on plates

hour broth culture

of vibrio Metchnikovi i.o c.c.

0.3 c.c.

Many thousands ;

2

0.5 c.c.

0.3 c.c.

Many thousands

3

0.25 c.c.

0.3 c.c.

Many thousands

4

O.I C.C.

0.3 c.c.

Few hundred

0.05 c.c.

0.3 c.c.

About 100

6

0.025 c.c.

0.3 c.c.

About 50

7

O.OI C.C.

0.3 c.c.

None

8

0.005 c.c.

0.3 c.c.

None

9

0.0025 c.c.

0.3 c.c.

About 100

10

O.OOI C.C.

0.3 c.c.

Many thousands

ii

" 0.0005 c.c.

0.3 c.c.

Many thousands

148 THE PRINCIPLES OF IMMUNOLOGY

NEISSER-WECHSBERG PHENOMENON (Continued)

Amount Inactivated Fresh guinea- Number of colonies

Controls of culture immune serum pig serum on plates

1 1/5000 c.c. ... ... Many thousands

2 1/5000 c.c. o.oi c.c. . . . Many thousands

3 o.oi c.c. . . . None

4 1/5000 c.c. ... 0.3 c.c. Many thousands

5 ... 1.0 c.c. None

The broth culture is so diluted that 0.5 c.c. are added to each tube. All tubes
are made up to constant volume with 0.85 per cent, salt solution. Incubation is
for three hours at 37° C, after which five drops from each tube are added to
a tube of melted agar for plating.

The Neisser-Wechsberg method not only presents a means of work-
ing with bactericidal sera but also demonstrates both the necessity for
the presence of complement to complete the bactericidal amboceptor
and the appearance of inhibition zones in the stronger concentra-
tions of the immune serum. Neisser and Wechsberg interpreted the
inhibition zone as illustrating what they called " complement devia-
tion," a term frequently used incorrectly as synonymous with com-
plement fixation. They believed that if an excess of amboceptor units
is present, a certain number of these units will combine with the avail-
able complement units, thus leaving a number of amboceptor units
unsaturated with complement. The amboceptor is present in amounts
too large to be entirely absorbed by the antigenic bacteria and therefore
it is assumed that a certain number of the free amboceptor units
combine with a number of complement units, thus preventing a suf-
ficient amount of complement to combine with the amboceptor units
already absorbed by the bacteria for the process of bacteriolysis. In
tubes four to nine of the preceding protocol the amboceptor units and
bacteria are closely enough balanced to ensure complete absorption
of amboceptor and thus permit of full action of complement; there
being no free amboceptor, there is no " deviation " of complement.
Except for the possible evidence afforded by the Ehrlich and Sachs
experiment (see page 125) there is no other experimental evidence sup-
porting the view that free amboceptor may enter into combination
with complement. Gay has suggested that the inhibition may be due to
precipitation by the interaction of the immune serum and bacterial
protein which may have gone into solution, the precipitate operating
to fix complement and prevent its combination with bacteriolytic am-
boceptor. Whilst precipitation may undoubtedly be of significance in
this connection, we are of the opinion that the resemblance to col-
loidal reactions as described in connection with precipitation and agglu-
tination, wherein excess of one colloid may prevent the occurrence of
precipitation or flocculation, offers an equally satisfactory explanation
ifor the Neisser-Wechsberg phenomenon and that we are therefore
justified in regarding the reaction as illustrating " inhibition zones "
where the concentration of amboceptor is great. The failure of bac-
teriolysis in tubes eleven and twelve is due, of course, to insufficient
amount of amboceptor. The control tubes show that neither ambo-
ceptor nor complement alone is capable of producing bacteriolysis.
Buxton's Method for Bacteriolysis. — Buxton determined that

CYTOLYSINS 149

active immune serum shows the same inhibition zones and also simplified
the method. By allowing the original tubes to incubate twenty-four
hours at 37° C, the degree of clouding of the medium by bacterial
growth gives an excellent indication of the degree of bacteriolysis. He
found that normal rabbit serum shows bacteriolytic powers in strong
concentration, gradually diminishing as dilution proceeds. Thus the
low titer normal amboceptor fails to show inhibition zones, as is true
of low titer agglutinins and precipitins. A protocol from Buxton's
work shows the difference in activity of normal serum and immune
serum as well as the correspondence between the results of plating and
observation of original tubes.

BUXTON EXPERIMENT

Dilution Count of colonies on plates Observation of original tubes

of sera Normal serum Immune serum Normal serum Immune serum

i o Many thousand Clear Cloudy

-2 o Many thousand Clear Cloudy

-5 2 Many thousand Clear Clear

-20 2500 4-5000 Cloudy Clear

-40 Many thousand 4-5000 Cloudy Clear

-80 Many thousand Many thousand Cloudy Cloudy

-100 Many thousand Many thousand Cloudy Cloudy

Teague and McWilliams have confirmed the work of Buxton and
others showing that normal rabbit serum is capable of killing large
numbers of typhoid and paratyphoid bacilli, but that the sera of rabbits
highly immunized against these organisms do not kill these bacilli.
These investigators have emphasized further that the normal bacteri-
olytic activity of rabbit serum for typhoid and paratyphoid bacilli is
not materially altered by immunization. In human typhoid fever the
blood serum normally shows bacteriolytic activity, but in spite of this bac-
teria multiply in the tissues apparently because the lymph does not pos-
sess bacteriolytic powers. Stone more recently made similar observations
but found further that fresh immune typhoid serum in vivo has appar-
ently a high bactericidal power, while fresh normal serum in vivo
has no protective power. Typhoid bacilli disappear more quickly from
the organs of immune animals than from normal animals, but macerated
organs from immune animals, cut sections, or their extracts are not
bactericidal even on the addition of fresh immune serum. This work
indicates that the destruction of typhoid bacilli in the immune animal
is due to some interaction between the tissue cells and plasma in vivo
or other unknown factor.

The Bioscopic Method for Bacteriolysis. — Neisser and Wechsberg
also devised the so-called bioscopic method of studying bacteriolysis.
They took advantage of the fact that living cells possess the power of
converting methylene blue into its colorless leucobase. By careful
adjustment of the number of bacteria it was possible to mix the various
agents together, add a very dilute alcoholic solution of methylene blue,
cover with paraffin and incubate. The degree of decolorization indi-
cates the relative amount of bacterial growth.

Summary of Cytolysis. — In summary it may be said that the phe-

150 THE PRINCIPLES O*F IMMUNOLOGY

nomenon of cytolysis represents a general biological phenomenon ap-
plicable to vegetable cells, exemplified by bacteria, and also to a wide
variety of animal cells. In both kingdoms there is a marked species
specificity exhibiting, as do other immune processes, the phenomenon
of group reactions. In so far as bacteriolysis is concerned, inhibition
zones appear, apparently similar to the inhibition zones of precipitation
and agglutination. Two bodies interact to produce cytolysis, a ther-
mostable body, the amboceptor or sensitizer, which may be increased
by immunization, and a thermolabile body, the complement or alexin,
which does not react to immunization. The amboceptor appears to
act by preparing the antigenic cells for the lytic action of the com-
plement rather than by furnishing a two-armed link between cell and
complement. The reaction takes place more nearly according to the
physical chemical laws of colloidal reactions than the simpler laws of
reactions between inorganic chemicals. The protection afforded an
animal which possesses bacteriolytic immune bodies is obvious, and
the role these bodies play in natural and acquired immunity to disease
must be of great importance.

CHAPTER VII
CELLULAR RESISTANCE

PHAGOCYTOSIS.

INTRODUCTION.

THE PROCESS OF PHAGOCYTOSIS.

MUTUAL APPROACH (CHEMOTAXIs).
INGESTION.
DIGESTION.

CELLS WHICH PARTICIPATE.
FUNCTIONS OF THE PROCESS.
EXPERIMENTAL DEMONSTRATION.
MECHANISM OF PHAGOCYTOSIS.

PHYSICAL BASIS.
OPSONINS.

INTRODUCTION AND DEFINITION.
EXPERIMENTAL DEMONSTRATION.
NORMAL OPSONINS.
IMMUNE OPSONINS.

B ACTERIOTROPI N S .

OPSONINS FOR CELLS OTHER THAN BACTERIA.
SPECIFICITY AND OTHER CHARACTERS.
INFLUENCE OF PHAGOCYTE AND INGESTED ELEMENTS.
RELATION OF BACTERIAL VIRULENCE.
INFLUENCES OPERATING UPON PHAGOCYTES.
ANALYSIS OF MECHANISM OF PHAGOCYTOSIS.
OTHER MANIFESTATIONS OF CELLULAR RESISTANCE.

INTRODUCTION.
THE LEUCOCYTES.

BACTERICIDAL EXTRACTS.

LEUCOCYTE ANTIBODY.
LEUCOCYTE ENZYMES.

LEUCOCYTE EXTRACTS FOR THERAPEUSIS.
SPECIFIC HYPERLEUCOCYTOSIS.
THE LYMPHOCYTES.
THE PLATELETS.
THE INFLUENCE OF INFLAMMATION.

PHAGOCYTOSIS

Introduction. — MetchnikofT has defined the phagocyte as a cell
capable of ingesting foreign bodies. Similarly the process of phago-
cytosis can be referred to as the process of ingestion of foreign bodies
by a cell. In the study of unicellular organisms and of certain lower
forms of multicellular organisms it has been found that the process
of phagocytosis is an important means of obtaining nutrition. That
such a simple process could have any bearing on the resistance of
vertebrates to disease was not pointed out for many years. The very
earliest study of bacteriology and immunity led to the knowledge of the
fact that the injection of bacteria into animals led, under favorable
conditions, to the disappearance of these bacteria. The investigation
of the cause of this disappearance resulted first in the belief that it was
due to solution of the bacteria by body fluids, more especially the blood.
Certain early investigators had noticed that following such injections
of bacteria the bacteria might appear within tissue cells, but Panum

151

152 THE PRINCIPLES OF IMMUNOLOGY

was the first to interpret the phenomenon in an immunological sense.
He pointed out that the penetration of bacteria into living cells, as
previously maintained by Birch-Hirschfeld, probably had much to do
with the disappearance of bacteria following injection. Subsequently,
it was shown that bacteria do not penetrate into cells but rather are
taken up by the cells. This work of Panum did not lead to any direct
result in the study of immunology, for Metchnikoff in his early work
on the subject was ignorant of it. Roser had also stated that according
•to his opinion the immunity of healthy animals and plants against in-
fectious organisms rests upon (a) the relative salt content of their
fluids and (b) the capacity of their contractile cells to take up the
invading organisms. Roser, however, did not support this statement in
later studies, and again Metchnikoff was ignorant of this work when
he took up his great work on phagocytosis. Metchnikoff had studied
extensively the nutrition of certain of the lower forms of animal and
vegetable life and also their defenses against the invasion of harmful
parasites. From this work he was led to the conclusion that the defense
of higher animals depends in great part upon the phenomenon of
phagocytosis. This statement was magnified into a conflict between
the so-called cellular theories of immunity and the humoral theories
of immunity. At the present time, however, such a conflict does not
exist because the two theories of immunity are in perfect harmony with
one another, and it is known that they are dependently interrelated.

The process of phagocytosis involves three steps; first, the ap-
proach of the cell and the material to be taken up ; second, the ingestion
of the material, and, third, the destruction of such material as may be
dissolved by the digestive fluids of the cell. The problem of the
approach of the cell and material to be ingested is one fundamentally
of irritability. The irritability of living tissue is in response to certain
stimuli and such stimuli include chemical, thermal, osmotic, photic,
mechanical and other physical agents. In the early studies of the
physiology of stimulation the response of a cell to a stimulus was
believed to be governed by the Weber-Fechner law, which states that
the intensity of sensation varies with the logarithm of the intensity
of the stimulus or, in other words, as the stimulus increases by geometric
progression the response increases by arithmetical progression. This
law has been found by further study to be untenable, for it has been
shown that logarithmic functions are not applicable to very strong
stimuli. In phagocytosis chemical stimuli are the most important, and
the response to such stimuli is referred to as chemotaxis.

Mutual Approach (Chemotaxis). — Chemotaxis may be positive or
negative, according to whether it attracts the two bodies or repels them.
Such attraction or repulsion does not depend essentially on the acidity
or alkalinity of the medium but does depend in certain measure upon
its concentration. Not only does variability of concentration play a
part, but the adaptability of the cell itself is of importance ; for example,
myxomycetes plasmodia exhibit negative chemotaxis in the presence
of sugar in certain concentrations, but after the organism becomes

CELLULAR RESISTANCE 153

accustomed to the presence of the sugar a positive chemotaxis appears.
The lower animal and vegetable cells exhibit a certain amount of selec-
tion in the material which they take up, and the leucocytes of higher
organisms may in a similar manner exhibit selectiveness. According
to our present-day physical conception of the activity of living proto-
plasm, this selectiveness would depend in all probability upon varying
sensibility to chemotactic influences or variation in the intensity of the
chemotactic stimuli.

Ingestion of Foreign Body. — The actual ingestion of the foreign
body depends upon the motility of the cell protoplasm, and this motility,
of course, is a function of the irritability of the protoplasm. Such
motility determines the ameboid movement of the cell to the material
to be ingested. Having approximated itself to the foreign material,
the cell throws out pseudopodia in such a fashion as to encircle the
foreign body ; as opposite pseudopodia meet the cell resumes in so far
as possible its normal form and the material is enclosed in the cell
protoplasm. These two stages in the process of phagocytosis have
been reduplicated in experiments with non-living material.

Digestion. — The third stage of phagocytosis is the digestion of the
foreign material. Such digestion is accomplished by secretions which
are poured out by the cell protoplasm so as to constitute a small area
of fluid about the ingested particle. By staining with dyes which show
the acid reaction it has been found that although the cell protoplasm
does not show acidity the fluid within the digestive vacuole is definitely
acid in character. Attempts to extract this digestive fluid from protozoa
have not been highly successful, but Mouton was able to extract from
a symbiotic culture of amebse and colon bacilli an enzyme which is
feebly proteolytic. This enzyme is capable of digesting colon bacilli
which have been killed but does not act upon living colon bacilli. The
intracellular digestion of these particles depends upon their solubility
by the digestive fluids. The cell may take up insoluble particles, in
which case the particles remain within the cells or are extruded with
the excreta of the cells.

Types of Phagocytic Cells. — It is incorrect to think of leucocytes
as identical with phagocytes, for numerous other body cells show this
capacity, including the eosinophiles, the endothelial cells, the pulp cells
of spleen and lymph-nodes, connective tissue cells, including bone cells,
striated muscle cells and giant cells. It is probable that the lymphocytes
and the mast cells exhibit no phagocytic activity. Metchnikoff divided
phagocytes into microphages and macrophages. The microphages
include particularly the neutrophilic leucocytes and the eosinophilic
leucocytes, the important phagocytes of the circulating blood. The
macrophages include the other cells mentioned above, the most important
being the endothelial cells. It is perfectly true that the endothelial cell
circulates in the blood, but apparently its most important activity is in
the tissues and body spaces. The microphages are the more sensitive
of the two groups and react not only to chemical stimuli but also to
tactile and physical influences.

154 THE PRINCIPLES OF IMMUNOLOGY

Functions of Phagocytosis. — Phagocytosis plays an important part
in the entire life of the mammalia, even though the differentiation of
many cells excludes them from this function. The destruction of
erythrocytes in spleen, liver and bone marrow is in part due to phago-
cytosis. Involution of the uterus after pregnancy, involution of senile
ovaries, decrease in substance of the brain and other organs in old age
are due to phagocytosis. Metchnikoff has laid considerable stress upon
the activity of phagocytes in the atrophic processes of old age. Rind-
fleisch claims to have demonstrated that phagocytes are active in the
breaking down and removal of gouty deposits in and about joints. The
fixed tissue phagocytes which play a part in the physiological destruc-
tion of red blood-corpuscles have been designated by Kyes as
hemophages. In various animal species the blood destruction accom-
plished by the hemophages may be carried on predominately in one
organ or another, the site of destruction, however, being constant for
a given species under normal conditions. Pearce and his co-workers
have shown that extensive blood destruction increases the phagocytic
activity in the spleen and liver. They have also shown, as has been
confirmed by us, that the removal of the spleen results in an assump-
tion of hemophagocytic activity by the endothelial cells of the lymph-
nodes. Gary has demonstrated that the injection of foreign red
blood-corpuscles markedly increases the hemophagocytic activity of the
recipient, not only in the spleen, which normally plays the important
part in destruction of the red cells, but also in other organs. The
resistance of the organism to foreign bodies, either living or inert,
is partly the result of the same process.

Under abnormal circumstances the removal of tissue and cell
detritus is due in part to phagocytosis. In the inflammatory reaction
following the introduction of foreign bodies, especially infective bac-
teria, phagocytosis is the first line and most important defensive
mechanism against invasion. Dusts inhaled into the lungs are taken
up by mononuclear phagocytes or macrophages and conveyed to neigh-
boring lymphatics and lymph-nodes, thus preventing accumulation on
the respiratory membrane. In inflammation the circulation is slowed
in the small vessels of the neighborhood, thus permitting the accumu-
lation of leucocytes on the inner wall of the vessels. They then migrate
through the vessel walls by ameboid movement and because of chemo-
tactic attraction continue through the tissues to the irritating sub-
stances. If the latter are bacterial the leucocytes attempt to ingest
and destroy them. Thus it can be seen that phagocytosis is an im-
portant process in the normal physiology of the body and perhaps
even more so in the pathological physiology of defense against disease.

The material to be ingested by phagocytes in part determines the
type of cell which participates. The microphages are especially active
in taking up bacteria, whereas the macrophages are active in ingesting
inert tissue detritus. Nevertheless, macrophages often take tip bacteria,
as in tuberculosis, and, as has been shown by Kyes, by Bull and by
Hopkins and Parker, pneumococci, typhoid bacilli and streptococci are

FIG. 16. — Microscopic drawing showing the phagocytosis

of gonococci by the polymorphonuclear leucocytes in

urethral pus.

CELLULAR RESISTANCE 155

removed from the circulation by endothelial cells lining blood-vessels.
Microphages may also play a large part in the removal of tissue detritus
and may take up pigment as in malaria.

Experimental Demonstration. — The experimental demonstration of phago-
cytosis in mammals is comparatively simple, as the following experiment from
Metchnikoff will show. The blood of a bird, such as goose, hen or pigeon, is
selected because of the fact that the nucleated erythrocytes are easily distinguished
from those of mammals. Defibrinated bird blood mixed with equal parts salt
solution is injected (about 3.0 c.c.) into the peritoneum of a healthy guinea-pig.
Material is removed for study by means' of finely drawn out glass pipettes, drops
being placed on slides for study with or without subsequent staining. Within
the first hour the leucocytes seem to have disappeared from the peritoneum.

The disappearance is particularly striking when bacteria are injected and
was interpreted by Metchnikoff as a destruction of the phagocytic cells, a
phenomenon which he called phagolysis. At the end of from one to two hours
exudate may be withdrawn which shows numerous cells, particularly macrophages.
The macrophages show ingestion of the nucleated erythrocytes and at from
twenty-four to forty-eight hours exhibit digestive vacuoles and partial digestion
of the erythrocytes.

At the end of three days the digestion is practically complete.
Metchnikoff has shown that immunization will definitely limit the ap-
pearance of phagolysis. Sanarelli, however, maintains that the disap-
pearance of the leucocytes is not due to phagolysis but rather to the
fact that the leucocytes of the peritoneal cavity and of the blood
accumulate in the epiploic appendages into which the bacteria are
likely to be carried by the lymphatic stream. Here, he asserts, bac-
teriolysis and phagocytosis progress actively. Hence the disappearance
of the cells from the exudate.

A similar experiment may be performed with a suspension of pigment, as
for example 5.0 c.c. finely-divided suspension of cinnabar (red mercuric oxide).
This shows no digestion but active phagocytosis and a rapid transfer to re-
gional lymph-nodes.

Phagocytosis of bacteria may be very well demonstrated with colon bacilli.
It is desirable in this instance to excite some exudation before the introduction
of the colon bacilli. This may be produced by injecting about twelve hours
previously 10.0 c.c. sterile bouillon or aleuronat suspension. This may be done
in the evening and the following morning the guinea-pig is ready for the injection
of a 24-hour bouillon culture or a 24-hour slant agar culture suspended in salt
solution. The subsequent phenomena are similar to those following the injection
of bird blood.

The Mechanism of Phagocytosis. — In earlier experiments of this
sort several questions as to the mechanism of the process arose. That
the bacteria do not actively penetrate into the phagocytes has been
demonstrated by direct observation of the ameboid action of the cells
and is concluded also by analogy from the fact that non-motile bacteria,
non-motile cells, such as erythrocytes, and inert bodies, such as cinna-
bar, are readily ingested by the phagocytic cells. That the bacteria are
not killed before ingestion is shown by the fact that cultures may be
successful in the case of anthrax bacilli shortly after they have been
taken up by phagocytes. This may also be illustrated by the following
experiment with the use of neutral red as a vital stain. This stains only
dead cells and imparts no color to living cells. A warm hanging drop
preparation of the exudate from a guinea-pig injected with colon

156 THE PRINCIPLES OF IMMUNOLOGY

bacilli as outlined above may be mixed with a drop of i per cent,
neutral red in isotonic salt solution. At first the extracellular bacteria
show no stain, and but few of the intracellular bacilli take the stain.
As time goes on the number of intracellular organisms taking the stain
increases until they are completely digested. Metchnikoff interpreted
the coloration of the bacteria as being due to an acid digesting fluid
formed by the cell, but we are unable to state at the present time
whether the digestion of the bacteria is due to a special ferment or
due to the same ferments that digest the cells themselves after they
are destroyed.

The Physical Basis of Phagocytosis. — The mechanism of phagocy-
tosis both as regards immunity and biology in general has been the
the subject of much investigation. There are those who have main-
tained that the ameba or the leucocyte, in spite of the absence of a
nervous system, exhibits individual intelligence in the selection of the
material it takes up, but the bulk of experimental evidence would place
the phenomenon largely on a physical chemical basis. There are im-
portant differences between free living amebse and the phagocytes of
higher animal life, such as the ectosarc and endosarc of the amebse,
its pulsating vacuoles, variety of pseudopodia, conjugation and cyst
formation, but there are resemblances in movement, form, nutrition and
ultimate genesis which form a basis for many comparisons. That the
life activities of the ameba can be closely simulated by non-living
materials has been known for many years, but the most important
stimulus to these studies in recent years has been given by the work
of Jennings. A fundamental conception necessary to understanding
the physical basis of ameboid motion and phagocytosis is that of the
phenomena of surface tension. Wells expresses the matter most
clearly and concisely as follows : " Imagine a drop of fluid suspended
in water — let it be a drop of protoplasm, or oil, or mercury ; the drop
owes its tendency to assume a spherical shape to the surface tension,
which is pulling the free surface toward the center and acting with the
same force on all sides. The result is that the drop is surrounded by
what amounts to an elastic, well-stretched membrane, similar to the
condition of a thin rubber bag distended with fluid. If at any point
in the surface the tension is lessened, while elsewhere it remains the
same, of necessity the wall will bulge at this point, the contents will flow
into the new space so offered and the rest of the wall will contract;
hence the drop moves toward the point of lowered surface tension.
Conversely, if the tension is increased in one place the wall at this
point will contract with greater force than elsewhere, driving the con-
tents toward the less resistant part of the surface, and the drop will
move away from the point of increased tension." The experimental
demonstration of this phenomenon is relatively simple. A drop of mer-
cury is placed in a nitric-acid solution and near it is placed a crystal of
potassium dichromate. A yellow color diffuses out from the dichro-
mate; as the color reaches the mercury the latter begins to move
toward the crystal. This is the result of oxidation of the adjacent

CELLULAR RESISTANCE 157

surface of the mercury drop whereby the surface tension of this side
is lowered, thus causing the progressive movement in the direction of
the dichromate crystal. Similarly a drop of clove oil in a mixture of
glycerol and alcohol will move about and send out pseudopodia in much
the same manner as an ameba. The movement depends upon the solu-
bility of the clove oil in alcohol, but the glycerin retards the diffusion
and thus determines a certain degree of irregularity in the movements.
If strong alcohol be introduced near the clove oil the surface tension
of the oil is reduced and it moves toward the alcohol. Heat applied
near one side of the drop will also lower the surface tension, and it
moves toward the point of heat — positive thermotaxis. These experi-
ments illustrate the physical basis of ameboid movement, but do not
explain ingestion of particles. For this purpose a drop of chloroform
may be placed in water and brought near a variety of objects, such as
glass particles and small pieces of shellac, paraffin and glass. Such a
drop will flow around a piece of shellac and dissolve it. A piece of
glass covered with shellac will be taken up, the shellac dissolved and
the piece of glass then extruded. If a long " hair " of shellac is
brought into contact with the chloroform, the former will be bent in
the middle, pseudopodia will extend along it and it will finally be
curled up inside the drop and dissolved. These various activities of
the oil drop or chloroform drop resemble in detail the activities of
amebse under similar circumstances and may be understood as indi-
cating that the process of phagocytosis is based on definite physical
laws. The experiments do not explain all the phenomena, however,
and must be interpreted as solving the problem only partially. Various
food particles are not soluble in the cytoplasm of the ameba, bacteria
are not soluble in the cytoplasm of the leucocytes, but in each instance
must be digested in some way. Furthermore, the phagocytes have the
property of taking up inert and insoluble particles such as coal dust
and other pigments, substances which cannot exert chemotaxis nor
alter surface tension. The artificial ameba does not assimilate, it
merely dissolves. An additional differentiation between the leucocytes
and the ameba is the fact that the ameba is a free living organism
capable of nourishing itself independently of life within another or-
ganism. On the other hand, the leucocyte depends upon the blood for
its nutrition and differs in ameboid movement and irritability from
the free living ameba. Thus we must conclude that the problem of
phagocytosis is not solved by these experiments and that the life activ-
ities of these cells are not as yet explainable on a purely physical basis.
Influence of Temperature on Phagocytosis. — Madsen and his
school have made accurate studies of the influence of temperature
on phagocytosis. They have shown that within certain limits the
phenomenon of phagocytosis increases with the degree of tem-
perature. Starting at a point of =±= 5° C, the phagocytic power
increases with temperature up to the normal temperature of the species
from which the phagocytic cells are derived. In cold-blooded animals,
on the other hand, the temperature of the environment within certain

158 THE PRINCIPLES OF IMMUNOLOGY

limits appears to have no influence whatever on the phagocytic activity
of their cells. Calderone and Runfola have recently studied the in-
fluence of temperature upon phagocytosis in the frog and find that
phagocytosis proceeds actively between 5° and 40° C, but ceases when
a temperature of 42° C. is reached.

OPSONINS

Introduction. — Early in the study of phagocytosis it was noted
that immune animals respond to the introduction of the antigenic
bacteria by a greater degree of phagocytic activity than normal animals.
This was interpreted by Metchnikoff as being due to " stimulins "
which were supposed to augment the activity of the phagocytic cells.
The first study of importance in contraindication of MetchnikorFs con-
ception of stimulins was that of Denys and Leclef in 1895. They
showed in a study of streptococcus immunity in rabbits that the leuco-
cytes of normal and immune animals took up the bacteria equally
well, but that both varieties of leucocytes acted much more powerfully
when immune serum was added. They indicated that the process of
immunization did not augment the phagocytic power of the leucocytes
and concluded that in their opinion the antitoxic substance acts not
upon the leucocyte but upon a poison enclosed within the bodies of the
microbes or dissolved in the medium, the poison acting to protect the
bacteria against the attacks of the leucocyte until neutralized by the
immune substance in the serum. The observations were confirmed by
other investigators and later Denys and Leclef showed that whereas
extremely virulent bacteria are taken up by leucocytes in normal serum
to only a slight degree, the addition of an immune serum markedly
increases the phagocytosis. Little progress was made until after the
discovery by Leishman whereby the study of phagocytosis could be
carried out in vitro. Modifying this method, Wright and Douglas in
1903 published the first of a series of experiments which have built up
in large measure our modern conception of the influence of serum on
phagocytosis and the practical use of bacterial vaccination in the treat-
ment of disease. They showed conclusively that it is the activity upon
the bacteria of some substance in the blood which favors phago-
cytosis and they named the substance opsonin. By treating the bacteria
with serum, then washing them to remove the serum, from the sur-
rounding medium and finally mixing with a leucocyte emulsion in salt
solution they showed that phagocytosis proceeds actively. Similar
treatment of the leucocytes by serum produces no augmentation of their
phagocytic activity. Thus it was shown that the serum does not stimu-
late the leucocytes but rather prepares the bacteria so that they may
more readily be ingested, hence the term opsonin (Gr. opsono — to pre-
pare food). There is some variation, however, in the way the serum
operates in the case of different bacteria. Tunnicliff and Davis have
shown that fusiform bacilli and influenza bacilli can be taken up readily
independently of the presence of serum. There are, of course, different
degrees of facility with which bacteria can be taken up, varying from

CELLULAR RESISTANCE 159

those which absolutely require the intervention of an opsonin
and those mentioned above, which apparently need little or no par-
ticular opsonization.

Experimental Demonstration. — For the experimental demonstration, of
opsonization it is necessary to have washed leucocytes, bacterial suspension and
blood serum. Large quantities of leucocytes may be obtained by injecting 5.0
c.c. aleuronat suspension into a guinea-pig's peritoneum and withdrawing the
exudate at the end of twelve to twenty-four hours. These may be suspended
in five to ten volumes normal saline, gently mixed and centrifuged, the process
being carried out three times, when the cells are said to have been washed three
times. If human leucocytes are desired, 10.0 c.c. saline sodium citrate are placed
in a centrifuge tube and 2.0 c.c. blood added. The tube is centrifuged at high
speed, whereupon a layer or " cream " of leucocytes collects at the upper level
of the cell mass. The cream can be removed by a drawn-out nipple pipette
and the cells washed as indicated for the peritoneal exudate. The bacterial
emulsion can be made from a twenty-four-hour slant agar culture of staphylo-
coccus pyogenes aureus by adding 10.0 c.c. salt solution, allowing to stand for
ten to fifteen minutes and then rotating the tube between the palms of the hands.
This is pipetted into another tube and for safety may be killed by heating in a
water bath at 55°-6o° C. for two hours. The serum may be obtained by allowing
blood to clot and then drawing off the serum. Small quantities may be obtained
by the use of a tube such as shown in Fig. 8. Having these ready, 0.5 c.c.
bacterial emulsion are mixed with o.i c.c. serum and incubated for one-half
hour, then washed three times and the organisms resuspended in 0.5 c.c. saline.
Several capillary pipettes are made from glass tubing (5 mm. bore) and the
upper end flanged so as to take a rubber nipple. A mark is made with a grease
pencil about 2 cm. from the tip, which serves as a volume indicator. (Fig. n.) In
the experiment one volume bacterial suspension, one volume bacterial emulsion and
one volume serum or saline are drawn into the pipette each in succession to
the mark, permitting a small amount of air to enter before the next volume is
taken up so as to permit of exact measurement of the volume. These are blown
into a watch crystal and mixed by blowing in and out several times ; then taken
into the capillary again and the end sealed. After incubation at 37° C. for
fifteen minutes the tip is broken, the mixture dropped on slides or cover slips,
spread, dried and stained with Wright's stain or some other modification of
the Romanowsky stain. Then the number of bacteria in a given number of
leucocytes (20 to 50 or more) are counted and the average calculated. A sample
protocol follows :

Average

phagocytosis

per cell

1. Leucocytes (washed) -f bacteria (untreated) -f serum 22

2. Leucocytes (washed) -J- bacteria (untreated) -j- NaCl I

3. Leucocytes (washed) -j- bacteria (treated) -j- NaCl 14

In the above protocol it is seen that the leucocytes exhibit a slight capacity
for taking up bacteria independently of the presence of serum, but that this is
much augmented either in the presence of serum or by previously treating the
bacteria with serum.

Normal Opsonins. — As has been indicated, the phagocytosis of bac-
teria and other cells is greater in immune than in normal animals, the
difference being due to increase in the opsonin content of the serum
of the immune animal. It was soon observed that the opsonin of the
serum of normal animals could be destroyed by heat to 60° to 65° C.
for 10 to 15 minutes; whereas the opsonin of immune animals is not
destroyed by heat of 62° to 63° C. for forty-five minutes. Similarly
exposure to light at room temperature leads to deterioration of normal
opsonin in a few days but has practically no effect on immune opsonin.
These differences in behavior were at first thought to constitute an

160 THE PRINCIPLES OF IMMUNOLOGY

actual difference in the nature of normal and immune opsonin, but
this view has now been almost entirely abandoned. In the discussion
of this change of view it is essential to present first the development of
work in regard to the normal opsonin. Conservative workers were
not disposed to accept the opsonin as a new form of antibody and
from the ease of deterioration of the normal opsonin thought that it
was identical with complement. Furthermore, it was shown that fixa-
tion of complement by a hemolytic system or sensitized bacteria re-
moves the opsonin, that yeast cells, cell detritus and bacteria will absorb
both opsonin and complement, that blood serum and edema fluids
contain parallel amounts of opsonin and complement, that certain body
fluids, such as the aqueous humor of the eye, contain neither complement
nor opsonin. Nevertheless, the removal of complement, as by heating,
does not, as Hektoen has shown, remove all the normal opsonic power
of the serum; and the fixation of complement by a hemolytic system
or by sensitized bacteria still leaves slight opsonic power in the serum.
The addition of fresh serum to a slightly active heated serum restores
the activity practically to normal in much the same manner as a
hemolytic amboceptor may be reactivated by complement. The fol-
lowing example taken from Cowie and Chapman and slightly modified
serves to illustrate this reactivation. The substances indicated in the
protocol are added to leucocyte and bacterial emulsions and the figures
given are for the bacterial count per leucocyte:

1. Unheated (normal) serum 1544

2. Salt solution 0.18

3. Heated serum 57° C 1.08

4. Normal unheated serum, diluted i to 15 1.56

5. Heated serum -f- normal serum diluted i to 15 12.40

6. Two volumes unheated normal serum 16.08

Thus it will be seen that heating the serum reduces the phagocytic
index from 15.44 to 1.08; that normal serum, diluted so that its
phagocytic index is reduced to 1.56, added to heated serum, raises
the index to 12.40, much higher than can be accounted for by the
total indices of the two components. It can then be concluded that
the normal opsonic power of serum depends upon two factors, a weakly
acting thermostable element and a thermolabile element which markedly
adds to the combined power of the mixture. Cowie and Chapman
have shown that at o° C. the thermostable element of opsonin is ab-
sorbed by the bacteria, but that the thermolabile element remains in
the supernatant fluid and is capable of reactivating a heated serum.
It has also been demonstrated by absorption experiments that the ther-
mostable element is specific. Numerous continental workers contradict
this statement, but their studies have, for the most part, ignored the
existence of the thermostable element of normal opsonins. Hektoen
has shown that saturation of the bacteria with opsonin and heating so
as to destroy the thermolabile part leaves the bacteria in such condi-
tion that they cannot absorb any more opsonin from another serum.

CELLULAR RESISTANCE 161

Moore has found that in guinea-pigs " the complement titer varies
with the opsonic index and in the same direction." These facts, to-
gether with the fact that vaccination with bacteria will increase specifi-
cally the opsonic content of the blood suggest a close similarity of
opsonins to agglutinins and amboceptors. The resemblance to agglu-
tinins is only relative for as we have seen the thermostable element of
opsonin is markedly augmented in activity by the addition of fresh
serum, whereas agglutinins are not affected in any way by the addition
of complementary substance. Hektoen has shown that in the process
of immunization the curves of opsonin and agglutinin production are
nearly parallel, but that heating does not influence the agglutinin and
markedly depresses the opsonic action, the latter being restored by the
addition of fresh normal serum. Levaditi, in a study of the site of
formation of opsonins, showed that certain organs rich in agglutinin
contain no opsonins. The thermostability and specific absorption of
opsonins suggest similarity to amboceptors, but the amboceptors are
not capable of acting without complement whilst the opsonin is capable
of acting independently of fresh serum. The fresh serum augments
the activity of the thermostable element of opsonins but is not an
absolute essential for activity. That the opsonin is not identical
with hemolytic and bactericidal amboceptors is indicated by the fact
that there are such amboceptors in sera which have no opsonic power ;
that in sera which show both amboceptors and opsonins there is no
parallelism between the activity of the two. Sera may be strongly
opsonic for certain bacteria and yet contain no bactericidal amboceptor.
Much of the material quoted above has been worked out iri connection
with immune opsonins, but nevertheless it is safe to conclude that the
opsonic action of normal serum depends upon the operation of two
elements, a thermostable element which behaves as a " facultative "
amboceptor and a thermolabile element which, if not identical with,
resembles complement most closely.

Immune Opsonins. — As has been indicated, it is possible by im-
munization to increase to a very considerable degree the opsonic activity
of serum. The immune opsonins were considered as of a constitution
different from the normal opsonin because of the claim that the appli-
cation of heat did not alter their activity. Dean showed, however, that
this assumption is not true for he found that heating to 60° C. definitely
though not very markedly reduces the opsonic activity of immune serum,
and that reactivation takes place on the addition of a fresh normal
serum. The following protocol shows the phagocytic index as deter-
mined by the use of various sera and mixtures :

Normal serum 1 1.9

Heated immune serum 7.1

Heated immune serum + normal serum 33.0

Hektoen reached the same conclusion with the hemopsonic power of
rabbits immunized to goat erythrocytes, diluting the serum so that it
ii

1 62 THE PRINCIPLES OF IMMUNOLOGY

showed minimal opsonic power and no hemolytic action. One protocol
from his work serves to illustrate.

Heated immune serum Fresh guinea-pig serum Phagocytosis

o.ooi 4

0.001 0.01 20
O.OI O

Levaditi and Koessler showed that a serum which contained anti-
complement by virtue of immunization with complement, when added to
an immune opsonin, noticeably reduces the opsonic power.

The full activity of the immune opsonin depends, as can be seen,
from the above experiments, upon a thermostable and a thermolabile
element, as is true of the normal opsonin, but the activation by fresh
serum in case of the thermostable element of immune opsonin is pro-
portionately much less than activation of thermostable normal opsonin
by fresh serum. Reference to the activation of a hemolytic amboceptor
by complement shows that a given amount of complement will activate
a very small amount of amboceptor in greater proportion than a large
amount of amboceptor. The thermostable fraction of opsonin has
been referred to as a facultative amboceptor, because the action of the
thermolabile part is not essential. Assuming this interpretation to be
correct and assuming that the thermolabile element operates as a com-
plement, it is a simple matter to infer that this complement would have
a proportionately larger action on the facultative amboceptor of normal
opsonin, which is present in very small amount, than on the similar am-
boceptor of immune opsonin, which is present in relatively large amount.

Bacteriotropins. — Neuf eld and his school maintain that the immune
opsonin is a body which operates only in the presence of complement
and that the tropins, bacteriotropins and cytotropins are bodies appear-
ing in serum which has been rendered complement-free, and which
exhibit a capacity for so altering bacteria or cells that they are easily
taken up by phagocytes. Levaditi and numerous other authors agree
that Neufeld has shown that the tropins are not identical with those
amboceptors which lead to cytolysis, but also agree that Neufeld has
not succeeded in demonstrating that the tropins are antibodies distinct
and apart from the thermostable element of immune opsonin.

Opsonins for Cells other than Bacteria. — Numerous substances,
including vegetable cells, such as yeasts, and bacteria, as well as a
variety of animal cells, may undergo phagocytosis when influenced by
opsonins. In connection with phagocytosis of animal cells the work of
Hektoen and his collaborators has been most extensive. The investiga-
tions have thrown much light on the general study of opsonins and, di-
rected particularly toward erythrocytes, have shown that the same
general laws governing the phagocytosis of bacteria operate in the
phagocytosis of erythrocytes. Neufeld and Handel have shown that
emulsions of fat droplets in protehvcontaining media can serve as
excitants of the formation of specific opsonic sera but conclude that in
these instances the protein capsule of the fat droplets which serves to
stabilize the emulsion is the important factor in the phenomenon. Led-

CELLULAR RESISTANCE 163

ingham has also shown that the injection of melanin produces a specific
opsonic serum and others have shown that carbon granules, cinnabar,
carmine, etc., are phagocyted much more readily in the presence of
serum than otherwise. In these latter instances it seems probable that
the serum provides a protein capsule for the pigment granules, thus
facilitating the action of opsonin, but at the present time no satisfactory
explanation has been offered for the production of a specific immune
opsonin following the injection of melanin. Neufeld and Ungermann
point out the difficulty of satisfactory measurement of phagocytic action
against pigment granules, and it is possible that this source of error
may be sufficient to throw doubt on the results claimed to have been
obtained with insoluble pigments.

Specificity and other Characters of Opsonins. — The specificity of
the immune opsonins is clear-cut, as has been shown by numerous in-
vestigators. An immune opsonin produced by vaccination with staphyl-
ococci shows a marked influence on the phagocytosis of the antigenic
organisms but none whatever on non-related organisms such as colon
bacilli. As in the case of other immune bodies, group reactions are
demonstrable. Vaccination with typhoid bacilli leads to the formation
of immune opsonins which operate in high degree on the antigenic
organism and also to less degree on closely-related organisms such as
those of the paratyphoid groups. Dean, in working with serum dilu-
tions in order to demonstrate that an optimum concentration of opsonin
may not necessarily be found in undiluted serum, reports the following
experiment. This may be interpreted as showing an inhibition zone in
the stronger concentrations, although the differences are so slight as
to fall within the limit of experimental error.

Dilution of serum Phagocytic index*

o 97

1-2 9.6

1-4 i o.o

1-8 8.2

1-16 8.5

1-32 6.4

* Average number of bacteria ingested per leucocyte.

Influence of Phagocyte and Ingested Elements. — The foregoing
paragraphs have considered the influence of serum on phagocytosis, but
detailed studies have shown that certain considerations in regard to both
the bacteria and the leucocytes exercise some influence. Neufeld pointed
out that bacterial cultures from ten to twenty-four hours old are best
for in vitro experiments. The reaction takes place best when the bac-
teria are suspended in equal-parts broth and physiological salt solution,
but in ordinary laboratory practice salt solution is used without the
addition of broth and whatever deterring action is exercised by the salt
is constant in the series of experiments. The thickness of the sus-
pension is of importance since very thin suspensions determine a
reduction in phagocytic index as compared with thicker suspensions.
The optimal density of the suspensions varies with different bacteria

164 THE PRINCIPLES OF IMMUNOLOGY

and must be determined, in exact work, for the organisms under investi-
gation. The more homogeneous the emulsion, the better the phago-
cytosis observed. Numerous investigators have shown that under
experimental conditions, bacteria killed by chemicals or by heat are
phagocyted at precisely the same rate as living organisms. Further-
more, the previous staining of the organisms has no deterrent action
on phagocytosis.

Relation of Bacterial Virulence. — The relation of bacterial viru-
lence to phagocytosis has been the subject of much research since
Marchand first showed that virulent streptococci are taken up hardly
at all under conditions where avirulent streptococci are phagocyted
with avidity. He demonstrated that this difference is not due to the
vitality of the bacteria, for when killed by heat at 60° C, 1.8 per cent.
HC1, 2.5 per cent. Na2CO3 or 90 per cent, alcohol, the virulent forms
show the same resistance to phagocytosis. Wright and also Levaditi
showed that the same difference is observable in the case of phago-
cytosis, without the intervention of opsonins. Rosenow confirmed
Marchand's results by the use of freshly-isolated virulent pneumococci.
Reduction of virulence of thirty-six strains by repeated cultivation on
media resulted in increased susceptibility to phagocytosis; and a res-
toration of virulence by animal passages led again to decreased phago-
cytosis. There is, however, no absolute parallelism between virulence
and susceptibility to phagocytosis. Markl, von Gruber and Futaki, as
well as Lohlein and others, found that anthrax bacilli and plague bacilli
when taken from culture material are actively phagocyted in vitro even
though highly virulent for animals. If removed from a guinea-pig's
peritoneum after having grown there for several hours, they are no
longer phagocyted in vitro. In animal experiments they are at first the
victims of active phagocytosis in vivo, but after several hours are re-
sistant to phagocytosis. Proper staining shows that in the resistant
stage the organisms show definite capsule formation. These experi-
ments indicate that the resistance is entirely a function of the bacteria,
but that there is some interdependence between the bacteria and the
opsonin is indicated by the experiments of Ungermann, who worked
with pneumococci virulent for mice in doses as small as 0.000,001 c.c.,
but not injurious for rabbits in doses as large as i.o c.c. He found
that mouse serum has no opsonic action and that rabbit serum acts
energetically. After repeated cultivation so as to reduce virulence for
mice the organisms are opsonized by mouse serum. Von Bockstaele
and also Denys and von den Bergh were able to see leucocytes in the
presence of a normal serum approach and even break up chains of viru-
lent streptococci without engulfing them; if a strong immune serum
were added, there resulted active phagocytosis. In summary, these
various experiments show that the possession of virulence by an organ-
ism confers upon it the power of resisting opsonization, that this power
has some relation to the susceptibility of the particular animal whose
serum is used for opsonization, that the resistance to opsonization is
not lost on the death of the bacteria, and that in certain instances this

CELLULAR RESISTANCE 165

resistance is accompanied by capsule formation. Levaditi believes that
the resistance of virulent bacteria is dependent upon some alteration
of the bacterial membrane (which alteration determines in all prob-
ability the virulence of the organism) and also perhaps on the formation
by the bacteria of an anti-opsonic or anti-phagocytic substance. In the
latter connection Tschistowitsch and Jurewitsch claim to have shown
that on washing, virulent pneumococci lose their resistance to phago-
cytosis, but that submitting the organisms to the action of the material
in the washings restores them again to their resistant state. They con-
sidered that the salt solution removed in the washing a secretion which
they called antiphagin. This work has not been confirmed and can-
not be regarded as establishing beyond question the existence of
an antiphagin.

Influences Operating upon Phagocytic Cells. — In the preliminary
paragraphs of this discussion the stimulin theory of Metchnikoff was
dismissed with a simple statement that such a theory exists. Never-
theless, the leucocytes and their possible alterations are of considerable
importance in phagocytosis, and while it is true that increased phago-
cytosis resulting from immunity is not the result of stimulins, neverthe-
less, it is possible to augment the activity of these cells. Neisser and
Guerrini gave the name Icuco stimulants to certain substances which
directly act upon the leucocytes. According to Manwaring and Ruh,
numerous antiseptics in proper concentration exhibit a stimulating
action. According to others, calcium chloride, magnesium salts,
potassium iodide, iodoform, fat soluble substances (except cholesterol),
substances facilitating oxidation, pepton, quinine in certain low con-
centrations, nucleinic acid, similarly excite increased phagocytosis.
Marbe has extracted a thermostable body from the thyroid gland which
excites phagocytosis. The demonstration that the action of these various
substances is upon the leucocytes depends upon the use of decreasing
dilutions of the substances in the presence of sensitized bacteria and
washed leucocytes.

Metchnikoff showed the influence of heat on the leucocytes in
experiments which are tabulated as follows :

Degree of heat Time of heating Phagocytic index

40° C. 15 minutes 18

45° C. 15 minutes 8

50° C. 15 minutes 3

55° C. 5 minutes 1.2

60° C. 5 minutes o

60° C. 30 minutes o

In addition to heat, alterations of OH ions, alterations of osmotic
pressure, cholesterol, reduction in amount of electrolytes, potassium
ions, alcohols, ether, quinine and certain other of the leucostimulants
in high concentrations act upon the leucocytes to depress their phago-
cytic activity.

Analysis of Mechanism of Phagocytosis. — The mechanism of
phagocytosis includes the approach of phagocytes and the object to be

1 66 THE PRINCIPLES OF IMMUNOLOGY

phagocyted, the ingestion of these objects and in the case of living
objects their death ; finally the digestion of bacteria and other suitable
objects. The approach of the cells and the phagocytable objects is,
according to Mesnil and his co-workers and also Levaditi, due to a
physical chemical reaction and not dependent on the life of the leu-
cocyte. If leucocytes are injured by heat to 45°, 50° or 60° C, by refrig-
erator temperature, by shaking, by grinding, and then mixed with bacteria
and inactivated immune serum, the bacteria become clumped about
the leucocytes. This reaction may be observed even if the tubes are
laid in melting ice. The leucocytes that have been killed or paralyzed
will not ingest the bacteria. The " anchoring " of leucocytes and
bacteria will not occur unless specific opsonin is present in the serum.
It occurs with fragments of leucocytes as well as other cells of the
leucocyte series, such as myelocytes and myeloblasts. Thus the affinity
may be expressed as existing between the protoplasm of the phagocytic
cell and the sensitized bacteria or other phagocytable object.

Although actual ingestion of objects may be shown in the case of
artificial amebse it does not occur in the leucocyte unless the cell is
alive and in possession of its capacity to project pseudopodia. Hence
this stage of phagocytosis must be bound up with the life processes of
the phagocyte.

From the earlier studies of Metchnikoff it has been known that the
bacteria, after phagocytosis, are killed and digested. The influence
of the blood fluids in this phenomenon has been the subject of much
study and conflicting results. Metchnikoff and his co-workers were of
the opinion that the leucocytes contain complement, which, as has been
shown in previous chapters, is required for the action of bactericidal
and bacteriolytic amboceptors. They believed that this complement is
liberated only upon the destruction of the leucocytes as seen in phag-
olysis for they were unable to find complement in plasma. They inter-
preted the presence of complement in serum as due to the death of the
leucocytes during clotting of the blood. This interpretation has been
combated by numerous observers who have been able to demonstrate
complement in plasma. In support of the conception that the death of
the bacteria is due to completion of the bactericidal amboceptor-antigen
complex by complement in the leucocyte, is Bordet's work with cholera
vibrios. Using immune sera which contained bacteriolytic amboceptor,
he found no lysis except in those bacteria that were within phagocytic
cells. As opposed to this conception, the work of Neufeld and his
collaborators has shown that sera may be richly opsonic without con-
taining lytic amboceptors, and in these instances the bacteria are
destroyed and digested by the phagocytes. The destruction varies with
different organisms and with the virulence of the organisms, the more
virulent being less readily killed than the avirulent strains. Bacteria
may be cultivated on artificial media after having been ingested, a
certain amount of time being necessary to kill the organisms. The
act of digestion is closely bound up with that of killing the organisms.
The presence of a proteolytic ferment in leucocytes has been known

CELLULAR RESISTANCE 167

since the work of Mueller and Jochmann, who placed the leucocytes
of animals upon plates similar to those used for bacterial cultivation.
At incubator temperature, the leucocytes exhibit distinct proteolytic
power. Recent studies by Van Calcar would appear to indicate that
the organs of the body which secrete digestive ferments have some
influence over the ferments existing within the leucocytes. He found,
for example, that after the removal of the stomach the leucocytes of
the animal were unable to act as peptic digesters. Similarly the
removal of the pancreas destroys the ability of the leucocytes to act
as tryptic digesters. In summary it is necessary, in order to accom-
plish destruction and digestion, to sensitize the organisms and to have
present active living leucocytes. Opsonization will not in itself kill or
digest the organisms; therefore, the phagocyte must furnish some
substance which either completes the action of the opsonin or of itself
can kill and digest the organisms. The fact that phagocytosis in all
its stages may occur in slight degree independently of opsonin would
indicate that the phagocyte is the important element in death and diges-
tion of the phagocyted object. Recent work by Bachmann would indicate
that the leucocytes of normal and immune animals have a different
capacity for protecting against disease. Sixty times more leucocytes
from a normal animal were needed to save a guinea-pig against typhoid
infection than the number required from an immune animal. In the
case of anthrax the leucocytes from immune animals were eighty times
more active than those from normal animals. That these studies can
be interpreted as indicating a variation in the actual phagocytic power
of leucocytes is open to considerable question.

It is probable that the affinity of the phagocyte and phagocytable
object is, in large part if not entirely, a physical chemical phenomenon
entered into on the one hand by the cytoplasm of the leucocyte and
other cells and, on the other hand, the opsonized organisms or other
object. The ingestion, death and digestion are dependent upon the life
function of the phagocyte, which is capable of liberating a microbicidal
and microbilytic substance capable of combining with the microorgan-
ism to bring about its death and destruction.

OTHER MANIFESTATIONS OF CELLULAR RESISTANCE

Introduction. — rStudies of inflammation and of other cellular activ-
ities have made it clear that body cells play an important part in resist-
ance to disease that is not entirely explained by the phagocytic capacity
of certain of the cells. As has been indicated, cells other than the
polymorphonuclear leucocyte and the large mononuclear cell possess
the property of phagocytosis, but this is occasional and presumably
not of great importance. It seems desirable, however, to discuss the
mechanisms of resistance as influenced by properties of the leucocytes
other than phagocytosis, by activities of the lymphocytes and by the
cells and fluids which play a part in inflammation.

Bactericidal Extracts of Leucocytes. — The destruction of bacteria
within the phagocyte so impressed Metchnikoff that he assumed that

1 68 THE PRINCIPLES OF IMMUNOLOGY

extracellular destruction is accomplished by identical destructive
agents. The demonstration that extracellular destruction of bacteria
(bacteriolysis) requires the participation of amboceptor and comple-
ment had little influence on Metchnikoff's views, inasmuch as he was
convinced that complement originates solely in the leucocytes. As we
have stated (page 129) the more recent examination of this problem
makes it certain that complement exists free in the blood. Further
study, more particularly of opsonins and bacteriotropins, has made
it apparent that the mechanism of intracellular digestion is quite differ-
ent from that of extracellular lysis. Nevertheless, the leucocytes may
contribute to the extracellular destruction of bacteria. Buchner showed
that the exudation, produced in the pleura of rabbits and dogs by injec-
tions of aleuronat, removed and killed by freezing and thawing, pos-
sesses the property of killing bacillus coli. Denys and Kaisin produced
pleural exudates by injection of killed staphylococci and removed the
cells by centrifugation. The clear supernatant fluid was actively bac-
tericidal. Others have made extracts of exudates, and of leucocytes
obtained from the blood, and have demonstrated that a bactericidal
substance is to be obtained. Certainly these substances are yielded up
after the destruction of the cells and, according to Petterson, they
may be secreted by the cell during its life. The substances are resistant
to a temperature of 56° C, but after inactivation by heat to 75° to 80°
C. they cannot be reactivated by the addition of fresh extracts. This
substance or group of substances has been called endolysin by Petterson
and leucine by Schneider. It is not identical in all animals since that
from dogs, rabbits and guinea-pigs kills bacillus proteus and bacillus
anthracis, but that from the guinea-pig and cat fail to kill the bacillus
typhosus and the spirillum cholerae.

Bachmann has recently reported on a so-called leucocyte antibody,
" cmticorps leucocytaire" which is distinct from the bactericidal endo-
lysin. It appears in the leucocytes of immunized animals and may serve
to produce passive immunity in other animals. It is found only in the
polymorphonuclear leucocytes and may be extracted in normal serum.
It is effective in protecting guinea-pigs against intraperitoneal injection
of the specific organism and also acts beneficially and specifically upon
established infections. A temperature of 75° C. destroys this substance,
but if the material is well diluted and gelatin added, the same degree
of heat serves to destroy the non-specific bactericidal substances (endo-
lysins) but permits the specific leucocyte antibody to remain active.
Bachmann believes that the persistence of this antibody in the leuco-
cytes explains the fact that individuals retain immunity to certain
diseases after the serum antibodies are no longer demonstrable.

Leucocyte Enzymes. — In contrast to the bactericidal substances
extracted from leucocytes it is possible to obtain enzymes. Leber, in
a study of inflammation, found that sterile pus can liquefy gelatin and
the study of this proteolytic enzyme, the leucoprotease, has been ex-
tended by Miiller and Jochmann, Opie, Longcope and others. This
leucoprotease may be purified by precipitation with alcohol more par-

CELLULAR RESISTANCE 169

ticularly from glycerol extracts and the desiccated precipitate may be
preserved almost indefinitely. In the moist state temperatures of from
50° to 65° C. increase its activity, but at 70° to 75° C. it is destroyed.
It acts best in weakly alkaline or neutral medium, and is inhibited by
acid. It differs from trypsin in that it is much less active ; it does not
require activation by any such substance as enterokinase, and exists
within the cells in an active state rather than in the form of zymogen.
It differs from the bactericidal extracts in that it cannot kill bacteria,
but may digest them after their death. The blood possesses an anti-
enzyme, but when the cells accumulate in bulk, as in the case of inflam-
matory exudates, the anti-enzyme is overbalanced and the protease
dissolves necrotic cells, dead bacteria and other detritus. It is of
considerable importance in the resolution of lobar pneumonia. In
addition the leucocytes are stated to contain amylase, diastase, catalase,
oxidase, peroxidase, nuclease and an ereptic ferment, but there appears
to be a difference of opinion in regard to lipase.

Opie has described an additional ferment in areas rich in large
mononuclear cells, which acts best in a very weak acid medium. It is
inhibited by temperatures of 50° to 65° C., by alkali and by the con-
centration of HC1 (0.2 per cent.) favorable for the action of pepsin.
He was able to demonstrate this ferment in hyperplastic lymph-nodes
rich in large mononuclear phagocytes. It is closely related to the
enzymes of tissue autolysis. The acid medium which favors the action
of this enzyme inhibits the activity of anti-enzyme.

Leucocyte Extracts for Therapeutic Purposes. — Petterson noted
that when leucocytes are placed in contact with blood serum for several
hours the mixture is more actively bactericidal than the serum alone
or salt solution extracts of the leucocytes. This led to experiments in
which he injected leucocytes simultaneously with anthrax bacilli into
dogs and found a moderate protection by this treatment. Opie similarly
observed that the injection of leucocytes and tubercle bacilli into the
pleura of dogs led to less severe manifestations than when tubercle
bacilli alone are injected. Probably the most important contributions
to the treatment of disease by leucocyte extracts are the studies of Hiss
with the collaboration of Zinsser, Dwyer and others. Hiss obtained the
leucocytes from pleural exudates produced by the injection of aleuronat
suspensions. This was centrifuged before clotting occurred and the
cells emulsified in distilled water. Either the leucocytes or the leu-
cocytes and supernatant fluid were employed for treatment. From
experiments with staphylococcus, pneumococcus, streptococcus, meningo-
coccus and typhoid bacillus infections in rabbits, it was determined that
protection was afforded by the extracts and that the infection was
favorably influenced if therapeutic doses were given as late as twenty-
four hours after infection. Encouraging results were also obtained in
the treatment of human cases of pneumonia, meningitis, staphylococcus
infections, erysipelas and other diseases. In analyzing the beneficial
effects of this form of treatment, it was found that the bactericidal
properties of the extracts are not sufficiently great to explain their

170 THE PRINCIPLES OF IMMUNOLOGY

influence, they do not materially favor phagocytosis but appear to
augment the migration of leucocytes to a slight degree and possibly are
of importance in this way because of the fact that they exert positive
chemotaxis. Zinsser states " we are inclined to believe at present that
the beneficial effects of leucocyte extracts are based on the same prin-
ciples as those which determine the reactions following on the injection
of bacterial and any other protein." To us it appears that this method
is to be included in the category of non-specific therapy previously
discussed (page 30).

Specific Hyperleucocytosis. — Following upon the earlier sug-
gestion of Bordet, Gay and his collaborators found that immune animals
exhibit a much higher degree of leucocytosis following the injection
of the organism to which they had been immunized than do normal
animals. For example, rabbits immunized to typhoid bacilli reacted
to subsequent injections of typhoid bacilli with blood counts of as high as
150,000 leucocytes per cmm., whereas normal rabbits showed a reaction
of only 40,000 to 50,000 leucocytes per cmm. This phenomenon of spe-
cific hyperleucocytosis has been contradicted by McWilliams, who found
no important difference in response between normal and immune animals
and further states that typhoid immune rabbits react in essentially the
same degree to colon bacilli as to typhoid bacilli. Others have confirmed
the work of McWilliams. Zinsser and Tsen found a slight favorable
difference in animals immunized to Gram negative cocci and a somewhat
more marked difference in those immunized to Gram positive cocci, not
in any case, however, to the degree indicated by Gay. There seems
little reason for believing that a specific hyperleucocytosis plays any
important part in resistance to infection. This, however, is not to be
construed as an argument against vaccination, since the latter procedure
is important in the production of specific opsonins, agglutinins and other
immune bodies. Any response to vaccination in the form of leucocytosis
must be regarded as only in small part if at all specific and is probably
of the same nature as the leucocytic response to the injection of non-
specific proteins and their products.

The Lymphocytes. — Lymphocytes appear in inflammatory areas as
the result of infection, but accumulate in largest amounts in chronic
inflammatory areas where, in most instances, the active infective agent
is no longer present. The part they play in the phenomenon of inflam-
mation and in protection against infection is not understood. From
the work of Opie it seemsi probable that the lymphocytes may be, in
part, the source of the ferment which he describes as operating in
weakly acid media. As pointed out above, this ferment was obtained
from hyperplastic lymph-nodes. The lymphocytes are said to contain
a lipase, and it is suggested that the large collections of these cells
about tuberculous foci may serve by the action of the lipase to break
down the waxy shell of the bacilli. The lymphocyte is stated to possess
phagocytic properties, but these are at best very slight and probably
play no important part in resistance to disease. It has long been noted
that the presence of tumors in the body often excites a neighboring

CELLULAR RESISTANCE 171

chronic inflammatory reaction in which lymphocytes appear in con-
siderable numbers. J. B. Murphy and his collaborators have put to
the test of experiment the hypothesis that lymphocytes are of import-
ance in resistance to cancer. By the use of the X-ray they were able
to destroy practically all the lymphoid tissue of the body of animals and
found in these animals a decreased resistance to transplanted cancer.
Immunity already established to cancer was also destroyed by this
procedure. Similarly there was a lowered resistance to tuberculosis
and to anterior poliomyelitis. In tuberculosis the lymphocyte constitutes
a large element in the inflammatory reaction, and this is true also in the
later stages of acute anterior poliomyelitis. Although small doses of
X-ray may stimulate lymphocyte production, Murphy and his asso-
ciates found that dry heat produces a more durable increase in the
circulating lymphocytes. By increasing the lymphocytes in this fashion
they demonstrated " the establishment of a high degree of immunity
to certain transplantable cancers in mice," regardless of whether these
cancers naturally showed a high or low percentage of successful inocula-
tion. The same was found to be true in regard to the implantation of
grafts from spontaneous cancers into the animals from which the
grafts were removed. This subject has also been studied by F. C.
Wood and associates in the Crocker Laboratory. They found that mice
with lymphatic leucemia show no demonstrable immunity to tumors.
They found that reduction of the total leucocyte count by means of
X-ray or radium produces no increase in the successful transplanta-
tion of normal tissues. They found further that successful transplan-
tation of the guinea-pig fibrosarcoma is not influenced by the use of
X-ray. They selected an immune strain of rats, exposed them to X-ray
and found no change in susceptibility to transplantable tumors. They
found that the use of X-ray on rats in which a highly virulent tumor had
been implanted did not prolong the life of the tumor. Wood states
that " it is, therefore, evident that the lymphocyte is in no way corre-
lated with cancer immunity." Sittenfield also found that artificial
lymphocytosis has no effect whatever on tumor growth. It is of further
interest that in human cancer the lymphocytes collect about the slowly-
growing rather than the rapidly-growing tumors and that the metastases
are frequent in the lymph-nodes. The later experiments of Murphy
on the lymphocytosis induced by heat have not received as yet extensive
examination ; therefore, the question remains open. Murphy's experi-
ments are so well conducted that it is difficult to be assured that the
lymphocytes play no part. The work of Wood carried out on a large
number of animals is of especial significance and would indicate that
the lymphocyte plays no such important part in resistance to cancer as
Murphy's work appears to indicate.

Platelets. — In 1901 Levaditi noticed that following the injection of
cholera vibrios they were often found clumped around small masses
of platelets. The phenomenon was called thigmotropism. Govaerts
subsequently demonstrated that the clumping is influenced by the action
of opsonins. LeFevre found that anti-bacterial immunization increases

172 THE PRINCIPLES OF IMMUNOLOGY

thigmotropism because of the increase in activity of opsonins. Further
study may throw light on the mechanism of the process, but at present
its function is obscure.

The Influence of Inflammation. — Infection always produces some
degree of inflammatory reaction, but this varies considerably with the
type of infectious organism and with the capacity of the host to react.
The exudate comprises the polymorphonuclear leucocyte, the lympho-
cyte, the plasma cell, the large mononuclear cell, certain other less
important cells, the red blood-corpuscles, serum and fibrin. The part
played by the more important of these cells is indicated above. As far
as we can determine, the red blood-corpuscles appear more as an
accident of the process than as an essential part of it. The fluid part of
the exudate rapidly coagulates with the formation of fibrin and serum.
There can be no doubt that the serum serves in certain measure to
concentrate in the inflammatory areas those immune bodies qualified
to offer resistance to the invader and its products. In case toxic
products are present, these are diluted by the serum and the subsequent
absorption of the serum with this diluted poison aids in its elimination
from the body. The fibrin network probably serves in a certain measure
to wall off and limit the growth of the invading organism. It also
serves as a scaffolding for the support of newly-growing fixed tissue.
Very early in the course of an acute inflammation the connective tissue
cells proliferate. They may be phagocytic, but this property is of
little significance. Certainly the most important function of the con-
nective tissue in resistance to infection is the formation of a tissue
which serves to limit the advance of the infection. The newly-growing
connective tissue, with its capillaries, constitutes granulation tissue and
the resistance of granulation tissue to infection is a matter of common
observance. As the inflammation becomes chronic the connective itssue
becomes denser and thereby provides a much less permeable wall than
is found in the earlier stages of the process. The production of a local
inflammation leads to the formation of an exudate which by virtue of
the polymorphonuclear leucocytes opposes to infection the important
process of phagocytosis; the liberation of bactericidal substances and
of enzymes from the leucocytes serves to aid in resistance and to liquefy
dead tissues and dead bacteria. Under favorable circumstances addi-
tional enzymes are provided by the large monuclear cells and lympho-
cyte. The large mononuclears aid in the removal of dead material by
virtue of their phagocytic powers. The fluid part of the exudate brings
into the process the immune bodies of the circulating blood, serves to
dilute toxic products and favors their absorption and elimination in
dilute form. The fibrin, granulation tissue and cicatrization act as de-
limiting elements and operate toward the localization of the process.

CHAPTER VIII
COMPLEMENT FIXATION

INTRODUCTION.

THE BORDET-GENGOU PHENOMENON.
LABORATORY DEMONSTRATION.

ANTI-COMPLEMENTARY AND HEMOLYTIC TITER OF ANTIGEN.
THE TEST.

SPECIFIC CHARACTER OF THE TEST.
INHIBITION ZONES.
GROUP REACTIONS.

RELATION OF COMPLEMENT-FIXING BODIES TO OTHER IMMUNE BODIES.
IS THE COMPLEMENT-FIXING BODY AN AMBOCEPTOR ?
ACTIVATION BY COMPLEMENT.

FIXATION OF THE COMPLEMENT OF NATURAL HEMOLYSINS.
INHIBITION OF COMPLEMENT OTHER THAN BY FIXATION.
ANTI-COMPLEMENTARY CHEMICAL AGENCIES.

ANTI-COMPLEMENTARY ACTION OF CELLS, TISSUE EXTRACTS AND BODY FLUIDS.
ANTI-COMPLEMENTARY ACTIVITY OF IMMUNE SERA.

Introduction. — A summary of the hypotheses concerning the con-
stitution of complements shows that there are three important views
offered, namely the " pluralistic " conception of Ehrlich and Morgen-
roth, the " dualistic " of Metchnikoff and " unitaristic " of Bordet.
As has been explained, the view of Metchnikoff that complement might
be a " macrocytase " or a " microcytase " depending upon its cellular
origin has been abandoned by most immunologists. Thus the conflict
has been, and in certain measure still is, between the views of Ehrlich
and of Bordet. Bordet and Gengou in demonstrating that the same
complement is called on for bacteriolysis as for hemolysis, discovered
the phenomenon named by them complement fixation (" la fixation
d'alexine ") which we employ in sharp contradistinction to complement
deviation. The latter term implies the anchoring of complement by
free amboceptor units, whereas fixation signifies the entrance of the
complement into combination with antigen and amboceptor. In brief,
they showed that if complement is utilized in the process of bacteriolysis
it is not available for hemolysis.

The Bordet-Gengou Phenomenon.-^The primary experiment was
performed with plague bacilli, the serum of a horse immunized to
plague bacilli, fresh guinea-pig serum (complement) and sensitized red
blood-corpuscles, i.e., corpuscles saturated with a specific hemolytic
immune serum. They mixed an emulsion of plague bacilli, the anti-
plague horse serum and complement. This mixture was left at room
temperature for five hours and then the previously-sensitized erythro-
cytes added, the mixture incubated and observed. No hemolysis
appeared, although the corpuscles were often agglutinated by the hemo-
lytic (and hemagglutinative) immune serum. Naturally, such an ex-

173

174 THE PRINCIPLES OF IMMUNOLOGY

periment required numerous controls, the complete series being indi-
cated in the following protocol :

1. Plague bacilli -j- immune horse serum -f- complement +v sensitized cells =

No hemolysis.

2. Plague bacilli + normal horse serum -f complement + sensitized cells =

Hemolysis.

3. — — — — immune horse serum -f~ complement + sensitized cells —
Hemolysis.

4. — — — — normal horse serum + complement + sensitized cells =
Hemolysis.

5. Plague bacilli -f- immune horse serum — — -f* sensitized cells =

No hemolysis.

6. Plague bacilli + normal horse serum + sensitized cells =

No hemolysis.

Throughout the experiment all the sera were inactivated except the
fresh guinea-pig complement and all mixtures stood at room tempera-
ture for five hours before the addition of the sensitized erythrocytes.
Hemolysis in tube 2 shows that normal horse serum does not serve
as an amboceptor or sensitizer for the plague bacilli and therefore
does not prevent the complement from entering into combination with
the sensitized erythrocytes. Tubes 3 and 4 contain no bacterial
antigen, cannot utilize complement and therefore hemolysis appears.
Tubes 5 and 6 show that the bacteria are not hemolytic and that
neither of the inactivated immune sera nor the inactivated normal horse
serum contain complement for the completion of the amboceptor-cell
complex. Bordet and Gengou showed that the same phenomenon could
be observed with a wide variety of bacteria and specific immune sera
both of human and lower animal origin; these operate to fix both
guinea-pig and human complements, so as to prevent combination of
these complements with hemolytic immune sera from several species.
Furthermore, the immune sera so fixed might be specific for several
varieties of erythrocytes. Muir and Martin found, however, that
whereas most complements can be fixed in such experiments, this is not
universally true and occasional complements are met with which do
not enter into certain combinations. Furthermore, the process could
•be reversed so that the fixation of complement in hemolysis prevented
its action to bring about bacteriolysis of sensitized bacteria. Thus it
appeared that one and the same complement operates for the produc-
tion of both bacteriolysis and hemolysis. This demonstration of the
unity of complement has been combated by later work, and it now
appears that there are certain exceptions to the rule, although it can
generally be accepted.

Laboratory Demonstration of the Bordet-Gengou Phenomenon. — In
order to demonstrate the phenomenon it is not necessary to use plague bacilli, as
others serve the purpose equally well. The readily obtainable typhoid bacillus
and typhoid immune serum can be used with good results. In setting up the test
it is important to bear in mind the fact that numerous substances may interfere
with the activity of complement, and among these are certain concentrations of

COMPLEMENT FIXATION

175

bacterial emulsions and extracts. Therefore, it is necessary to be sure that the
amount of bacterial emulsion used in the test is not " anti-complementary," but
yet in sufficient concentration to operate well. The emulsion is made from a
twenty-four-hour slant agar culture (see page 81 for preparation) and may be
killed by heat or formalin. The preliminary titration may be set up as follows :

Bacterial
emulsion

Complement
i-io dilution

1

Hemolytic
amboceptor

Erythrocyte
suspension

I

Result

0.5 c.c.
0.4 c.c.
0.3 c.c.

0.2 C.C.
O.I C.C.

o 5 c.c

0.5 c.c.
0.5 c.c.
0.5 c.c.
0.5 c.c.
0.5 c.c.

incubate one

0.5 c.c. (2 doses)
0.5 c.c. (2 doses)
0.5 c.c. (2 doses)
0.5 c.c. (2 doses)
0.5 c.c. (2 doses)
0.5 c.c. (2 doses)

0.5 c.c.
0.5 c.c.
0.5 c.c.
0.5 c.c.
0.5 c.c.
0.5 c.c.

incubate one

P.H.
C.H.
C.H.
C.H.

Each tube should be made up to a volume of 2.0 c.c. with salt solution before
primary incubation. If convenient the erythrocytes may be sensitized by the
previous addition of amboceptor. In the protocol C.H. indicates complete
hemolysis, P.H. partial hemolysis and — no hemolysis.

The Test. — The results given indicate that 0.5 c.c. bacterial emulsion is defi-
nitely anti-complementary, but the 0.3 c.c. has no such influence. The last tube
excludes hemolytic activity on the part of the emulsion. In order to be abso-
lutely sure that the final test will not be misleading through the anti-comple-
mentary action of the bacterial emulsion it is advisable to use the next smaller
amount than the titration shows to be free of anti-complementary activity, which
in this case is 0.2 c.c. This being the case 2.0 c.c. bacterial emulsion may be
diluted with 3.0 c.c. salt solution, whereupon 0.5 c.c. of the dilution will contain
0.2 c.c. original emulsion. The complement-fixation test may then be set up
as follows :

Bacterial
emulsion
(2-5)

Anti-
typhoid
immune
serum

Normal
rabbit
serum

Complement

Salt
solution

L

Sensitized
erythrocytes

£

Results

O 5 C.C.

(l-IO

dilution)

O.5 C.C.

(l-IO

dilution)

(l-IO

dilution)
0.5 c.c.

1

1

I.O C.C.

&

a>

0.5 c.c.
0.5 c.c.

0.5 c.c.

0.5 c.c.
0.5 c.c.

0.5 c.c.

<D

I.O C.C.
I.O C.C.

$
1

C.H.
C.H.

0.5 c.c

0.5 c.c.

0.5 c.c.

I

I.O C.C.

C.H.

0.5 c.c.

0.5 c.c.

0.5 c.c.

£

• I.O C.C.

a

C.H.

0.5 c.c.

I.O C.C.

I.O C.C.

C.H.

1.5 c.c.

I .O C.C.

The first incubation permits of fixation of the complement by the bacteria
and their specific immune serum and the second determines whether or not
complement is free to act upon the sensitized erythrocytes. For sensitization of
erythrocytes the hemolytic immune serum should be diluted so that 0.5 c.c.
contains two units hemolytic amboceptor, then added to an equal volume 5 per
cent, erythrocyte suspension. In the above test the immune serum is diluted, so
that 2.5 c.c. contain ten units amboceptor; it is then added to 2.5 c.c. 5 per cent,
erythrocyte suspension and the mixture allowed to remain at room temperature
for one hour. The protocol given above shows, reading from below upward,
that the hemolytic immune serum used for sensitization is not of itself hemolytic,
that the complement is in sufficient concentration for hemolysis, that neither the
bacterial emulsion nor the typhoid immune serum is anti-complementary in the
amounts used. In the first tube the bacterial emulsion, specific anti-bacterial
serum and complement interact so that the complement is not free to combine with
the sensitized erythrocytes, whereas tube 2 shows that normal rabbit serum will
not fix complement.

Specific Character of the Test. — In order to elaborate the test and to show
its specificity it is well also to titrate an emulsion of some other organism, for
example, colon bacilli for anti-complementary activity at the same time the
typhoid emulsion is titrated and in the same manner. If this shows anti-comple-

176

THE PRINCIPLES OF IMMUNOLOGY

mentary activity in a dose of 0.3 c.c., then O.I c.c. is used in the te<st. The fully
controlled test would then be set up as follows :

Typhoid

emulsion

(2/5)

Coli
emulsion
(i/5)

Anti-typhoid
immune serum
(i/io dilution)

Complement
(i/io dilu-
tion)

Salt solu-
tion

u

Sensitized
erythro-
cytes

w,

I

Results

0.5 c.c.

0.5 c.c.

0.5 c.c.

rCJ

.0 c.c.

^H

0.25 c.c.

o.s c.c.

0.5 c.c.
0.5 c.c.

0.5 c.c.
0.5 c.c.

0.25 c.c.

8

o

.0 c.c.
.0 c c

CH

0.5 c.c.

0.5 c.c.

0.5 c.c.

ctf

.0 c.c.

3

C.H.

0.5 c.c.

0.5 c.c.

0.5 c.c.

1

.0 c.c.

.g

C.H.

0.5 c.c.

0.5 c.c.

0.5 c.c.

§

.0 c.c.

i

C.H.

0.5 c.c.

I.O C.C.

H- 1

I.O C C.

d

CH

I e c.C

I O C C

•Reading from above downward, the second tube shows 0.25 c.c. typhoid
emulsion diluted 2 to 5, thus corresponding in bulk of original emulsion, to the
bulk of coli 'emulsion in 0.5 c.c. of a 1-5 dilution. It is necessary to use the
smaller bulk of coli emulsion in order to prevent anti-complementary activity.
Both quantities of typhoid emulsion are sufficient to fix the complement, whereas
the coli emulsion (tube 3) does not. The next three tubes which are controls show
that neither typhoid emulsion, coli emulsion nor anti-typhoid immune serum
have any anti-complementary activity. The last two tubes show that the com-
plement is in sufficient concentration to operate and that the sensitized erythrc-
cytes will not of themselves hemolyze under the conditions of the experiment.
If the results prove to be confusing it is necessary to make additional controls
to determine if any of the reagents is hemolytic. This contingency is extremely
rare if proper care is given in their preparation. The test in this form shows
that the reaction is specific.

Inhibition Zones. — The phenomenon of complement fixation ex-
hibits certain of the characters noted in regard to other immune reac-
tions, not only in the titration of the reacting bodies but also in the
formation of the so-called inhibition zones and in the group reaction.
These latter features are best illustrated with the fixation of comple-
ment by immune sera prepared from the use of dissolved protein.
Gengou, a year after the publication of Bordet and Gengou, showed
that the inoculation into an animal of dissolved proteins, such as egg-
white, could lead to the formation of bodies which participate in com-
plement fixation with the specific antigen. This was confirmed by
Moreschi and later by Neisser and Sachs. The latter authors applied
the reaction to the forensic determination of protein. Gengou was of
the opinion that the immunization of animals with dissolved protein
led to the formation not only of precipitins but also of complement-fixing
bodies. The relation between these two immune substances will be
discussed after presenting data concerning inhibition zones and group
reaction. An experiment from the work of Neisser and Sachs serves
to illustrate the fact that immune serum may be used in the reaction
in such strong concentration as to inhibit fixation of the complement.
For this purpose they arranged two series of tubes. In series A they
placed decreasing amounts of the specific immune serum, a constant
quantity of 0.2 c.c. of 1-2000 solution of the antigenic human serum
and o.i c.c. fresh guinea-pig complement. In series B the same con-
stituents were placed with the exception of the immune serum which
was replaced in each tube by 0.2 c.c. salt solution. These mixtures
were incubated at 37° C. and then sensitized red blood-corpuscles were

COMPLEMENT FIXATION 177

added, followed by another period of incubation. In this particular
instance they employed ox blood-cells and immune serum prepared by
injection of ox blood-cells into the rabbit.

MODIFIED PROTOCOL FROM NEISSER AND SACHS

Immune serum Complement fixation

i- 10 dilution Series A Series B

I.O C.C. +

0.75 c.c.

0.5 c.c.

0.35 c.c.

0.25 c.c. +++ —

0.15 c.c. ++

O.I C.C. + —

0.0 C.C. —

The above protocol shows that in the strong concentration of im-
mune serum the fixation of complement is not as marked as in some-
what weaker concentration. Nevertheless, there also comes a point
when the concentration is too dilute to permit of fixation. The tubes
in series B show that the concentration of human serum in itself is not
sufficiently great to prevent the hemolytic reaction. Two points are
of interest in this connection. In the first place, it is possible to add
immune serum or other serum in amounts so large that the serum itself
will have inhibitory action upon the complement. Under optimal con-
ditions immune serum may be diluted to an extreme degree and still _
act as a complement-fixing body; for example, Friedberger, by the
use of a well-prepared serum was able to demonstrate complement ;
fixation by an immune serum diluted 1-1,000,000,000. The same deli-
cacy has not been confirmed by other investigators and must be regarded
as a scientific curiosity. The dilution of the antigenic protein can be
carried to a considerable degree but not usually to the same degree as
is possible with antiserum.

Group Reactions. — In the application of the complement-fixation
test to the forensic determination of dissolved protein, Neisser and
Sachs showed that the group phenomenon also appears. They also
showed that the antigenic serum could be very much reduced in amount
and still give complement fixation. The following protocol illustrates
the manner in which such a demonstration may be made. In setting
up the test there was used throughout a constant quantity of o.i c.c.
immune serum prepared by the injection of human serum. The anti-
genic serum was added according to the amounts indicated in the
protocol. Complement was used in amounts of 0.05 c.c. The mixtures
were incubated and then beef blood-corpuscles which had been sensitized
with a specific anti-beef corpuscle serum were added, the mixtures
again incubated and the degree of fixation determined.

GROUP REACTION MODIFIED FROM NEISSER AND SACHS

Amounts of Fixation with serum of

antigenic serum Man Monkey Goat

0.01 +++

0.001

o.oooi ++4-

O.OOOOI -j-

O.OOOOOI
0.0
12

178 THE PRINCIPLES OF IMMUNOLOGY

The above protocol shows that anti-human serum is capable of
fixing complement in the presence of an amount of antigenic serum,
which is considerably less in the case of human antigenic serum than
in the case of monkey antigenic serum. Thus the group reaction is
indicated by the fact that the serum of a closely-related species is in
certain doses sufficient to produce fixation. Muir and Martin, also,
were able to demonstrate similar reactions in several different
animal groups.

Relation of Complement-fixing Bodies to Other Immune
Bodies. — The fact that the treatment of animals with a protein in
solution can lead to the development in the animal's serum of a capacity
both for precipitating the antigen and combining with the antigen to fix
complement suggests naturally that there may be some relationship
between the two phenomena. Gay and Moreschi independently were
able to show that precipitates formed by the action of a specific immune
serum can so bind complement as to prevent its action upon a hemolytic
system. The assumption is justified, therefore, that the two phenomena
are very closely related and may indicate that complement fixation
depends in part at least upon fixation of the complement by a precipitate.
The question naturally arises then whether or not there may be com-
plement fixation without precipitation or precipitation without com-
plement fixation. Furthermore, a fundamental problem is whether or
not the two activities of the antiserum depend upon two different
immune bodies in the serum or upon the double capacity of the same
immune body. Neisser and Sachs were able to show that complement
fixation occurred with very much smaller amounts of antigen than does
visible precipitation. As has been mentioned before, in reference to the
delicacy of the reaction, it was pointed out that fixation of complement
may occur with dilutions of 1-1,000,000,000, whereas visible precipita-
tion has never occurred in such marked dilution of antigenic or of
immune serum. Thus it can be concluded that the presence of a visible
precipitate is not necessary for the fixation of complement, a statement
amply corroborated by Muir and Martin. Wassermann and Bruck
found that by permitting bacterial extracts to stand for a considerable
time, the extracts were no longer precipitable in the presence of specific
precipitating immune sera, whereas fresh extracts show beautiful pre-
cipitation. Nevertheless, both new and old bacterial extracts were
found to fix complement in the presence of the specific immune serum.
Liefmann further showed that the action of heat may so alter the
antigenic protein as to lead to differences in complement fixation and
precipitation. He immunized rabbits with egg-white and found that
after heating the egg-white it could be so changed that it was no longer
precipitable by the immune serum but could still operate with the
immune serum in complement fixation.

Felke and also Garbat have found that anti-typhoid vaccination in
man leads to the production of agglutinins, but to no or very slight
production of complement-fixing bodies. Felke found that in the course
of typhoid fever and during convalescence complement fixation could

COMPLEMENT FIXATION 179

be demonstrated in addition to agglutination. Most of the preceding
experiments indicate that the phenomena of precipitation and comple-
ment fixation are not necessarily associated, but, on the other hand,
cannot be interpreted to indicate that the immune serum contains two
different immune bodies. Friedberger and Liefmann, working inde-
pendently, showed, however, that heating an immune serum to 67° C.
can destroy the precipitin in the serum without altering the capacity
of the serum for participating in complement fixation. This experiment
has been interpreted as indicating that precipitating and complement-
fixing bodies represent independent activities but not necessarily that
they are different bodies. Muir and Martin found that upon immuniz-
ing animals they were able to demonstrate that the serum of these
animals contained complement-fixing' powers earlier than precipitins
could be demonstrated. Altmann found that complement-fixation
bodies appeared earlier than agglutinins for paratyphosus B and colon
bacilli but with the use of typhoid bacilli both bodies appeared about
the same time. As a converse of this demonstration, Moreschi im-
munized birds with rabbit serum and found in contravention to his
earlier work that he was able to produce a precipitin of very high titer
without being able to demonstrate the power of complement fixation on
the part of the immune serum. This was corroborated by Sobernheim.
Liefmann was able to show a certain amount of difference in the activity
of immune serum. He brought the immune serum in contact with the
antigen at o° C. for sufficient time to produce a considerable amount
of precipitate. He then centrifuged the precipitate and found that the
supernatant fluid at 37° C. was capable of fixing complement. Lebailly,
by the fractional addition of antigen to the precipitating immune
serum, was able apparently to separate the precipitating and comple-
ment-fixing bodies. Arlo precipitated the antigenic and immune sera
by means of CO2, thereby obtaining the globulins in the precipitate.
In both instances the complement-fixing body was found in the redis-
solved globulin fraction and the precipitating body was found in the
supernatant fluid. This has been controverted by Bruynoghe, who
maintains that euglobulins are capable of producing non-specific fixa-
tion. Reviewing all this experimental evidence, it seems perfectly
clear that complement fixation can and does occur independently of
visible precipitation, a statement supported by a great mass of more
recent investigation of the subject. None of these experiments, how-
ever, can be safely interpreted as indicating that there are two separate
bodies in the immune serum. Neufeld and Handel, however, appear to
be definitely of the opinion that there are two separate bodies concerned.
They found that sensitized cholera spirilla are capable of fixing the
hemolytic complement at o° C. but that at 37° C. the organisms will
fix both hemolytic and bacteriolytic complement. They explain this
by assuming that the fixation at higher temperature is due to the bacteri-
cidal amboceptor but that the fixation at o° C. is due to a separate sub-
stance which they named the Bordet antibody. Such experimental
evidence cannot be accepted as final. Sachs states that a priori it can

i8o THE PRINCIPLES OF IMMUNOLOGY

be supposed that the antigenic protein can simultaneously combine with
precipitins and with amboceptor. He offers the hypothesis that one
immune molecule may contain different binding complexes, one, for
example, combining with precipitins to produce a precipitate and the
other combining with the antigen and the complement to produce fixa-
tion. If this view be accepted, the experiment of Friedberger and Lief-
mann, in which the immune serum was heated, indicates that of the
two binding complexes the precipitating one is the more labile. It can
very readily be seen that the interpretation of Sachs depends almost
entirely upon an acceptance of the Ehrlich hypothesis of the structure
of immune bodies.

Dean has examined the question and finds that the optimal rela-
tionship between antigen and immune serum for the production of
precipitation is by no means necessarily the optimal relationship for
complement fixation. Therefore, the two phenomena, as has already
been pointed out, are by no means parallel. He is of the opinion,
however, that this lack of parallelism is not necessarily an indication
that the two things are entirely distinct and separate. He is of the
opinion " that they represent two phases of the same reaction." The
complement fixation represents the earliest and more delicate stage
of a reaction which, in its more marked manifestation, is seen by the for-
mation of a precipitate. Zinsser has studied the matter carefully and has
come to the conclusion " that the precipitation is merely a secondary,
colloidal phenomenon, which may, or may not, coincide with the phase
of greatest alexin (complement) fixation, according to other fortuitous
conditions which may favor or retard flocculation." He found that a
mixture of sheep serum and its specific immune serum showed com-
plement-fixing activity only in the precipitate. On the other hand, in
a mixture of a filtrate of typhoid bacilli and a specific immune serum
both the precipitate and the supernatant fluid were capable of fixing
complement. " From this it seems to follow that immunization with
the more complex cellular elements has given rise to the precipitating
antibodies present also in the anti-sheep serum, and in addition to
this to sensitizers which are not precipitable (remaining in the super-
natant liquid) and not present in the anti-sheep serum." He, therefore,
is of the opinion that since both the antigen and the immune body are
colloidal in character they may be expected to follow the laws of
colloids. This may be interpreted to indicate that the contact of the
mutually precipitating colloids must be present in optimal concentration
in order to show a visible precipitation, but, on the other hand, .the
interaction of the two bodies which, in the quantities employed,
show no visible precipitate, may be demonstrated by the comple-
ment-fixation test. He states " that the visible precipitation would
seem, therefore, to be a secondary phenomenon, the essential one
being the union of an antigen with a sensitizer by which it is ren-
dered amenable to the action of the alexin " (complement).

Is the Complement-fixing Body an Amboceptor? — There arises
further the question as to whether or not the body, which, in combina-

COMPLEMENT FIXATION 181

tion with antigen, serves to fix complement, is to be regarded as an
amboceptor (sensitizer). As has been shown in the discussion of
cytolysins, it is possible at o °C. to bring about a selective combination
of hemolytic amboceptor with its antigen. Liefmann attempted to
bring about a union of complement-fixing body and its antigen in this
way but was unsuccessful. It is known that if a considerable excess
of antigen or antiserum is present, complement may also be absorbed
at o° C. and in such an experiment as Liefmann's it is impossible to
say that such an excess did not exist. Therefore, the experiment is
not conclusive. Neufeld and Handel also attempted selective absorp-
tion at 37° C. They showed that cholera vibrios and their specific
immune sera fix the hemolytic complement at o° C., whereas the
bacteriolytic complement remains active. At 37° C. both complements
are fixed. They are of the opinion that at o° C. the complement-fixing
amboceptor is bound to the hemolytic complement and that at 37° C.
both the complement-fixing and bacteriolytic amboceptors are active.
This experiment has been held to support the hypothesis of the multi-
plicity of complements. They also found that an immune serum pro-
duced by the injection of a certain water vibrio acted as a complement-
fixing body with cholera spirilla but did not serve as a bacteriolytic
amboceptor. This may be interpreted as indicating that the two im-
mune bodies are distinct but does not prove the amboceptor nature of
that body which enters into the phenomenon of complement fixation.
It may very well be that the experimental conditions were not optimal
to the reactions and that while investigators sought to separate two
forms of complement they were working with one and the same body
which operates somewhat differently under the diverse conditions.
Sachs interprets the amboceptor as a body which brings about the union
between antigen and complement but states that certain amboceptors
may be toxic (lytic) and others, for example, those serving to fix com-
plement, may be considered as atoxic. He considers that the differences
in effect may be the result of a number of factors, including mass action
and differences in combining avidity of the various reacting bodies.
It would appear to us that Zinsser's interpretation in regard to pre-
cipitins might also be applied here and that the lysis of cells may be an
incident in complement fixation, certain conditions favoring lysis, others
merely fixation of complement. If this be accepted, the complement-
fixing body must be regarded as an amboceptor or sensitizer in the
same sense as are the cytolysins.

Activation by Complement. — -The utilization of complement in
hemolysis serves so to fix complement that it cannot activate a bac-
teriolytic amboceptor. Therefore, hemolysis exhibits the fixation of
complement in association with lysis of the cells. Handel found that
hemolytic and complement-fixing properties of an immune serum were
parallel, but Muir and Martin observed marked differences. The latter
investigators produced two immune sera, one against ox serum and the
other against ox cells, both of which exhibited hemolytic and comple-
ment-fixing properties. The immune serum prepared against ox cells

182 THE PRINCIPLES OF IMMUNOLOGY

laked the antigenic cells in doses of 0.0015 c.c. and fixed complement in
the presence of o.ooi c.c. ox serum. The immune serum prepared
against ox serum hemolyzed ox cells in doses of 0.05 c.c. but fixed
complement when combined with only 0.000,001 c.c. of ox serum. The
immune serum against ox serum had only about one-thirtieth the
hemolytic power of the immune serum prepared against ox cells but was
looo times more powerful in fixing complement. They found that
ox cells can absorb hemolysin from an immune serum without removing
the precipitating or complement-fixing activity and conclude, in oppo-
sition to the hypothesis offered at the end of the preceding paragraph,
that the complement-fixing body and the hemolysin are distinct and
separate immune bodies.

Fixation of the Complement of Natural Hemolysins. — In the case
of natural hemolysins the complement in many instances is apparently
in a state of close combination with the thermostable lytic body. The
entrance of such complements into the phenomenon of complement
fixation has only rarely been demonstrated and then only in the case
of those naturally hemolytic sera in which it is possible to absorb
hemolytic amboceptor at o° C. without at the same time removing
the complement.

Nature of Antigen and Amboceptor. — The chemical character of
the antigen and amboceptor have been studied more particularly in
connection with investigations of the Wassermann test and will be
considered in the discussion of that application of complement fixation.
It may be said at this place, however, that the complement-fixing
immune body will resist the ordinary inactivating temperature of 56° C.
and is therefore to be regarded as thermostable but is destroyed by
75° C. for one hour. The antigen is thermostable in the same sense
but is reduced in activity at 75° C. but not destroyed until 100° C.
is reached!

Inhibition of Complement other than by Fixation. — Of great im-
portance are the factors that exercise an influence upon complementary
activity. Those which operate on the living animal have been discussed
in the chapter on Cytolysins (see page 127). There was also presented
a brief discussion of physical conditions such as heat, exposure to light,
desiccation, etc. All these factors must be considered in interpretations
of complement fixation, and in addition it is considered desirable to
present certain other conditions which may be gathered into three classes
(a) chemicals, (b) various tissues and fluids, (c) antisera.

Anti-complementary Chemical Agencies. — The salt concentration
of the media for complement fixation is extremely important and reaches
its optimum at a point isotonic with the body fluids. The action of
complement is decreased in hypotonic and absent in salt free media.
Examination of this phenomenon leads to the conclusion that such
action is upon complement rather than upon amboceptor, and Ferrata is
of the opinion that the important change is the splitting of the comple-
ment into mid-piece and end-piece. Under these circumstances the
mid-piece may be bound to the amboceptor-antigen complex, but as

COMPLEMENT FIXATION 183

the end-piece remains free, complementary activity does not appear.
This explanation, however, is only hypothetical, is not entirety supported
by other experiments and fails to take into account the influence of salts
on colloidal suspensions and solutions. Excesses of salts also interfere
with the action of complement, but on dilution to isotonicity the function
is immediately restored. Therefore, the salts do no permanent injury
to complement. Hektoen and Reudiger, as well as Manwaring, offer
the explanation that ionization of the salt permits of a union with com-
plement which is easily reversible. Certain salts, such as those of
bile acids, as well as sodium oleate, permanently injure complement.
The salts are of themselves hemolytic, but serum inhibits their hemo-
lytic activity. The amounts which are hemolytic in themselves com-
pletely inhibit complement and by virtue of the presence of serum
cannot produce lysis.

Acids and alkalis in considerable concentration permanently destroy
complement, but if the injury be due to a dilute alkali the comple-
mentary activity may be restored by neutralization. It appears that
moderate concentrations of acids destroy complement without restora-
tion by neutralization. Dilute acids accelerate hemolysis and for this
reason are to be avoided in accurate work with complement fixation.
Certain protein products, such as urea (also urea sulphate) and guani-
din are anti-complementary.

Colloids may also inhibit complement as, for example, the organic
colloids, glycogen, inulin, pepton, albumose, gelatin, etc., as well as
inorganic colloids, such as quartz sand, kaolin and carbon. Numerous
indifferent chemical precipitates, such as colloidal iron hydroxide and
protein precipitates inhibit complementary activity. It is possible that
in certain measure this may depend upon their interference with the
complement amboceptor and antigen behaving as interacting colloids.

The influence of lipoids on complementary activity is of great im-
portance, particularly in the Wassermann test, but we may mention at
this point that lecithin, cholesterol, protagon and tristearin in sufficient
concentration are anti-complementary as well as certain lipins, including
the neutral fats, olive oil, triolein, etc. Added, finally, to the list of
chemical agents are boric acid, benzoic acid, formalin, sodium fluoride,
sodium sulphite and extracts of certain spices.

Anti-complementary Action of Cells, Tissue Extracts and Body
Fluids. — As was pointed out by von Dungern, most animal cells either
in the form of emulsions or cells may inhibit the activity of comple-
ment. Muir found that the stroma of red blood-corpuscles enters into
fixed combination with complement and that if washed red corpuscles
are heated to 55° C. for twenty-four hours they also will combine
directly. The union does not take place at o° C. but occurs readily at
37° C. The combination is apparently not dissociable. Not only animal
cells but also a wide variety of bacterial emulsions or their filtrates
as well as yeast cells fix complement. On the basis of the Ehrlich
hypothesis this may be due to the union of complement with the com-
plementophile groups of those sessile receptors of cells which by im-

184 THE PRINCIPLES OF IMMUNOLOGY

munization are overproduced and become free in the blood. Other
investigations, particularly those of Landsteiner and von Eisler, indicate
that the cell lipoids play a part in the union with complement. . The
material extracted from the cells by petroleum ether was found to be
definitely anti-hemolytic and furthermore this was especially true if
the cells used in hemolysis were from the same species as the lipoidal
extracts. Landsteiner and von Eisler demonstrated in addition that
cells treated with fat-dissolving agents were less susceptible to
hemolysis than normal cells. They suggested the possibility that the
fixing substance may be a lipoid protein combination. Bang and
Forssman extracted cells with ether and found that an acetone soluble
material could be recovered that was definitely anti-complementary.
Dantivitz and Landsteiner confirmed this but found in addition that the
fraction remaining in the ether, the acetone insoluble fraction, could
fix normal amboceptor but not immune amboceptors. Thus it will be
seen that the finer details of the anti-hemolytic powers of lipoidal
extracts are still unsettled. As to the anti-complementary action of bac-
terial extracts Zinsser suggests that it may be non-specific and com-
parable to the anti-complementary activity, mentioned in the previous
paragraph, of such inert substances as kaolin and quartz sand.

The body fluids of importance in this connection are the tissue
juices, certain pathological exudates and more particularly the blood
serum. Camus and Gley found that a normal hemolysin may be in-
hibited by the addition of a similar serum which had been inactivated.
Miiller showed that a heated serum may inhibit the activity of other
sera, and concluded that this was due to an anti-complementary activity.
Extreme instances of this action have been reported by Kenneway
and Wright. Muir and Browning demonstrated that inactivated sera
homologous with those used as complement were more strongly anti-
complementary than heterologous sera. They concluded that the action
was due to the presence in the heated sera of complementoid which,
at least partly, excluded the complement from union with the ambo-
ceptor. Bordet and Gay found that a sufficient dilution of inactivated
sera removed the anti-complementary action and therefore consider
concentration of the serum a most important factor. This would indi-
cate that the inhibition is, in general, against the reaction, although
Sachs offers the suggestion that the dilution provides for a dissociation
of complement and anti-complement. More proof than is now at hand
is necessary in order to admit the existence of an anti-complement in
the sense in which Sachs uses the term Of great importance is the
work of Noguchi, who found that whereas heating the serum to 56° C.
permits of the demonstration of anti-lytic powers, a temperature of
70° C. considerably augments this activity. Noguchi was able to extract
from both serum and cells by means of ether a substance, highly ther-
mostable (90° C.), which exhibited the same anti-lytic properties
as the serum. The removal of the ether extract left the serum free
from anti-lytic activity. He named the substance " protectin " and
believed it to be the source of the inhibiting action of serum. Noguchi's

COMPLEMENT FIXATION 185

opinion is that the inhibiting action of serum is largely anti-com-
plementary in nature, although in part the action may be upon the
amboceptor. The great thermoresistance of the body in the serum
argues against the assumption of an anti-complement in the strict
immunological sense. The action may well be anti-complementary,
but from the work of Bordet and Gay, as well as of Noguchi, it would
appear that the concentration of colloids associated with a disturbance
of lipoidal balance or combination must occupy a most important place
in hypotheses concerning this phenomenon. Of practical importance
is the fact that prolonged preservation of serum increases its anti-
lytic capacity.

Anti-hemolytic Activity of Immune Sera. — In discussing the prop-
erties of complement (see page 137) we mentioned the experimental
evidence concerning the production of anti-lysins and anti-comple-
ments by the injection of immune and normal sera. The anti-lytic
activity of such immune sera was thought at first to be due to an
anti-complement, but later was thought to be the result of action upon
the amboceptor or sensitizer. It must be recognized, however, that the
injection of a serum, whether it contain complement or immune ambo-
ceptor, leads to the production of a precipitin and that such precipitins
can be demonstrated in the immune sera containing the so-called anti-
complement. As has been pointed out in the preceding discussion on
complement fixation the presence of precipitates serves to fix com-
plement and this probably accounts for the anti-lytic and anti-comple-
mentary powers of the immune sera.

The fact that agglutination of the red cells inhibits their lysis was
pointed out independently by Handel and by Karsner and Pearce. This
renders inadvisable the use for complement-fixation tests of sera which
are strongly hemagglutinative.

CHAPTER IX

APPLICATION OF COMPLEMENT FIXATION TO THE
DIAGNOSIS OF DISEASE

THE WASSERMANN REACTION.
INTRODUCTION.
THE ANTIGEN.

NATURE OF THE SYPHILITIC ANTIGEN.
THE SYPHILITIC " AMBOCEPTOR."

NATURE OF THE SYPHILITIC " AMBOCEPTOR."
THE COMPLEMENT.
THE HEMOLYTIC SYSTEM.

PRESERVATION OF ERYTHROCYTES.

INFLUENCE OF TEMPERATURE UPON THE REACTION.
TECH NIC OF THE TEST.
THE ANTIGEN.
OTHER REAGENTS.
THE TEST.

MODIFICATIONS OF THE TEST.
SPECIFICITY OF THE REACTION.
DIAGNOSTIC VALUE OF THE REACTION.
INTERPRETATION OF RESULTS.
DEPENDABILITY OF THE TEST.
QUANTITATIVE RESULTS WITH THE TEST.
TEST OF SPINAL FLUID.
POST-MORTEM WASSERMANN TESTS.
COMPLEMENT FIXATION IN TUBERCULOSIS.

" ACID FAST FIXATION."

COMPLEMENT FIXATION IN GONOCOCCUS INFECTIONS.
OTHER COMPLEMENT-FIXATION TESTS.
GLANDERS.
TYPHOID FEVER.
SMALLPOX.
WHOOPING COUGH.
ECHINOCOCCUS CYST.
MALIGNANT TUMORS.
SPOROTRICHOSIS.

THE WASSERMANN REACTION

Introduction. — The demonstration of complement fixation employs
five reagents, syphilitic antigen, red blood-cells, syphilitic serum, hemo-
lytic serum and the complement. Having any four of these known it is
possible to determine the immunological nature of an unknown fifth
reagent. This unknown may be an antigenic substance or may be an
amboceptor. In the forensic tests for species proteins the unknown is
the questionable protein which is employed as an antigen; in other
tests the unknown may be bacteria or bacterial proteins. In the Was-
sermann and other clinical tests the unknown is an amboceptor or
similar substance, produced in the blood and other body fluids of the
diseased subject.

After preliminary experiments on animals, Wassermann, Neisser,
Bruck and Schucht published in 1906 the results of a series of com-
plement-fixation tests in cases of human syphilis and demonstrated the
186

APPLICATION OF COMPLEMENT FIXATION 187

clinical value of the reaction. The widespread use of the reaction has
led to marked advances in the understanding of this disease, its sequelae
and its treatment. This application of the Bordet-Gengou phenomenon
has enabled science to progress far toward the elimination of one of
the greatest plagues of mankind. Wassermann and his collaborators
had first shown that the Bordet-Gengou phenomenon was applicable
not only to bacterial suspensions but also to bacterial extracts and from
this developed the proposition that the causative agent of syphilis
might act as an antigen in extracts from syphilitic organs. The test
was originally performed with a salt solution extract of the liver or
spleen of a syphilitic fetus (rich in treponema pallidum), inactivated
human serum, guinea-pig complement, an inactivated hemolytic im-
mune serum and sheep erythrocytes. All the reagents were tested and
titrated to avoid factors of error and proper controls were instituted in
each experiment. Much has been accomplished by further study in the
hands of numberless investigators, but we shall limit our discussion
to those features which are of fundamental importance in the under-
standing and application of the test.

The Antigen.— The preparation of the antigen is one of the most
important features of this test. It would be supposed that an extract
of a pure culture of the treponema pallidum should give the most
specific results. This, however, has not proved to be the case. It is
difficult to grow the organism in pure culture and the method of culti-
vation interposes difficulties in the way of obtaining pure extracts.
Results are variable and therefore not so specific as with the use of
other antigens. Until recently the organism had not been cultured in
vitro and Wassermann and many of his successors were unable to
utilize the method. Wassermann selected the organs of syphilitic
fetuses, because they were known to contain large numbers of tre-
ponemata, and from these made extracts in physiologic salt solution.
He cut syphilitic fetal liver in fine pieces and mixed 100 grams liver
with 360 c.c. physiologic salt solution and 40 c.c. 5 per cent, phenol
solution. This was shaken for twenty-four hours, centrifuged and the
supernatant fluid employed as antigen. Practical experience shows that
these antigens vary considerably in strength and rapidly lose fixing
power. Deterioration may result from light, air, warmth and freezing,
so that the extract must be kept tightly stoppered in the dark at low
but not freezing temperature. Marie and Levaditi dried and pulverized
the liver in order to preserve it and made up salt solution extracts when
needed. Morgenroth and Stertz preserved the organ in the frozen
state. The subsequent work of Weil and of Lansteiner and their col-
leagues indicated that tumor extracts, extracts of animal tissues and
of normal human tissues would operate as antigens. More recently
Varney and Baeslack have employed extracts of experimentally inocu-
lated testes of the rabbit in that stage of infection when the organs are
richly infiltrated with the treponema.

Landsteiner, Miiller and Potzl found that alcoholic extracts of
guinea-pig heart serve admirably as antigen. Independently Porges

i88 THE PRINCIPLES OF IMMUNOLOGY

and Meier showed that alcoholic extracts of normal or syphilitic fetal
organs operate equally as well as the watery extracts of syphilitic or-
gans. These studies demonstrated that the antigen in the Wassermann
test is not necessarily derived from the .treponema pallidum, is alcohol
soluble and therefore is largely of lipoidal nature. Landsteiner, Muller
and Potzl extracted I. gram heart with 50. c.c. absolute alcohol but
this method has been somewhat modified. For practical purposes 50 c.c.
absolute alcohol are placed in a wide-mouth amber bottle and as
guinea-pigs are killed in the laboratory the heart is freed from blood
and connective tissue, cut into a few pieces and placed in the alcohol.
When ten hearts are so collected they are dried and ground in a mortar.
Ten grams of the dried powder are returned to the alcohol and the
volume made up to 100. c.c. This is shaken for twelve hours and placed
either at 60° C. for about twelve hours or at 37° C. for about five days.
It is then filtered and the filtrate preserved in a cool, dark place. Further,
a second extraction with alcohol of the first dried extract yields an
antigen of greater value because it contains less lytic and anti-lytic
substance, although it may be slightly weaker in fixing power. Appar-
ently, however, the alcoholic extracts of syphilitic organs produce more
specific antigens. To prepare such an antigen 100 grams syphilitic
liver are freed from surrounding tissue, washed free of blood and cut
into fine pieces- This is extracted in 1000 c.c. absolute alcohol for a
week at 37° C., the flask being shaken several times daily. It is
then filtered and titrated.

Porges and Meier found that lecithin could, within certain limits,
be substituted for the antigenic extracts. This naturally led to extensive
investigation of the nature of the substance or substances concerned.
The fact that ether extracts of alcohol soluble antigen, according to
Levaditi and Yamanouchi, did not contain antigen led to the thought
that salts of bile acids might serve as antigens. Neither lecithin nor
salts of bile acids give consistent results in the actual test and at the
present time no pure substance serves well as antigen. The importance
of lecithin was further emphasized by the refined technic of Noguchi
in preparing the so-called acetone insoluble antigen. This method ap-
pears to be especially adapted to the use of normal human organs,
particularly heart. The tissue is cut into fine pieces^jnixed with five
times its weight of absolute alcohol and placed at '37° C. for from
five to seven days. It is then filtered and the clear filtrate evaporated
in a dish by means of an electric fan or in a vacuum desiccator. The
residue is taken up in as small a volume of ether as will permit solution
and allowed to stand overnight. The clear supernatant fluid is decanted
and slightly evaporated. To it is added four volumes of acetone. The
supernatant fluid is poured off and the precipitate allowed to evaporate
to a resinous consistence. Three-tenths of a gram of this mass is
added to a mixture of i.o c.c. ether and 9.0 c.c. pure absolute methyl
alcohol and preserved in a dark, cool place. According to certain re-
ports, it would appear that this antigen gives positive results in cases
which are negative with other antigens and in which syphilis has not

APPLICATION OF COMPLEMENT FIXATION 189

been demonstrated by clinical examination. It is useful, how-
ever, as a control of other antigens with which doubtful results have
been obtained.

The source of the lecithin appears to play some role in its value as an
antigen; that from heart is most active, while that from liver, brain
and egg yolk follow in the order named. An extract such as that
recommended by Noguchi contains in all probability a mixture of lipoids
and unsaturated fatty acids; Noguchi and Bronfenbrenner found the
fixing capacity of such extracts to vary in accordance with the content
of unsaturated fatty acids. Browning and Cruikshank found that the
addition of cholesterol to the antigen augments the delicacy of the re-
action and this method has found widespread use in this country,
particularly through the work of Walker and Swift. The latter investi-
gators recommend the addition to alcoholic extracts of human or
guinea-pig hearts of 0.4 per cent, of cholesterol. In the hands of sev-
eral workers this has so increased the fixing power of the antigen as
to give positive results in the presence of non-syphilitic serum, the
so-called false positive reactions, and with the development of the
method of fixation at refrigerator temperature, to be described subse-
quently, it has been discarded in several laboratories. Nevertheless, the
cholesterolized antigens are found, in the hands of numerous workers,
to show much less variation in fixing capacity than the non-cholesterol-
ized extracts and for this reason are recommended for routine
laboratory work.

It would appear that the antigenic substance in the Wassermann test
is not an antigen in the biological sense, for it can be obtained from
tissues not the seat of a syphilitic infection and as has been shown by
Fitzgerald and Leathes, upon injection into animals it does not lead to
the formation of immune substances.

The methods of preparing syphilitic antigens have been multiplied
in great number and cannot be included in the scope of this book. Sim-
plification of preparation has been attempted with variable results.
Of interest is the method suggested by Ecker and Sasano. They quote
Neymann and Gager to the effect that primary extraction of the tissue
with ether removes substances of anti-complementary power but only a
small amount of the lecithin. Ecker and Sasano suggest three ten-
minute extractions with ether in the proportion of 25. grams ground
and dried heart muscle to 50. c.c. ether. The material is then extracted
for one hour with 75. c.c. 95 per cent, ethyl alcohol at boiling tempera-
ture (78° C.) in a flask connected with a reflux condenser. An antigen
of this sort has retained its original fixing power in this laboratory
after more than a year.

Nature of Syphilitic Antigen. — Extracts of the treponema pallidum
may serve as antigens and are true antigens in the biological sense.
Craig and Nichols, however, found that alcoholic extracts of organ-
isms closely related to treponema pallidum, as the treponema per-
tenue and the treponema microdentium, may fix complement in the
presence of syphilitic serum. Extracts of animal and human

190 THE PRINCIPLES OF IMMUNOLOGY

organs, particularly when prepared by alcoholic extraction appear to
be distinctly more dependable than treponema extracts in the reaction
of complement fixation. The exact nature of the substances in the
alcoholic and in the acetone insoluble extracts is not definitely known
except that lecithin constitutes a large part and that it is associated
probably with other lipoids of the diaminophosphatid group, unsat-
urated fatty acids and certain proteins or protein fractions. That
physical conditions are of importance has been known since Wasser-
mann's early work, for it is established that a certain degree of turbidity
of the antigen or its dilutions is necessary. The watery extracts are in
a state of finely-suspended colloidal emulsion and, as Reudiger and
others have pointed out, the dilutions of the alcoholic or acetone insol-
uble extracts by means of salt solution can be demonstrated to have an
optimal degree of turbidity.

The Syphilitic "Amboceptor." — This is contained in the blood
serum, the cerebro-spinal fluid and other juices of syphilitic patients and
experimental animals. In the usual technic the blood serum is inac-
tivated for one-half to one hour at 56° C. in order to remove comple-
ment, but in certain modifications of the test the serum is used fresh in
order to utilize human complement in the reaction. Bronfenbrenner,
Reudiger and others have shown that inactivation of the serum reduces
its fixing power. Bronfenbrenner recommends the use of unheated
serum because of the greater delicacy of the reaction. In this way it is
possible to use for the test 0.04 c.c. or 0.05 c.c. serum, instead of the
usual o.i c.c. With such small amounts of serum the human comple-
ment is a negligible factor. Long preservation or excessive heating
of the serum may render it anti-lytic or anti-complementary. Con-
tamination from unclean skin and glassware may make it either anti-
lytic or lytic. The ingestion of alcohol, the presence of bile in the
blood in jaundice or fat in the blood after a heavy meal or in cases
of lipemia may all interfere with the activity of the fixing body in a
syphilitic serum; sera in lipemia may be markedly anti-comple-
mentary. There has been much discussion of the fact that human
serum may contain natural hemolysins for sheep corpuscles. In such
an instance the corpuscles may be dissolved by the excess of ambo-
ceptors in spite of slight fixation of complement by the syphilitic ambo-
ceptor, thus transforming a weakly positive into a negative reaction.
Sasano has found, however, that the use of an excess of immune
hemolytic amboceptor, for example, ten to twenty units and one and one-
half units of complement, determined by careful titration does not influ-
ence the result. Thus the factor of hemolysis by the normal anti-sheep
amboceptor of human serum, which amboceptor is practically never pres-
ent to the extent of more than four units, is practically negligible.

The serum may be preserved for a considerable time if kept cool and
in sealed ampoules or in tightly-stoppered bottles. Reudiger has found
that mixing equal parts of fresh inactivated serum and pure sterile
glycerol preserves the so-called syphilitic antibody for as much as
two years. Under these circumstances the sera may become anti-

APPLICATION OF COMPLEMENT FIXATION 191

complementary, but this property can be removed by heating to 56° C.
for thirty minutes, and the sera are then satisfactory for use. He
maintains that heated glycerolated sera give much stronger positive
results than fresh unheated sera and somewhat stronger than fresh
heated sera. This method is valuable for preserving known positive
and negative sera as controls for the Wassermann test.

Nature of the Syphilitic Amboceptor. — The substance in the blood
which acts as amboceptor is apparently closely related to the globulins,
especially the euglobulin. Recently, however, Duhot has suggested that
the albumin is of importance. Pfeifler, Kober and Field, as well as
Rowe, have shown an increase of globulins in syphilitic blood and
spinal fluid. Noguchi has taken advantage of this fact in his butyric
acid test of spinal fluid, but diseases other than syphilis may lead to
increase of globulins in the spinal fluid. Peritz states that the lipoid
content of syphilitic serum is increased, but Bauer and Skutezky found
no parallel between lipoid content and Wassermann reaction. Klaus-
ner believes that the flocculent precipitate which appears on addition
of 0.6 c.c. distilled water to 0.2 c.c. fresh syphilitic serum is due to the
high lipoid content of the serum. Weston has found no definite increase
of serum cholesterol in late syphilis and no parallelism between the
serum cholesterol content and the Wassermann reaction. ' According
to Wells, " a favorite interpretation of the Wassermann reaction, which
seems to harmonize with the facts, is that there is a precipitation of
serum globulin by the lipoidal colloids of the antigen and adsorption of
the complement by this precipitate." This is supported by the work
of Jacobsthal who has demonstrated such precipitates by use of the
ultra-microscope even when they are invisible to the naked eye. Holker
has recently studied the colloidal phenomena and finds that the addi-
tion of antigen to syphilitic sera produces a turbidity the curve of
which is steeper and higher than with normal sera. He finds that the
serum is an emulsoid and the antigen a suspensoid. Salt solution dis-
perses the serum and precipitates the antigen, thus increasing the pro-
tective state of the serum. Negative sera are much more protective
than positive sera in preventing the antigen from being precipitated
by salt solution.

The Complement. — As has been pointed out in the general discus-
sion of complement, guinea-pig complement is most widely useful in
immunological work. It was used by Wassermann in his original test
and is extensively employed to-day. From the practical viewpoint it
has certain objections. Animals are expensive and for a small number
of tests it is undesirable to sacrifice an animal. This objection may
be overcome by bleeding from the heart or from an ear vein (Rous),
but the technic of both these operations is somewhat difficult. Owing
to the lability of complement, it cannot be well preserved and the serum
must be used soon after collection. The use of dried complement in
filter paper has been abandoned, although such complement papers
may be preserved for a few weeks in vacuum desiccators or in tubes
containing calcium chloride. Drying in the frozen state in vacuo has

192

THE PRINCIPLES OF IMMUNOLOGY

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APPLICATION OF COMPLEMENT FIXATION 193

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194 THE PRINCIPLES OF IMMUNOLOGY

been recommended by Shakell, but Karsner and Collins found that the
activity was lost in eleven to fifteen days. Moledzky states that com-
plement in the frozen states retains its strength indefinitely, but Reu-
diger found that although its strength is somewhat augmented at the
end of one week, it deteriorates after the second week of preservation.
Preservation for even these periods involves a good laboratory equip-
ment and considerable skill. Kolmer recommends the addition of
chemically pure sodium chloride to pooled guinea-pig sera in the pro-
portion of 0.425 gram salt to 10. c.c. serum. This is effective for sev-
eral weeks' preservation, and dilution is so adjusted as to restore the
serum to practical isotonicity. Detre used rabbit complement, but it is
not as desirable as guinea-pig complement and has the same objections.
Human complement is employed in several modifications of the Was-
sermann test, but is present in human serum in extremely variable
amounts and is difficult to titrate. It is, however, easily accessible,
as it is present in the serum to be tested for syphilitic amboceptor.
The selection of the complement to be used depends to a certain
extent upon the hemolytic system and the modification of the test
which is employed.

Complement should be used in accurately-determined amounts.
Therefore, titration is of the utmost importance. Guinea-pig serum
shows individual variation in complement, but in large laboratories
this may be in part overcome by the " pooling " or mixing of the sera
from several guinea-pigs. Such pooling, however, does not remove the
necessity for titration. In some laboratories the complement is diluted
i to 10, and the hemolytic amboceptor titrated each day by testing.
We are of the opinion from experience and in view of the work of
Sasano that the complement should be titrated in various dilutions
(see page 190) against a constant amount of previously titrated hemo-
lytic amboceptor. In either case the titration should take place on the
same day as the Wassermann tests are made. The use of a single unit
of complement does not allow for the presence of anti-lytic bodies in
the reagents nor for possible deterioration of complement. At 37° C.
the use of one and one-fourth units appears to be most satisfactory,
whereas at ice-chest temperature the use of two units appears to be
more desirable. Titration of the complement should be most accurate
and the end-point be determined only by absolutely complete hemolysis.

The Hemolytic System. — Sheep erythrocytes and the correspond-
ing hemolytic immune serum obtained from the rabbit were used by
Wassermann and are widely used at the present time. Other systems
include the use of goat, horse, ox, human and fowl corpuscles, with
specific antisera obtained by immunizing rabbits. Certain investi-
gators have depended upon the normal hemolysin for sheep erythro-
cytes often found in human serum. This is a variable quantity and
almost never very large. Noguchi has summarized the hemolytic sys-
tems in a table giving all the essential data (see pages 192, 193).

The preparation of a hemolytic immune serum has been discussed
(see page 117). The preservation of this serum in the moist state is

APPLICATION OF COMPLEMENT FIXATION 195

highly satisfactory if placed in amber ampoules in a refrigerator. If
considered advisable preservatives may be added such as 0.5 per cent,
phenol or 50 per cent, glycerol. If the serum is of high titer it may
be preserved by desiccation, particularly if frozen and dried in a vacuum
desiccator. Noguchi has obtained good results by drying the serum in
filter paper. The filter paper is subsequently cut into strips and titrated
by cutting measured lengths of the strips. We have found that this
does not permit of sufficiently accurate titration and also that the titer
is not well maintained.

Preservation of Erythrocytes. — If kept in a cool place without freezing,
sheep erythrocytes show slight hemolysis in a few days and well-marked
hemolysis in about a week. The cells oi other animals show variable degrees
of fragility, those of the dog being especially fragile. Various methods of
preserving sheep erythrocytes for the Wassermann test have been studied
by Reimann in this laboratory. The methods of particular value are preserva-
tion with formalin (Bernstein and Kaliski) and with the solutions of Rous
and Turner. For formalization the sheep blood is allowed to run directly
into formalin solution in the proportion of 0.5 c.c. of 40 per cent formaldehyde
solution to 400 c.c. blood. The blood is then defibrinated by shaking with glass
beads, preserved in the refrigerator and before use washed three times with
saline for use. The method of preservation as worked out by Rous and
Turner is carried out in the following manner: The sheep is bled directly into
Locke's solution containing i per cent, sodium citrate, in the proportion of
i part of blood to 4 parts of solution. The corpuscles are separated by rapid
centrifugalization and carefully washed three times in Locke's solution containing
0.25 per cent, gelatin. The cells are then placed in ampoules in a layer not
more than 2 mm. in depth and covered with saccharose-Locke solution to a
depth of about 2 cm.; the ampoules are sealed and stored at a temperature of
5° C. to 6° C. Just prior to use the cells are washed with .85 per cent, saline
to remove the saccharose solution, and proper dilution effected with saline.
Strict asepsis is to be observed.

THE SOLUTIONS

Locke-sodium citrate solution :

Sodium citrate 10 grams

Sodium chloride 9.2 grams

Sodium bicarb 0.05 gram

Potassium chloride o.i gram

Calcium cholride o.i gram

Aq. dest. q.s. ad 1000 c.c.

Locke-gelatin solution:

Gelatin 2.5 grams

Sodium chloride 9.2. grams

Sodium bicarb .* 0.05 gram

Potassium chloride o.i gram

Calcium chloride o.i gram

Aq. q.s. ad 1000 c.c.

The Locke and saccharose solutions are sterilized separately and used in
the proportion of 2.8 c.c. of the saccharose solution and 7.5 c.c, of the
Locke's solution.

Saccharose solution :

Saccharose 103.0 grams

Aq. q.s. ad 1000 c.c.

Locke's solution:

Sodium chloride 9.2 grams

Sodium bicarb 0.05 gram

Potassium chloride o.i gram

Calcium chloride o.i gram

Aq. q.s. ad 1000 c.c.

196 THE PRINCIPLES OF IMMUNOLOGY

Reimann found that the cells can be preserved for use in the Wassermann
test for 3 to 4 weeks by formalization and for 21 to 25 days by the Rous and
Turner method. " The readings obtained differ from those obtained with fresh
cells only in so far as some s*era produce slightly different results when used
with cells from the same specimen of sheep blood." An excellent control for the
usefulness of preserved blood is suggested by Kolmer, who maintains that there
should be no discoloration of supernatant fluid after the second washing and
that the blood should become brighter in color than the dark color it possesses
after standing.

When extreme accuracy is desired cell emulsions are made to
contain 1,000,000,000 cells per cubic centimeter. Such emulsions are
being more widely adopted, but many laboratories still use 5 per
cent, or 10 per cent, emulsions calculated either from the original
blood volume or the bulk of the centrifuged cells. The cells should
always be most carefully washed, so as to avoid precipitin reactions
which may appear if the serum is not entirely removed and to wash
out antilytic substances which may appear if the blood is old.

Influence of Temperature upon the Reaction. — This influence may
be determined as regards the velocity of the reaction and the amount
of complement fixed. The earlier work with complement fixation was
based on the general assumption of immunologists that a temperature
of 37° C. represents the optimum. In 1912, however, A. McNeil pointed
out that ice-chest temperature favors the completeness of complement
fixation in the Wassermann test, provided the time of exposure is from
eight to twelve hours. This was confirmed by Coca and 1'Esperance,
Smith and W. J. McNeil, Berghausen and others, and the ice-chest
method has now been adopted by a large number of laboratories as a
standard method. The time, however, has been reduced to from three
to four hours and the results appear to be entirely satisfactory. The
antigen, .serum to be tested and complement are mixed and placed in
the ice-chest for the required time ; the mixture is then brought to about
37° C. in a water bath, the sensitized erythrocytes added and the whole
incubated at 37° C. for one hour.

Dean has investigated the influence of temperature and finds that
fixation proceeds most rapidly at 37° C. Noguchi confirms this but
finds that at the lower temperature of 23° C, fixation will reach a maxi-
mum but proceeds more slowly. He states that with the acetone insol-
uble antigen " a serum containing one unit of fixing substance will
complete the reaction within thirty minutes at 37° C., sixty minutes at
30° C., and two hours at 23° C., irrespective of whether human or
guinea-pig complement is used." Dean, however, finds that at o° C.
the amount of complement fixed is much greater than at 37° C., and this
accords with experience in the use of the ice-chest method. Certain
unknown factors may delay the action of the complement, as has been
pointed out by McConnell, and a second incubation may accordingly
have to be prolonged beyond the usual time.

The Technic of the Wassermann Test. — For the demonstration of the
method we may use an alcoholic extract of ox heart as the syphilitic antigen,
inactivated human serum from a normal individual and from a known victim
of syphilis, guinea-pig complement and a sheep hemolytic system.

APPLICATION OF COMPLEMENT FIXATION 197

The antigen may be made by weighing 10 grams ox heart which has been
freed from blood, fat and connective tissue, ground in a meat grinder and dried
under a current of air from an electric fan or in a 'desiccator. It is then
extracted in 100 c.c. 95 per cent, ethyl alcohol, first by shaking 18 hours in an
electrical shaker and then standing for 5 days at 37° C. It is filtered and kept
tightly stoppered in an amber glass bottle in the refrigerator. For use, slowly
add 9.0 c.c. physiologic salt solution to i.o c.c. alcoholic extract. This constitutes
the "antigen dilution" of the charts. It must then be titrated to determine
its antilytic properties as well as its lytic powers. The following tests, of the
antigen may be set up after previously determining the titer of the hemolytic
amboceptor and complement. In the following titrations the complement is
diluted so that i.o c.c. contains I unit, the amboceptor so that i.o c.c. contains
2 units. In the first series, volume is made up by addition of salt solution, so
that each tube contains 4.0 c.c. and in the second series so that each tube
contains 2.0 c.c.

TlTRATION OF ANTIGEN FOR ANTILYTIC PROPERTIES

Antigen dilution

Complement

1

Hemolysin

5 per cent, cell
suspension

|

Result

I.O C.C.

0.8 c.c.
0.6 c.c.
0.4 c.c.

O.2 C.C.

unit
unit
unit
unit
unit
i unit

ncubate one ]

2 units
2 units
2 units
2 units
2 units
2 units

I C.C.
I C.C.
I C.C.
I C.C.
I C.C.
I C.C

ncubate one ]

P.H.
P.H.
P.H.
C.H.
C.H.
C H

TlTRATION OF ANTIGEN FOR LYTIC PROPERTIES

Antigen dilution

5 per cent, cell suspension

IH

rs
o

Result

I.O C.C.

0.8 c.c.
0.6 c.c.
0.4 c.c.

O.2 C.C.

C.C.
C.C.
C.C.
C.C.
C.C.
C.C.

[ncubate one h

P.H.
P.H.

In the protocols P.H. indicates partial hemolysis, C.H. complete hemolysis
and ( — ) no hemolysis. Thus it is seen that 0.6 c.c. is the smallest amount of
antigen which is antilytic and 0.4 c.c. the largest amount which is not. For
practical purposes one-half the latter amount, or 0.2 c.c., is the largest amount
which may be used. This is considerably smaller than the amount of antigen
which possesses hemolytic properties in itself, as shown in the second protocol.

After obtaining this information, the antigen should be titrated to determine
the smallest dose that fixes complement in the presence of a known syphilitic
serum. A strong serum (H — h++) may be obtained from a laboratory or if
not available a serum may be secured from 9. patient in the florid secondary
stage of the disease. This serum is used in constant amounts of 0.2 c.c. More
delicate titration is accomplished by the use of a known ++ serum either
alone or in addition to the H — h+H~ serum. Knowing1 that 0.2 c.c. is the largest
dose of antigen dilution that may be employed the test is set up with that as the
maximum amount of antigen, followed by decreasing doses. (See table on page 198.)

The protocol includes the necessary controls, showing that neither antigen
(12), syphilitic serum (7), nor non-syphilitic serum (19) exhibits antilytic
powers. It shows that antigen (13), syphilitic serum (15) and non-syphilitic
serum produce no hemolysis. It shows that non-syphilitic human serum
(15-18) fails in the presence of the antigen to fix complement. It shows that
in the presence of syphilitic; serum the antigen solution in amounts as small as
o.oi c.c. fixes complement. That amount, o.oi c.c., is the fixing dose and is
doubled for the actual Wassermann test.

198 THE PRINCIPLES OF IMMUNOLOGY

TlTKATION OF ANTIGEN FOR FIXING PROPERTY.

Tube

Antigen
dilution

Syphilitic
serum

Complement

Hemolysin

5% Cell
suspension

Result

O2 C C

O 2 n C

2 units

2 units

T P f*

O I C C

O 2 C C

2 units

2 units

3

4

5

O.OO5 c-c-

0.2 C.C.

2 units

2 units

C.C.

P. H.

6

O.OOI C.C.

0.2 C.C.

2 units

2 units

C.C.

C. H.

7

O.2 C.C.

2 units

^2

2 units

C C

J3

C H

8

2 units

o

2 units

C C

o

P Ii

2 units

C C

xo

g

C C

C

ii

2 units

o

C C

o

12

O.2 C.C.

2 units

g

2 units

C C

C H

1 1

O 2 C C

^

ia

14.

O.2 C.C.

i

ICC

8

j-j

r)

Non -syphili-

HH

tic human

serum

15

0.2 C.C.

O.2 C.C.

2 units

2 units

C.C.

C. H.

16

O.I C.C.

O.2 C.C.

2 units

2 units

C.C.

C. H.

i?

0.05 c.c.

O.2 C.C.

2 units

2 units

C.C.

C. H.

18

O.OI C.C.

0.2 C.C.

2 units

2 units

C.C.

C. H.

19

0.2 C.C.

2 units

2 units

C.C.

C. H.

20

0.2 C.C.

C.C.

Other Reagents for the Test. — The methods of obtaining guinea-pig blood,
the hemolytic amboceptor and sheep blood have been described (see pages
117 and 127). Various methods are in vogue for obtaining human blood. In adults
the simplest satisfactory method is to obtain the blood by puncture of one of
the large veins in the cubital fossa anterior to the elbow-joint. The needle should
have a calibre of about i. m.m. and although sharp should not have an elongated
point. The fossa is cleansed with soap and water followed by alcohol. A tourni-
quet is applied at the middle of the upper arm and the patient instructed to
" make a fist " several times until the veins stand out prominently. The sterile
needle is inserted and the blood collected in amounts of 5 to 10 c.c. in a 15 c.c.
centrifuge tube. The tourniquet is released before the needle is withdrawn
and the wound sealed with collodion. The blood is allowed to clot, the clot
separated from the side of the tube by means of a sterile needle, and allowed
to contract for several hours in the refrigerator. The tube is then centrifuged
and the serum pipetted into another tube so as to avoid hemolysis. The serum
is inactivated at 56° to 6p° C. for one-half hour before testing, unless the test
is to be made by a modification which employs human complement. Methods
have been suggested in which the amount of blood obtained by puncture of finger
tip or ear lobe provides sufficient blood. In infants or obese adults blood may be
obtained by the use of a scarifier and cupping. Bleeding from the longitudinal
sinus, from the great toe and from the heel are also practised in infants.

The Test. — With the reagents at hand the test is set up with one or more
antigens. In many laboratories different types of antigen are employed, as for
example an acetone insoluble antigen, a cholesterolized alcoholic extract of
heart muscle and a non-cholesterolized alcoholic extract of heart muscle. Others
are employed as the operator sees fit. Antigens may deteriorate, so that it is
wise to have several on hand and under observation in the test. The protocol
shows 2 antigens of the same strength. All the elements in the test are to be
controlled to prove that they are not antilytic and to show that the hemolytic
system operates properly. In addition it is essential to have controls with a
known positive and a known negative serum. The antigen, complement and
hemolysins are diluted so that the proper quantity of each is contained in i.q c.c.
It appears to be desirable to add the human serum without dilution. This is
done with a i.o c.c. pipette graduated in hundredths of a cubic centimeter.
The dotted lines in the body of the protocol indicate that salt solution is to be
substituted in quantities of i.o c.c.

APPLICATION OF COMPLEMENT FIXATION 199

THE WASSERMANN TEST

Tube

Antigen
No. i
dilution

Antigen
No. 2
dilution

Human serum

Comple-
ment

Hemolysin

S % cell
suspension

Result

I

O.O2

0.2

2 units

2 units

C.C.

2

0.02

O.I

2 units

2 units

C.C.

P. H.

3

O.O2

O.2

2 units

2 units

C.C.

4

O.O2

O.I

2 units

2 units

C.C.

P. H.

Known

positive

5

O.O2

O.2

2 units

2 units

C.C.

6

O.O2

O.I

2 units

2 units

C.C.

7

O.2

O.2

2 units

f-H

3

2 units

C.C.

f-H

a

8

....

O.2

O.I

2 units

1

2 units

C.C.

0

^

Known

<u

0

negative

8

d

o

9

O.O2

....

0.2

2 units

<D

2 units

C.C.

S

C. H.

10

O.O2

O.I

2 units

1

2 units

C.C.

j-i

C. H.

ii

O.O2

O.2

2 units

vU

2

2 units

C.C.

3

C. H.

12

O.O2

O.I

2 units

o

c

2 units

C.C.

o
d

C. H.

13

O.O2

2 units

t— <

2 units

C.C.

1— 1

C. H.

14

O.O2

.

2 units

2 units

C.C.

C. H.

15

....

0.2 (test

2 units

2 units

C.C.

C. H.

serum)

16

0.2 (positive)

2 units

2 units

C.C.

C. H.

i?

....

0.2 (negative)

2 units

2 units

C.C.

C. H.

18

....

. . .

2 units

2 units

C.C.

C. H.

IQ

211T1 li~C

f\ f\

Ly
2O

. . .

14.111 to

C.L..

C.C.

The results of the Wassermann test are usually indicated by plus signs ; the
following diagram indicates the interpretation of the results:

DEGREE OF HEMOLYSIS
WITH
0.2 c.c. human serum o.i c.c. human serum

Result

P.H.

P.H.
CH.
C.H.

++

+

In these readings the partial hemolysis is relatively small in amount. If
with 0.2 c.c. human serum the hemolysis is well advanced without being complete
and is complete with o.i c.c. serum, the result is indicated by the sign +. Other
symbols are used, but the results are indicated in the same general way.

Reference to the protocol shows that the serum in tubes I, '2, 3, 4 is positive
for syphilis and would be signified as a three plus (H — h+) serum. The known
positive is a four plus (++++) and the known negative reacts properly. Tubes
13 and 14 show that the antigens are not antilytic, and tubes 16, 17, 18 show that
the sera are not antilytic. Tube 18 shows that the hemolytic system operates
properly. Tube 19 shows that the hemolysin does not produce hemolysis with-
out complement, and tube 20 shows that the corpuscles do not hemolyze without
the other agents.

The quantities given in the protocol are based on a unit of i.o c.c. to simplify
the explanation. In order to save reagents the quantities are usually divided in
half, so as to be on a 0.5 c.c. basis. The directions for the United States Army
in France called for quarter quantities, so as to save reagents. The latter direc-
tions also call for half saturation of the alcoholic heart extract with cholesterol
(0.2 per cent.). Bronfenbrenner has suggested the use of o.i c.c. amounts of the
reagents. Methods of measuring by drops have been employed, but are inaccurate
because of the possible variation in the size of drops unless a stalagmometer or
similar instrument is employed.

200 THE PRINCIPLES OF IMMUNOLOGY

Modifications of the Tests. — Numerous modifications of the test
have been recommended. These are based on variations in syphilitic
antigen, various ways of treating the human serum, differences in
selection of the complement and in selection of the hemo-
lytic system. These are indicated in the chart on page 192. It
is our opinion that any method, to be acceptable, must per-
mit of accurate measurement of the reacting bodies. The possi-
bilities as to methods of preparing antigen and human serum have been
discussed. The use of human complement in the test interposes errors,
which we believe have not been overcome. The titration of human
complement must differ with different specimens and in the Gradwohl
method fails to take account of the variable content of natural hemo-
lytic amboceptor in human serum. Of the hemolytic systems recom-
mended, the most satisfactory are the sheep or goat and the human
systems. In most laboratories the sheep system appears to be most
accessible and the factor of error introduced by the presence of normal
anti-sheep amboceptors in human serum can usually be overcome by
absorption with sheep erythrocytes or can be controlled by the use of
one and one-half units of complement. The human hemolytic system
largely obviates this objection, but it is sometimes difficult to obtain
enough blood to immunize animals: for the production of the specific
immune hemolysin. We also suggest the possibility that an unusually
strong natural iso-hemolysin in the tested serum may confuse the
results. Kolmer, in a recent study, has found that the human hemo-
lytic system considerably increases the delicacy of the reaction, espe-
cially when small amounts of the patients' sera are employed. In
positive cases he found 10 per cent, more positive reactions by the use of
the human system than with the sheep system.

The Specificity of the Wassermann Reaction. — Numerous studies
have been made as to the specificity of the test in the different stages
of syphilis. In evaluating such figures certain factors of error in the
actual performance of the test must be considered. Unless the worker
is familiar with the many factors which may influence the reaction
of hemolysis and the fixation of complement, as pointed out briefly
in the chapter on hemolysis and the discussion of complement fixation,
the results may -be misleading. The type of antigen employed is also
of significance as influencing the results. Of no small importance is
the operator himself, for although the Wassermann test may properly
be regarded as a physico-chemical test rather than a strictly biological
or immunological reaction, nevertheless it requires a thorough under-
standing of immunological procedures. Tests made in the hands of
persons trained to perform this test, without broader training, are not
to be given the same value as tests in the hands of broadly-trained
immunologists. The subject of specificity of the test is closely bound
with the clinic, in which certain factors of error in clinical diagnosis
must be accepted. Until more satisfactory methods are provided for in
the post-mortem room, the factor of error there is almost as large as
in the clinic. Warthin, by particularly refined methods applied to

APPLICATION OF COMPLEMENT FIXATION 201

cases which have been examined shortly after death, has shown the
presence of the treponema in lesions which previously had not been
positively known to be syphilitic. Symmers, Darlington and Bittman
found a considerable divergence between ante-mortem Wassermann
tests and the post-mortem evidence of syphilis, but Turnbull finds a
striking agreement. Certainly syphilis can progress for a long time
without gross morbid anatomical manifestations, and it seems possible
that the pathologist cannot be sure of excluding syphilis in his ana-
tomical diagnosis. Improved technic is the only way of reducing this
factor o>f error and thereby providing an accurate control of the Was-
sermann and other clinical tests.

The Diagnostic Value of the Wassermann Test. — Naturally this
subject has been studied extensively and figures vary as the technic is
improved. In 1914 Boas published an analysis of over 8000 cases
reported in the older literature and tabulates them as follows :

Number positive Per<*nt.
of cases positive

Primary syphilis 1060 629 59

Secondary syphilis 3526 3181 90

Tertiary syphilis 1212 1020 84.1

Early latent syphilis 983 504 51

Late latent syphilis 1520 605 39

Tabes dorsalis 159 115 72

Paresis 405 402 99.2

These figures are sufficient to indicate that the Wassermann test is
of distinct value in the diagnosis of syphilis. More recent statistics
offered by Craig as the result of tests carried out by himself illustrate
the accuracy of the reaction as applied under excellent conditions.
In interpreting the following figures from Craig, given as the result
of a single test on each of 4658 cases diagnosed as syphilis, it must
be remembered that there is at least a small factor of error in the
clinical diagnosis. The table follows:

Number pnci>:w Per cent.
of cases Positive positive

Primary syphilis 908 813 89.5

Secondary syphilis 1889 1817 96.1

Tertiary syphilis 638 558 87.4

Latent syphilis 1 173 790 67.3

Congenital syphilis 28 25 89.2

Parasyphilis 22 7 68.1

4658 4010 86.2

Tests made by Craig on 2643 individuals, either not diseased or vic-
tims of disease other than syphilis, showed the reaction to be positive in
eleven instances (0.4 per cent.). These eleven instances included four
cases of malaria, three of tuberculosis (two of which ultimately gave a
clinical history of syphilis), three cases of pityriasis rosea and one case
in which the diagnosis was not established. It is to be considered pos-
sible that diseases other than syphilis may produce those changes in the
blood which lead to fixation of syphilitic antigen and complement ;
among these are occasional cases of leprosy, scarlatina, malaria, try-

202 THE PRINCIPLES OF IMMUNOLOGY

panosomiasis and certain skin diseases. Gordon, Thomson and Mills
have recently insisted that malaria will not produce a positive re-
action unless complicated by syphilis or as the result of faulty technic.
Although a controversial point, we believe that occasional cases of
tuberculosis may give a positive Wassermann test. That this is not
necessarily due to coincident infection with syphilis is shown by the
experience of Petroff, who found a positive Wassermann in a
tuberculous cow.

Interpretation of Results. — Craig and others are of the opinion that
a strongly positive result, such as would be indicated by -| — | — | — |- in
our schedule, is conclusive evidence of syphilis, whether there are
symptoms or not. Other degrees of fixation must be interpreted with
the aid of clinical history and symptoms. A single negative reaction
does not exclude syphilis. In doubtful cases the so-called provocative
treatment should be applied. This means that a short course of mer-
cury or preferably half the usual dose of salvarsan or neosalvarsan
should be given and the Wassermann test made subsequently. It is
advisable to test the blood twelve, and twenty-four hours after pro-
vocative administration of salvarsan as well as every day for at least
ten days. If the reaction is to become positive, it usually does so in
from a few hours to five or six days, but may be delayed for ten days
or even more. That this is an absolutely specific effect of the drug is
contradicted by the report of Wildgren, who found that the injection
of milk may produce similar results. Endless discussion might be pre-
sented as to the interpretation of the Wassermann test in the clinical
diagnosis of syphilis, but we incline to the view that this test, as is
true of many laboratory examinations, is to be regarded as important
evidence in clinical diagnosis, is of striking specificity when properly
performed, but is not absolutely pathognomonic.

Dependability of the Test. — Criticism has been directed against
the test because of the fact that results do not always agree with
clinical findings and because of differences in results upon the same
serum in different laboratories. It must be admitted that the factors
of error in the test are greater than in clinical diagnosis of the disease.
Discrepancies in reports from different laboratories may, in part, be
due to inherent faults in the test, to faults in technic, to faults in selec-
tion of materials and to insufficient training of the worker. The older
literature contains serious criticisms of the test, as for example the
papers of Wolbart and of Uhle and Mackinney. Under the direction
of the Medical Research Committee of Great Britain in 1918 the results
obtained independently by Dr. C. H. Browning, Dr. J. Mclntosh and
Col. L. W. Harrison upon the same specimens are in very close agree-
ment. More recently Solomon has analyzed the results of 3000 tests
carried out in two different laboratories by skilled workers, Dr. Hinton
and Dr. Castleman. There was complete agreement of results in 9344
per cent, of this large series of tests. This study demonstrates that
with modern methods and skillful performance of the test results are
highly dependable.

APPLICATION OF COMPLEMENT FIXATION 203

Quantitative Results with the Wassermann Test. — For various
purposes, more particularly the observation of the results of treatment,
it may be desirable to titrate accurately the amount of patient's serum
which serves as an amboceptor. This may be done by using different
quantities of the serum. Dilutions of the serum are made with salt
solution, i to 4, i to 8, i to 16, i to 32, i to 64, or are measured as

o.i c.c., 0.05 c.c., 0.03 c.c., 0.02 c.c., o.oi c.c., etc. The tubes are treated

w
in the usual fashion and the results recorded as-g-, indicating complete

p

fixation in dilution I to 8, -^ indicating partial fixation in dilutions of

TT

i to 16, — indicating hemolysis or no fixation in dilutions of i to 32.

O

Wassermann Test on Spinal Fluid. — Spinal fluids are not inac-
tivated and are employed in larger volumes than blood serum, up to as
much as i.o c.c. Hauptmann and Hossli were the first to insist upon
the use of large quantities of spinal fluid, and this modification changed
the entire conception of the frequency of positive results in the spinal
fluid of such diseases as paresis and tabes dorsalis. The test with spinal
fluid is of particular value in syphilis of the central nervous system,
where it is somewhat more specific than the test with blood serum.
The test has also been used with success with transudates and exudates
from the peritoneum, pleura and pericardium. Apparently of value
in examination of the spinal fluid is the Lange colloidal gold test,
described in texts of clinical pathology.

Post-mortem Wassermann Tests. — In a certain number of cases,
death ensues too soon after the patient comes under observation to
secure blood for the Wassermann test. Not infrequently the result of
a Wassermann test may aid the pathologist in morbid anatomical diag-
nosis and may furnish information of value to the clinician in the con-
sideration of doubtful cases. The question arises as to whether or not
post-mortem changes in the blood will invalidate the Wassermann test.
Valuable information has been collected by Graves. In a series of
400 cases he found that only 0.46 per cent, of sera from cadavers were
antilytic and only 0.58 per cent, of sera were hemolyzed, coagulated
or otherwise unfit for use. The post-mortem and ante-mortem results
were the same in 97 per cent, of sixty-eight controlled cases. " The
reactions conformed to the anatomic and historical evidence in 304 of
378 cases, or 80.4 per cent." Contradictory findings are recorded in
less recent literature, but we believe that valuable results may be ob-
tained with blood taken after death.

COMPLEMENT FIXATION IN TUBERCULOSIS

The advantage of a complement-fixation test in the diagnosis of
early pulmonary tuberculosis and in concealed or suspicious lesions is
obvious, Certain authors, Craig, Miller, von Wedel, report a high
percentage of positive reactions in tuberculous individuals, whereas
others, Cooper and Lange, report relatively few positive results. Petroff
is of the opinion that these differences may be due to lack of complete

204 THE PRINCIPLES OF IMMUNOLOGY

and careful study of the cases clinically, as well as a failure to observe
minute details of the test.

Antigens are of the utmost importance, and numerous forms have
been suggested. There appears to be well-founded evidence for using
several strains of the human type bacillus associated with one or more
strains of bovine type. The methods of making antigen vary and
include the use of saline suspensions or extracts of tubercle bacilli,
living or dead, intact or pulverized ; filtrates of broth cultures ; ether
alcohol extracts of whole or autolyzed bacilli, and extracts of tubercu-
lous organs. Apparently those extracts which contain both lipoids and
proteins are most satisfactory. The antigenic substance is thermostable.

The human serum is inactivated, and in Petroff's hands appears to
be most satisfactory if collected one or two days before the test.
Accurate titration of complement, to be used in doses of two units,
and of hemolytic amboceptor is essential. Guinea-pig complement and
a sheep-rabbit hemolytic system are satisfactory. It is absolutely essen-
tial that glassware be perfectly clean and that measurements be accur-
ate. The incubation of the mixture of antigen, tuberculous amboceptor
and complement should be from one and one-half to two hours at the
optimal temperature of 35°-4O°.

Wilson, using a lipoid-free bacillary antigen, attaches great im-
portance to the complement and finds that there is not a universal
adaptability of guinea-pig complement. That from some guinea-pigs
appears to be fixed more readily than that from others. Therefore, in
general, pooled complements are likely to give the best results. If a
single complement is used tests should be made to determine the extent
of fixation. Von Wedel states that preservation of the patient's serum
in the ice-box for five to seven days favors the reaction, but Petroff
found that fresher sera are preferable. It is desirable to make several
tests at intervals upon the same patient. As the result of 1555 tests on
713 cases Petroff obtained the following results:

Cases Positive Negative Percent.

Clinically active tuberculous 212 199 13 93.9

Quiescent tuberculous 158 89 69 56.3

Apparently cured more than two years 58 5 53 8.5

Normals 78 3 75 3.8

Suspected 166 65 101 39.1

Other diseases 41 6 35 14.6

An -analysis of these figures shows that under proper conditions
complement fixation is of distinct value in the diagnosis and prognosis
of tuberculosis. Basing his conclusions on experimental data Petroff
considers " the complement-fixation test in tuberculosis more specific
than the Wassermann test " in syphilis, an opinion in which we concur.
Nevertheless, its most ardent advocates do not regard the test as pathog-
nomonic and Petroff regards it as " only one of the many links in the
tuberculosis diagnostic chain." It is unfortunate that the complement-
fixation test gives the highest percentage of positive results in cases in
which the need for such diagnostic aid is least evident, namely in those

APPLICATION OF COMPLEMENT FIXATION 205

cases of active tuberculosis in which the diagnosis on clinical and
bacteriological grounds is reasonably certain.

"Acid-Fast Fixation." — Of great interest is the fact as shown by
Cooke and others that the complement-fixation test affirms the close
biological relationship of the acid-fast bacilli. From rabbits immunized
with various acid-fast bacilli Cooke obtained sera which reacted inter-
changeably with each member of the group employed in the experiment.
In certain instances the immune sera reacted somewhat more strongly
with their own antigenic organism than with others of the group. Cooke
also found that the sera of tuberculous patients react not only with
the tubercle bacillus but also with other acid-fast bacteria. Sera from
cases of leprosy also contain complement-binding substances which
react with antigens made from several members of the acid-fast group,
the cases of nodular leprosy giving more striking fixation than those
of anesthetic type. According to Cooke, the Wassermann test gives
crossed reactions in tuberculosis which are too frequent to be ex-
plained by the coincidence of syphilis.

COMPLEMENT FIXATION IN GONOCOCCUS INFECTIONS

As with other immune reactions of the animal body, time plays an
important part in the production of complement-fixing bodies in gono-
coccal infections. Acute gonorrhea is usually diagnosed with ease
by bacteriological methods, but it is not until the disease has persisted
several weeks that complement-fixing bodies are likely to be demon-
strated. The value of complement fixation appears in those cases where
simpler bacteriological methods are not adaptable, such as gonorrheal
rheumatism and endocarditis, as well as infections of deeper parts of
the genital tract, such as the Fallopian tube, Cowper's glands and
prostate. The test is also useful in determining the cure of the disease.

Miiller and Oppenheim in 1906 reported favorable results with the
gonococcus complement-fixation test. Bruck and subsequently
Meakins had a similar experience, but more recent study indicates
that the older methods possess little specificity. The work of Teague
and Torrey, Wollstein, Watabiki and Schwartz and McNeil demon-
strated the occurrence of numerous immunologically distinct forms of
gonococcus and the necessity for using several strains in the antigen.
It now appears that from ten to fourteen strains are desirable.

The production of antigen has been extensively studied. Salt
solution extracts appear to be satisfactory. Alcohol extracts have no
value, and Wilson believes that a lipoid-free antigen presents an im-
provement in titer, stability and freedom from anti-complementary
activity. Warden claims good results with an antigen composed of
salts of the fats of the gonococci. Thomson, working under the direc-
tion of Col. L. W. Harrison, reports excellent results by dissolving
the organisms in decinormal sodium hydrate solution and restoring
to the neutral point by decinormal hydrochloric acid. In the hands of
most workers the sheep-rabbit hemolytic system appears to be satis-

206 THE PRINCIPLES OF IMMUNOLOGY

factory, but it has the same objections as obtain in the Wassermann
test. A human-rabbit system may be substituted if desired.

The test appears to be highly specific and of great clinical value
when properly performed. Although the gonococcus and meningo-
coccus are closely-related organisms and may, according to Wollstein,
give crossed complement-fixation reactions there is no satisfactory
evidence that epidemic cerebrospinal meningitis in man produces con-
fusing complement-fixing bodies. Dixon has recently studied 840
tests made by Dixon and Priestly on 625 individuals. Of fifty-three
strongly positive reactions 90.4 per cent, had gonorrhea or a history
of the disease, of sixty-six moderately strong reactions 86.3 per cent,
were confirmed clinically, of seventy-five weakly positive reactions 72
per cent, were confirmed clinically, of ninety doubtful reactions 58.9
per cent, were clinically cases of gonorrhea. Of 341 negative reactions
26.1 per cent, were cases of gonorrhea in some form; of these only
one case was positive to a second test. Therefore, a positive test is
to be regarded as strong presumptive evidence of the disease, but
both positive and negative reactions should be controlled by sub-
sequent tests.

OTHER COMPLEMENT-FIXATION TESTS

Glanders. — The complement-fixation test in this disease appears
to be highly specific independently of the strain of the antigenic
organism. Its principal application is in the disease as it affects horses.
The mallein test and the agglutination test are satisfactory but can be
supplemented by complement fixation. Occasionally it may be ser-
viceable in human medicine.

Typhoid Fever. — Although earlier workers obtained variable re-
sults, later investigations in the hands of Garbat and of Kolmer with
salt solution extracts of numerous strains of the bacillus, the so-called
polyvalent antigen, have given excellent results more particularly in
the second or third week of the disease or later. Blood cultures, the
Widal and the Dreyer tests are so much more easily performed that
the complement-fixation test is to be regarded as only supplementary.
Nevertheless, complement fixation is more likely to occur in the course
of the disease than as the result of prophylactic vaccination and accord-
ingly may gain diagnostic value.

Smallpox. — Positive results have been obtained in this disease by
Jobling, Sugai, Dalm, Klein, Kolmer and others. The antigen has been
obtained either from the lesions of vaccinia in calves or from human
smallpox lesions. Salt solution extracts appear to be better than alcoholic
extracts. In addition to the diagnostic value, the reaction adds to the
evidence concerning the biological identity of smallpox (variola) vari-
oloid and vaccinia. Our interpretation of Xylander's results indicates
that vaccinia in man does not lead to the establishment of complement-
fixing bodies over a long period of time and therefore in all probability
is not a true index of immunity to smallpox. The great diagnostic

APPLICATION OF COMPLEMENT FIXATION 207

value lies in the differentiation of smallpox from syphilis and from
chicken-pox (varicella).

Whooping-Cough. — With antigens made from the pertussis bacil-
lus of Bordet-Gengou the reaction appears to have considerable diag-
nostic value.

Echinococcus Cyst. — The antigen is obtained by filtering the cyst
fluid of .man or sheep and preserving with 0.5 per cent, phenol in a cool
place. Varying results have been reported, but the test appears to be
worthy of further investigation where material can be obtained for
its use.

Malignant Tumors. — Numerous attempts have been made to aid
in the diagnosis of malignant tumors by the complement-fixation test,
using antigens prepared from tumor material. The results have been
conflicting. Von Dungern has devised a test using, on empirical
grounds, an antigen prepared by making an acetone extract of normal
human red blood-cells. He has obtained fixation in as high as 90 per
cent, of known cases of malignant tumors. The test, however, has not
as yet been sufficiently widely applied to justify recommending it as
of clinical value.

Sporotrichosis. — Widal, Abrami, Joltrain and Weil have obtained
excellent results using as antigen the sporotrichum Beurmanni. Moore
and Davis have recently demonstrated fixation with a human serum in
the presence of Schenck-Hektoen, Beurmann and Davis strains of
the organism. This reaction, in addition to agglutination, is of distinct
diagnostic value.

CHAPTER X
HYPERSUSCEPTIBILITY

DEFINITION.
OCCURRENCE.

ANAPHYLAXIS.

SENSmZATION.
PERIOD OF INCUBATION.
INTOXICATING INJECTION.
THE REACTION.

CLINICAL PHENOMENA.
DISTENTION OF LUNGS.
FALL IN BLOOD-PRESSURE.
METABOLIC AND BLOOD CHANGES.
DECREASED COAGULABILITY OF BLOOD.
DESENSITIZATION.
PASSIVE ANAPHYLAXIS.
SPECIFICITY OF ANAPHYLAXIS.
THEORIES OF REACTION.

ANAPHYLACTIC POISONS.
CELLULAR THEORIES.
PHYSICAL THEORIES.
ANAPHYLACTOID PHENOMENA.
SUMMARY.
THE RELATION OF ANAPHYLAXIS TO IMMUNITY.

Definition. — On casual consideration hypersusceptibility appears to
be a condition exactly the opposite of immunity. If by immunization
an animal becomes more than normally resistant to a poisonous or in-
fective agent so in the state of hypersusceptibility it is more than nor-
mally susceptible to poisons, to infective agents and to agents which in
the normal animal appear to be entirely innocuous. More critical
examination of the phenomenon, however, has led to the conception
that hypersusceptibility is but one manifestation of the intricate
mechanism of immunity. The reasons for this latter conception will
appear in the subsequent discussion. The term hypersusceptibility is
not to be confused with anaphylaxis, with which, in our judgment, it
is not synonymous. We prefer to limit the term anaphylaxis to that
state of hypersusceptibility to a given substance which has been induced
by a previous injection of the same substance. The reaction is limited
to proteins or protein fractions. Natural hypersusceptibility to non-
protein substances may occur, but this condition cannot be induced by
a previous administration of such substances.

Occurrence. — Hypersusceptibility may be natural or acquired.
Undoubtedly certain individuals in whom the condition is supposed to
be natural have acquired the state by preliminary inoculation of the
substance to which they are susceptible. This may be an unconscious,
forgotten or concealed acquisition. The introduction of practically any
protein into the tissues of the body may lead to the acquisition of a
hypersusceptibility of long duration unless the primary inoculation is
succeeded by others at proper intervals and in proper amounts to produce
208

HYPERSUSCEPTIBILITY 209

immunity. In man natural hypersusceptibilities are believed to be
manifested upon the introduction of the special proteins or similar sub-
stances into the respiratory tract, the alimentary canal, into the skin and
by injection into the tissues, body spaces or circulation. Man may
exhibit respiratory symptoms in the presence of vegetable effluvia, as in
" hay fever," " rose fever," and of the effluvia of certain animals, such
as the horse and guinea-pig1. In individuals thus susceptible, local or
general reactions may occur following inoculation with the specific
animal or vegetable protein. The ingestion of animal proteins, such as
egg, or vegetable proteins, such as strawberry, may produce severe
gastro-intestinal disturbances sometimes accompanied by general symp-
toms. In certain cases this hypersusceptibility may have been acquired
by previous sensitization, but in the greater number no such explanation
is to be offered. In babies susceptible to egg-white there is no prob-
ability that preliminary direct sensitization occurs, but it is possible
that the tendency to hypersusceptibility may have been inherited. The
instances mentioned are examples of individual hypersusceptibility.
Although less clear cut there are also evidences of species hypersuscepti-
bility, as, for example, the fact that ox serum is distinctly toxic for
guinea-pigs and much less so for man. The acquired forms of
hypersusceptibility will be considered under the general discussion
of anaphylaxis.

Anaphylaxis. — Following the introduction of the serum treatment
of disease, disturbing elements appeared, the most striking of which
were the frequent production of " serum rashes " and the reports of
occasional severe constitutional reactions and even sudden death. Von
Pirquet and Schick pointed out on the basis of a clinical investigation
in connection with the serum treatment of diphtheria and scarlatina,
that in from seven to twelve days following a single injection of serum
or several injections on successive days, a so-called " serum disease "
appears. This is characterized by macular or maculo-papular eruptions
of urticarial type, malaise, fever and other symptoms. After this period
a subsequent injection of the same protein leads to the appearance of
similar symptoms and signs usually within twenty-four hours. After
the lapse of months or years the reaction may be delayed and fail to
appear for several days, but is only rarely as late as that following the
primary injection. In otfrer words, the patients appeared to have been
sensitized by the primary injection. Not being able to define exactly
the nature of this condition, the name allergy was suggested, indicating
an " altered state " of the animal body. The usage of the term at the
present time is confusing and definitions vary ; we, therefore, prefer not
to employ it.

Experimentally similar phenomena had been noted in the course of
other studies as far back as Magendie in 1839, but it remained for
Richet and Portier in 1902 to point out the fact that an animal may be
rendered hypersusceptible to a poison, by the previous injection of a
small dose. They used actino-congestine, a toxic protein extracted
from the tentacles of actinia. Because the phenomenon indicates a
14

210 THE PRINCIPLES OF IMMUNOLOGY

condition the opposite of prophylaxis they named it anaphylaxis. As
a result of the reports of accidents following the use of diphtheria
antitoxin, Rosenau and Anderson investigated the problem experi-
mentally and found that the danger lies in the serum rather than in its
content of antitoxin. They demonstrated that the reaction is specific
for the protein employed, that the period of " incubation " is about six
days and that once established the sensitive state persists for many
months with but slight reduction in intensity. In the same year, 1906,
Otto entirely independently published similar findings in Ehrlich's
laboratory as the result of an interview between Ehrlich and Theobald
Smith. Smith had noted that animals used for the titration of diph-
theria antitoxin were subsequently extremely sensitive to horse serum.
Otto, accordingly, employed the name Theobald Smith phenomenon.
Of somewhat similar significance, but for the time without the same
direct application to medicine, were the investigations of Arthus, who
in 1903 published a study in which he showed that if repeated sub-
cutaneous injections of protein are given, the fourth and subsequent
injections may lead to severe local reactions which may go on to
gangrene. If a later injection is given intravenously death may result.
Arthus also recognized the specificity of the reaction. The year 1906
marked the beginning of a period of widespread investigation
of anaphylaxis. Much has been learned in regard to the mechanism
of the process, but the fundamental principles are still in the form
of hypotheses.

The Sensitization. — The substances necessary for the demonstra-
tion of anaphylaxis are proteins. These need not contain all the amino-
acids, for Wells has shown that certain vegetable proteins, zein, hordein,
gliadin, lacking " one or more such amino-acids as glycocoll, tryptophane
or leucine produce typical reactions " and Abderhalden claims to have
demonstrated anaphylaxis with a compound polypeptid made up of four-
teen amino-acid molecules, which include only two of the amino-acids,
leucine and glycocoll. The sensitizing substance is extremely thermo-
resistant. Wells finds that proteins such as casein and ovO-mucin
which are not heat coagulable are active after heating to 100° C. and
Besredka has found that if a coagulable protein is so diluted as to
prevent coagulation it withstands temperatures up to 120° C. Rosenau
and Anderson found that if the protein be in the dry state it may be
heated to 170° C. for ten minutes and upon re-solution will serve for
the production of anaphylaxis. Heat or chemical agents which render
the protein insoluble destroy its sensitizing properties. Trypsin diges-
tion has the same effect. Gay and Adler reported that upon frac-
tioning serum with ammonium sulphate the euglobulin contains the
sensitizing substance, but not that substance which intoxicates at the sec-
ond injection. Kato, however, finds that the globulins possess the largest
content of both sensitizing and intoxicating substances. Bogolomez
and subsequently Meyer claimed that anaphylaxis could be produced
with lipoids but this has failed of confirmation in the hands of Wilson
and of White and others ; it is not generally accepted. The chief dif-

HYPERSUSCEPTIBILITY 211

ficulty in the work with lipoids lies in the fact that it is practically
impossible to obtain the lipoids in pure form ; an extremely small amount
of adsorbed protein may produce the reaction.

The method of sensitization is by parenteral routes, although
Rosenau and Anderson in their original communication state that they
had been able to sensitize guinea-pigs by feeding horse serum. Bes-
redka was unable to confirm this, but in a few dogs we have obtained
results which have been highly suggestive. A question of fundamen-
tal importance in this connection is whether or not proteins may be
absorbed through an intact intestinal mucosa without digestion. Ac-
cording to the work of Van Alstyne and Grant, such absorption may
occur. Absorption of the whole protein through the intestinal mucosa
or the mucosa of other surfaces might well serve to sensitize animals
or man, but as yet the problem is not conclusively settled. If
the inoculation be by parenteral routes there is apparently little
difference in outcome whether administered subcutaneously, intra-
venously or intraperitoneally.

The amount of protein necessary for sensitization is extremely
small. In their original work, Rosenau and Anderson found that in
guinea-pigs 0.000,001 c.c. horse serum suffices. Wells succeeded in
sensitizing guinea-pigs with 0.000,000,05 gram crystallized egg albumin.
Larger sensitizing doses are necessary in order to produce subsequent
death from anaphylactic shock. Such minute doses are not applicable
in the case of rabbits, dogs and monkeys, in which it may be necessary
to inject the material on two or three successive days in order to
sensitize. The minimal sensitizing dose in man is not known. In
experimental animals there is an optimal sensitizing dose which bears
a certain relation to the subsequent intoxicating dose, as has been shown
by White and Avery. In a general way, the smaller the sensitizing
dose, the larger the minimum intoxicating dose and vice versa, but a
sensitizing dose may be too large for satisfactory sensitization. Accord-
ing to Besredka the larger doses also require a longer time for sensi-
tization to appear.

Period of Incubation. — For a period of eight to twelve days after
the sensitizing dose, subsequent injections of the same material produce
no evidence of hypersusceptibility. Gay and Adler reported that if the
euglobulins of serum are employed for sensitizing, the period of incu-
bation may be shortened to four or five days. If during this period
a second injection be given the animal is more likely to become immune
than hypersusceptible. Rosenau and Anderson found that the state
of hypersusceptibility increases until the twenty-first day, after which
it very gradually diminishes but persists in modified form probably
throughout the life of the animal.

Intoxicating Injection. — The French term, injection dechainante,
is highly descriptive of this part of the process, as it indicates the
explosive character of the manifestations that are likely to occur. This
injection may be intravenous, intrameningeal, intraperitoneal or sub-
cutaneous. The rapidity of reaction and severity of symptoms are most

212 THE PRINCIPLES OF IMMUNOLOGY

marked in intravenous injection and exhibit decreasing severity in the
order named. Besredka estimates that by the use of serum, approxi-
mately equivalent reactions may be produced by intravenous injections
of 0.05 to o.i c.c., intrathecal injections of 0.066 to 0.125 c.c. and intra-
peritoneal injections of 5.0 to 6.0 c.c. Subcutaneous injections in experi-
mental animals rarely produce severe or fatal reactions.

When used for the intoxicating dose the proteins are subject to the
same physical and chemical agents as have been discussed in connec-
tion with the sensitizing injection. The statement of Gay and Adler
that the sensitizing agent is contained in the globulin fraction of serum
and the intoxicating agent in the whole serum and albumin fractions
is not generally accepted and has recently been contradicted by Kato.
Kato found that guinea-pigs sensitized to any of the serum fractions
respond to intoxicating doses of any of the fractions but most strongly
to that fraction to which they were sensitized. The aging of serum
has an important influence. The toxicity of fresh serum decreases
rapidly during the first ten days of preservation to about half its
original power. A slight decrease occurs during the first two
months, after which the deterioration is extremely gradual. Besredka
has found that a serum twenty years old produced anaphylactic shock
in a sensitized animal. Uhlenhuth states that he has produced ana-
phylactic shock with proteins from mummies.

The selection of the route of intoxicating injection depends on the
character of the protein; the intravenous route is undesirable with
solid proteins and even with bacteria because thrombosis and embolism
confuse the picture of anaphylaxis. The minimal intoxicating dose is
larger than the minimal sensitizing dose in the ratio of about 100 to i.
Wells has obtained fatal reactions with 0.000,001 gram crystallized
egg-white. Fatal reactions are rarely obtained with less than 0.025 c.c.
serum and, as a rule, 0.05 c.c. to o.i c.c. is required. We find that for
laboratory demonstrations the use of 0.05 c.c. serum given subcu-
taneously for sensitization and o.i c.c. fresh serum given intravenously
practically always produces fatal reactions. Of great importance is the
fact that there is considerable individual variation not only in the
sensitivity of the experimental animals but also in the sera employed
for experiments. Wells has stated that blood serum contains so many
substances that it is in reality an " extract of the animal " ; hence
variations such as are found in serum are not present in pure iso-
lated proteins.

The Reaction. — The phenomena of the reaction may be discussed
under three heads, the objective manifestations, the morbid anatomical
changes and the functional disturbances. The reaction may be imme-
diate or delayed, depending upon the sensitiveness of the animal, the
size and mode of administration of the toxic dose and the state of
deterioration of the intoxicating substance. The immediate reaction
is called anaphylactic shock. In the guinea-pig the objective manifesta-
tions include rubbing of the nose, ruffling of the fur, evacuation of
urine and feces, spasmodic movements of increasing severity, including

PLATE II.

Drawing of the gross appearance of the lungs of the
guinea-pig in anaphylactic shock, showing marked
distention and pallor. Note the overlapping of the
lobes and the almost complete masking of the heart.

HYPERSUSCEPTIBILITY 213

violent general convulsions, marked inspiratory and expiratory effort
with cyanosis, exhaustion and death from respiratory failure with the
heart still beating. In the dog the respiratory and convulsive phe-
nomena are not so marked ; there is violent precordial activity, marked
fall in blood-pressure, diarrhea and vomiting. In man the phenomenon
may show predominance of the respiratory and convulsive symptoms
or of the cardio-vascular and the gastro-intestinal symptoms. Other
animals show variations of the general picture outlined. The necropsy
on a guinea-pig shows large, pale, distended lungs filling the thoracic
cavity, cardiac dilatation, particularly of the right side, passive con-
gestion of the abdominal viscera sometimes associated with minute
hemorrhages in the gastro-intestinal tract. The lungs may show con-
gestion, edema and small hemorrhages, but, as a rule, the distention
is so marked that there is little blood in these organs. Microscopically
there is marked distention of the alveoli, with rupture of their walls,
constriction of the bronchioles and frequently of the small arteries.
Gay and Southard describe fatty degenerative changes in capillary
endothelium near small hemorrhages, as well as fatty changes in heart
muscle, skeletal muscle and peripheral nerves. Beneke and Stein-
schneider found Zenker's degeneration particularly of the respiratory
muscles, but Wells believes this to be the result of asphyxia which pro-
duces Zenker's hyalin through the increase of lactic acid in the muscle.
In dogs and other animals the pulmonary distention is not marked ; the
important features are dilation of the heart, marked congestion and
multiple hemorrhages. None of these anatomical changes is charac-
teristic or to be distinguished from other toxic conditions. The pul-
monary distention is more distinctive than any of the other changes.

From the functional point of view there have been extensive in-
vestigations of the distention of the lungs, the fall of blood-pressure,
fall in temperature, delayed coagulability of the blood and alterations
of the nitrogen metabolism. Auer and Lewis found that in guinea-pigs
death is due to asphyxia " apparently produced by tetanic contraction
of the smooth muscles of the bronchioles." This is independent of
pithing, section or degeneration of the vagus, and is therefore periph-
eral, either in the nerve terminals or the muscle itself. Auer has
shown that atropin reduces this effect, thus indicating the action upon
nerve terminals. Karsner and Nutt found that there is a definite
quantitative relation between the intoxicating dose of serum and the
protective dose of atropin and this fact, together with the protective
action of anesthetics such as ether, indicates that there may be factors
involved other than mere physiological antagonisms. The exciting
action on smooth muscle is not confined to the bronchiolar muscle for
Schultz demonstrated a similar action in vitro on smooth muscle of the
intestine and bladder of sensitized guinea-pigs and this has subsequently
been extended to include other smooth muscle such as uterus. Pelz
and Jackson have recently observed broncho-constriction in dogs during
the acute shock, but although this is severe we have been unable to
demonstrate acute emphysema in dogs.

214 THE PRINCIPLES OF IMMUNOLOGY

The fall in blood-pressure appears in anaphylactic shock in the dog
and cat but is not so highly characteristic of the reaction in the guinea-
pig or rabbit. Biedl and Kraus described the condition, and it has
since been studied extensively. Pearce and Eisenbrey note that it
amounts to a fall of from 20 to 30 mm. mercury in the dog and believe
it to be due to vaso-dilatation, particularly of the splanchnic area, due
to action upon the nerve endings rather than upon the muscle. Schultz
was of the opinion that the fall in pressure is due to direct action upon
the heart by the toxic agent. He expressed the opinion that in the cat
the fall in general pressure is due to vaso-constriction in the pulmonary
circuit so that the right heart cannot empty itself. Eisenbrey and
Pearce in a further study on dogs found that the functional activity
of the myocardium is not primarily affected, that there is no satisfactory
evidence of pulmonary vaso-constriction and that the later changes
in the myocardium with the fall in general pressure result from incom-
plete filling of the heart consequent on the accumulation of blood in
the larger venous trunks, particularly of the splanchnic area. Simonds
finds that with the fall in arterial pressure, there is a fall in pressure in
the superior vena cava and a rise in portal vein pressure, associated
with an increase in the volume of the liver. Upon examination of the
hepatic vein of the dog the vessel shows a very heavy musculature as
compared with that of the herbivorous animals, and Simonds con-
cludes that spasm of the hepatic vein and its tributaries explains the
phenomena observed. Manwaring and subsequently Voegtlin and
Bernheim had previously found that exclusion of the liver from the
circulation prevented the appearance of anaphylactic shock, observations
well in accord with Simonds' hypothesis. However, Pelz and Jackson
excluded the entire abdominal circulation, and in spite of this demon-
strated broncho-constriction and marked fall in blood-pressure. Thus,
although numerous factors may play a part, the only fact that we can
bring forward as generally accepted is that the fall in arterial pressure
is associated with peripheral vaso-dilatation. Davis and Petersen
observed an increase in the volume of lymph for a short time imme-
diately following injection and again for a longer period beginning
about one hour after injection. The antiferment increases in the lymph
without any change in the blood serum.

There can be no doubt that the gaseous interchange in the
convulsive phases of anaphylactic shock is increased. Varying reports,
however, have appeared as to the influence of anaphylactic shock upon
nitrogen metabolism. Major found an inconstant decrease of nitrogen
output in rabbits during shock, but this increased in the animals that
survived the immediate shock to such a degree as to exceed the intake.
Zunz and Gyorgy found a definite increase in amino-acids, which
Jobling, Petersen and Eggstein confirmed, with the additional informa-
tion that the total non-coagulable nitrogen is increased. Hisanobu
found a marked increase of urea nitrogen, as well as of the non-urea
and amino-acid nitrogen. He concludes, as would also be apparent
from Major's work, that there is an abnormally rapid destruction of

FIG. 17. — Drawing of the microscopical appearance of the lung of the guinea-pig
in anaphylactic shock, showing the alveolar emphysema, constriction of the bron-
chioles and of an arteriole.

HYPERSUSCEPTIBILITY 215

tissue proteins. Jobling, Petersen and Eggstein found an increase in
non-specific protease with a decrease of antiferment and an associated
decrease of serum proteoses ; this is followed by a progressive increase
in non-coagulable nitrogen, proteoses and serum lipase. They, there-
fore, conclude that " the acute intoxication is brought about by the
cleavage of serum proteins (and proteoses) through the peptone stage
by a non-specific protease." Modern opinion thus favors an increase
in nitrogenous metabolism in anaphylactic shock and this may well
be due to a liberation or mobilization of proteases ; that the action of
the latter is limited to the blood appears to us not to be conclusively
proven. In spite of the increase in metabolism there is a fall in body
temperature ; therefore, there must be an increase in heat radiation.
In lower animals the respiratory function is of great importance in heat
radiation, and we suggest that the marked increase of respiration in
anaphylactic shock has some bearing on this problem, but we by
no means wish to exclude other factors that may play a part in
the phenomenon.

The decrease in coagulability of the blood was first observed by
Biedl and Kraus and since has been amply confirmed. They believed
the change to be due to either a decrease of thromboplastin or an
increase in antithrombin. The salts of the blood apparently are un-
changed. Achard and Aynaud, as well as Lee and Vincent, found
a decrease in the number of platelets, but this was not found by Biedl
and Kraus. Shattuck found a delay in action of prothrombin. Pepper
and Krumbhaar reached the same conclusion as Biedl and Kraus con-
cerning a decrease of thromboplastin or an increase of antithrombin.
Bulger concludes, in terms which summarize our knowledge at the
present time, that the decrease in coagulability is " due to changes in
that stage of the coagulation process at which thrombin is formed
through the interaction of prothrombin, calcium, thromboplastin
and antithrombin ( ?) . These changes are probably due to variations
in thromboplastin."

Desensitization or Anti-anaphylaxis. — If an animal recovers from
anaphylactic shock its earlier hypersusceptibility is replaced by a period
of resistance during which injections of the specific protein produce no
demonstrable reaction. This refractory period lasts for a varying
period of time up to several weeks, and although the animal subse-
quently becomes hypersusceptible, it rarely reaches the same degree of
hypersusceptibility which it primarily exhibited. These facts were
pointed out in the original investigations of Rosenau and Anderson
and of Otto. Besredka has studied the matter extensively and has
found that very small doses of the protein may desensitize, doses in
themselves too small to lead to any observable symptoms. By repeated
injections it is possible to produce such a degree of resistance that the
animal may withstand doses 1000 times as great as that which proves
fatal if desensitization has not occurred. The rapidity with which
desensitization appears depends upon the route of injection. After
subcutaneous injection it may not appear for twenty-four hours ; intra-

216 THE PRINCIPLES OF IMMUNOLOGY

peritoneal injections may require three or four hours for results,
whereas intravenous injections may be effective in a few minutes. An
experiment quoted from Besredka illustrates the rapidity and extent
of the process. Guinea-pigs sensitized with egg-white exhibited fatal
reactions with a toxic dose of 0.002 c.c. egg-white. An animal of this
group was given 0.0005 c-c- egg-white intravenously without reaction.
It did not react to 0.005 c-c-> tne ^ata^ dose, given two minutes after
the first injection nor to doses of 0.02' c.c., or 0.2 c.c., given at ten-
minute intervals. Ten minutes later it was given 2.0 c.c. and, although
visibly uncomfortable for a time, recovered. Desensitization may also
be practised by four or five repeated subcutaneous or intraperitoneal
injections at intervals of about two hours, the subcutaneous route
requiring a longer time to be effective than the intraperitoneal route.
We have found relatively large, but still sub-lethal, single doses to be
most effective by intravenous injections, but less so by intraperitoneal
and least by subcutaneous injection. Besredka also reports desensitiza-
tion by introducing the protein into the gastro-intestinal tract but as
yet this has not received widespread confirmation. Desensitization may
be effective at any period during the hypersusceptible state. If a second
dose be given before hypersusceptibility appears, the condition may,
by subsequent injections, become one of increased resistance
or immunity.

Other methods of preventing shock include the use of atropin as
suggested by Auer and Lewis, of adrenalin, of chloral hydrate, admin-
istration of ether, alcohol, atoxyl and numerous other drugs. Pelz and
Jackson found adrenalin most satisfactory in dogs. Karsner and Nutt
found that atropin sulphate is satisfactory in guinea-pigs provided the
toxic dose of serum is not too large. The use of adrenalin in guinea-
pigs is unsatisfactory because of the pulmonary hemorrhage and
edema which it produces. Drugs which depress the excitability of
the smooth muscle of the bronchioles, those which depress nerve activity
generally as the anesthetics, and those which tend to maintain blood
circulation, are pharmacologically adapted to the prevention of ana-
phylactic shock. They do not operate as effectively after the toxic dose
of protein has been given, as they do when given in time to produce
physiologic effects before the onset of shock. Thomson found that
exposure to the X-ray inhibits anaphylactic shock. Friedberger and
Mita have suggested that anaphylactic shock may be inhibited by very-
slow administration of the protein. Lewis has investigated this problem
experimentally and by the use of the Woodyat pump has found that
" acute anaphylactic shock can be prevented in sensitized experimental
animals by giving otherwise fatal doses of diluted antigen intravenously
at very slow rates."

Passive Anaphylaxis. — As was shown in 1907 by the independent
investigations of Nicolle, Richet, Otto and Friedemann, the serum of
a hypersusceptible animal, when injected into a normal animal, will
render the latter also hypersusceptible. This condition is transferred
in the serum rather than in corpuscles or tissue cells. Passive ana-

FIG. 18. — Tracing from the dog in anaphylactic shock. From
above downward the tracings are: myocardiograph, blood-
pressure, base line, membrane manometer, base line, signal, time
in seconds. The down strokes of the myocardiograph tracing
represent cardiac contractions. The perpendicular line was
drawn arbitrarily through the blood-pressure curve at the point
where the fall began. Corresponding points in the myocardio-
graph and Hurthle manometer tracings were measured and are
indicated by the cross where the recording levers were not in
accurate alignment. (From Eisenbrey and Pearce. A study of the
action of the heart in anaphylactic shock in the dog, Journal of
Pharmacology and Experimental Therapeutics, 4, 21. 1912.)

H YPERSUSCEPTIBILIT Y 2 1 7

phylaxis may be demonstrated about four hours after intravenous
administration of the serum from the hypersusceptible animal, about
twenty-four hours after intraperitoneal injection and from twenty-four
to forty-eight hours after subcutaneous administration. It remains
at its height for about three or four days, gradually disappears in a
few weeks and never exhibits the permanence of active anaphylaxis.
Further study by numerous investigators has shown that passive ana-
phylaxis arises as the result of injection of serum from an animal in
the hypersusceptible state or from an animal in the " incubation "
period before sensitization can actually be demonstrated; it may also
follow the injection of the serum of an animal in the anti-anaphy lactic
state and may be produced by the injection of an immune precipitating
serum. In the last-named instance an animal is immunized to the
particular protein for which passive anaphylaxis is to be produced.
Doerr and Moldovan pointed out this fact, and it has been repeatedly
confirmed. Scott demonstrated that the intensity of the anaphylactic
shock parallels the titer of the precipitating serum. The young of
sensitized female guinea-pigs are sensitive, as has long been known.
The recent work of Reinals confirms this fact, but does not definitely
settle the question as to whether the sensitization of the young is active
or passive.

Specificity of Anaphylaxis. — That the process is specific was
pointed out by the earliest investigators. It is undoubtedly one of the
most specific of the biological reactions, as is emphasized by its extreme
delicacy in regard to sensitizing dose. Nevertheless, group reactions
appear as in the reactions of immunity. For example, a guinea-pig
sensitive to sheep serum will react somewhat less violently to goat
serum. Wells and Osborne have shown that cross reactions occur
between gliadin from wheat and rye, and hordein from barley. The
reactions, however, are strongest with the homologous protein. Never-
theless, Wells was able to separate ovovitellin and crystallized egg-white
by the anaphylaxis reaction and is of the opinion that where group
reactions occur the reactions are the result of common groups in the
protein molecules even though the proteins may appear to be chemi-
cally distinct. If guinea-pigs are sensitized to several proteins simul-
taneously they will react to any of the proteins employed, but after an
animal has reacted to one of the proteins, subsequent reactions to the
others are less severe. Investigations conducted in this laboratory with
serum proteins indicate that although desensitization is best produced
by homologous sera it may be effected by biologically-related sera and
by non-related sera. For such purposes considerably larger doses of the
heterologous sera are necessary than of the homologous serum. Against
the assumption that desensitization indicates the specificity of the reac-
tion is the fact claimed by Banzhaf and Steinhardt that lecithin protects
against anaphylactic shock. Rosenau and Anderson failed to confirm the
work of Banzhaf and Steinhardt, but it may well be that a colloidal dis-
turbance of some sort may prevent the appearance of shock and that

218 THE PRINCIPLES OF IMMUNOLOGY

some similar disturbance may appear as the result of injection of heter-
ologous sera.

In view of the great specificity of anaphylaxis it was hoped that
by this means organ specificity might be demonstrated. Ranzi used
extracts of liver, kidney, spleen and ovary and found that these gave
species reactions with serum, but that there was no evidence of organ
specificity. Pfeiffer pointed out that the organs employed by Ranzi
contained blood and therefore could not be expected to show more
than species reactions. He washed the organs apparently free from
blood and found that animals sensitized to a given organ extract respond
somewhat more markedly to that extract than to extracts of other
organs, i.e., there is a relative specificity. This was found to be true
in somewhat lesser degree by Pearce, Karsner and Eisenbrey. Minet
and Bruyant desensitized with -serum and then attempted to produce
shock by the organ extracts ; they failed to demonstrate organ specificity.
Bell has pointed out the fact that the most careful perfusion of organs
fails to remove the blood completely, and it appears that Minet and
Bruyant's conclusions must hold for the present. Extracts of sperma-
tozoa and of ovary fail to exhibit organ specificity, but crystalline lens
behaves as it does in the reactions of precipitation and cytolysis.
Numerous investigators have shown that the lenses of different species
react with each other but that serum fails to interact as either sensi-
tizer or intoxicating body with the serum of the species from which
the lens was taken.

There is no doubt that anaphylaxis produced by bacterial
emulsions or extracts is specific, but reports vary as to the presence
of group reactions. Delanoe holds that group reactions appear,
whereas Kraus and his collaborators maintain the absolute specificity
of bacterial anaphylaxis.

Theories of the Reaction of Anaphylaxis. — In order to avoid any
more confusion than is necessary it seems well to review these theories
in groups rather than in historical sequence. The most important
difference of opinion is as to whether or not a poisonous substance is
produced in the reaction. If not it would appear to be necessary to
suppose that some sort of reaction occurs in the cells of the body or
in the body fluids, perhaps in the nature of a liberation of energy on
the part of the cells or in some form of disturbed colloidal or enzymatic
balance of the fluids. If a toxic substance is formed it may be pro-
duced in the cells or in the circulating fluids. This may be the result
of partial destruction of the proteins of the body or of the introduced
protein, or it may appear as a new body which is formed by substances
produced by the first injection coming in contact with the antigen upon
second injection. This summary gives the essentials of the con-
troversy, and a further elaboration follows.

Anaphylactic Poisons. — Richet formulated the hypothesis that the
primary injection of protein produces a substance in the body which
he named toxogenine. Upon second injection the antigen is supposed to
combine with the toxogenine which has been produced during the period

HYPERSUSCEPTIBILITY 219

of incubation and forms a toxic substance named apotoxlne. He com-
pares the reaction to the combination of amygdaline and emulsine to
produce hydrocyanic acid. This hypothesis resembles somewhat that
of Friedberger, which has been investigated intensively by many
workers. Friedberger prepared a toxic substance, which he named
anaphylatoxin, by mixing antigen, precipitating serum and complement.
He obtained a precipitate by mixing sheep serum with a specific immune
precipitating serum from the rabbit. This precipitate was washed, sus-
pended in fresh guinea-pig serum for twelve hours, then centrifuged.
The supernatant fluid was found to be extremely toxic for guinea-pigs.
The reduction of complement in anaphylaxis has been emphasized by
Friedberger in the development of his hypothesis concerning ana-
phylatoxin. Thomson, however, has found that this reduction is not
constant and that it is in proportion to the quantity of the free antibodies
in the circulation. It is insignificant when the animal has been sensi-
tized with a small single dose of antigen, but if the animal has been
sensitized by repeated doses and the precipitin content of the blood is
high, the diminution in complement is likely to be marked. The symp-
toms following injection of anaphylatoxin include the usual clinical
manifestations, with fall of temperature, retardation of coagulation of
the blood and leucopenia. The poison resists heat at 56° C. for one-half
hour, resists desiccation and is precipitated by alcohol. Subsequently
it was found that bacteria and their antisera could be employed in the
same fashion as the precipitinogen and precipitin. Doerr and Russ
found that precipitates are toxic without the addition of complement,
and in view of this fact and the production of passive anaphylaxis by
precipitating sera, reached the conclusion that precipitin and the sub-
stance produced by the primary injection in anaphylaxis are inseparable.
Kraus and his co-workers have contradicted this parallelism and point
out that the guinea-pig is a poor producer of precipitin; rabbits may
produce a powerful anaphylactic substance without producing precipi-
tins; goats produce precipitin readily but have a serum incapable of
conferring passive anaphylaxis. Biedl and Kraus pointed out the fact
that injections of pepton produce symptoms similar to anaphylaxis in
the dog. Karsner has confirmed this in the guinea-pig. Biedl and
Kraus found that following injection of pepton into a dog the animal
subsequently fails to react to anaphylaxis and hence they formulatedV.
the hypothesis that the poison of anaphylaxis is a pepton-like body.
Doerr offered the hypothesis that the actual disturbance is in the physi-
cal character of the blood. He assumes, however, that this disturbance
is produced by a toxic agent originating in complement. The com-
plement is supposed to contain the toxic substance held in check by an
antagonistic substance ; the latter is adsorbed by precipitates or bacteria,
thus liberating the toxic substance. The further investigation of the
so-called anaphylatoxin led to the discovery by Keysser and Wasser-
mann that a similar substance could be produced by the action of com-
plement on barium sulphate or kaolin. Besredka then found that placing
fresh serum upon pepton agar produces a toxic fluid which induces

220 THE PRINCIPLES OF IMMUNOLOGY

symptoms identical with those from anaphylatoxin. Bordet found that
the action of fresh serum upon agar in solution produces a similar
toxic substance. Novy and De Kruif have published very extensive
studies upon toxic materials in a measure similar to the anaphylatoxin.
It is found that the action of serum upon agar intensifies the toxic
power of the agar. They have shown that agar and other non-protein
colloids produce anaphylactoid symptoms.

The poison, if there be such in anaphylaxis, is not dependent on the
presence of any antibody or other substance within the cells of the sensi-
tized animal, because it can be produced in vitro; neither is it dependent
on antigen, inasmuch as barium sulphate and kaolin serve a similar pur-
pose; nor is it dependent on complement, for, as Doerr has shown, it
can be produced without the action of fresh serum. Besredka maintains
that the anaphylatoxin produces no symptoms by sub-dural injection
and that it kills only upon intravenous injection. Besredka has found
that pepton does not interfere with true anaphylaxis in the guinea-
pig, but that it does inhibit the action of anaphylatoxin. Furthermore,
the state of anti-anaphylaxis which protects an animal against a massive
dose of the antigenic substance and therefore prevents anaphylactic
shock has no such protective influence upon anaphylatoxin. These
arguments as well as those presented in the subsequent section on the
cellular theories of anaphylaxis serve to show that there is prob-
ably no poison, which can be produced in vitro, that leads to the
development of a condition identical with true anaphylaxis. Certain
features of this discussion will be referred to under the heading of
Anaphylactoid Phenomena.

Cellular Theories. — The conflicting views are that either a poison
is produced within cells, or that some disturbance of cells appears inde-
pendently of the production of a poisonous substance. Gay and
Southard, influenced perhaps by the prevailing conceptions of immune
reactions and impressed by the cellular degenerations seen in their
animals, emphasized the intracellular character of the reaction. They
assumed that the injected protein contains a substance, anaphylactin,
which is eliminated from the body extremely slowly, in contrast to the
fairly rapid elimination of the other constituents of the protein. " The
anaphylactin, however, remains and acts as a constant irritant to the
body cells, so that their avidity for the other assimilable elements
of the horse serum (or protein), which have accompanied the ana-
phylactin, becomes enormously increased. At the end of two weeks of
constant stimulation on the part of the anaphylactin, and of constantly
increasing avidity on the part of the somatic cells, a condition has
arrived when the cells, if suddenly presented with a large amount of
horse serum, are overwhelmed in the exercise of their increased assim-
ilating functions and functional equilibrium is so disturbed that local
or general death may occur." This theory was supported by their
statement that the sensitizing fraction of serum is contained in the
globulin fraction and that the other elements of serum may serve to
produce shock. They could not produce a toxic body by mixing the

H YPERSUSCEPTIBILIT Y 22 1

serum of sensitized guinea-pigs and horse serum. The fact that
further investigation, as for example that of Wells and of Kato, has
failed to demonstrate a manifest difference between sensitizing and
intoxicating fractions of the protein, is an argument against this
hypothesis. Friedberger's original conception was that the primary
injection leads to the development of receptors in the cells but in such
small amounts as not to be liberated into the blood stream. These
" sessile " receptors are responsible for an increased affinity of the
cells for the antigen, the consequent disturbances resulting from the
rapid anchoring of the protein by the cells. If injections are repeated
before the anaphylactic state is developed the receptors are formed in
large amounts and appear in the blood stream as precipitins. This
hypothesis accords well with the modern conception of immunity and
anaphylaxis save for the assumption that the sensitizing substance and
precipitins are identical. This theory was followed by Friedberger's
anaphylatoxin theory. Somewhat more concrete is the hypothesis of
Vaughan and Wheeler. After a long period of study of toxic frac-
tions of bacterial and other proteins by Vaughan and his co-workers,
the following statement in regard to anaphylaxis was made. " When
a foreign protein is introduced into the blood or tissues it stimulates
certain body cells to elaborate the specific ferment which will digest
that specific protein. When this protein first comes in contact with
the body cells, the latter are unprepared to digest the former, but this
function is gradually acquired. The protein contained in the first
injection is slowly digested, and no ill effects are observable. When
subsequent injections of the same protein are made, the cells prepared
by the first injection pour out the specific ferment more promptly, and
the results are determined by the rapidity with which digestion takes
place. The poisonous group in the molecule may be set free rapidly,
and in amounts sufficient to produce symptoms, or to kill the animal."
Jobling and his co-workers, however, have reached the conclusion that
the development of proteases in the blood is not dependent upon anti-
bodies and is not specific. Vaughan replies to this objection that " we
have only transferred the problem of specificity from the development
of a specific enzyme to the specific uncovering of a non-specific
enzyme." Undoubtedly, the bodies studied by Vaughan are extremely
toxic. As an example, he found that the product of I gram of casein
is sufficient to kill 800 guinea-pigs. We are not ready to admit that
toxic substances of this sort produce clinical and pathological changes
that are identical with anaphylaxis. Weil has given the participation
of the cells most extensive study. He considered that the cells are of the
utmost importance in the destruction and elimination of foreign protein
and that in the course of this process they construct an antibody. The
union of antigen and antibody within the cells gives rise to the serious
disturbances which constitute anaphylaxis. His excellent work was
interrupted by his death in the service of his country, but his hypothesis
is one which serves equally well in the phenomenon of desensitization
and in anaphylaxis. In support of the assumption that the primary

222 THE PRINCIPLES OF IMMUNOLOGY

change is in the cells may be considered the work of Schultz, Dale,
Woods and others with isolated sensitized organs containing smooth
muscle. These organs, washed free of blood, responded specifically to
the protein with which the animal was sensitized. Of considerable
value was the experiment of Pearce and Eisenbrey, who transfused
dogs so that the blood of a sensitized dog circulated in the body of a
normal dog and vice versa. Under these circumstances the intoxicating
dose of the antigenic protein produced symptoms in the sensitized dog
with normal blood, but no symptoms in the normal dog provided with
blood from its sensitized fellow. Coca confirmed this with the guinea-
pig. Although Manwaring and collaborators have found that per-
fusion of rabbit heart indicates that anaphylactic shock is entirely
humoral, subsequent work of Manwaring and Kusama with perfusion
of guinea-pig lungs showed that the cells of the* lungs of sensitized
animals respond by bronchiolar constriction to perf usion with antigenic
serum. They also found that perfusion of normal lungs with a mixture
of the blood of a sensitive animal and antigen also produces bronchiolar
constriction. None of the experiments so far outlined establishes
definitely the parts the cells play, for, as Bell points out, none of these
methods has completely removed the native blood from the organs.
We know that minute amounts of certain protein poisons are highly
toxic, and it may be that the amount of blood left in a perfused organ
is sufficient for the production of a humoral poison. Nevertheless,
studies of passive anaphylaxis tend to confirm the conception of cellu-
lar participation. Weil has pointed out that simultaneous injection of
a serum, capable of producing passive anaphylaxis, and its antigen fails
to produce symptoms. A certain interval of time must elapse before an
animal becomes passively anaphylactic, an interval in which it is pre-
sumed the cells either anchor or develop the sensitizing substance.
Isolated organs fail to respond to the antigen until a certain time has
elapsed. The time element depends to a certain extent upon the mode
of injection, but is never less than several hours even with intravenous
injection. This fact, in association with the experiments in active
anaphylaxis, in vitro, with perfusion and with isolated organs, all tend
to support the conception that the participation of the cells is of funda-
mental importance in the reaction. Weil has studied further the phe-
nomenon of desensitization and finds that the reaction between the
cellular antibody and the antigen follows in a general wray the Danysz
phenomenon (see page 50). By' the fractional injection of antigen the
substance in the cells takes up the antigen so that subsequent additions
of antigen produce little effect. He states that "partially combined
cellular antibody manifests a marked diminution in its affinity for fresh
antigen." Thus the conception of cellular participation fits the demon-
strated facts of passive anaphylaxis.

Physical Theories. — These have been less susceptible to experi-
mental proof than other theories because of the limitations of technic.
As has been mentioned, Doerr conceived the idea that adsorption of the
supposed antagonistic substance of complement by bacteria or precipi-

HYPERSUSCEPTIBILITY 223

tates liberates the toxic substance of complement. This theory omits
consideration of the cellular participation and needs further elaboration
to be acceptable. Of more significance is the fact demonstrated by
Jobling, Petersen and Eggstein that anaphylactic shock " is accom-
panied by (a) the instantaneous mobilization of a large amount of
non-specific protease, (b) a decrease of antiferment, (c) an increase
in non-coagulable nitrogen of the serum, (d) an increase in amino-
acids, (e) a primary decrease in serum proteoses." They conclude that
the " intoxication is brought about by the cleavage of serum proteins
(and proteoses) through the pepton stage by a non-specific protease"
and that " the specific elements lie in the rapid mobilization of this
ferment and the colloidal serum changes which bring about the change
in antiferment titer." From our discussion of the cellular participa-
tion in anaphylaxis the conception of Jobling cannot be accepted as
entirely satisfactory, but it has more ground in demonstrated fact than
any of the other physical theories. Support for Jobling's conception
is furnished by Bronfenbrenner and others. Bronfenbrenner finds,
however, that the state of dispersion of the colloids is important in
maintaining the ferment-antiferment balance and that simply bubbling
ether through serum decreases the antitryptic activity probably because
of an increased dispersion of colloidal particles. A similar decrease
of antitryptic activity of the blood follows a mixture of antibodies and*
antigen. This theory may be applied to desensitization by assuming
that the small intoxicating dose inhibits antiferment, that the proteases
then operate and that the split products act as antitrypsin, thus prevent-
ing the toxic effects of subsequent injections. Danysz hypothesizes that
anaphylaxis is an intracellular or intravascular disturbance of digestion
or a combination of the two. The disturbance of digestion consists in an
inability of the organism rapidly to transform the colloid antigen into
crystalloids. The symptoms are produced by a sudden alteration of
equilibrium between the sol. and gel. state of the colloids which enter
into the composition of the cells and of the blood. His conclusion that
acute anaphylaxis is due to intravascular changes in the animal is in
contradiction to what we believe to be well demonstrated facts. Krits-
chewsky found that the sap of a certain plant, cotyledon scheideckeri,
precipitates blood proteins, agglutinates and hemolyzes erythrocytes.
Symptoms in animals following injections into the circulation or sub-
cutaneously resemble anaphylaxis and are due, Kritschewsky believes, to
a change in degree of disperseness of the plasma colloids, and he
therefore assumes that anaphylaxis is of the same nature. We do not
concede that Kritschewsky worked with true anaphylactic shock, as
the pathological findings in his animals lack the uniformity of those
seen in true anaphylaxis. Similarly we object to the experiments of
Doerr and Moldovan who produced toxic symptoms by the injection
of water colloidal solutions of silicic acid, also of nucleinic acid and
of dialyzed iron hydroxid. Kopaczewski found that the injection of
serum rendered toxic by addition of bacterial suspension or colloidal
gels., when injected into animals reduces the surface tension of their

224 THE PRINCIPLES OF IMMUNOLOGY

blood three or four dynes, from which reduction the animal gradually
recovers. Upon investigation of the electrical potential of sera it was
found that a current of eight volts shows a precipitate at both electrodes
in the case of normal serum, but that with a so-called anaphylactic serum
the precipitate collects almost entirely in the negative pole. Although
Besredka's former idea that the injected serum contains a separate
sensibilisinogen which leads to the formation of sensibilisin in the cells,
and an antisensibilisin which combines with sensibilisin upon the second
injection of the serum is not in accord with prevailing ideas, yet he was
one of the first to propose a physical theory. Thus he stated in a gen-
eral way the majority of the facts seem to indicate that the phenom-
ena of anaphylaxis and anti-anaphylaxis are reduced to the action of
precipitation and adsorption which upset the mutual relations of the
colloids. Besredka no longer insists upon the separation of the two
elements of protein, but is of the opinion that the important site of
reaction is in the nerve cells. He believes that the second injection
of the protein meets with the preformed sensibilisin in the cells and
produces there either a liberation or absorption of energy, thermal or
otherwise, and that this reaction leads to the phenomena O'f anaphylactic
shock. He compares the reaction to the mixing of water and sulphuric
acid. If the water is added suddenly to the acid there is an explosive
liberation of the heat of hydration. If the water is added slowly, the
heat is generated more gradually and no serious manifestations take
place. So with anaphylaxis, if the injection is in a single large dose,
anaphylactic shock is produced, but if several small doses are given,
there is a series of very slight shocks leading to no serious disturbance
and so desensitizing the body that serious results cannot follow a
subsequent large injection. Besredka argues that the inhibitory effect
of anesthetics supports his contention that the nerve cells are of great
importance in production of anaphylactic shock, but the work of numer-
ous investigators shows that the broncho-constriction and fall in blood-
pressure occur in spite of anesthesia, and that the reaction may be fatal
if the intoxicating dose be sufficiently large. Bronfenbrenner points
out that anesthetics increase the antitryptic power of the blood 100
per cent, or more, thus inhibiting the liberation of proteases and the
consequent production of toxic split products. As a further objection
to Besredka's conception is the fact that the experiments with isolated
organs and perfusion demonstrate that smooth muscle reacts and that
the phenomenon is by no means confined to the nerve cells. When
calorimetric and metabolism experiments can be performed with nerve
tissues, definite information can be obtained in regard to energy changes
in these tissues in anaphylaxis.

Anaphylactoid Phenomena. — In the discussion of the theories of
anaphylaxis references have been made to anaphylatoxin and certain
similar substances. As has been pointed out, these substances may be
protein in character, may represent certain decomposition products of
protein, or may be non-protein colloids. It is even maintained that
arsphenamine is to be included in this category of colloids. The in-

HYPERSUSCEPTIBILITY 225

vestigation of these substances has had an important bearing on the
development of theories of anaphylaxis, because if these can be com-
pared to the supposed toxic substance of anaphylaxis it would seem
reasonable to suppose that anaphylaxis has as its basis a colloidal dis-
turbance. Many of those who have worked with these substances
have not been strict in their use of the term anaphylaxis and have
depended in large part on the clinical manifestations following the injec-
tions of these agents. From time to time certain investigators have
indicated that more intimate study would prove that anaphylaxis and
the phenomena following the injection of these colloids are not identi-
cal. Manwaring and Crowe, for example, found that occasionally there
appears in anaphylaxis occlusion of pulmonary blood-vessels by thrombi
and used the term pseudo-anaphylaxis. The problem has recently been
investigated extensively by Hanzlik and Karsner. The experiments
in this series oi studies were controlled by gross and microscopic studies
of the viscera of the animals after death. More than thirty colloidal
agents were studied by a variety of methods, including intravenous
injection, studies of perfused organs, protection by atropin and epi-
nephrin, as well as test-tube studies of the action of the agents upon
blood-corpuscles. Many of the agents studied produce serious dis-
turbances of circulation and others produce equally serious disturb-
ances of respiration. In the case of none of these colloids was it
possible to demonstrate that the clinical and morbid anatomical phe-
nomena, taken collectively, are identical with those of anaphylaxis.
The symptoms provoked can all be explained on grounds other than
the assumption that we are dealing with anaphylaxis. Even in the case
of agar, where bronchial constriction and pulmonary distention are well
marked, the common occurrence of thrombi both in the living animal
and in perfused lungs definitely excludes an identity with anaphylaxis.
These phenomena may, therefore, be considered as of colloidal nature
and may well be referred to as " colloid shock." Pepton produces symp-
toms and signs more nearly like those of true anaphylaxis than the other
substances studied, but the fact that pepton more frequently produces
thrombosis, hemorrhage and edema of the lungs than is the case in true
anaphylaxis, would place pepton poisoning in the group of ana-
phylactoid rather than anaphylactic phenomena. Similarly the injec-
tion of primarily toxic sera such as ox serum and eel serum into the
guinea-pig produces certain circulatory disturbance with hemorrhage
and edema. It seems probable that the toxicity of some of the sub-
stances of protein nature or the decomposition products of protein may
depend for their activity upon the presence of histamine. The poison-
ous character of histamine depends in no way upon previous sensitiza-
tion, is primarily toxic and therefore may be included as anaphylactoid
in its action.

Summary. — With a strict adherence to the conception that ana-
phylaxis constitutes that state of hypersusceptibilityto a given substance,
which has been induced by a previous injection of the same substance
we may conclude that the mode of the second injection determines the
15

226 THE PRINCIPLES OF IMMUNOLOGY

particular manifestations observed. The sensitizing fraction of the
protein, if there be any such fraction, has not been isolated nor has
the intoxicating substance. If the second dose be given in mass, ana-
phylactic shock results. If, on the other hand, divided small doses are
given the state of the organism is so changed that severe anaphylactic
shock does not appear. In agreement with Besredka, we believe that
desensitization produces a series of minor shocks but believe that the
explanation lies rather in the work of Weil than in the hypothesis of
Besredka. In other words, there is a partial saturation of the sensi-
tizing substance within the cells, so that any subsequent union cannot
produce the intensity of reaction that would have been produced by a
massive injection. The time that must elapse for the production of
passive anaphylaxis, as well as the other experiments offered in evi-
dence, support the conception that some change must occur in the cells
in order to produce sensitization. The nature of the combination
between the specific protein and the substance within the cells or the
influence of the protein upon the cells is not definitely known, but the
data offered in review appear to rule out the probability that definite
toxic bodies are formed. Similarly the nature of the primary changes
in the cells upon second injection cannot be identified; as to whether
there is a liberation of energy of some sort or a disturbance of colloidal
relations must still be the subject of investigation. The specificity of
the reaction is similar to that of other biological reactions and is subject
to similar limitations of the group phenomenon. Nevertheless, we find
in anaphylaxis a most specific phenomenon, which is approached
in delicacy only by the reactions of precipitation and of com-
plement fixation.

The Relation of Anaphylaxis to Immunity. — If desensitization of
an anaphylactic animal is carried on for only a short time the period of
desensitization is relatively brief, but, on the other hand, if the vac-
cination be continued the animal may be rendered resistant. This indi-
cates a close relationship between the two phenomena. We do not
propose to discuss this at length because of the intricacy of the subject.
Weil pointed out in his earlier experiments by saturation of the animal
with proteins that although the animal may become immune in so far
as his body fluids are concerned he may remain hypersensitized in so
far as his cells are concerned. Manwaring and Kusama found that
the lungs of guinea-pigs immunized to a certain protein, when washed
free of blood, were still sensitive to perfusion with the protein in
question. We, therefore, revert to the conception of Weil that im-
munity is in large part exhibited in protective power of the blood and
body fluids. In the state of anaphylaxis this immunity has not been
established in the fluids and therefore the cells can be directly operated
upon by the antigen. If, on the other hand, the animal is immune his
blood and fluids combine with the antigen so as to protect the cells.
The direct bearing of this upon diseases in man is a matter of specula-
tion. It seems possible, however, that during the period of incubation
of an infectious disease the animal, as suggested by Danysz, likewise

HYPERSUSCEPTIBILITY 227

passes through the period of sensitization to the infecting organism.
When, therefore, the infecting organism or its products are present in
sufficient amounts the manifestations of disease appear in the form of
what may be termed an acute or sub-acute anaphylaxis. As time goes
on this process is transformed into an immunity and the disease under-
goes cure. In this latter state it may be assumed that the body fluids
have developed a sufficient amount of protective substance so that the
cells are no longer susceptible to attack. This would satisfactorily
explain the self -limitation of infectious disease. By assuming that
injury of the cells may have become so serious as completely to
interfere with life processes, death may ensue, or if the disturb-
ance is not so severe the condition may exhibit the chronic com-
plications which so frequently follow acute infection.

CHAPTER XI
HYPERSUSCEPTIBILITY IN MAN

INTRODUCTION.

SERUM DISEASE.

THE DELAYED REACTION.
THE ACCELERATED REACTION.
ANAPHYLACTIC SHOCK IN MAN,
NATURAL HYPERSUSCEPTIBILITY.

TESTS FOR HYPERSUSCEPTIBILITY.

TOXINS IN HAY FEVER.
TECHNIC OF CUTANEOUS TESTS.
DELICACY OF TESTS.
THE REACTION.

THEORIES OF CUTANEOUS REACTION. ^

DRUG IDIOSYNCRASIES.
THE TUBERCULIN TEST.

GENERAL REACTION.
CUTANEOUS REACTION.
INTRACUTANEOUS TEST.
CONJUNCTIVAL TEST.
THEORIES OF TUBERCULIN TEST.
SPECIFICITY.
UTILITY.

THE LUETIN REACTION.

CUTANEpUS REACTIONS IN TYPHOID FEVER.
CUTANEOUS REACTIONS IN GONOCOCCUS INFECTIONS.
CUTANEOUS REACTIONS IN MENINGOCOCCUS INFECTIONS.
CUTANEOUS REACTIONS IN PNEUMOCOCCUS INFECTIONS.
CUTANEOUS REACTIONS TO VACCINE VIRUS.
CUTANEOUS REACTIONS IN GLANDERS.
OTHER CUTANEOUS REACTIONS.

Introduction. — The manifestations of hypersusceptibility in man
can be classified into two groups, those in which a definite previous sensi-
tization has been effected and those in which no such sensitization is
known or can be conclusively proven. In the former group are
included a relatively few cases of anaphylactic shock and the widely-
observed phenomenon called serum disease. In the latter group are
those individuals who are abnormally sensitive to a wide variety of sub-
stances. These may gain access to the body from the air, through the
respiratory tract, skin or conjunctiva or through ingestion of foods
which contain the specific substance. In addition to air contacts, direct
contacts with plants and animals, which may or may not serve to produce
dusts, may also lead to dermal manifestations of hypersenstiveness.

Serum Disease. — The Delayed Reaction. — The serum treatment of
various diseases has given ample opportunity for the study of the
symptom complex called by von Pirquet and Schick serum disease.
This follows with extreme frequency upon subcutaneous, intravenous or
intrathecal injections of animal sera employed for therapeutic pur-
poses and may be delayed or accelerated. The symptoms may develop
after a primary or series of primary injections and constitute the delayed
reaction. These symptoms appear from six to twelve days after the
228

HYPERSUSCEPTIBILITY IN MAN 229

injection, and in our experience have been most frequent after ten to
eleven days. The most noticeable and most common symptom is a skin
eruption which is usually urticaria! but may be a patchy or diffuse
erythema, a scarlatiniform or a multiform eruption. Edema may
appear in the lips, eyelids, face or other parts of the body and rarely
may effect the larynx. According to Longcope, " in one instance a
transient hemiplegia was supposed to be caused by local edema .of the
meninges." We have seen one case in which a broncho-pneumonia,
following a prophylactic injection of serum appeared to be the sequence
of an edema of the bronchi. There is often lymph-node enlargement,
which may precede the eruption and may be accompanied by enlarge-
ment of the spleen. There is likely to be a moderate fever, headache,
malaise and occasionally nausea and vomiting. Multiple joint pains,
increased by motion, but without tenderness, redness or swelling, are
common in severe cases. Albuminuria appears in 5 to 9 per cent,
of the cases, and Longcope has found that there is likely to be salt
and water retention with little or no disturbance of nitrogenous elim-
ination. There may be a primary leucocytosis, followed by a leuco-
penia, which latter shows an absolute increase of lymphocytes. The
condition usually lasts twenty-four, forty-eight or seventy-two hours
and occasionally is prolonged to twenty days or more. Relapses may
occur, more particularly after the use of large amounts of serum.

Several factors enter into the occurrence, severity and duration of
the disease, the larger doses giving more frequent occurrence, greater
severity and longer duration. There are certainly individual differences
in the resistance of patients and probably individual differences in
specimens of serum. Sera from different species exhibit differences
in toxicity for man, that of the ox, according to Kraus, being less likely
to produce serum disease than that of the horse. The globulin pre-
cipitation or so-called concentration of horse serum in the preparation
of antitoxins reduces the toxic manifestations in man.

The Accelerated Reaction. — Frequently repeated injections of serum
at properly spaced intervals may lead to a state of resistance or im-
munity, but this is practically never permanent. Following a primary
injection or series of injections, there usually develops a state of hyper-
susceptibility. This condition does not precede the appearance of the
delayed reaction and does not precede the tenth day after injection,
even if the delayed reaction fails to appear. Repeated doses of serum at
short intervals delay the appearance of hypersusceptibility. The height
of sensitiveness is reached in from two to three months, after which it
slowly subsides but probably never entirely disappears. We have
observed accelerated reactions nine and fourteen years after primary
injection. The hypersusceptibility exhibits itself only on injection of
the protein and is specific for the species from which it originated.
Following the second injection of the protein or serum there is occa-
sionally no acceleration of reaction, but if accelerated it may be mod-
erately or markedly so, the last producing the so-called immediate
reactions. These immediate reactions may be local, appear about the site

230 THE PRINCIPLES OF IMMUNOLOGY

of injection in from a few minutes to an hour or two and show edema,
erythema or urticaria. There may also be a general immediate reaction
which appears in from twelve to twenty-four hours and in addition to
the usual symptoms and signs of serum disease may be accompanied
by severe asthmatic form of dyspnea, cardio-vascular disturbances with
cyanosis, collapse, chills, nausea and vomiting and renal disturbances
including complete suppression for several hours. If the second injection
is given when hypersusceptibility is not marked, as for example after
a small primary dose, or several years after a primary dose, the accel-
erated reaction is not likely to be immediate but appears in from two
or three to five or six days. Under these circumstances the reaction
may appear as an ordinary case of serum disease or may be more severe.

Anaphy lactic Shock in Man. — There is little doubt that the accel-
erated reactions of serum disease bear in some way a relation to ana-
phylactic shock. During the period of hypersusceptibility in man the
subcutaneous administration of serum rarely if ever produces death,
in spite of the fact that the clinical symptoms may be extremely severe.
On the other hand, intravenous injections have been reported to produce
death following symptoms closely resembling those of anaphylactic
shock in animals. Reports of accidents of this sort led to the funda-
mental investigations of Rosenau and Anderson, which have been
described. Injections of serum into the spinal canal have been followed
by fatalities, but an analysis by Auer of the reported cases leads him
to believe that for the most part these deaths were due to other causes
than anaphylaxis. Miller and Root, in analysis of death following
subcutaneous administration of horse serum, find that death in some
instances was probably caused by status thymo-lymphaticus and that in
other cases the cause of death had not been demonstrated to be ana-
phylactic. The clinical and pathological picture of fatalities has in most
instances not been clearly described. Nevertheless, Boughton has
recently reported a case in which a man, the subject of bronchial
asthma when near horses, died upon being given intravenously one
minim of normal horse serum,. Autopsy showed enormous distention
of the lungs with congestion of other viscera and numerous small hemor-
rhages. This apparently is an instance of true anaphylactic shock in
man, and it cannot be doubted that such accidents occur. Caution
must be exercised, however, in attributing death to anaphylaxis because
of the numerous other conditions which may lead to sudden death,
particularly in the course of acute infectious diseases.

Natural Hypersusceptibility. — The recent scientific investigations
of hay fever and its various modifications, as well as asthma, eczema,
other diseases of the skin, angio-neurotic edema and certain gastro-
intestinal disturbances, have shown that a considerable number of these
cases are hypersusceptible to proteins of various origins. The skin
reactions, to be described subsequently, and the effect of specific treat-
ment both demonstrate the etiological influence of the special proteins.
The evidence presented from large clinics devoted to the study of these
conditions leaves no doubt concerning the fact that many of these cases

HYPERSUSCEPTIBILITY IN MAN 231

are instances of hypersusceptibility. The sensitive state appears to be
inherent in the cells of certain individuals, and although not directly
inherited, Cooke and Van der Veer have found that the tendency to
spontaneous sensitization appears to be heritable, that it follows the
law of Mendel and appears as a dominant character. Nevertheless,
there is a possibility that sensitization may be acquired in some manner.
Cooke, Flood and Coca maintain that artificial sensitization cannot be
produced by pollens. Heyl, however, has obtained from the pollen of
ragweed an albumin, a proteose and a globulin and found that mixtures
of the albumin and proteose possess definite sensitizing properties upon
animal inoculation. Individuals who have been given horse serum
therapeutically become somewhat sensitive to subsequent injections of
horse serum, but only in rare instances is the sensitiveness shown as a
coryza or asthma when near horses. The chance of sensitization by in-
jection of other proteins is not great. The possibility of sensitization
by virtue of the material gaining access to the body through the respira-
tory or intestinal surfaces is apparently remote. There is little satis-
factory evidence that protein materials in the form of dust gain access
to the circulation through the respiratory membrane. Ulrich, however,
reports the experimental sensitization of guinea-pigs by nasal insuffla-
tion of pollens and of horse serum, but reports that rabbits cannot be
so sensitized. It is impossible, under these circumstances, to exclude
the possibility that the material is ultimately swallowed, and sensitiza-
tion effected through the intestinal tract. The ingestion of proteins as
foods ordinarily leads to such changes in the protein in the process of
digestion that the absorption of the products cannot produce sensitiza-
tion. On the other hand, it is known that if given in large amounts and
given under certain circumstances native protein may gain access to
the blood stream through the intestinal tract. Rosenau and Anderson
maintained that sensitization could be effected by feeding horse serum
to guinea-pigs, but the failure of Besredka, as well as of other investi-
gators, to confirm this leaves the matter in some doubt. As against the
acquisition of hypersusceptibility in hay fever, Dunbar and also Cooke,
Flood and Coca have found that patients may be sensitive to the pollens
of plants indigenous to foreign countries and with which the patients
have never come in contact.

Hay fever, rose fever and similar disturbances are due to the pol-
lens of certain plants and the flowering period of these plants deter-
mines the seasonal prevalence of the disease. The pollens responsible are
those which are disseminated by winds ; those plants which are pollin-
ated by insects do not produce hay fever. Scheppegrill points out
also that the direct effects of pollens are. of importance as they may
be locally irritant to both normal and hypersusceptible individuals,
either because of the mechanical effect of spiculated pollens or because
of the discharge from the pollen of irritant juices. Local reactions may
be increased by anatomical malformations in the nose and pharynx, such
as deviation of the septum, polyps, adenoids, and the condition may
entirely subside following correction of these abnormalities. Strouse

232 THE PRINCIPLES OF IMMUNOLOGY

and Frank claim that the attacks may be intensified and prolonged
because of a concurrent acute or sub-acute bacterial infection, which
perhaps permits greater absorption of the pollen protein. The condition
may be so severe as to be called asthma, and in addition to respiratory
phenomena may show erythematous and urticarial eruptions. Similar
conditions are met with in certain individuals sensitive to the effluvia of
horses, rabbits and other animals. It is well known that the ingestion
of certain foods, such as egg albumin, shell fish, strawberries, may give
rise to serious intestinal disturbances and that these may occasionally be
associated with skin eruptions or respiratory disturbance. In sensitive
individuals contact of the skin with plants or animals, to the protein of
which the individual may be sensitive, leads not uncommonly to cutane-
ous eruptions. These, however, are not likely to be very severe or of
long duration. The inflammation of the skin in ivy or sumac poisoning
is not to be included in this group, because the irritant agent is prob-
ably not of protein nature, but rather an acid-resin. Eczema and per-
haps certain other skin diseases may also be due to hypersusceptibility,
and it is found that this is exhibited rather toward food products than
toward other forms of protein. Furthermore, certain of these cases
of asthma, eczema, etc., may be due to bacterial proteins as well as
those of higher plants and of animals.

The hypersusceptibility of the sort discussed in this section differs
from induced hypersusceptibility in two important respects. In the
first place, the degree of sensitiveness is extreme. This may be illus-
trated by the case reported by Boughton, quoted above, in which one
minim of horse serum produced death. It is further illustrated by the
fact that hay fever, asthma and other similar conditions are induced
by what must necessarily be an extremely small amount of protein
in the atmosphere. In the second place, the sensitization is not limited
strictly to a single protein. Longcope classifies these individuals roughly
as those " who react to the sera of animals ; those who react to eggs,
or the sera of fowls ; those who react to the extracts of shell fish and
those who react to the protein of plants." Within each group the
individual may be sensitive to the protein of several species. As has
been pointed out by Walker, those who react to bacteria frequently
react to several varieties of organisms. Furthermore, individuals may
occasionally show reactions to two or three of the large groups indicated
by Longcope. Of further interest in regard to specificity is the fact
that apparently within a given species, proteins of somewhat different
origin may not produce identical reactions. For example, skin icac-
tions may demonstrate sensitiveness to horse dandruff and not to horse
serum. Desensitization may be produced by careful and prolonged
vaccination, but as in experimental animals the desensitized state does
not persist for a very extended period, it may be necessary to repeat
the vaccinations every six months, every year, or at such other periods
as the individual case requires. The possibility of passive sensitizition
in natural hypersusceptibility is illustrated by a case reported by
Ramirez. A man who had never shown any hypersensitiveness to proteins

HYPERSUSCEPTIBILITY IN MAN 233

was transfused with the blood of a donor who was a victim of horse
asthma. The recipient, two weeks later, while driving in a carriage,
was seized with a typical asthmatic attack and subsequently showed a
positive skin reaction to horse dandruff. This apparently is a case of
passive transfer of a natural hypersusceptibility. No data have been
collected to show whether such a variety of passive sensitization is per-
manent in man or exhibits the same evanescent character as occurs in
animals. Passive sensitization of animals has been produced by Koess-
ler, but Cooke, Flood and Coca, as well as Ulrich, have been unable
to confirm this. Our own experience with one case of human hyper-
susceptibility to rabbit serum failed to demonstrate passive transfer
into guinea-pigs.

Tests for Hypersusceptibility. — The manifestations of hyper-
susceptibility may be general or lo'cal, depending on the mode of in-
oculation and the amount of material employed. If the dose can be
carefully regulated, the hypersusceptible state may be demonstrated by
inducing a general reaction, as in the tuberculin reaction. Owing to the
fact that individuals may be extremely sensitive to certain proteins, as
in the case reported by Boughton (see page 230), the use of the general
reactions for diagnostic purposes is limited to those in which severe
general reactions are not likely to appear. The local reactions give
equally satisfactory information in man and are devoid of serious
results. These local reactions are based fundamentally upon the studies
of Arthus, published in 1903. He found that if animals are given
several subcutaneous injections of normal horse serum at three- or
four-day intervals, the first three injections are absorbed readily, but
the fourth is followed by a local inflammatory reaction and subsequent
injections are likely to be followed by more severe inflammation, necrosis
and gangrene. Animals rendered sensitive by these first two or three
injections could be killed by intravenous or intraperitoneal injections.
The Arthus phenomenon was early employed as a means of detecting
hypersusceptibility resulting from bacterial invasion, but it has now
found widespread employment in the detection not only of the presence
of changes incident to infectious disease but also for the determination
of sensitiveness to a large number of proteins of animal and vegetable
origin. Although hypersusceptibility may exhibit itself in respiratory
phenomena as in hay fever, horse asthma, etc., or in gastro-intestinal
disturbance, as in sensitiveness to egg-white, definite local reactions
may be evoked by the introduction of the proteins into or under the
skki and these local reactions may be accompanied by general symp-
toms, such as fever, headache, malaise and transitory leucopenia fol-
lowed by slight leucocytosis with an associated esinophilia.

Toxins in Hay Fever. — The studies of Dunbar assumed that the irri-
tant agent in pollens is a toxin. He based this conclusion on the fact that
he could prepare a so-called antitoxic serum " pollantin " by immunizing
anjrnals and subsequently claimed that he could demonstrate antibodies
by precipitin and complement-fixation tests. Clowes found positive
precipitation and complement fixation in some but not all cases before

234 THE PRINCIPLES OF IMMUNOLOGY

the beginning of the hay-fever season, which disappeared for a few
weeks after specific desensitization. On the other hand, numerous
other investigators have failed to demonstrate such reactions, and this
phase of the question must be considered unsettled. Dunbar claimed
that treatment with the anti serum " pollantin " produced specific effects,
but Weichardt maintains that equally good results are obtained with
the serum of normal animals taken in the summer season. Cooke,
Flood and Coca were unable to demonstrate immune reactions in the
sera of rabbits inoculated repeatedly with the pollens of ragweed and
of redtop. Other objections to the toxin conception include the fact
that the majority of normal individuals are, practically speaking, abso-
lutely resistant to the pollens and fail to react to doses 1000 times the
dose which produces reactions in susceptible cases. This is not in
accord with the finding in regard to any other of the known toxins.
Apparently normal individuals may resist diphtheria toxin, but Cooke
and Van der Veer have pointed out that such resistance depends upon
the presence of demonstrable antitoxin, which is not true in resistance
to pollens. By mixing the " pollantin " and pollens and then testing
by an ophthalmic reaction in sensitive individuals Dunbar's assistant,
Prausnitz, plotted a curve of neutralization, but Wolff-Eisner found
that this curve does not follow the law of multiple proportions and is
therefore not similar to other toxin-antitoxin combinations. There
seems, therefore, little ground for assuming that the pollens contain
a special toxin and the subsequent work with hay fever and similar
conditions indicates that they represent a condition of hypersuscepti-
bility to proteins or to protein decomposition products.

Technic of Cutaneous Tests. — If the antigenic proteins are already in
solution, as is the case with blood serum, no especial treatment is required
other than suitable dilution under strictly aseptic precautions. If the protein
is in solid form, as in the case of vegetable proteins and other cellular forms,
extracts are required. The studies of Walker and of Wodehouse on the
preparation of materials for the tests have been of the utmost importance.
These are independent of the preparation of the various tuberculins, which
will be discussed subsequently. They found that an excellent dried prepara-
tion of serum could be obtained by precipitating with several volumes of
acetone, washing the precipitate centrifugally twice with alcohol and with
ether, and drying to a powder. The powder may be applied to an incision in
the skin and dissolved with N/io NaOH solution. Bacteria are cultivated on
solid media, washed centrifugally in salt solution, then twice in absolute alcohol
with 0.5 per cent, phenol added, twice in ether and then dried to a powder, which
may be used as is the serum powder. Cereals, nuts and other seeds, roots
and tubers, fruits, leaves and stems are extracted in water, precipitated with
95 per cent, alcohol, washed with 95 per cent, alcohol, absolute alcohol, ether
and desiccated over hydrochloric acid. Hair and dandruff of animals may
be employed as a dissolved extract in 14 per cent, alcohol, but for more
accurate studies, dried preparations of acid metaprotein, alkali metaprotein
and pepton extracted from the material are employed.

The methods of inoculation include introduction of the protein into
abraded surfaces and intracutaneous injection through a fine needle. In
special instances, as, for example, in the use of tuberculin, the material may
be incorporated in an ointment and carefully rubbed into the skin; this is
the so-called percutaneous test. Somewhat similar to the cutaneous tests is
the ophthalmo-reaction, more particularly applied in tuberculin tests, where
the material is instilled into the opnjunctival sac. Subcutaneous injection of
material is also resorted to, again with tuberculin rather than with other

HYPERSUSCEPTIBILITY IN MAN 235

substances, but the determination of results is by means of the general
rather than the local reaction. As with other reactions, controls are a neces-
sary part of these tests. The cutaneous test, by which is meant introduction
of material into an abrasion, is performed as in smallpox vaccination. Any
part of the body may be selected, but we have found the arm most con-
venient. Walker advises making small incisions in the skin, deep enough to
permit absorption, but not deep enough to cause bleeding. A small dental
burr may be used, as in the Schick test. The material is placed on the
abrasion or incision and allowed to remain one-half hour. If a powder, a
solvent should be added after the powder is placed on the skin. If not com-
pletely soluble in water, a weak solution of sodium hydroxide, either o.i per
cent, or N/io may be employed, as it does not affect the reaction. Walker's
studies show that for detecting hypersensitiveness in cases of asthma, hay
fever, etc., the cutaneous test is more delicate and yields fewer false positive
reactions than the intracutaneous test.

The delicacy of these tests is probably greater than that of any
other biological reaction. As has been stated, patients sensitive to
extracts of hair of an animal may not be sensitive to the serum proteins
and vice versa. Very small amounts of antigen suffice to produce reac-
tions; alkali metaprotein and pepton from hair and dandruff give
reactions commonly in dilutions of I— 10,000 and Wodehouse reports
one case in which reactions were obtained with dilutions of 1-1,000,000.
Clowes reports reaction by means of the ophthalmic test to 0.000,000,05
gram pollen. The fact that positive reactions are found with cutaneous
tests in individuals whose serum fails to exhibit antibodies by pre-
cipitation, agglutination and complement-fixation tests, is a further
indication of the delicacy of the reaction. The accuracy of the reac-
tions is supported by the .beneficial results of specific vaccination or
desensitization. The treatment is usually by means of subcutaneous
injections of the protein to which the patient is sensitive. In cases of
sensitiveness to food products, as well as in other cases, patients may
be vaccinated by giving the protein by mouth. In either method the
amounts are extremely small, and in most instances the course of treat-
ment must be repeated at intervals which may vary from a few months
to a year or more. The intracutaneous test appears to be the most
delicate in producing local reactions, but unfortunately is more likely
to produce confusing traumatic and non-specific reactions to be de-
scribed subsequently. Details of treatment are given in numerous
articles, such as those of Blackfan, Talbot, Goodale, Berger and others
in the recent literature.

The Reaction. — This depends to a certain degree upon the particu-
lar cutaneous test employed and the sensitiveness of the patient, but in
a general way the description applies to all the methods. An urticarial
wheal may appear within a very few minutes and may persist for from
several minutes to several hours, elevated, firm, pale and itching. Either
with or without this preliminary reaction, the passage of a few hours,
six, twelve, twenty-four or more, reveals a local area of inflammation
about 10 m.m. in diameter, elevated, papular, red, firm and tender. In
severe reactions the area may reach a diameter of several centimetres,
may be surrounded by an areola of subcutaneous edema, may show
fine punctate hemorrhages and may ultimately show vesicles and

236 THE PRINCIPLES OF IMMUNOLOGY

crusts. In unusually sensitive individuals the local reaction may be
accompanied by systemic manifestations. Less severe but sometimes
confusing reactions may appear in the form of pseudo-reactions
which are non-specific in nature and probably due to the
action of body proteases upon introduced proteins. The reaction
to the traumatism from the introduction of the protein may at times
be somewhat confusing but in most instances is slight. Certain drugs,
such as iodides and bromides, appear to increase the intensity of reac-
tions whether they be specific or non-specific. Iodides are known to
reduce the antiferment titer of the blood, and it is possible that the
use of these drugs therefore liberates protease and in this way acceler-
ates the non-specific local reaction. The increase of the specific local
reactions is probably due to the increase of the non-specific interaction
of protease and the introduced protein.

Theories of Cutaneous Reactions. — The appearance of local reac-
tions in hypersusceptibility may be explained according to any of the
theories offered for anaphylactic shock. If the mechanism of ana-
phylaxis involves the formation of poisons these may be concentrated
in situ because of the irritation produced by introducing the antigen.
The irritation leads to a slight local inflammation with its incident vaso-
dilatation and edema. Thus there is a local concentration of antibody,
which in reaction with the introduced antigen produces a hypothetical
poisonous substance. If the sensitizing substance is within cells, the
local contact of antigen in the tissues of the skin explains the local
reaction. Similarly the physical theories are adaptable. Stokes, for
example, has found that agar will produce a local non-specific reaction.
This is probably the result in part of a local loss of balance between
ferment and antiferment due to adsorption of the latter by the agar.
Similarly any of the physical theories might apply, but the acceptance of
the importance of the cells in the reaction, whether physical or other-
wise, offers an excellent reason for the early appearance and severity
of the local reaction without general manifestations. Cooke, Flood and
Coca state that antibodies are not demonstrable in the blood of naturally
sensitive persons and therefore emphasize the essential importance of
the cells. While agreeing that the cells play a most important part,
the experiments of Koessler and the case reported by Ramirez suggest
that natural sensitization is of essentially the same nature as anaphylaxis,
with marked differences only in the degree of cellular and humoral
participation. Therapeutic desensitization of man lasts for a relatively
short period of time and differs only in duration from desensitization
in experimental animals. In both cases the phenomenon is specific
for the antigen employed.

Gay and Force, Gay and Claypole, and Gay and Minaker, in their
work with cutaneous reactions in typhoid fever and in the carrier state
in meningococcus infections, have expressed the opinion that positive
reactions are an indication of resistance on the part of the body against
infection by the organisms concerned. Nichols studied the typhoidin
test (see page 242) in individuals who had survived typhoid fever and

HYPERSUSCEPTIBILITY IN MAN 237

found that only 75 per cent, of these reacted positively, whereas experi-
ence has shown that at least 90 per cent, of such individuals are
immune to reinfection. He also pointed out that the immune state
following an attack is of much greater duration than is indicated by the
typhoidin test. Furthermore, those who have survived typhoid fever or
have been vaccinated with bacillus typhosus react positively to para-
typhoidin, but it is known that these individuals are not immune to
paratyphoid fever. Kolmer and his associates have found no con-
stant parallelism between the presence of positive cutaneous tests
and those circulating antibodies, whose presence is indicative of im-
munity. " The positive anaphylactic skin reaction is, therefore, evidence
of infection or sensitization to a particular protein without bearing any
direct relation to resistance to infection or reinfection."

Drug Idiosyncrasies. — It is well known in connection with certain
drugs, such as morphin, that prolonged use makes it necessary to in-
crease the dose in order to obtain physiological effects. This increase in
resistance to morphin is specific, but in the case of chronic alcoholism
the individual's resistance to somewhat related substances, such as
chloroform and ether, is also increased. Experiment fails to show that
this resistance is a state of immunity, and no immune reactions
in the ordinary sense of the term have been demonstrated. The use
of certain drugs, such as iodof orm, iodides, bromides, coal-tar products
and quinine, sometimes gives evidence on the part of the patient of
a special susceptibility or idosyncrasy in the form of cutaneous erup-
tions and more or less severe general symptoms. Both Bruck and
Klausner expressed the view that this is an evidence of hypersuscepti-
bility similar to or identical with anaphylaxis. Inasmuch as anaphylaxis
is a phenomenon concerning proteins, Bruck offered the hypothesis
that the drugs enter into combination with the body proteins, so that
a new drug-protein complex of specific character is formed. This pro-
tein complex may act as a sensitizer, and upon subsequent injection of
the drug there occurs a combination with blood proteins to produce
a similar complex which reacts with the sensitizer to produce symptoms.
Bruck and Klausner claimed to be able to sensitize guinea-pigs passively
with the blood of susceptible patients, so that the animals reacted with
the symptoms of anaphylaxis. The autopsies on these animals failed
to show the characteristic findings of anaphylactic shock. Cole studied
patients sensitive to iodides and to copaiba but failed to obtain results
justifying the conclusion that the phenomenon should be included among
anaphylactic manifestations. Specific cutaneous reactions to such drugs
as quinine and aspirin have been described, and it is maintained that
small doses by mouth may desensitize, but no widespread confirmation
has been recorded. None of the drugs studied is without some essential
toxicity and the idiosyncrasies in some instances, according to Sollmann,
" are doubtless due to differences in the strength or constituents of
drugs." He further states in regard to increased susceptibility that it
" may be due to very rapid absorption, or slow elimination ; to the

238 THE PRINCIPLES OF IMMUNOLOGY

presence of synergistic substances in the body; or to increased func-
tional susceptibility."

The Tuberculin Test. — In the course of his studies on the treatment
of tuberculosis, Koch devised the method of diagnosis which we now
speak of as the general tuberculin reaction, in contrast to the local
reactions subsequently discovered. It is now known that the introduc-
tion of tuberculin into the body may lead to local reactions both at the
site of inoculation and in the neighborhood of a tuberculous focus as
well as a general reaction which manifests itself in fever, headache
and malaise. Numerous methods of preparation of tuberculin for
therapeutic and diagnostic purposes have been described, but at the
present time the diagnostic methods, in' the hands of the majority of
workers, depend upon the use of original or old tuberculin of Koch.

For the preparation of this tuberculin now designated as tuberculin O. T.
the organisms are grown for six to eight weeks on the surface of 5 per cent,
alkaline glycerine broth at 37° C. At the end of this time the entire contents
of the flask are sterilized and concentrated to about one-tenth of the original
volume by means of a current of live steam and a water bath. The glycerine
does not evaporate, and as a result of the concentration constitutes 50 per cent,
of the final mass. This is filtered through porcelain and the filtrate employed.
Koch subsequently made other preparations, particularly the tuberculin
known as T. R. and that known as B. E. The T. R. or tuberculin residue is
prepared by growing virulent tubercle bacilli on nutrient glycerine broth for
four to six weeks at 37° C. The bacilli are obtained by filtration, dried, and
ground in a mortar. One gram is washed with 100 c.c. distilled water, the
precipitate is again dried, powdered and repeatedly washed in small volumes
of water until no sediment results. The watery extract constituted by this
second series of washings, which should not exceed 100 c.c., is preserved with
20 per cent, of glycerine and constitutes the T. R. The bacillus emulsion
(B. E.) is prepared by growing the organisms as indicated in the preparation
of the original or old tuberculin. The bacilli are obtained by filtration, ground
in a mortar and emulsified in 100 parts of distilled water to which is added an
equal amount of glycerine.

Numerous other methods of preparing extracts of the tubercle
bacillus have been described but are, in essential, modifications of
the methods of Koch. At the present time the original or old tuber-
culin is used most widely.

The General Reaction. — As a general rule, the old tuberculin is put on
the market in the form of ampoules of fluid, i.o c.c. of which represents i.o
gram of pure tuberculin. This may be diluted for the actual test. Inasmuch
as individual sensitiveness varies considerably, the primary dose should be
very small. According to Hamman and Wolman, three classes of patients
may be recognized, (a) children, (b) patients who have a slight fever or are
not in good general condition, (c) patients in good condition. The smaller
doses are given to children and the largest dose to patients in good general
condition. Upon this basis the initial dose of old tuberculin should be
0.000,000,1 c.c. to 0.000,001 c.c.; failing to obtain reactions with these doses,
subsequent tests may be made at intervals of about a week, increasing the
dose each time. Although it is possible to give a maximal dose of i.o c.c. of
the dilution, it is rarely advisable to exceed 0.05 c.c. The injections should
be given under strict aseptic precautions, and appear to be most satisfactory if
given at the lower angle of the scapula. They are probably best given in the
afternoon, after the patient's afternoon temperature has been taken, so as
to avoid the confusion of an unusually high elevation of temperature on the
day selected. The reaction appears as a rule in from twenty-four to thirty-
six hours. It may appear as late as forty-eight to sixty hours. A positive
reaction is indicated by an elevation of temperature of about 2° to 4° C. In

HYPERSUSCEPTIBILITY IN MAN 239

addition there is likely to be headache, malaise, and sometimes a loss of
weight. At the site of inoculation there may be pain, tenderness, redness,
swelling, sometimes associated with tenderness and enlargement of the
regional lymph nodes. The contraindications to the employment of the test
include the presence of fever, if fairly high and continued, nephritis, gen-
eralized miliary tuberculosis, intestinal ulceration, epilepsy, acute infectious
diseases, either during the course of the disease or its convalescence.

The Cutaneous Reaction. — Von Pirquet, who first described this modifi-
cation of the tuberculin test, originally recommended the use of 25 per cent,
solution of the old tuberculin, but subsequently found that the undiluted
material is more suitable. He recommends the inner (flexor) surface of the
forearm, and suggests the use of three points of scarification about 4 to 5 cm,
apart. The skin is cleaned with ether or alcohol before making the abrasions.
These may be small scratches with a needle, a knife or with an instrument
which he describes as a borer, which has a sharp chisel point and is rotated
in order to make a small circular abrasion. A drop of tuberculin is rubbed
into the upper and lower abrasions; the middle one remains as a control. In
positive cases, the reaction about the point of inoculation is considerably
greater than that about the control point. The traumatic reaction in the
control may reach a diameter of 3 to 5 mm. in twenty-four hours and then
rapidly disappears. The positive reaction usually appears within twenty-four
hours, but may be somewhat delayed. Its diameter is ordinarily about
10 mm., but may reach 30 mm. It appears as a red, somewhat tender papule,
which in severe reactions may show small vesicles. According to Kolmer, it
is not to be interpreted as positive unless its diameter exceeds by 5 mm. that
of the control. Occasionally the so-called scrofulous reaction appears, in
which papules develop upon other parts of the extremities and the trunk.

The Intracutaneous Tuberculin Tests. — This was described by Mendel
and also by Mantoux. For this purpose old tuberculin is injected into the
corium in doses whose bulk is 0.05 c.c. Two injections are necessary, one
with salt solution and the other with tuberculin. The injection of tuberculin,
however, may include three doses of different strengths. The reaction is
very similar to that of the cutaneous test. Following a subcutaneous tuber-
culin test, a similar reaction may appear in the track of the needle.

The Percutaneous Tuberculin Test. — This test was devised by Moro and
Doganoff and is frequently spoken of as the Moro skin test. For this pur-
pose 5.0 c.c. of old tuberculin are thoroughly mixed with 5 grams of anhydrous
lanolin. This may be preserved for a long time in a light-proof container in
the refrigerator and may be obtained on the market in collapsible tubes.
About 0.5 gram of this ointment is rubbed into the skin of the abdomen, or
breast near the nipple, rather vigorously for one minute. The reaction usually
appears within twenty-four hours, but may be delayed from four to six days,
and it subsides in three to ten days. It usually appears as a number of small
papules reddened at the base. In severe cases the papules may become con-
fluent and vesicles may form.

The Conjunctival Tuberculin Reaction. — This is also referred to as the
ophthalmo-reaction and was described independently by Calmette and Wolff-
Eisner. Calmette recommended a special aqueous extract of the bacilli but
at the present time the test is usually applied with a I per cent, solution of
old tuberculin. One drop of this solution is instilled into the conjunctiva
near the inner canthus. The opposite eye serves as a control. Even in
normal individuals the instillation may induce a slight reddening of the
conjunctiva within six hours, but the positive reaction appears in from six to
eight hours, reaches its height in from twenty-four to forty-eight hours, and
then subsides in a few days or a week. The reaction may include simply a
slight reddening and swelling of the caruncle, including the neighboring
part of the lower lid, may extend over the scleral conjunctiva or may lead
to a purulent conjunctivitis. Following the introduction of this test, unfavor-
able reports were made because of the seeming danger of producing perma-
nent injury to the eye, but Hamman and Wolman state that this danger is
not considerable, provided proper precautions are taken in the selection of
patients. Diseases of the eye or of the skin near the eye, obvious scrofula in
children, and arterio-sclerosis are contraindications. The test should never
be applied twice in the same eye, and no stronger solution than I per cent,
should be employed for the first test. If the first test is negative a 5 per cent.
solution may subsequently be employed in the opposite eye.

24o THE PRINCIPLES OF IMMUNOLOGY

Theories of the Tuberculin Reaction. — Koch is of the opinion that
the amount of tuberculin introduced when added to that already present
in the body provides a .sufficient amount of toxic substance to produce
a definite general reaction. Koehler and Westphal thought that a
toxic body is formed in the tuberculous focus by the union of tuberculin
and the products of the tubercle bacillus. Marmorek suggested that
the tuberculin excited the tubercle bacilli to produce in excess those toxic
bodies which lead to fever. Von Pirquet and Schick were the first to
suggest that this is a phenomenon related to hypersusceptibility. This
conception fits very well the view of the relation of anaphylaxis and
immunity which we have indicated above (page 226). Individuals who
have markedly active tuberculosis are not likely to react, whereas those
who have quiescent or cicatrized lesions almost always react. If the
presence of tuberculosis leads to the formation of a sensitizing sub-
stance, this can well be absorbed by the cells and be responsible for the
local and general reactions. The tuberculin, upon local application,
may react with a sensitizing substance in the situation concerned, or
upon entrance into the circulation may similarly react with the sensitiz-
ing substance in more widely distributed cells, thus producing a general
reaction similar in principle to anaphylactic shock. If, on the other
hand, the tuberculous process is so active that immune bodies can be
found in the circulating blood, combination may be effected in that situ-
ation and the cells protected. The study of complement fixation in
tuberculosis indicates that this latter assumption is true, namely, that
those who have active tuberculosis are more likely to react positively
by the complement-fixation test, thereby indicating the presence of cir-
culating antibodies in the active stages of the disease.

Krause has studied this problem extensively, particularly in experi-
mental animals and finds no reason for associating skin hypersensitive-
ness and anaphylaxis. The anaphylactic state may be induced in
animals by parenteral injection of tuberculo-protein, but they do not
acquire cutaneous hypersensitiveness. Only by establishing a focus
of infection, is it possible to demonstrate a skin reaction. Although
during the period of anaphylactic shock an animal may appear to be
somewhat less resistant to infection, the state of anaphylaxis produces
no alteration in its resistance. Krause is of the opinion that tissue and
cutaneous hypersensitiveness' and immunity to infection occur under
the same conditions, and that one may probably be a function of the
other. In the experimental animal the degree of cutaneous hyper-
susceptibility and immunity parallel each other. He suggests that the
local reaction may also appear in the neighborhood of foci of infection
and thus aid in walling off the infecting agent. Krause's opinion,
based on much admirable work, is worthy of the highest consideration,
but in so far as we can determine, it is not in accord with studies of
immunity and cutaneous reactions in many other conditions, as pointed
out in our discussion of cutaneous hypersusceptibility in general. Peter-
sen considers the tuberculin reaction as a two-phase phenomenon. The
primary alteration of the ferment-antiferment balance brings about a

HYPERSUSCEPTIBILITY IN MAN 241

medium favorable for proteolysis in and about the tubercle. Digestion
and the liberation of toxic material result and are reflected in the con-
stitutional effects. In the non-tuberculous individual it is probable
that the primary serum alterations also occur, but the digestive ferments,
finding no focus to attack, liberate no toxic material and no general
reaction is elicited. Any agent that brings about a f erment-antif erment
ratio favorable for proteolysis will effect a general reaction provided
the focus be sufficiently unstable. Conditions such as pregnancy, acute
infections, protein shock, in which there is an increase of antif erment,
will inhibit the reaction. In late stages of tuberculosis there is also
increased antiferment and therefore less marked local reactions but
more marked general reactions.

Specificity of the Tuberculin Reaction. — The tuberculin tests have
probably been more carefully controlled by autopsy than any of the
other clinical tests, and we therefore are able to state with considerable
assurance that a positive reaction indicates the presence of tuberculosis
in the vast majority of cases, but, on the other hand, gives no very
precise information as to the degree of activity of the process. Factors
of error are more particularly found in the personal equation of the
examiner. Leprosy and actinomycosis, however, may give confusing
results. In a very large series of tests, more than 15,000, the percentage
of error is very small, varying from 2 to 3 per cent. Negative reactions
may appear in markedly active tuberculosis, in the very early stages of
the infection, in those small cicatrized lesions of the lung so firmly
encapsulated that no absorption takes place, during continued treatment
with tuberculin ; also during the course of measles, typhoid fever,
acute articular rheumatism, pneumonia, diphtheria, pertussis, serum
disease and during pregnancy.

Some authors have such confidence in the specificity of the tuber-
culin reaction that they consider it possible to determine the strain of
the organism concerned, but others deny that this delicacy is attainable.
The recent work of Petersen would indicate that there is a large non-
specific element in the tuberculin reaction. Tuberculous patients may
react to the following substances with local and even general reactions :
hypertonic salt solution, distilled water, iodides, some colloidal metals,
protein split products, etc. Non-tuberculous individuals will tolerate
equal doses without reaction. The relation of this type of reaction to
the true test has been indicated in discussion of the theories of the
tuberculin test.

Utility of the Tests. — At the present time in clinical practice the
subcutaneous or general reaction is not very widely employed, because
of the prejudice that has been aroused by the possibility of exciting the
lesion to renewed activity. Similarly a prejudice exists somewhat
unjustly against the use of the conjunctival reaction. Although Ham-
man and Wolman indicate that the intracutaneous test is the most sensi-
tive, our observation is to the effect that the cutaneous test is most
widely employed. It is simple, free from danger, well controlled,
16

242 THE PRINCIPLES OF IMMUNOLOGY

easily read and is sufficiently sensitive to provide all the information that
can reasonably be expected to accrue from the tuberculin test.

Luetin Reaction. — Numerous attempts were made following the
announcements of the Von Pirquet cutaneous tuberculin test, to devise
a similar test for syphilis. It was found, however, that extracts of
normal organs produced the same effects as those from syphilitic organs.
It was not until Noguchi cultivated the treponema pallidum in -vitro that
a preparation of the causative agent could be prepared. Noguchi pre-
pared a suspension of the organisms together with the ascites-kidney
agar upon which they were grown. Cutaneous reactions were unsat-
isfactory, and it was found necessary to make the test by intracutaneous
injection of the material. The reaction appears in papular or pustular
form in from twenty-four to forty-eight hours or later. It was found
by Sherrick that patients receiving potassium iodide give positive reac-
tions and by Cole and Paryzek that similar reactions follow the ad-
ministration of bromides. Although Noguchi found that injection of
the culture medium without the organisms did not produce reactions,
Stokes as well as Kolmer, Matsunami and Broadwell were able to pro-
duce reactions by injecting agar. Although Noguchi and others re-
ported high percentages of positive reactions in known syphilitics, yet
in the hands of some workers the number has been only about 50 per
cent. The test is not widely employed and apparently gives no informa-
tion that cannot be obtained equally well or better from the Wasser-
mann test. It has been suggested that the luetin test may be of value
in late syphilis, where the Wassermann test is negative, but the large
non-specific element of this skin reaction does not tend to place much
reliance upon the test

Cutaneous Reactions in Typhoid Fever. — Several of the earlier
studies on this subject were concerned with reactions in the conjunctiva.
Chatemesse and also Austrian were able to obtain positive ophthalmo-
reactions in a large percentage of cases of typhoid fever and prac-
tically no positive reactions in other individuals. Although Kraus could
not obtain skin reactions, Zupnik and also Floyd and Barker obtained
encouraging results. Gay and Force have employed a substance which
they name typhoidin, prepared from bacillus typhosus, according to the
method employed for the preparation of old tuberculin. The prepara-
tion was modified subsequently by Gay and Claypole. The typhoidin
is applied in abrasions of the skin as with the cutaneous tuberculin test.
These investigators found a high percentage of positive reactions in
individuals who had recovered from typhoid fever as well as those
who had been vaccinated and recommend it as a method for determining
the presence of immunity to typhoid fever. Kilgore has studied the
test clinically and finds that the test is unreliable because of unavoidable
variations in the application of the test, indefiniteness of the readings
and the large non-specific element in the reaction.

Cutaneous Reactions to Gonococcus Infections. — These reactions
are particularly applicable to deep-seated and chronic infections with
the gonococcus. Irons found local and general reactions following the

HYPERSUSCEPTIBILITY IN MAN 243

subcutaneous injection of gonococcal vaccine and subsequently pre-
pared a glycerol extract of the organism for cutaneous tests. He
instituted controls with equal quantities of glycerol and obtained dis-
tinctly encouraging results even to the point where one strain of or-
ganism produced stronger reactions than other strains.

Cutaneous Reactions to Meningococcus Infections. — Recently Gay
and Minaker have employed the intracutaneous reaction for the detec-
tion of meningococcus carriers. They prepared a salt solution emulsion
of carefully washed and thoroughly dried cultures of five strains of
meningococcus and injected 0.000,006 gram of the dried powder in a
total volume of 0.05 c.c. They obtained reactions in 64.5 per cent, of
known carriers and 26.4 per cent, in individuals known not to be
carriers. They do not think that the reaction serves any important
purpose in diagnosis but suggest that it may indicate a systemic reaction
and possibly a certain degree of acquired resistance to the organism.

Cutaneous Reactions to Pneumococcus Infections. — Earlier in-
vestigations with salt-solution extracts were not particularly satis-
factory in regard to the early diagnosis of pneumonia, although after
the crisis reactions were obtained. Weiss and Kolmer prepared a
solution of Type I pneumococci in sodium choleate which they designate
pneumotoxin. The test is performed by intracutaneous injection. By
careful study of animals, on the basis of both gross and microscopic
examination of the site of reaction, as well as of human patients with
pneumonia, they obtained distinctly encouraging results during the
course of the disease and state that although the test does not seem
to be of distinct value in differentiating the type of organism con-
cerned, yet it may aid in differential diagnosis between appendicitis,
tuberculosis and pneumonia.

Cutaneous Reactions to Vaccine Virus. — Jenner noted that in cer-
tain individuals who had previously been vaccinated against smallpox,
a second vaccination might produce a local reaction which did not go on
to produce vaccinia. This has been observed by numerous investi-
gators since then and Force has given the subject close study. For
this purpose Force produced three abrasions on the arm, into two of
which vaccine virus was rubbed and made observations at the end of
twenty-four, forty-eight and seventy-two hours. " If either of the vac-
cinated spots showed an areola of 5 mm. or over (with or without
papule) at the end of twenty-four hours, which areola (or papule) had
decreased at the tim-e of the seventy-two-hour observation, it was con-
sidered a reaction of immunity due to the presence in the blood of the
individual of antibodies against vaccine virus." " If either of the vac-
cinated spots showed an areola at the end of twenty-four hours which
developed into a small vesicle, maturing on the fifth or sixth day and
then rapidly subsiding the reaction was considered a v&ccinoid" a con-
dition in which it is supposed antibodies are not present but are rapidly
formed because of a previous vaccination, thus leading to the small
size and rapid subsidence of the vesicle. " If there was no change until
the third day, and then a small areola began <to form, the case would be

244 THE PRINCIPLES OF IMMUNOLOGY

vaccinia" This description indicates the changes that may appear fol-
lowing an uninfected vaccination with smallpox virus. There appar-
ently occurs, following smallpox and vaccinia, an altered state which
determines these local reactions, but the interpretation offered by Force
that some of these reactions are immune reactions still lacks satis-
factory confirmation and is not consistent with other studies of
cutaneous reactions (see page 237).

Cutaneous Reactions in Glanders. — The Mallein test devised by
Kelmann and Kelming is widely employed in veterinary practice, either
in the form of subcutaneous injection which produces a general reaction
as is the case with tuberculin, or in the form of conjunctiva! test which
produces local and often general reactions.

Other Cutaneous Reactions. — As can very well be understood the
encouraging results with such a large number of skin reactions has
led to the investigation of similar tests in other diseases and the reac-
tion has been applied in leprosy, sporotrichosis, hyphomycetes infec-
tions, pregnancy, canine distemper and numerous other conditions.
The Schick test for diphtheria is not to be included among the skin
reactions indicating hypersusceptibility, for, as has been shown pre-
viously, this test depends upon the presence or absence of antitoxin in
the circulating fluids of the body.

CHAPTER XII
DEFENSIVE FERMENTS

INTRODUCTION.
SPECIFICITY OF FERMENTS.
IMMUNE FERMENTS.
FERMENTS IN THE BLOOD.

FERMENT-ANTIFERMENT BALANCE.

ANTIFERMENT.

THE ABDERHALDEN TEST.

Introduction. — The relation of ferments to immunity and ana-
phylaxis has long been the subject of discussion. In the chapters on
special immune bodies we have discussed the similarities and differences
between ferments or enzymes and antibodies. Special consideration has
been given to certain phases of ferment activity, particularly in the
chapter on Cellular Resistance and that on Hypersusceptibility. Ap-
parently the first work to prove that digestion takes place outside the
intestinal tract was that of Hammersten, who showed in 1885 that
washed leucocytes increase the solubility of fibrin. This was followed
by more comprehensive studies on cellular ferments as have been pre-
viously outlined (page 167). In addition to those ferments which exist
in the cells, ferments have been discovered in the blood and other circu-
lating body fluids. Therefore, we may classify the ferments as intra-
cellular and extracellular. The scope of this book is too limited to
permit of any general discussion of ferments as a group and the
reader is referred to the sections on this subject in Wells' " Chemical
Pathology." Many of the earlier workers assumed that ferments in
the body fluids are derived essentially from the leucocytes. A recent study
of considerable significance is that of Boldyreff. He maintains that the
glands of the alimentary canal, with the exception of those of the
3tomach, are not at rest between the digestive periods and that they
exhibit a periodic function. As a result of this periodicity, secretions
are discharged into the empty intestine from which they are absorbed
and at times are demonstrable in the blood. Van Calcar claims that
the leucocytes are incapable of producing their own ferments and that
these ferments are derived from special glands. He found that extir-
pation of the stomach is followed by a decrease or absence of that
ferment of the leucocytes which acts best in acid medium and that
extirpation of the pancreas similarly is followed by a loss of tryptic
powers on the part of the leucocytes. Abderhalden believes that invertin
also is derived from the intestinal glands. This conception would indi-
cate that the appearance of ferments in the circulating body fluids is to
be regarded as a mobilization of ferments from the cells which
formed them.

Specificity of Ferments. — It is well known that the body ferments
act specifically upon certain chemical substances, as exemplified by the

245

246 THE PRINCIPLES OF IMMUNOLOGY

digestion of starch by amylase and of protein by pepsin. The question
as to whether or not specificity in the immunological sense can be demon-
strated has been the subject of much discussion. Claims have been
made for specificity of ferments not only in regard to animal species
but also in regard to specificity for the cells of particular organs. The
chief proponent of the specificity of ferments for cells and proteins is
Abderhalden. He was stimulated to this view by the work of Schmorl
and others, who demonstrated that during pregnancy fragments of the
syncytium of the chorionic villi often enter the circulation and by the
claims of Weinland that specific reducing ferments are produced fol-
lowing the parenteral introduction of cane sugar. Abderhalden there-
upon examined the blood serum of pregnant animals and found that
the serum contained a ferment capable of splitting placental pepton
into amino-acids and of digesting coagulated placental tissue into pep-
ton, polypeptids and amino-acids. These decomposition products are
diffusible and also alter the axis of optical rotation of the mixture. The
detection of the diffusible products of protein decomposition was made
by means of " ninhydrin " or triketohydrindenhydrate, which reacts
with alpha amino-acids so as to produce a blue or violet color. The
practical application of a test of this sort is obvious and the method
has been employed to detect specific ferments in pregnancy, in car-
cinoma, in sarcoma, in diseases o/f the brain, of the eye and of numerous
other organs. Practical experience with the test, as well as further
scientific study, has made it seem probable that the specificity claimed
by the Abderhalden school does not exist. This will be further dis-
cussed in connection with the Abderhalden test.

Immune Ferments. — Numerous investigators have published re-
ports indicating that the parenteral introduction of special sub-
stances leads to production of special ferments or at least to an increase
of preexisting ferments in the form of a mobilization. Delezenne re-
ported in 1900 that the injection of animals with gelatine produces a
blood serum capable of liquefying gelatine. Weinland in 1907 showed
that although normal dog serum cannot reduce cane sugar the immuniza-
tion of a dog by several injections of cane sugar leads to the formation
of a ferment capable of reducing cane sugar in vitro. Similarly, im-
munization with edestin produces a serum capable of splitting this
substance. The more recent investigations of the subject would make
it appear that the immunization leads rather to mobilization of non-
specific ferments than to the production of a specific immune body.

Ferments in the Blood. — Wells states that the blood contains di-
astase, glucase, lipase, thrombin, rennin and proteases. In addition,
the blood possesses oxidizing properties due presumably to the presence
of oxydase, peroxydase and probably also due to catalase. The pro-
teases have been given particularly careful study. Petersen divides
these ferments into the leucoproteases, serum proteases and serum pep-
tidases. The leucoproteases include (a) an active ferment operating in
alkaline medium and capable of digesting native protein to the proteose
stage, (b) an active ferment capable of operating in acid medium with

DEFENSIVE FERMENTS 247

a similar range of activity, and (c) an ereptase active in both acid and
alkaline media and capable of splitting partially hydrolized proteins into
amino-acids. Of these ferments only the ereptase is able to act in the
presence of blood serum and tissue fluids because the others are inhibited
by the activity of antiferment constantly present. The serum protease
is a polyvalent trypsin-like ferment active in neutral, in slightly acid,
or in slightly alkaline media ; it is completely inhibited by the antifer-
ment of the circulating fluid. It becomes active only when the anti-
ferment is removed and is capable of digesting native protein to the
amino-acid stage. It is present in fairly large amounts in sera of the
lower animals, but is found in only small quantities in human serum.
The serum peptidase is a polyvalent ferment which operates in the
same type of media as the protease ; it is present in normal human serum
in small amounts, is not inhibited by antiferment and digests partly
hydrolized proteins to the amino-acid stage. Since the toxic fractions
of proteins are principally in the form of proteoses and pepton, the
peptidase apparently is the most important ferment in destroying such
toxic bodies.

The importance of esterases in the blood is not at all clear. It is
known that lymphocytes contain a lipase, and it has been suggested
that the accumulation- of these cells about tuberculous foci may indicate
the importance of this ferment in breaking down the waxy capsule of
the bacilli. Jobling, Petersen and Eggstein recommend the following
methods for the determination of serum protease and serum esterase
(Journal of Experimental Medicine, vol. xxii.).

" The technic for proteases is as follows : The clear hemoglobin-free serum
is measured with an accurate i.o c.c. pipette into a rather wide test-tube (about
18 mm.). To the tube 0.5 to 0.75 c.c. of chloroform is added and the tube is
sharply shaken, at intervals, until a milky emulsion is formed. We prefer chloro-
form because the emulsion is more stable than with ether or other lipoid solvents.
A control tube is inactivated at 60° C. for thirty minutes and a drop of toluol is
then added in place of chloroform. Both tubes are then incubated over night
(fifteen to sixteen hours at 37°). In the morning about i.o c.c. of a mixture
of 10 per cent, acetic acid plus 20 per cent, salt solution is added, and the tubes
are then gently warmed in a water bath until the chloroform has been evaporated.
About 2 or 3 c.c. of distilled water are then added slowly and the tubes boiled
for at least ten minutes. The coagulated protein is filtered off by means of
hard filter paper, previously moistened, the filtrate being permitted to filter
directly into the large tubes used for oxidizing. The tubes are then oxidized
and Nesslerized according to the usual Folin method, the readings being made
against varying dilutions of the I mg. standard, so that test readings are made
against standard of apparently equal color."

"Serum esterase has been determined as follows: To i.o c.c. of the serum,
i.o c.c. of neutral, redistilled 'ethyl butyrate and 0.5 c.c. toluol are added, the
volume being brought to 10.0 c.c. with physiological salt solution. The flasks
are then shaken 100 times and incubated for four hours ; 25 c.c. of neutral 95 per
cent, alcohol are then added to each flask and the acidity which has developed is
titrated with N/SO sodium hydrate (alcoholic) to a faint pink with phenolphtha-
lein. After deducting the proper controls, i.e., serum alone, ethyl butyrate alone,
etc., the esterase index is expressed in terms of c.c. of N/ioo sodium hydrate used to
neutralize the acidity developed by i.o c.c. of serum from i.o c.c. of ethyl butyrate."

Ferment-Antiferment Balance. — The activity of various ferments
in the body is probably effective in various degrees at all times, and
this activity probably plays a certain part in normal metabolism. Cer-

248 THE PRINCIPLES OF IMMUNOLOGY

tain of the ferments become active only when suitable material is pre-
sented, as is the case with the serum peptidase; others operate only
when the .surrounding medium reaches suitable reaction; still others
operate only if the antiferment content is sufficiently reduced. There-
fore, the preservation of the body tissues against destructive action of
'ferments and the normal processes of metabolism depend in considerable
part upon the neutralizing activity of antif erments.

Antiferment. — Certain investigators have reported the production
of specific antibodies following the injection of ferments. Morgen-
roth claimed to have produced a specific antirennin, Sachs and Achalme
an antipepsin and an antitrypsin, Schultze an antisteapsin and an
antilactase, Gessard an antityrosinase and Moll an antiurease. The
recent studies of antif erments, however, indicate that inhibitory activity
is not specific and this subject has been contributed to particularly by
Jobling and his collaborators. They are of the opinion that anti-
i erment activity depends upon the highly dispersed unsaturated lipoids
of the serum and lymph and that the titer varies with the amount of
lipoids, their dispersion and chemical structure. In studying anti-
trypsin, they found that the inhibitory substances are of the nature
of soaps and that the ability to inhibit ferment activity depends upon the
degree of unsaturation of the carbon bonds in the fatty acid. They
made soaps from olive oil, cod-liver oil, linseed and other oils and
found that these soaps inhibited the action of trypsin and leucoprotease.
They determined further that extraction of the blood serum with such
fat solvents as chloroform and ether removes the antitryptic activity.
Soaps prepared from the extracts restored the antitryptic activity. The
serum residue, after extraction, was found to be highly toxic for
guinea-pigs. If, however, the soap prepared from the extract were
added to the residue, the toxicity was neutralized. Jobling and his
collaborators attributed the toxic action of the serum residue to (a)
alteration of the mechanism of intravascular coagulation, (&) exposure
of native serum proteins to the action of ferments and (c) the resulting
formation of toxic split products. These workers isolated unsaturated
fatty acids from tubercle bacilli and found that when these were sapon-
ified they inhibited the action of trypsin but lost this power when satur-
ated with iodine. They were able to obtain similar soaps from caseous
lymph-nodes and suggest that the soaps prevent the activity of ferments
which would normally digest the necrotic material. This failure of
digestion leads to the formation of the partly-digested and fatty sub-
stance which is spoken of as the caseation necrosis.

The antif erments are greatly augmented in certain diseases, such as
acute infections, carcinoma, cachexias in general, anaphylactic shock,
certain degenerative changes of the nervous system and in pregnancy.
Jobling explains the crisis of pneumonia as being due to an alteration
in the ferment-antif erment balance ; that there is a decrease in the anti-
ferment with a corresponding mobilization of protease, an increase in
the serum lipase with a resulting decrease in the non-coagulable nitrogen
and proteoses of the serum.

DEFENSIVE FERMENTS 249

Determination of Antiferment in Blood Serum. — The determination of
antitrypsin by the Fuld-Gross method is satisfactory for this purpose. This
requires in addition to blood serum taken preferably in the morning before
the patient's breakfast, solutions of casein, acetic acid, and trypsin. The
solution of casein is made by dissolving i gram casein in 100 c.c. N/io NaOH
with the aid of slight heating; the solution is neutralized with N/io NaCl and
made up to 500 c.c. with 0.85 per cent. NaCl. The acetic acid solution is made
by mixing 5.0 c.c. acetic acid with 45.0 c.c. alcohol and 50.0 c.c. water. The
trypsin solution is made by dissolving 0.5 gram trypsin (Griibler) in 50.0 c.c.
0.85 per cent. NaCl and 0.5 c.c. normal soda solution; this is diluted ten times
with saline. The patient's serum must be fresh and should be diluted with
salt solution so as to make a 2 per cent, solution.

The trypsin is titrated as follows: Place in a series of test tubes o.i, 0.2,
0.4, 0.6, 0.8, and i.o c.c. of the trypsin solution. Add 2.0 c.c. of casein solution
to each tube, shake and incubate for one-half hour at 50° C. Add three or
four drops of the acetic acid solution to each tube and note the precipitation
(cloudiness) which appears in the course of a few minutes. The tube which
remains perfectly clear contains enough trypsin to digest 2.0 c.c. of the casein
solution. For testing the antitryptic content of the serum add 0.5 c.c. of the
2 per cent, solution of serum to each of six small test tubes. Then add to
each tube in series, increasing amounts of the trypsin solution, beginning with
the largest dose that completely digested the casein, and increasing in each
tube by o.i c.c. Add 2.0 c.c. of casein solution to each tube and make up to
equal volumes with normal saline. Shake and incubate for one-half hour at
50° C.; then add three or four drops of the acetic acid solution to each tube
and observe as before. The amount of trypsin which is inhibited by the
serum is determined by the lack of complete digestion, as shown by the acetic
acid precipitation. A control series should be set up with the pooled sera of
normal individuals. Jobling and his associates made the test somewhat more
accurate by filtering after the incubation and then determining quantitatively
the non-coagulable protein nitrogen.

The Abderhalden Test. — In the discussion of the specificity of fer-
ments, we pointed out that Abderhalden had assumed that the entry of
cellular and other proteins in the circulation could lead to the formation,
or increase, of ferments which have as their specific character the prop-
erty of digesting the antigenic protein. The test is based fundamentally
upon a mixture of serum and antigenic substance and the determina-
tion of the formation of diffusible protein products. He regarded the
ferments as protective, inasmuch as they could break down and aid
in the elimination o>f substances essentially foreign in nature. The
technic of the test has been carefully reviewed by Bronfenbrenner in
Vol. I of the Journal of Laboratory and Clinical Medicine, and we call
particular attention to certain modifications that have been offered by
Retinger in Volume XXII of the Archives of Internal Medicine. The
following brief description of the test is given in order to provide an
outline of the general principles.

The materials essential for the test are the serum or plasma of the
patient, the substratum, dialyzing tubes and flasks, carefully distilled water,
clean test tubes and ninhydrin. The blood is withdrawn before the patient's
Ibreakfast in order to obtain blood at a time when no dialyzable products of
intestinal digestion are present. It may be taken into paraffin-coated centri-
fuge tubes for the preparation of plasma or may be allowed to clot and
the serum centrifuged so as to be absolutely clear. The substratum is the
material to be digested. As a rule, the tissue is cleared of connective tissue
in so far as possible, is perfused with salt solution and subsequently washed
several times with distilled water until it is absolutely free from blood. It is
then placed in a suitable container, coagulated by boiling, and repeatedly
washed with boiling water until the fluid gives no ninhydrin test. It is then

250 THE PRINCIPLES OF IMMUNOLOGY

preserved under toluol. The dialyzing thimbles are especially prepared for
work of this kind. They are kept in distilled water for at least a week and
are carefully tested before use so as to be sure that protein does not pass
through and also to be sure that pepton will pass through. For the actual
test a dialyzing thimble is placed in a clean, dry, sterile Erlenmeyer flask.
About 0.5 gram of dried substratum is placed in the bottom of the thimble
and 1.5 c.c. of serum then introduced. The thimble is withdrawn, closed at
the top by means of a forceps and the outside washed carefully with sterile
water so as to remove any adherent protein. The thimble is then replaced in
a flask containing about 20 c.c. sterile distilled water. The contents of the
thimble and the water in the flask are covered with toluol and the flask
incubated for sixteen to eighteen hours. The dialyzate is examined by
means of the nmhydrin test. For this purpose, 0.2 c.c. of i per cent, ninhydrin
solution is placed in a clean dry test tube and 10.0 c.c. of the dialyzate added,
the mixture boiled for one minute and the color observed. The development
of a blue or violet color indicates the presence of diffusible protein products
and constitutes a positive test. Proper controls of all the reagents are essential.

Since the earlier work of Abderhalden appeared, numerous articles
have been written and there has been much discussion concerning the
alleged specificity of the reaction. The protective ferments of Abderhal-
den are assumed to possess the property of directly digesting the antigen
and the appearance of the products of such direct digestion constitutes
the fundamental principle of the Abderhalden test. Stephan, Haupt-
mann, Bronfenbrenner and others have shown that these ferments lose
their activity after heating to 58° C. for one-half hour, but they can
be reactivated by the addition of fresh serum. This suggests a parallel
with the activity of complement and amboceptor, but Frank and Rosen-
thai point out that in hemolysis there is no indication that the action
of complement is accompanied by proteolysis. Therefore, although the
ferment may be reactivated after heating, this does not necessarily
indicate that it is of the nature of an amboceptor or other immune
body. Flatow, Plaut and others have reported that positive results
can be obtained by the manipulation of material and that positive or
negative reactions can thus be found with almost any serum. De Waele
found that he could demonstrate a digesting substance within a few
minutes after the parenteral introduction of foreign protein, a time
interval too short for the production of specific ferments. Heilner and
Petri regard this, however, as a sort of mobilization of ferment and not
the result of new formation. Bronfenbrenner found that the serum of
highly immunized animals* failed to digest the protein used for im-
munization. He determined, however, that such a serum gave a positive
Abderhalden test and with his collaborators has demonstrated that the
dialyzable substances do not originate from the substratum. He showed
also that the ferments responsible for the cleavage of protein during
the reaction are not specific. Positive results with placenta are to be
obtained with the serum of males as well as of females, but the protein
digested is that of the serum. The work of Jobling and his collabor-
ators favors the view that proteolytic activity of the serum is not
specific. Plaut, Bronfenbrenner and others found that positive
Abderhalden tests may be obtained by the use of kaolin, starch, barium
sulphate and chloroform, all of which probably absorb the inhibiting
substance or anti ferment of the blood. Van Slyke and his associates,

DEFENSIVE FERMENTS 251

by means of determining the amino-nitrogen, found that practically every
serum shows some degree of protein digestion when incubated with
placental tissue. Van Slyke's methods are so accurate that it seems
probable that the ninhydrin tests with dialyzates must vary consider-
ably, depending upon the amount of dialyzable substance which may
pass through any given thimble. Elsesser worked with the purified
vegetable proteins of Osborn and found that at best the specificity of
the reaction is less than that of anaphylaxis and that there are many
non-specific results. Boldyreff found that the ferments act not only
upon placental proteins but also upon other varieties of protein; he
believes that the method is excellent for detection of proteolytic enzymes
in the blood but as a distinctive sign of pregnancy it is useless. Against
these views are the recent results of Retinger, who claims that it is
not only possible to demonstrate lesions of the brain by this test but
further to define within fairly small limits the localization of the
lesion. It may be that with further modifications a test of some clini-
cal value can be developed upon the basis of the Abderhalden test. At
the present time, there is little reason for accepting the conception of
specific ferments and the test has been entirely discarded in
many laboratories.

APPENDIX A
THERAPEUTIC EMPLOYMENT OF BLOOD SERUM

INTRODUCTION.

SERA PREPARED BY USE OF BACTERIA OR BACTERIAL EXTRACTS.

ANTI-STREPTOCOCCUS SERUM.

ANTI-MENINGOCOCCUS SERUM.

ANTI-PNEUMOCOCCUS SERUM.

ANTI-CHOLERA SERUM.

ANTI-ANTHRAX SERUM.

ANTI-PLAGUE SERUM.

ANTI-BACTERIAL SERUM FOR DIPHTHERIA CARRIERS.

ANTI-GONOCOCCUS SERUM.

ANTI-TUBERCULOSIS SERUM.

ANTI-TYPHOID SERUM.
AUTO-SERUM THERAPY.

GENERAL USES.

SYPHILIS.

HUMAN IMMUNE SERUM.
SERUM THERAPY IN INFECTIONS OF UNDETERMINED ETIOLOGY.

INTRODUCTION.

ANTI-POLIOMYELITIS SERUM.
ANTI- HOG-CHOLERA SERUM.
THERAPEUTIC USE OF NORMAL SERUM.

THE development of immunology has resulted in extensive study
of the treatment of disease by sera prepared according to a variety of
methods. We have considered in other chapters the value of certain
sera, more particularly those which possess a demonstrable content of
antitoxin. In this chapter there is presented a brief statement as to the
methods oi preparation and use of other types of sera with the idea
of illustrating how widely this form of therapeusis has extended and
the principles upon which the methods are founded. Certain of these
sera have given excellent results, but others have failed utterly and still
others are yet in the stage of experiment and investigation. The judg-
ment as to the value of many of the sera rests upon statistical evidence
collected on a clinical basis. The use of man for investigation intrudes
into the results obtained a wide variety of sources of error, many of
which can be excluded in investigations upon the lower animals. Dif-
ferences in hygienic surroundings, conditions of exposure, presence of
diseases other than that treated, differences in weight, age and sex
must all be considered. The stage of increase or decrease of the epi-
demic must be included in the final judgment since the virulence of
infections is likely to be greater at the beginning of an epidemic than
during its decline ; this may be due to exhaustion of the causative agent,
but is more probably accounted for in that the less resistant individuals
succumb early in the epidemics and the more resistant are attacked sub-
sequently. The factor of error in random sampling must be calculated
as closely as possible and it must be recognized that the greater the
number of cases studied, the more conclusive are the results. The
252

EMPLOYMENT OF BLOOD SERUM 253

investigator is always actuated by the hope that the particular method
he fosters will be of value in the alleviation of human disease, and
this fact may determine a subconscious selection of cases and perhaps,
equally subconscious, somewhat superior nursing and better care of the
cases under special treatment than of the controls. Thus the analysis of
statistical evidence must be made with rigid consideration of the various
factors of error. Minor differences in percentages of cure or of im-
provement may be within carefully computed factors of error and still
not take sufficiently into account a considerable factor of error resulting
from our ignorance of the intricacy of biological phenomena.

Immune sera for therapeutic purposes have been prepared by the
injection of bacteria, their toxic or non-toxic extracts, or by combina-
tions of these substances. Some of these sera exhibit a variable content
of antibodies of the first, second or third order of Ehrlich. The most
important laboratory test, however, appears to be the protective value
of the sera in preventing* infection in animals or their curative value
after the infection is established. There is no necessary parallel between
the content of special antibodies and the protective or curative value, ex-
cept in the case of antitoxic sera. Thus a serum may exhibit a low ag-
glutinin or bacteriolysin content and yet protect animals when used in
extremely small amounts. The converse is also true, namely, that rela-
tively high content of agglutinin or bacteriolysin does not necessarily
presuppose a great capacity for protection. Furthermore, it cannot be
assumed positively that because animals are protected or cured, the
serum will be of equal value in human medicine ; hence the necessity
for carefully studied experiments on man.

Not only have immune sera been employed, but many attempts at
treatment of disease by means of normal sera have been made. This
procedure is based in part upon the principles of non-specific immun-
ological treatment which have been previously discussed. Such sera
may be obtained from man, horse, goat, ox or other animal. In the
treatment of certain hemorrhagic disease the purpose of the serum
may be physiological rather than immunological, inasmuch as the serum
is believed to provide certain essentials for the process of clotting which
the patient provides in insufficient amounts or not at all.

In the following discussion it will be noted that there are first taken
up those sera prepared by immunization with bacteria; second, those
prepared by immunization with bacterial extracts, either with or without
the bacterial bodies; third, treatment with the patient's own serum;
fourth, treatment with sera from convalescent human cases; fifth, spe-
cific serum therapy in diseases of unknown origin, and finally, treat-
ment by normal sera.

SERA PREPARED BY USE OF BACTERIA OR BACTERIAL EXTRACTS

Anti-streptococcus Serum. — The protection afforded by the use of
streptococcus immune serum is still problematical. The reason for this
lies partly in the fact that there are several different types of streptococci
concerned in human infection. Some of the strains occur frequently and

254 THE PRINCIPLES OF IMMUNOLOGY

others only rarely. It is, therefore, advisable to determine as soon as
possible the type of the organism, and then combat it with its special
antiserum. Havens succeeded in dividing these organisms into three
distinct groups by means of cultural and immunological examination
and found that an immune serum can be produced for each of the three
groups. The serum is specific for its own group and protects mice
against infection with homologous organisms, but furnishes no pro-
tection against infection with organisms from the other groups. From
this work it is evident that the utilization of specific sera is of para-
mount importance in the treatment of streptococcus infections. The
oldest serum is that of Marmorek. This serum was produced by im-
munization with a strain which was made highly virulent by animal
passage and the serum was found to be protective experimentally when
administered twelve to eighteen hours before the bacteria were injected.
This serum was used in erysipelas, puerperal septicemia and scarlatinal
angina with favorable results. Lenhartz, Baginsky, Zangemeister and
others, however, failed to obtain definitely good results with anti-strepto-
coccus sera. Sera were later produced by Aronson and Tavel, Van de
Velde, Meyer, Ruppel, Menzer and Moser for use in puerperal sepsis,
scarlatina, erysipelas and acute articular rheumatism. In puerperal
infection a fresh polyvalent anti-streptococcus serum should be given
daily in intravenous doses of 30. c.c. until marked improvement occurs.
These cases are usually slow in improvement, but the results so far
obtained seem to be encouraging. It is, however, of the greatest im-
portance to introduce serum treatment at the earliest possible moment.
In scarlatina Escherich found that if the serum be used on the first and
second days of illness recovery of the majority of cases is likely to
occur. Axenow, in fact, believes that it is the only means to ward off
a fatal outcome. In erysipelas and acute articular rheumatism the
results have been at variance. Park states that the injections should
be made before the infection has become advanced and before the
streptococci have acquired an increased resistance to the serum anti-
bodies and ferments. The repeated local bathing of exposed infected
tissues with the serum seems to have a beneficial result beyond that
exercised by a non-specific serum. The action of anti-streptococcal sera
is largely due to its opsonic powers. The hope for effective serum
therapy in streptococcus infection is at present based on the new
methods of serologic classification of the organism and further labora-
tory and clinical study is highly desired.

Anti-meningococcus Serum. — In 1906 Jochmann for the first time
treated cerebrospinal fever with serum of horses immunized to several
strains, of meningococci. This serum was highly agglutinative, some-
what bactericidal, but not antitoxic. The death rate among the treated
cases was 27 per cent, as compared to 53 per cent, among the non-treated
cases. In the earlier work Jochmann administered the serum subcu-
taneously but later advised its use by the intraspinal method. Almost
simultaneously Flexner and Jobling carried out extensive work on
monkeys. They demonstrated that the most beneficial effect of the

EMPLOYMENT OF BLOOD SERUM 255

serum follows intrathecal administration, and in 1907 successfully
applied serum treatment of the disease during an epidemic in Akron,
Ohio. The following table, taken from Worster, Drought and Mills
Kennedy, " Cerebrospinal Fever," London, 1919, gives the results ob-
tained by several investigators.

Author

Flexner . . .
Netter
Dunn

No.
of treated
cases

1294 (collected)

100
40

Serum
used '

Flexner
Flexner
Flexner

Serum
treated
mortality s

30.9%
28.0%

22.5%

Cases not
treated with
•erum mortality

70%
49%

70%

Robb

•!&•
3OO =*=

Flexner

•Ml*^ /L/

30.0%

/ V7 /U

72%

Dopter . . .

Lew

4O2

1 6s

Dopter
/ Kolle and \

\IT r r

16.4%

18.8%

65%

C2%

Steiner . . .
Schoene . .

•*• WO

2280 (collected)
30

. Wassermann J
Jochmann

37-0%
30.0%

O f**

77%
53%

Although many English investigators have been successful in the use
of anti-meningococcus serum, several experienced men have advised
against its use. This is largely because of 'the fact that Gordon, Ellis
and others have demonstrated several types or groups of the meningo-
coccus, and it is believed that sera should be prepared against each
type in order to obtain the best results. Rolleston has compiled the
following table of results with various types of anti-meningococcus sera :

Brand of serum Mortality Recoveries

Flexner 22.3 per cent. 77.7 per cent.

Gordon 18.7 per cent. 81.3 per cent.

Pasteur Institute 44.5 per cent. 55.5 per cent.

Burroughs-Wellcome ... 33.3 per cent. 66.7 per cent.

Mulford 50.0 per cent. 50.0 per cent.

Lister Institute 54.5 per cent. 45.5 per cent.

Gordon's and Flexner's sera have so far given the best results. It
is advisable to test the agglutinative power of a serum prior to its use,
using a strain freshly isolated during the epidemic. The serum should
agglutinate the organism in a dilution of at least one to five hundred.
The lack of a definite potency standard makes it impossible to judge
accurately the value of any given serum. As in diphtheria and other
diseases, the early use of serum is of the greatest importance. Flexner
found that if the serum was given in the first three days the mortality
was 1 8 per cent., if given from the fourth to the seventh day it was
27 per cent., and if given later 36.5 per cent. Similar figures were
obtained by Rolleston, Gray, Robb and Worster-Drought. If neces-
sary, the injections should be repeated. According to Park, it is advis-
able to give not less than four daily injections unless the case is already
convalescent when it comes under observation. If the organisms or
symptoms do not disappear, the injections of 10 c.c. to 25 c.c. of serum
should be continued for many days. Finally, as a result of army experi-
ence, Herrick believes that the disease is in most if not all cases a
general bloodstream infection with secondary meningeal involvement
and therefore advises the use of large doses of anti-meningococcus
serum intravenously as soon as the diagnosis is made in addition to

256

THE PRINCIPLES OF IMMUNOLOGY

intrathecal injections. The results so far obtained seem to be better
than when intraspinal injections alone are used. Frich also recom-
mends that all patients with positive signs and symptoms be given both
intraspinal and intravenous injections of serum. Large doses of serum,
both intravenously and intraspinally at frequent intervals apparently
do no harm, lower the mortality, prevent serious complications and
shorten the period of convalescence.

Anti-pneumococcus Serum. — Washburn, Mennes, Pane and numer-
ous other early investigators attempted to produce anti-pneumococcus
sera for the treatment of man, but their results were irregular and not
encouraging. An important advance in the production of anti-pneumo-
coccus serum was made when Neufeld and Handel in 1909 pointed out
that pneumococci can be divided into various immunological groups,
and that no curative properties can be expected from a given serum
unless it is homologous for the type that causes the infection. This
work has been confirmed and extended by Dochez and Gillespie, Cole,
Lister and many others. At present we recognize four groups. Groups
I and II are immunologically distinct groups, Group III is that of
the streptococcus or pneumococcus mucosus, and Group IV a heter-
ogeneous group of pneumococci which cannot be classified under the
other three groups. The following table, taken from Park ("The
Practical Application of Serum Therapy," Transactions of the Con-
gress of American Physicians and Surgeons, 1916, x, 118) gives the
group-incidence and mortality :

Type

Number

Percent, inci-
dence

Per cent, mor-o
tality.

University of Penna.
Hospital, Richardson

Cole

Longcope

Cole

Longcope

Cole

Longcope

No.

Per cent,
incidence

Per cent,
mortality

I

78
75

22

48
H

*d3)
(ii)
(7)

(21)

33
32
9

20

6

*(23)
(21)
(14)
(40)

25.0
29.0

45-o
12.5

*(I2.5)
(72.7)
(85-7)
(23.8)

60
39
13

83

31
2O

6
43

30
25
50
12

II

III

IV

Other bacteria ....

* Presbyterian Hospital, Longcope.

From this table it appears that about 30 per cent, of the cases of pneu-
monia and about one-third of the total deaths from the disease are
caused by Type I pneumococci. In the United States Cole claims that
75 per cent, of all pneumonia cases are caused by Types I, II and III, and
25 per cent, by Type IV. Lister, in South Africa, finds Type IV very
common among the negroes in the Rand. So far only Type I and
Type II sera have given encouraging results. The antigenic value of
Type III pneumococcus is exceedingly low, and that of Type IV vari-
able. From the more recent work of Raphael it would appear that
sera produced against various strains of pneumococci are in a sense
strictly monovalent and also that only virulent pneumococci are suf-
ficiently antigenic to produce antisera of distinct value.

The sera against infection with Type I organisms have been used
extensively and appear to have given especially good results. The

EMPLOYMENT OF BLOOD SERUM 257

Type II antiserum, however, is much less efficacious and thus far its
therapeutic value is questionable. The Types III and IV antisera
have no clinical value. Dochez reports sixty-five cases treated with
Type I serum, with a mortality of 6.6 per cent., as compared with a
mortality of 25 per cent, in Type I cases not treated with serum.
Type II cases treated with serum have a mortality of 25 per cent,
as compared with 61 per cent, without the use of specific sera. When
patients are treated early, they do well, and large doses of serum
should be given as soon as the type of infection has been determined.
Cole advises an intravenous injection of 80 to 100 c.c. of serum diluted
with an equal amount of salt solution and repeated every twelve hours
until improvement occurs. The average total amount of serum required
in the Hospital of the Rockefeller Institute was about 250 c.c. The
injection of such large doses of serum is not entirely without possible
harm to the patient because of reaction to the foreign protein. The
possibility of severe serum sickness should further be taken into con-
sideration. From evidence recently collected, more particularly in the
United States Army, the value of anti-pneumococcus sera has been
questioned. Parallel series of cases showed no important difference
in mortality between series receiving anti-pneumococcus serum, normal
horse serum or no serum whatever. The patients were young men who
had passed rigid physical examinations and therefore were good risks
in acute infections. It does not follow that other classes of patients
would show the same results. There is also great variation in the mor-
tality of different epidemics, and also normally in different ages, so
that only a sufficiently large number of treated cases extensively con-
trolled will form a trustworthy basis of actual comparison as to the
death rate, which is, after all, the final criterion as to the actual value
of the serum. Cecil and Blake have recently examined the question
on the basis of experiments with monkeys. They find that the admin-
istration of normal horse serum has no beneficial effect on experimental
pneumococcus Type I pneumonia but that the intravenous administra-
tion of specific Type I antiserum, particularly if given early and fre-
quently, " exercises a specific therapeutic effect, frees the blood
promptly and permanently from pneumococci, shortens the course of
the disease and greatly moderates its severity." The treatment of
lobar pneumonia with Cole's serum at present is best carried out in
institutions where it is possible to make accurate bacteriological diag-
nosis and differentiation of the types of the cocci, and where intra-
venous administration of large doses of sera can be accomplished with
the largest margin of safety to the patient.

Kyes has carried out extensive investigations on the clinical value of
a serum produced by injecting massive doses of virulent pneumococci
into the domestic fowl. The reason for the selection of the fowl as
supply animal is that no matter how virulent pneumococci are for other
species, they do not occasion disease in fowls, and therefore large doses
can be injected with impunity. The initial dose in most instances is a
surface growth equal to that of 240 test-tube slants. The average
17

258 THE PRINCIPLES OF IMMUNOLOGY

subsequent doses are approximately 400 test-tube slants each. All the
injections are made intraperitoneally. Injections are given every two
weeks over periods of from four months to two years. One week after
the sixth injection a trial bleeding is made and thereafter at intervals of
two weeks, alternating with the biweekly injections. The sera possess
a high content of agglutinins and bacteriolysins and also exhibit a
marked therapeutic influence upon infected animals. Clinically the
serum is used in doses of 2.5 c.c., and injections made slowly. A ma-
jority of the cases have received one injection daily, but not infre-
quently two injections are given the same day. The injections are
continued until the temperature remains below 100° F. Of 538 cases
not treated, 244 cases died, the death rate being 45.3 per cent. Of the
175 similar cases treated with serum the death rate was 20.8 per cent.
In the ward in which the serum was employed the death rate during
the six weeks prior to the introduction of the serum treatment was 55
per cent. During the six weeks subsequent to the withdrawal of the
serum treatment, the death rate was 51 per cent. These results are
distinctly encouraging. McClelland has recently reported the results
in 322 cases of lobar pneumonia in soldiers at Camp Grant in which
treatment with fowl serum was given and concludes that the low
mortality (7.7 per cent.) together with the favorable modification of
clinical symptoms by the serum would seem to indicate the extension
of its use in pneumococcus pneumonia. Considering the fact that
these cases were in selected young men of military age, and that the
author does not give a comparative mortality among non-treated cases,
much of the value of this paper is lost. The serum has also been used
by Litchfield with great benefit in a series of pneumococcus meningitis
cases. Gray employed the Kyes serum in 234 cases of pneumococcus
pneumonia with a mortality of 16.8 per cent., whereas in similar cases
treated in the same way except that they received no serum, the mor-
tality was 63.6 per cent. Much laboratory and clinical work remains to
be done before any definite conclusive evidence as to the value of
polyvalent or antigroup sera can be drawn with any degree of safety.
Pneumococcus sera act in part by opsotiization of the cocci, thus favor-
ing phagocytosis. The standardization requirements of the Hygiene
Laboratory, Washington, call for a serum that shall protect white mice
against Type I pneumococcus only. It is felt by Ferry and Blanchard
and many others that a potent polyvalent serum is an absolute necessity.
These authors recently succeeded in immunizing horses with Types I,
II and III and some strains of Type IV. This serum in doses of 0.2 c.c.
protected mice against infection of Types I, III and IV organisms (ten
million M.L.D.).

Anti-cholera Sera. — While antisera against cholera have been pro-
duced by several investigators, the treatment of the disease with these
sera has not given the best of results. Metchnikoff, Roux and others
have prepared sera against the toxins of organisms cultivated in col-
lodion sacs. McFadyen used ground organisms. Kraus used the toxin
of the El Tor vibrio as antigen. This organism was obtained by

EMPLOYMENT OF BLOOD SERUM 259

Gottschliech in 1905 from the intestinal contents of pilgrims who had
died at El Tor from dysentery, and is not a true cholera vibrio but
very closely related to it. Kraus recommended his antitoxin for the
treatment of the cholera. Schurupoff treated one-and-a half to two-
day-old cultures of the vibrio with alkali and injected this toxic material
into horses at six to ten-day intervals. Under Kolle's direction, Car-
riere and Tomarkin injected horses and goats with cholera cultures con-
taining also the toxic derivatives and used the mixed sera of these
animals. They found that these sera are more valuable against cholera
peritonitis of guinea-pigs than any other animal.

Ketscher and Kernig used Kraus' serum in 119 severe and mod-
erately severe cases with a death rate of 58 per cent, in those who
received subcutaneous injections, and 50 per cent, when used intrave-
nously, while among the non-injected cases the mortality was 63.4 per
cent. Others have found among the serum-treated cases a mortality of
57.5 per cent, and among the control cases 84.3 per cent. This serum
was administered by Jegunoff intravenously together with physiological
salt solution, giving at first 140 c.c. of serum with 500 c.c. to 700 c.c. of
physiological salt solution and subsequently a second injection of 80 to
1 20 c.c. of serum within seven and one-half to twenty-three hours after
the primary injection. During the Russian epidemics in 1908 and 1909
it was shown that large doses of sera did not harm the patients. It
was originally believed that large doses of sera lead to quick destruc-
tion of the vibrios with subsequent intoxication, but this has not proven
to be the case. During these epidemics Salimbeni's and Kraus' sera
did not give satisfactory results. SchurupofFs serum was considerably
better, and the best results were obtained with the serum prepared
according to Carriere and Tomarkin. This serum was given in doses
of 50 c.c. to 100 c.c. diluted with salt solution subcutaneously and intra-
venously and resulted in quick improvement. Von Stiihlern and
Tuschinski treated 149 algid cases with fifty-six deaths.; twenty-five
moderate and thirteen early cases were treated with no deaths. From
a total of 187 cases the mortality was 29.9 per cent. The serum should
be applied as early as possible. Cholera antisera contain bacteriolytic,
agglutinative, probably anti-endotoxic, complement-fixing antibodies,
and also tropins. Because of the variety of sera used and the incon-
clusive reports given it is exceedingly difficult to reach a definite con-
clusion regarding the curative value of anti-cholera sera. It seems to
us that Carriere and Tomarkin's serum is the most promising.

The Use of Anti-anthrax Serum. — The treatment of anthrax has
consisted mainly in excision of the pustule, application of chemical or
thermal cautery, and the injection of germicides as iodine, mercuric
chlorid or phenol in the regions of the pustule, but all these methods
are objectional because they are likely to produce scars and disfigure-
ment. Excision may furthermore increase the danger of systemic
infection. Sclavo, Deutsch, Sobernheim and others have produced
immune sera by immunization of the sheep, horse and ass with attenu-
ated culture of anthrax bacilli. From the work of Marchoux we know

26o THE PRINCIPLES OF IMMUNOLOGY

that this serum possesses prophylactic and therapeutic properties in
animals. Anti-anthrax serum has been used for several years, espe-
cially in Italy, France and England, with encouraging results. Sclavo
treated his cases without excision and in a series of 164 cases treated
with specific serum this author reduced the mortality from 24 per cent.
to 5.3 per cent. Sclavo recommends 30 to 40 c.c. serum administered
subcutaneously in doses of 10 c.c. on the first day and repeated if neces-
sary on the next day. In severe infections 10 c.c. were given by the
intravenous route. In severe cases it is advisable to give the injections
in massive doses of 80 c.c. to 100 c.c. and preferably intravenously.
Shera advises administration of 20 c.c. every twelve hours until pyrexia
ceases. Regan recently injected the serum (10 c.c. to 15 c.c.) into the
tissues surrounding the pustule and found that it possesses none of the
disadvantages of the previous methods of local treatment, and has a
very rapid and complete effect on the pustule, not only in arresting its
further development but also in producing a subsidence of all local
inflammatory symptoms. He advises also general treatment either
intramuscularly or intravenously. Local treatment in order to effect
a cure must anticipate the onset of an anthrax septicemia. In case
the organisms have been demonstrated in the bloodstream the prog-
nosis is usually grave. In this case 200 c.c. of serum is not excessive,
and if necessary should be repeated until a negative blood culture is
obtained. In intestinal anthrax large doses of serum should be given
by the intravenous route. Penna and Beltrami and Penna, Cuenca and
Kraus have obtained good results with normal beef serum. Their mor-
tality was 6.2 per cent, in 372 cases, while the mortality previous to this
period of treatment was about 10 per cent. These authors advise the
use of 30 to 50 c.c. of normal beef serum administered subcutaneously.
If no improvement occurs the injections should be repeated every
twelve, twenty-four or thirty-six hours, but it seldom happens that a
patient requires more than two or three injections. In severe cases
intravenous administration is recommended. Similar favorable results
were obtained by Solari and Langon, but Lignieres has reported un-
favorably upon the curative action of normal beef serum, stating that
it is inferior to horse anti-anthrax serum. He calls attention to the
prevalence of anthrax in cattle as evidence of the apparent lack of
natural resistance to the disease. More recently Kolmer, Wanner and
Koehler pointed out on the basis of their experiments that normal beef
serum as secured from animals under ordinary conditions is but feebly
protective or curative for anthrax and while its administration as
described by Penna and his associates may favorably influence the pus-
tule it is doubtful if the serum is sufficiently powerful to influence
anthrax bacteremia. According to Kolmer, cases with sterile blood
culture always recover. The potent factor of anti-anthrax serum
appears to be a thermostable opsonin.

The Serum Treatment of Plague. — The therapeutic value of anti-
plague serum is still a matter of dispute. Plague epidemics are exceed-
ingly variable in character. Irregularity in the gravity of the disease

EMPLOYMENT OF BLOOD SERUM 261

in different individuals is of common occurrence. A wide variety
of antisera has been employed, but no attempt has been made to stand-
ardize the different sera. In many instances the cases treated were
especially selected and moribund cases excluded. The result is that much
of the existing statistical data is unreliable. Yersin, Calmette and Borrel
were the first to show that the serum of an animal immunized to bacillus
pestis has protective qualities and Yersin is credited with the production
of anti-plague horse serum. This serum was prepared by immunization
of horses first with dead and subsequently with living bacilli. Tavell's
serum was prepared on the same principle, but Hata and also Kraus
immunized their animals with dead bacilli alone and claim that these
sera compare favorably with sera produced by the injection of living
bacilli. The use of dead bacilli minimizes the danger of laboratory in-
fections. Soon after the discovery of the nucleoproteins by Ferrannini,
Galeotti and Lustig employed nucleoproteins from plague bacilli as
antigen for the production of anti-plague serum. For this purpose the
bacilli were broken down in i per cent. KOH solution and the nucleo-
proteins precipitated by the addition of acetic acid and then suspended
in salt solution. Rowland also used a similar antigen, and others have
employed a variety of extracts as antigens.

The serum at present most commonly used is obtained from horses
after repeated intravenous injections of killed cultures sometimes fol-
lowed by living organisms. Experimentally the sera show considerable
strength in protecting animals against infection and exhibit specific
bacteriolytic, bacteriotropic, agglutinative and antitoxic qualities. The
antitoxic titer is usually very low. According to Kraus, Yersin's serum
is not any better than the sera prepared with dead bacilli or nucleo-
proteins. Yersin used his serum in twenty-six cases during the epi-
demics of 1896 in Canton and Amoy, China, with a mortality of 7.6
per cent., while the mortality in cases not treated with serum reached
80 per cent, to 90 per cent. In 1897 141 cases were treated in Bombay
and Cutch-Mandir with a mortality of 49 per cent. Of 685 cases not
treated 80 per cent. died. In 1898 thirty-three cases were treated in
Anam with Yersin's serum. The death rate among non-treated cases
was 100 per cent, but was 42 per cent, among the serum-treated cases.
It was found that the serum was entirely inefficient in cases with the
pneumonic form of the disease. The German Commission at Bombay
claimed that the low mortality (50 per cent.) o>f serum-treated cases was
due to the selection of mild cases or cases arriving at the hospitals
during the first or second day of their illness. Clemon also failed to
obtain results in his fifty cases in which he injected as much as 60 c.c.
of the Yersin serum. The Indian Plague Commission did not report
favorably on Yersin's serum. Calmette and Salimbeni obtained very
good results with serotherapy in Oporto, Portugal ; of 142 treated cases
twenty-one died, while of seventy-two not treated forty-six died. Kos-
sel and Frosch and others studied this epidemic and found it to be of
a mild type. During the Manchurian campaign serum treatments
were entirely inefficient. Choksy injected large doses (100 c.c)

262 THE PRINCIPLES OF IMMUNOLOGY

of the Parisian serum in his cases, repeating this six to eight
hours later, and if necessary followed again by another injec-
tion. The next two days he administered 20 to 50 c.c., so that
an adult received a total of 590 c.c. From a careful study he obtained
a mortality of 72.5 per cent, among the serum-treated cases, and 82.3
per cent, among his control cases. This author also emphasizes the
enormous advantage of early injections. In a series of 222 cases
treated on the second, third, fourth, fifth, sixth and seventh day of
illness he found the mortality as follows: 38.2, 56.7, 58.2, 50.8, 62.9,
60.0, and 75 per cent, respectively. According to Burnet, satisfactory
results have been obtained in Queensland at the Colmolie Plague Hos-
pital. Among 190 serum-treated cases during the period 1901 to 1907
the mortality was 29.7 per cent., while the mortality during the same
period among non-treated cases was 73.9. Penna in Argentina injects
massive doses 80 to 100 c.c. intravenously, and repeats the injection of
50 c.c. after twelve to twenty-four hours. Among 664 treated cases
during the period 1905 to 1912 he reported a mortality as high as 23
per cent, in 1906, and a mortality of 7.3 per cent, in 1912, the average
mortality was 12.5 per cent. From 1914 to the middle of 1919 Kraus'
serum was used with an average mortality of 7.8 per cent. Kraus'
serum, therefore, gave better results than Yersin's serum. Intravenous
or intramuscular injections can be employed to ensure rapid absorption
and the injections should be continued every twelve to twenty-four hours
for two or more days until diarrhea has been controlled and the disease
begins to subside. From all these studies we may conclude that although
serum therapy of plague has not given striking results as diphtheria
antitoxin in diphtheria, still it is the only specific means of combat-
ing the disease and when given early and in massive doses appar-
ently influences the disease favorably.

Anti-bacterial Serum in the Treatment of Diphtheria Carriers. —
Although Wassermann in 1902 recommended the use of a bactericidal
serum, Martin was the first to use anti-bacterial serum in the treatment
of diphtheria carriers. Martin injected diphtheria bacilli intravenously
or intraperitoneally into horses and obtained sera with marked agglu-
tinating properties. He claims that this serum has, when applied
locally, the property of causing a rapid decrease in number of living
bacilli in the throat. The best results were obtained by incorporating
the dried serum with gum and using it in the form of pastilles. Dopter
and many others have reported a decrease in the carrier period by the
use of anti-bacterial serum. More recently Roskam and Arloing and
Stevenin have called attention to the value of this method of treatment.
Ecker immunized sheep with various strains of diphtheria bacilli, and
by using massive doses obtained a potent agglutinative and lytic serum.
To this serum fresh guinea-pig complement was added and the mixture
sprayed by means of atomizers into the nasal passages, and over tonsils,
fauces and pharynx four and five times a day. A total of forty-eight cases
were treated, eighteen convalescent and thirty contact carriers. The
duration of the carrier state after the introduction of the serum was seven

EMPLOYMENT OF BLOOD SERUM 263

days, while the average duration of eighty-seven control cases was 18.6
days. A few cases proved to be persistent carriers. The less favorable
results obtained by Kretschmer, Blumenau and Nolf may be explained
by their small series of treated cases, weak sera and ineffective methods
of application of the serum. Although the results so far obtained are
not entirely convincing, the use of anti-bacterial serum in the treatment
of diphtheria still deserves careful consideration. It is not pos-
sible to resort to tonsillectomy or adenoidectomy in all instances,
and the majority of antiseptics are irritant. In many instances it is
practically impossible to reach the organisms because they are buried
in crypts, and tonsillectomy remains as the favored mode of treatment,
although even this method is not invariably successful.

Anti-gonococcus Sera. — The early work of Rogers and Torrey
has led to attempts at treatment of gonococcal infections by means of
immune sera. Torrey's serum is prepared by injecting sheep with
dead and subsequently living cultures of virulent strains of the gono-
coccus. Although efforts have been made to treat urethral, vulvar
and vaginal gonorrhea by local applications of serum the disposition
of the organisms in deep glands has been sufficient to result in the
failure of this method. Recent studies of Debre and Paraf offer some
encouragement for the treatment of gonorrheal rheumatism by the use
of polyvalent sera, but they find that local injections about the site
of the disease are more effective than general subcutaneous or intra-
venous injections. Further studies may demonstrate the value of
serum treatment of chronic gonorrheal infections, but at the present
time the method cannot be highly recommended.

Serum Treatment of Tuberculosis. — The best-known sera for use
in tuberculosis are those of Maragliano and Marmorek. The first is
prepared by immunizing horses with a mixture of a toxic filtrate of
the bacilli and an aqueous extract of killed virulent tubercle bacilli.
One cubic centimetre of the immune horse serum is injected into the
patient every other day for a period of one and one-half months. A
number of Italian workers found the serum effective, but other ob-
servers have not been convinced of its value. Marmorek's serum is
prepared by inoculating horses with young tubercle bacilli poor in acid
fast character. In addition, Marmorek immunized animals with pure
cultures of streptococci obtained from the sputum of tuberculous
patients. This serum is injected subcutaneously in daily doses of from
5 to 10 c.c. or intrarectally in doses of from 10 to 20 c.c. A number of
workers, as for instance Wohlberg, have reported a favorable influence ;
others deny this effect. Wohlberg found the best results in scrofulous
cases but not in cases of fully developed tuberculosis. The benefits of
serum therapy of tuberculosis have not been convincing.

Serum Treatment of Typhoid Fever. — Lewin and Yes, Beumer
and Pf eiffer and Chantemesse were among the first to produce antisera
for this disease. Chantemesse's antiserum was prepared by immuniz-
ing horses with soluble toxins of the typhoid bacillus. Balthasard
tested this serum and found it to agglutinate typhoid bacilli in very

264 THE PRINCIPLES OF IMMUNOLOGY

high dilutions and to protect animals under experimental conditions.
In 1000 cases of typhoid fever, Chantemesse reduced the mortality to
4.3 per cent, whereas the mortality among 5621 cases at the other
hospitals in Paris not treated with serum was 17 per cent. Similar
favorable reports were made by Brunon and Josias. Kraus and
Stenitzer also produced antitoxic sera by immunizing their animals
with soluble toxins and Cjaupp claims that the serum can be used with
advantage in the treatment of the disease. Besredka and Liidke pre-
pared sera by immunizing horses and goats with the endotoxin of the
typhoid bacillus, but it seems that the serum is not primarily an anti-
endotoxin but rather a bactericidal serum which neutralizes both the
exo- and endotoxins of the typhoid bacillus. According to Andriesen
and Cinca, it can be used clinically. Sera were also prepared by im-
munizing animals with sensitized cultures of the typhoid bacillus and
also with products obtained by digesting typhoid bacilli with trypsin.
This toxic compound is known as " Fermotoxin " (Gottstein and
Mathes). Rommel and Herman failed to obtain encouraging results
with serum prepared by immunization with sensitized bacilli. The most
favorable results, however, were secured by Rodet and Langrifoul.
These authors immunized horses intravenously with both living cultures
and old endotoxins, and in a summary of 400 cases Rodet finds that by
repeated injections of this serum in doses from 10 to 20 c.c. given sub-
cutaneously every other day the duration of the fever is markedly
reduced in cases that are treated early. Serum treatment appears to
reduce the bacteremia. It is also known that twenty-four hours after
the injection of serum a definite increase in splenic dullness is observed,
which presumably indicates a general stimulation of the lymphoid and
myeloid tissue. The self-limitation of the disease, in the absence of
complications, throws some doubt on the practical value of such sera.

AUTO-SERUM THERAPY

The use of the patient's own serum in the treatment of his disease
has been suggested and applied by a number of workers. Gilbert,
Marcon and many others treated tuberculous peritonitis and tubercu-
lous pleurisy with effusion, by the subcutaneous injection of I. c.c. to
2. c.c. of the patient's own serum and claim that the absorption of the
exudate is greatly increased and an immediate improvement occurs.
Eisner observed a leucocytosis following the injection of the serum
in experimental tuberculous infections of rabbits and guinea-pigs and
believes that this fact explains the favorable results reported in this
method of treatment. Other investigators believe that specific antibodies
favorably influence the process, but Levy, Valenzi and others are
inclined to believe that the results are independent of the injections.
It is possible that simultaneously with the transfer of the serum a
minute amount of tuberculin is introduced. The exact nature of the
phenomenon is, however, obscure. In influenza, Malta fever and
typhoid fever Modinos has also obtained beneficial effects and Jez
applied the treatment favorably in erysipelas. Capogrossi more re-

EMPLOYMENT OF BLOOD SERUM 265

cently treated two cases of cerebrospinal meningitis with fairly good
results. Hodenpyl treated a case of carcinoma with the patient's own
ascitic fluid with apparent success. He used this fluid in large quanti-
ties in a number of cases but only with transient success. Risley also
applied this method of treatment in sixty-five cases of cancer, using
ascitic fluid from cancer cases and also other body fluids from non-
cancerous cases. No encouragement for this method has been found
in experimentally inoculated mouse cancers and the subsequent history
of Hodenpyl's cases showed no permanent improvement. Auto-serum
therapy has further been applied in obstinate and chronic skin troubles,
such as psoriasis, dermatitis herpetiformis, pemphigus, lichen ruber,
lichen planus, urticaria and squamous eczema. The serum is used in
doses of 30 to 40 c.c. and repeated from two to six times at intervals
of from three to five days.

Auto-serum Therapy in Syphilis. — Perhaps the most widely
used auto-serum therapy is the salvarsanized auto-serum in the
treatment of parasyphilis. The treatment of syphilis of the ner-
vous system with salvarsan or neosalvarsan alone has not given
the results expected. This is because the choroid plexus filters
out these compounds, preventing their entry into the cerebrospinal
fluid. It has been shown by Plaut that the serum of patients who have
received salvarsan possesses antisyphilitic power, while normal serum
fails to display this characteristic. Similarly Meirowsky and Hart-
mann and Gibbs and Calthrop obtained good results in the subcutaneous
treatment of lues with serum of salvarsanized patients. According to
Swift and Ellis, salvarsanized serum inhibits the treponema more in-
tensively if heated to 56° C. for half an hour. These facts formed the
underlying principles for the treatment of late syphilis with salvarsan-
ized serum. Swift and Ellis injected salvarsanized serum intrathecally
in a number of cases of tabes dorsalis and in other manifestations of
neurosyphilis, and reported most encouraging results in both clinical
and immunological manifestations. This work has since been confirmed
by a large number of authors. The treatment is of special value in the
earlier stages of neurosyphilis. Unfavorable results have been ob-
served, as for instance the spasmodic retention of urine. As a result
of long standing of the salvarsanized serum prior to its use, the drug
may become oxidized with a marked increase in toxicity.

Method of Treatment. — Six-tenths to nine-tenths gram of salvar-
san or neosalvarsan is injected intravenously. One hour later 40 c.c. of
the patient's blood is withdrawn, allowed to coagulate and centrifuged.
Twelve cubic centimetres of the sterile serum is diluted with 18 c.c. of
sterile physiological salt solution to make it a 40 per cent, dilution and
heated for half an hour at 56° C. A lumbar puncture is then per-
formed, and 25 to 30 c.c. of fluid is withdrawn, and the serum very
slowly injected. Swift and Ellis recommend the gravitation method of
injection to prevent a sudden increase in intrathecal pressure. The
patient is then kept in bed for twenty-four hours and the foot of the
bed elevated for part of this time. The reaction is usually of a mild

266 THE PRINCIPLES OF IMMUNOLOGY

type, including slight fever, pain in the legs, but in rare instances violent
symptoms have been observed. Before and after the treatment a Was-
permann test, the globulin test and a cell count should be made. After
one week or more the treatment can be safely repeated until definite
improvement occurs.

TREATMENT WITH IMMUNE HUMAN SERUM

Weisbecker in 1897 appeared to be the first to have used blood
serum of convalescents, in cases of scarlet fever, but with little success.
Huber and Blumenthal, von Leyden and others renewed the interest
in convalescent serum therapy but failed to reach any definite con-
clusion probably because of the small doses employed. Reiss and
Jungmann, Koch, Zingher and Weaver more recently applied the
treatment with a fair degree of success. Reiss and Jungmann gave
intravenous injections of 40 c.c. to 100 c.c. and drew the blood from
scarlet-fever convalescents about the end of the third or beginning of the
fourth week of the disease, testing each serum for the possibility of
syphilis and for sterility. Zingher injected citrated whole blood intra-
muscularly in doses of 120 c.c. to 240 c.c. and repeated in two or three
days if necessary. Weaver drew the blood from convalescents between
the twentieth to twenty-eighth day, only such convalescents being selected
who had not been septic and who gave a negative Wassermann reaction.
The sera were tested for sterility and used pooled. Intramuscular in-
jections were given in doses of 25 c.c. to 90 c.c., 60 c.c. being the usual
amount. The effects of the serum are rapid and start with a sudden drop
in temperature and general improvement of the patient within twenty-
four hours after the administration of the serum. The best results
are obtained when the patients are treated early in the disease. Kling
and Widfeldt also reported favorable results in their series of cases
during an epidemic of 237 cases at Stockholm in 1918. This method has
not been widely adopted and there is still much question as to whether
improvement is due to the treatment or to the natural self -limitation
of the disease.

Monvoisin has recently reported encouraging results in typhus fever
by intravenous injections of human convalescent serum. One or two
cubic centimetres of serum brought a marked drop in temperature and
general improvement in the patient. Monvoisin noted a decrease in
mortality from 30 down to 10.34 per cent, by the use of convalescent
sera. The serum was obtained from a patient on the eighth day after
subsidence of fever. Favorable results were also reported by Teissier
in cases of severe and hemorrhagic smallpox. In leprosy the serum
obtained from cantharides blisters on lepers has been reported to
be of value.

Bleyer recently injected immune human blood into a series of forty-
five cases of whooping-cough in the early weeks of the disease. This
series was divided into three groups. The first group received blood
from persons who were convalescent or who had recovered from
whooping-cough within three months. In the second series the blood

EMPLOYMENT OF BLOOD SERUM 267

was from persons who had the disease at more remote periods, and the
third group from persons who, so far as they knew, had never suffered
with whooping-cough. The stage at which the treatment was given
was about the same in the three groups and the dosage depended upon
the body weight of the patient, varying between 40 c.c. and 125 c.c.,
divided into two, three or four doses and injected into the gluteus
muscles. In the first group there were no deaths and no complications,
and the course of the disease was in no definite way different from
the usual course. The second group showed quite as satisfactory im-
provement as in the first group. In the third group there were two
pneumonia cases with one death and one case which apparently was
favorably influenced by normal serum treatment. The groups are so small
and the difference so slight as to give no reason for regarding this
mode of treatment as particularly effective. Vaccine treatment of this
disease gives much greater promise of success.

During the recent great epidemics of so-called influenza, conva-
lescent serum was used in a considerable number of cases which
developed pneumonia. In many instances there was marked improve-
ment, but there is no clear indication that the results were specific or
that they depended absolutely upon the serum treatment.

SERUM THERAPY IN INFECTIONS OF UNDETERMINED ETIOLOGY

Introduction. — The preparation of the immune sera discussed above
depends not only upon knowledge of the etiological agent of the disease
concerned but also necessitates the isolation of the organism in pure
culture. Several infectious agents are known to exist in blood and
tissues, since the diseases may be transmitted by means of inoculation
of blood, organs or organ extracts. Many of these agents are so small
as to pass through porcelain filters and are spoken of as the filterable
viruses. Some of these viruses have been observed to contain minute
globoid bodies which have been obtained in pure culture, but under
such conditions that they have not served well as antigens for the
production of immune sera. If immunization be attempted by injec-
tions of the blood or tissues containing the infective agents, the result-
ing immune serum contains not only antibodies for the infective agent
but also for the tissues. If these tissues happen to be from the same
species into which the serum is to be injected the hemagglutinins,
hemolysins and cytolysins in the immune serum may seriously damage
or even kill the individual so treated. Active immunization by the use
of infected tissues appears to progress favorably in spite of the presence
of the tissues, as seen in the active immunization of man and other
animals by the use of the virus of rabies* contained in the dried spinal
cords of rabbits. It is in the production of sera for passive immuniza-
tion that the danger from simultaneously formed tissue antibodies ap-
pears. Rous, Robertson and Oliver have studied this problem with a
view to removing from the immune serum these harmful elements.
After the immune serum is prepared the tissue antibodies are removed
by selective absorption with red blood-corpuscles, since these cells re-

268 THE PRINCIPLES OF IMMUNOLOGY

move the most important source of danger, the hemagglutinins and the
hemolysins; undoubtedly many of the other tissue antibodies, as the
cytolysins, are reduced in amount. For example, they immunized a
goat with megatheriolysin and finely-ground liver, spleen and kidney,
as well as defibrinated blood, of guinea-pigs. The immune serum was
then repeatedly mixed with guinea-pig blood-cells until all the hemag-
glutinin and hemolysin had been removed. The process did not reduce
the titer of the special antilysin against megatheriolysin either in test
tube or animal experiments. Guinea-pigs were protected against
megatheriolysin by the use of this serum and the treatment of the
serum by selective absorption removed practically all the elements dan-
gerous for the guinea-pig. Similar experiments were performed using
as antigen the blood of rabbits suffering from pneumococcus
septicemia. It was found that absorption, by means of blood, of
anti-poliomyelitis serum produced no change in its protective value.
Experiments were also performed with the Rous chicken sarcoma, a
tumor caused by a filterable virus. The immune serum was prepared
by injecting into geese a mixture of tumor tissue and the blood of
moribund fowl since under these circumstances the blood contains the
causative agent. The immune serum was treated with fowl blood-
corpuscles to remove the tissue antibodies. The serum so treated, when
employed in proper ratio to the amount of tumor inoculated, served
to protect fowl against the subsequent growth and development of the
tumor, whereas growth proceeded regularly in the unprotected con-
trols. Rous makes no claim as to high protective value but that some
such power is developed is undoubted.

The work quoted above is of the utmost importance in establishing
the important principles that must be observed in the preparation of
immune sera against infective agents either known or unknown when
used as antigens in animal tissues. The studies are recent and have not
as yet been widely applied. The immune sera against infections of
undetermined cause to be described in this section were studied before
the work of Rous and his associates appeared, and it is probable that
the methods of preparation may be considerably modified in the course
of time. The inclusion of acute anterior poliomyelitis in this group
is justified only on the ground of dissension as to whether the disease
is due to the globoid bodies described by Flexner and his collaborators
or to the pleomorphic streptococcus studied by Rosenow, Nuzum
and others.

Anti-poliomyelitis Serum. — That one attack of poliomyelitis pro-
tects against subsequent infection has been known for many years.
Levaditi and Landsteiner and also Flexner and Lewis in 1910
demonstrated that the serum of convalescents and of monkeys recov-
ered from the disease protects against infection. Treatment of human
cases of the disease was applied by Netter in 1916. This author
injected intrathecally the serum of recovered patients in doses of 5 to
13 c.c. for a period of eight days with most encouraging results. He
believed that the best serum is found in individuals whose acute attack

EMPLOYMENT OF BLOOD SERUM 269

dates back from three months to four years. Flexner carried out
experiments with monkeys and proved that the serum of recovered
cases was efficacious in the cure of these animals. In 1916-1917 this
author used the serum extensively during the epidemic in New York
and recommends the combination of intraspinal and intravenous in-
jections. Children were given combined doses of 5 to 10 c.c. intra-
spinally and 30 to 40 c.c. intravenously. The possibility of conveying
the disease is not considered a danger, because the virus has never been
detected in the blood. The only difficulty encountered in this method
of treatment is that of securing sufficient quantities of serum. Pooling
of sera is of the greatest advantage, since the antibody content may
vary widely in the sera of different persons.

During the epidemic of 1917 Mathers, Rosenow, Towne and
Wheeler, Nuzum and Herzog, and later Nuzum reported the discovery
of a pleomorphic streptococcus which they had constantly observed in
the brain and spinal cord, and also in the cerebrospinal fluid in human
cases of poliomyelitis. Flexner and Noguchi, Smillie and many others
deny the etiological importance of this streptococcus. Rosenow, Nuzum
and Willy claim to have produced sera with definite protective and cura-
tive effects. In the hands of Nuzum and Willy serum treatment re-
duced the mortality in a series of 159 cases from 38 per cent, to
11.9 percent.

Amoss reported that only imperfect success in developing antibodies
in rabbits and monkeys has attended the repeated injection of cultures
of the globoid bodies of Flexner and Noguchi and also failed to find
evidence that Rosenow's serum is either therapeutically effective in
monkeys or possesses antibodies of the same nature as those present in
the blood of monkeys which have recovered from experimental polio-
myelitis. Since the antibodies in convalescent poliomyelitis serum in man
and monkey are identical, this author states that any antibodies present
in Rosenow's horse serum do not conform to those occurring in human
convalescent serum. Again Amoss and Eberson in a later paper con-
cluded that the anti-streptococcus serum of Nuzum and Willy fails to
show in the monkey neutralizing or therapeutic power against small
doses of the virus of poliomyelitis. Under the same conditions the
serum of monkeys which had recovered from experimental poliomye-
litis proved neutralizing and protective. These facts leave some doubt
as to the actual value of anti-poliomyelitis horse serum, and until
more conclusive evidence has been brought forward by the supporters
of the streptococcus as an etiological factor we believe that the only
effective serum existing is that of convalescent or recovered cases.
Neustadter and Banzhaf immunized horses against a filtrate obtained
from the digested brain and cord of a human case of the disease. The
immune serum gave encouraging results in a few experiments with
monkeys, but as yet data are too limited to justify a conclusion as to
the usefulness of this serum.

Rinderpest. — Kolle and Turner injected gradually increasing doses
of virulent rinderpest blood and also' bile of infected animals into oxen

270 THE PRINCIPLES OF IMMUNOLOGY

and obtained potent sera against the rinderpest virus. Of 3318 animals
treated with this serum 455 or 13.9 per cent, died, while the mortality
among non-treated animals averages between 85 per cent, and 95 per
cent. The serum can be used prophylactically in doses of 100 to 200
c.c. If the virus is simultaneously injected in small doses as advised '
by these authors, the results appear to be extremely satisfactory.
The serum for curative purposes should be employed within thirty days
after the onset of fever.

Anti-hog-cholera Serum. — Immunization against hog cholera has
an important historical as well as a practical bearing since it was in this
disease that the first attempt to immunize with bacterial products was
made. Salmon and Theobald Smith published in 1884 their account
of the production of immune sera in the pigeon by the inoculation of
killed broth culture of the bacillus of hog cholera. Subsequent studies
have made it appear that the disease is not due to the bacillus of hog
cholera and much evidence is at hand to support the view that the
etiological agent is a filterable virus. At the present time immunity is
produced in healthy hogs by the injection of blood obtained from
infected hogs, thus implanting the virus. It is necessary to protect
the animals employed by passive immunization with a previously-
prepared antiserum. The animals selected are injected subcutaneously
with 40 c.c. of anti-hog-cholera serum per hundred pounds of weight.
Two to three days later the animals receive intravenously 3 or 4 c.c.
of defibrinated blood obtained from an animal suffering from the
disease, or the animals may be exposed in infected pens. If the animals
survive, after a period of one month they are given 5 c.c. of the living
virus. This is repeated after two or three weeks. The immunized
animals are bled from the tail. Five cubic centimeters of blood per
pound of weight are usually withdrawn. The protective power of the
serum thus obtained is then determined in a series of hogs. For
prophylactic purposes the" animals receive 40 c.c. subcutaneously per
hundred pounds of weight or simultaneous injections of virus and
serum, but this combination is not without danger. For therapeutic
purposes several injections are necessary and the serum should be
administered as early as possible.

THERAPEUTIC USE OF NORMAL SERUM

Normal serum therapy in man has included the use of both human
and animal sera. In the treatment of natural or experimental disease
in man or animals the normal serum employed may be homologous or
heterologous. The basis of such method of treatment has often been
entirely empirical, but as serum therapy has been more carefully
studied the employment of normal serum may be placed in two cate-
gories, namely that of the non-specific protein treatment of disease
or that of providing the blood with certain elements necessary for the
process of clotting. It is to be conceded that a normal serum may be
employed because of some natural antibodies which it may contain,
but such a form of passive immunization is much improved if the

EMPLOYMENT OF BLOOD SERUM 271

natural antibodies are increased by specific immunization. The use of
normal serum in non-specific therapy probably increases those non-
specific factors of defense such as fever, serum enzymes, etc., that
have already been discussed. In hemophilia, purpura hemorrhagica,
melena neonatorum and similar hemorrhagic diseases there is a disturb-
ance of proper balance of those constituents of the blood and tissues
which provide for coagulation of the blood. Hypotheses differ as to
the exact mechanism of the process of coagulation, but fundamentally
it seems necessary to have an equilibrium of prothrombin and anti-
thrombin. This balance may be upset by an excess of antithrombin,
by a deficiency in prothrombin, fibrinogen, calcium salts or other ele-
ments. The interaction of prothrombin, thrombokinase (or thrombo-
plastin) and calcium salts results in the formation of thrombin.
Thrombin and fibrinogen interact to form fibrin, the essential element
of a clot. Blood serum is rich in prothrombin and if a hemorrhagic
disease be due to prothrombin deficiency, serum treatment is likely to
be beneficial. If, on the other hand, the disease be due to an excess
of antithrombin the introduction of prothrombin has little value.
Similarly hemorrhagic disease with low fibrinogen content is not bene-
fited by serum treatment. Whipple has found decrease of fibrinogen
in advanced cirrhosis of the liver with hemorrhage, excess of antithrom-
bin in aplastic anemia and leucemia and deficiency of prothrombin
in melena neonatorum. Duke holds that the lack of prothrombin
is due to a deficiency in the number of platelets, whereas Minot
and Lee believe that in hemophilia, at least, the slow clotting is due
to a hereditary defect in the platelets which renders them less avail-
able for the process of coagulation. Various studies have given different
results as to the changes found in the elements concerned in clotting.
Whipple points out that if the phenomenon is studied in the individual
case rational therapy may be applied. In melena neonatorum the ad-
ministration of blood serum often gives brilliant results. In other
hemorrhagic diseases the results are somewhat more variable. If
hemorrhage has been severe and anemia is marked, the double purpose
of favoring clotting and replacing lost blood may be served by trans-
fusion from a suitable and properly-tested donor. The more direct
the transfusion the less likelihood is there of alteration of the blood
due to beginning clotting and the greater is the probability of con-
tributing substances to replace or augment those which may be deficient
in the patient's blood.

APPENDIX B

PROPHYLACTIC VACCINATION

INTRODUCTION.

TYPES OF VACCINES.

LIVING VACCINES.
SENSITIZED VACCINES.
KILLED BACTERIAL VACCINES.
PREPARATION OF BACTERIAL VACCINES.
METHODS OF COUNTING.

HEMOCYTOMETER METHOD.
WRIGHT'S METHOD.
OTHER METHODS.
DOSAGE OF ORGANISMS.
LIPOVACCINES.
CONTRAINDICATIONS.
VACCINATION WITH LIVING VIRUS.
SMALL-POX VACCINATION.
PREPARATION.
METHODS OF INOCULATION.
LINEAR INCISION.
DRILL METHOD.
MULTIPLE PUNCTURE.
INTRACUTANEOUS METHOD.
VACCINIA.

IMMUNITY AS THE RESULT OF VACCINATION.
UNFAVORABLE RESULTS OF VACCINATION.
RABIES VACCINATION.

ACTIVE IMMUNIZATION (VACCINATION).
PREPARATION OF MATERIAL.
VACCINATION IN MAN.
EFFECTS OF VACCINATION.
PROTECTIVE RESULTS.
VACCINATION WITH KILLED ORGANISMS.
TYPHOID AND PARATYPHOID FEVERS.
PREPARATION OF VACCINES.
METHOD OF ADMINISTRATION.
PROPHYLACTIC VALUE.
DURATION OF PROTECTION.
COMPLICATIONS.
CONTRAINDICATIONS.

CHOLERA.
PNEUMONIA.
PLAGUE.
TYPHUS FEVER.
PERTUSSIS.
DYSENTERY.
INFLUENZA.
OTHER DISEASES.

VACCINATION

Introduction. — In contrast to the methods of passive immunization,
i.e., the parenteral introduction of immune sera, vaccine treatment aims
to increase the resistance to disease by the injection of the causal
organisms or their products. The duration of this increased resistance
varies in time according to species and types of organisms injected and
the individual characterisics of the subject. For instance, vaccination
272

PROPHYLACTIC VACCINATION 273

against smallpox immunization may last for a considerable number of
years, while with other organisms, such as the staphylococcus or pneu-
mococcus the immunity is of relatively short duration.

The aims of vaccination are either to cause prophylactic resistance
against disease or to increase an already established resistance. Proph-
ylactic vaccination against typhoid is an example of the former, while
the vaccine treatment of furunculosis or gonorrhea are examples
of the latter.

The term vaccine is derived from vaccinia or cowpox, and the method
of protective immunization against smallpox with vaccinia virus was
called by Jenner " vaccination." This great empirical work was placed
on a sound scientific basis by Pasteur after he had discovered the
method of protective inoculation against chicken cholera, and Pasteur
used the term vaccination for such inoculations. To-day the simple
term vaccine is loosely applied and should be restricted to cowpox
vaccine. Suspensions of bacteria such as typhoid bacilli or pyogenic
cocci should be designated bacterial vaccines. Wright defines a bac-
terial vaccine as follows : " Bacterial vaccines are sterilized and enumer-
ated suspensions of bacteria which furnish, when they dissolve in the
body, substances which stimulate the healthy tissues to the production
of specific bacteriotropic substances (or antibodies) which fasten
upon and directly or indirectly contribute to the destruction of the
corresponding bacteria."

Perhaps the first serious attempt to apply practically a bacterial
vaccine in the treatment of human disease was that of Koch, who in
1890 employed tuberculin in the treatment of tuberculosis. In 1893
Frankel treated thirty-seven cases of typhoid fever with subcutaneous
injections of killed typhoid bacilli. He reported that the course of
the disease was favorably modified and in a few instances terminated
by rapid lysis. Rumpf treated a series of cases of typhoid fever with
bacillus pyocyaneus and obtained equally favorable results, thus throw-
ing doubt upon the specific character of the treatment and leading into
the newer field of non-specific therapy. Wright and Douglas soon
after the discovery of opsonins demonstrated their method of treatment
by bacterial vaccines under the guidance of the opsonic index. Wright
stated that a patient who had become infected by an organism such as
the staphylococcus aureus or the tubercle bacillus would be found to
have a lowered resistance against these organisms; that this degree
of want of resistance could be accurately determined, and that the
resistance could be stimulated and controlled by measured doses of
a vaccine of the causative organism. Wright's method of treatment
was based on the principle of strict specificity. It was soon pointed out
that opsonins are only one link in the defensive chain of the host, and
the use of the method has been somewhat restricted. The measure of
opsonins in a given instance was subsequently found not to be a measure
of the existing degree of total immunity. In the majority of diseases,
therapeutic vaccination has not withstood the test of time. Wright
himself, after experiences in the World War, stated that it has been
18

274 THE PRINCIPLES OF IMMUNOLOGY

accepted that the inoculation of microbes into the already infected
system is as illogical as to instil further poison into an already poisoned
body. However, a wide field for prophylactic vaccination is still open.
Soon after Wright's work bacterial vaccines were applied in every
conceivable way and unfortunately much harm has been done to the
rational use of vaccines by reckless commercialism.

Wright and his collaborators have studied carefully the opsonic
index of patients the victims of infectious disease as well as that of
normal individuals. They found that phagocytosis is often depressed
in those who are unsuccessfully combating certain disease and that the
phagocytic power can be increased by specific bacterial vaccination.
They pointed out further that following the first dose of vaccine the
opsonic index is considerably depressed and spoke of this phenomenon
as the negative phase. This phase may last for several days and
numerous writers have thought that such a depression of phagocytic
resistance might indicate such a decrease of general immunity as to
render vaccination during an epidemic highly undesirable. The nega-
tive phase has been carefully investigated and many now believe that
it does not exist. The factor of error in the determination of the
opsonic index is considerable, owing to the variability of conditions
operating in vitro. Therefore, it is possible that the decrease of index
pointed to by Wright may fall within the limit of experimental error.
The recent observations of Balteano and Lupu indicate that no such
negative phase is demonstrable in cholera, and the careful investiga-
tions of Cantacuzene indicate that the negative phase does not occur
in other diseases.

Types of Vaccines. — Living Vaccines. — From animal experiments
it is generally admitted that the greatest and most lasting immunity is
produced by the injection of living bacteria. The killing of bacteria
apparently destroys certain thermolabile substances which possess anti-
genie properties. In human practice the use of living bacteria is not
without danger. One may at first inoculate with a single living organ-
ism and cautiously increase the number, but the virulence of the organ-
ism is not easily controlled and may be so great as to make such
inoculations dangerous. In addition there is a risk of establishing a
" carrier state " since the gradual increase of the number of organisms
may establish a mutual immunity on the part of both the parasite and
the host. If the virus of the disease can be so attenuated that danger
of producing an outspoken attack of the disease is eliminated, vac-
cination can be performed with great success. The outstanding ex-
amples of this method in human medicine are vaccination against
smallpox and against rabies. In smallpox the virus is attenuated
by animal passage through the calf and in rabies the virus is attenuated
by desiccation.

Sensitised Vaccines. — These are bacterial vaccines composed of
"bacteria which have been exposed to their specific immune serum. As
early as 1891 Babes mixed the blood of a highly refractory dog with
an emulsion of street virus in order to produce in other animals a more

PROPHYLACTIC VACCINATION 275

rapid development of immunity against rabies. In the only experiment
reported at this time it was shown that some protection was afforded
by the mixture, although the inoculated animal finally succumbed to
rabies. Lorenz in 1892 made similar observations in swine erysipelas.
Since this time numerous workers have used the method. The most
important advance was made when Besredka suggested the removal
of the excess of serum by centrifugally washing the sensitized bacteria.
Subsequent work has been carried on with killed bacteria treated with
their immune sera, washed and suspended in a suitable menstruum.
The ordinary non-sensitized bacterial vaccines injected into an animal
during the incubation period of a disease are likely to hasten the death
of the animal, or if the infection is already acquired, the injection of
the vaccine appears to lower the natural resistance. Besredka and
Metchnikoff believe that sensitized bacterial vaccines produce no nega-
tive phase, but only slight local and general reactions and facilitate the
production of antibodies. Kakechi has shown that the toxicity of
sensitized bacterial vaccines is less than that of the non-sensitized.
Sensitized bacterial vaccines have been employed in numerous infec-
tious diseases such as typhoid fever, asiatic cholera and bubonic plague
with varying degrees of success.

Killed Bacterial Vaccines. — These are suspensions of bacteria usu-
ally in salt solution but sometimes in other mentsrua such as neutral
oil. The organisms are usually killed after the suspension has been
made, but in making oil suspensions the organisms are killed before
the final suspension. Heat is usually employed for killing the bacteria
and the action is further supplemented by the addition of a bactericidal
preservative to the suspension. Under certain circumstances chemicals
such as formaldehyde or phenol may be employed both for killing and
preserving the vaccine. Autogenous vaccines are bacterial vaccines
prepared from bacteria which have been freshly isolated from the
individual patient. At times it is very difficult to isolate the organism as
for instance in gonorrhea. In these cases stock vaccines are usually
employed. Stock vaccines are made from strains of bacteria isolated
at some previous time aad kept in the laboratory stock. Stock vaccines
are used extensively in prophylactic vaccinations. Mixed vaccines are
composed of various kinds of bacteria. Their value is questionable
and their use unscientific, except on the basis of non-specific therapy.
Many efforts have been made to produce the bacterial antigen in a pure
form so as to obtain a minimum of local and general reaction, and to
immunize in the shortest space of time possible. Such vaccines have
been made from nucleoproteins, autolyzed bacteria, digested bacteria
and detoxicated organisms. It appears that some of these methods are
promising, especially for the production of antigens from spore-
bearing bacteria.

Preparation of a Bacterial Vaccine. — Under strict asepsis an emulsion of
the organism in question is prepared by adding 5 to 10 c.c. of physiological salt
solution to a twenty-four-hour agar slant culture. This is allowed to stand ten
minutes and then rotated actively in order to make a suspension of the organisms.
The suspension is now filtered through sterilized filter paper in a funnel into a

276 THE PRINCIPLES OF IMMUNOLOGY

sterile test-tube. In case of scanty growth the emulsion is directly transferred
to another surface culture, the growth in this tube suspended and the process
repeated with additional growths until a satisfactory 'emulsion is obtained.
Instead of filtering the emulsion one may shake the emulsion in a test tube
containing glass beads to break up the clumps. It is of great importance to have
a homogeneous suspension. Because of the presence of pepton or proteins
from the culture media some authors advise washing of the organisms until the
supernatant fluid gives a negative biuret reaction. The next step in the prepara-
tion is the counting of the -emulsion. This can be done by the hemocytometer
method, by Wright's method and other methods.

Hemocytometer Method. — (From Zinsser, Hopkins and Ottenberg, " A
Laboratory Course in Serum Study.") — A staining solution is prepared by adding
to 20 c.c. of i per cent, phenol i c.c. of a saturated alcoholic solkition of thionin.
A small amount of the carefully shaken bacterial suspension is removed to a
watch glass. A dilution of i-ioo is prepared in a red cell pipette with the staining
solution as diluent to the 101 mark. After carefully shaking and after blowing
out the portion of the fluid in the capillary end of the pipette a small drop is
placed in a counting chamber and covered with a flat coverslip. After allowing
fifteen minutes for the bacteria to settle a count is made, with 4. m,m. objective,
of a number of squares until 200 or more bacteria have been counted. It is best
to take this count from different portions of the ruled surface and from two
separate drops of the mixture. The small squares have an area of 1/400 of a
square mm., the depth of the chamber is o.i mm., the dilution is l-ioo. The
number of bacteria may be estimated by the following formula :

Number of bacteria counted X 400 X 10 X 100 X 1000

— 3 . = number of bacteria in i.o c.c.

Number of squares counted

Wright's Method. — A drawn-out capillary pipette is prepared and marked
with a grease pencil about 2. cm. from the tip. A small puncture is made in the
tip of the finger and a fresh drop of blood obtained. Three units of salt solution
are then drawn up in the pipette, admitting a bubble of air between each unit of
salt solution. The unit is the amount that is drawn up to the mark on the
pipette. Blood from the finger-tip is then drawn up to the mark, a bubble of air
admitted and the bacterial suspension drawn up to the mark. The mixture is then
blown out on a clean slide and drawn in and out of the pipette several times to
ensure even mixing of the blood and bacteria. A drop of this mixture is placed
on a second slide and carefully spread across the slide in the manner of making
blood smears. It is important that the film be thin and even, so that the ^ red
cells are not piled in masses in any portion of the film. This film is stained
with Wright's stain, or by any other simple method, and a differential count of the
number of bacteria and red cells in a number of fields in different parts of the
slide is made. For this a rule scale to be inserted in the eyepiece of the micro-
scope is very helpful. Fields are counted until 200 red cells have been counted.
The number of bacteria in the suspension may then be estimated from the number
of bacteria counted, using the following formula (assuming that the blood of the
worker contains 5.000,000 red cells per cmm.) :

Number of bacteria X 5,000,000 X i.ooo XT e ,

=r= — r P ; — TS — 7 v — = Number of bacteria per c.c.

Number of red cells (200)

Other Methods. — Among the other methods of standardization of the sus-
pension are the comparison of the emulsion with a known standard emulsion, the
estimate of the average number of organisms per slope grown in, say, eighteen
hours, or an estimate of the number of germs per loopful (Kolle's method).
Hopkins centrifugalized his suspension at high speed in a special tube with
graduated tip until the supernatant fluid was clear. The number of organisms
for a number of species in such a closely-packed sediment has been determined
and is as follows :

Staphylococcus aureus o.oi c.c. equals 10 billion

Streptococcus hemolyticus o.oi c.c. equals 8 billion

Gonococcus o.oi c.c. equals 8 billion

Pneumococcus (capsulated) o.oi c.c. equals 2.5 billion

B. typhosus o.oi c.c. equals 8 billion

B. coli o.oi c.c. equals 4 billion

PROPHYLACTIC VACCINATION 277

Gates recently standardized his bacterial suspension by measuring the opacity
of the suspension by the length of the column of the suspension required to cause
the disappearance of a wire loop. By a simple formula the measured opacity is
translated into terms of the concentration of bacteria per cubic centimeter and so
made comparable with that of other suspensions of the same organism.

The stock suspension after estimation of the number of organisms contained
is ready for dilution. Shera employs the following method for dilution. Suppose
the suspension is found to contain 6400 million organisms per cubic centimeter,
and that a vaccine of 1000 millions per cubic centimeters is required. Five cubic
centimeters are measured out accurately after shaking well, and they are made
up to 6400/1000 parts, i.e., 6.4 parts. Multiply 6.4 x 5 and the result 32 equals the
volume in cubic centimeters to which the 5 c.c. should be made up. The suspension
is sterilized by means of heat. For staphylococci and streptococci 59° to 60° C.
for half an hour is sufficient; for typhoid bacilli 50° to 56° C. for an hour is
usually employed. It is best to add some preservative as phenol or tricresol (0.3
to 0.5 per cent.) to the suspension and to have the suspension in sealed ampoules
preferably of brown glass before immersing in the water bath. Connor success-
fully sterilized his staphylococcic vaccines by means of fluorides. After steriliza-
tion an ampoule should be opened so that a culture may be made. No vaccine
should be used until a culture is found to show no growth. If ampoules are not
at hand they may be made from test tubes or the vaccine may be kept in sterile
bottles with rubber stoppers or caps.

Dosage of Organisms. — For gonococci, bacillus coli, streptococci
and pneumococci 5,000,000 to 50,000,000 are usually employed, while for
staphylococci 200,000,000 to 1,000,000,000. Wilson gives the following
minimum and maximum doses : Streptococcus, 6 and 68 millions ;
staphylococcus, 150 and 900 millions; gonococcus, 45 and 900 millions;
meningococcus, 300 and 900 millions; micrococcus melitensis, 700 and
1400 millions; bacillus coli, 16 and 240 millions; bacillus typhosus for
treatment, 100 and 250 millions; bacillus typhosus for prophylaxis, 500
and 1000 millions; bacillus pyocyaneus, 34 and 1000 millions; bacillus
pneumoniae, 44 millions; bacillus tuberculosis, 1/2000 to 1/200 mg.

Lipovaccines. — Recently LeMoignic and Sezary showed that it is
possible to obtain as highly hemolytic serum by injecting red cells sus-
pended in oil as by injecting them suspended in salt solution. They also
showed that the oil suspension gives slow absorption, and that the oil acts
as a detoxifying agent. As an example of the rate of absorption it was
shown that the injection 0.35 mgm. of strychnine in aqueous solution
kills a guinea-pig, but the injection of six times that amount is harmless
if the strychnine be dissolved in oil. This led LeMoignic and Pinoy,
Achard and Foix, and LeMoignic and Sezary to suspend bacterial
vaccines in oil, and to inject the entire vaccinating dose at one time.
Bacterial vaccines suspended in saline are rapidly autolized. As
autolysis advances, absorption following injection becomes more rapid
and the immediate reaction more severe. Oil vaccines are preserved
much more easily than saline vaccines and the reactions following
their injection are less severe. The oil vaccines are known as " lipo-
vaccines." The bacteria have been suspended in lanolin, lecithin,
sperm oil and many vegetable oils. Cotton-seed oil is at present widely
used. It is of great importance to use neutral oils. The sterilization
of these oils has been a difficult problem and a drawback in the prepara-
tion of the vaccines. The technic must be strictly aseptic. At present
lanolin and oils are sterilized in the autoclave at fifteen pounds for

278 THE PRINCIPLES OF IMMUNOLOGY

fifteen minutes. Ultra-violet rays have been used. Chlorine has also
been employed, but the resulting hydrochloric acid is difficult to remove.
Whitmore and Fennel used powdered potassium iodid. This was added
to olive oil and sweet almond oil; iodin was liberated in sufficient
amount to sterilize the oil, and was taken up in the oil molecule so that
no free iodin could be detected. Sweet almond oil is sterilized in about
three days, but it requires about ten days to sterilize olive oil. It is not
known, however, how the suspension in oil affects the antigenic power
of the vaccine, but certain workers claim to get better results than by
the use of saline vaccines. Against the use of lipovaccines is the
possibility of fat embolism from accidental entrance of the vaccine
into a vein, but Graham considers this factor of minor importance since
the amount of oil is small. He injected as much as 0.8 c.c. of oil into
the ear vein of a rabbit and observed only a slight passing dyspnea
and no other evidence of discomfort. Care should be taken, however,
to administer the vaccine subcutaneously and to avoid veins. Drawing
out the plunger of the syringe after the needle has been introduced
determines whether or not an important vein has been entered. The
upper arm beneath the insertion of deltoid muscle is usually selected
for the injection. The region of the scapular or pectoral muscles
may do as well.

Contraindications. — In prophylactic immunization it is of im-
portance to ascertain whether the patient has latent or active
infection. In active tuberculosis vaccination is considered danger-
ous. Caution should be observed in diabetes, parenchymatous nephritis
and carcinoma.

VACCINATION WITH LIVING VIRUS

Smallpox Vaccination. — Although inoculation with the virus in
smallpox in an attempt to produce a mild attack of the disease had
been practiced for centuries and although for many years it had been
observed that an attack of cowpox rendered man immune to smallpox,
it remained for Jenner in 1796 to furnish the scientific proof of the
efficacy of vaccination with cowpox in the prevention of smallpox.
Jenner's publications were so convincing that the method soon attained
widespread use and was introduced into America in 1800 by Dr. Benja-
min Waterhouse, of Boston. The work of the latter investigator was
especially well conducted and convincing. In 1894 Copeman demon-
strated the protection of monkeys against smallpox by vaccination with
cowpox, and this was subsequently confirmed by Brinckerhoff and
Tyzzer. The introduction of vaccination following Jenner's publication
immediately led to marked reduction in the incidence of this disease
and its mortality. The table on page 279 (taken from O'Connell, " Vac-
cination; What It Is, etc.," circular New York State Department of
Health, 1908) gives a clear indication of the reduction of mortality.

Up until fairly recent times vaccination was practiced by inocu-
lating patients with the fragments of the crust obtained from others
who had been successfully vaccinated. This method has been aban-

PROPHYLACTIC VACCINATION

279

doned because of the possibility of transferring infection in the crusts,
not the least important of which is syphilis. With the development of
important Board of Health laboratories and large commercial lab-
oratories it is now possible to secure cowpox virus in a form free
from infective organisms.

DEATH-RATE FROM SMALLPOX, AVERAGE PER 1,000,000 INHABITANTS.

Before vac-
cination

After vaccina-
tion

Territory

Smallpox death rate, per 1,000,000 pop.

Before vaccination

After vaccination

1777-1806

1807-1850

Austria (lower)

2,484

340

1777-1806

1807-1850

Austria (upper and

Salzburg)

1,421

501

1777-1806

1807-1850

Trieste

14,046

182

1777-1806

1807-1850

Tyrol and Vorarlberg

911

170

I777-l8o6

1807-1850

Bohemia

2,174

215

1777-1806

1807-1850

Moravia

5402

255

I777-l8o6

1807-1850

Silesia (Austrian)

5,812

I98

1776-1780

1810-1850

Prussia (East Prov-

ince)

3,321

556

1780

1810-1850

Prussia (West Prov-

ince)

2,272

356

1776-1780

1816-1850

Westphalia

2,643

114

1776-1780

1816-1850

Rhenish Provinces

908

90

1781-1805

1810-1850

Berlin

3,422

I76

I774-I80I

1810-1850

Sweden

2,050

158

I75I-I80O

1801-1850

Copenhagen

3,128

286

The Preparation of Smallpox Vaccine. — For this purpose cow-
pox is produced in young heifers from two to four months old. The
animals are taken from selected stock, carefully tested for the presence
of tuberculosis and observed for several days so as to< ensure perfect
health. The body is cleansed and the abdominal surface shaved from
the ensiform cartilage to the pubis, extending the area out on the
flanks and the inner surface of the thighs. The skin is washed with
soap and water, then with alcohol and finally with sterile water. About
100 small cuts through the epidermis are made under strictly aseptic
precautions. If bleeding occurs the blood is carefully wiped away.
Virus may be obtained primarily from smallpox patients who are
otherwise healthy. At the present time, however, the virus kept as
" seed virus " is obtained from previously inoculated animals. Virus
is introduced into the scarifications usually in the form of a glycerol
suspension. In about forty-eight hours a reaction appears and by the
sixth day the vesicles are well filled with semipurulent material. The
animal is killed and the vesicles carefully curetted away. After the
curettage, serum appears and this may be preserved in ampoules or
small tubes for subsequent vaccination. The pulpy mass obtained
by curettage is mixed with four times its weight of a mixture com-
posed of glycerol 50 per cent., water 49 per cent., phenol i per cent.
The glycerolated pulp is allowed to stand three or four weeks in order
to destroy any contaminating bacteria. The pulp is then triturated in

280 THE PRINCIPLES OF IMMUNOLOGY

special machines and sealed in capillary tubes. Formerly " ivory "
vaccine points were also charged from this pulp, but these have been
forbidden in interstate commerce (page 282). In all cases the material
before being prepared for distribution is carefully tested for the
presence of tetanus bacilli or their spores. Its potency may be deter-
mined by directly inoculating the inner surface of the ears of rabbits
and observing the rapidity of the reaction. A somewhat superior
method is to make dilutions of the virus and to note the effect of these
dilutions when inoculated on the ears of rabbits. A potent virus should
produce vesicles in a dilution of i to 500. Efforts have been made to
secure a virus in purer form and Noguchi has planted the virus in the
testicles of rabbits and of bulls. Virus recovered from this situation
is not subject to contamination in the same way as that obtained from
surface inoculations. The amount of material obtained, however, is
small and the method has not been used extensively enough to justify
an opinion as to its value. After preparation of a virus the date
should be indicated on the container and the material preserved in
the ice chest.

Methods of Inoculation in Man. — As a rule, vaccination is applied
on the upper arm over the point of insertion of the deltoid muscle.
This situation offers protection against injury and contamination such
as is not afforded by vaccination upon the leg or thigh. The area is
carefully cleansed with soap and water, followed by alcohol or ether
and then by distilled water. The last step is sometimes omitted. For-
merly the area was scarified in a criss-cross manner by means of a
needle or scalpel, but such extensive scarification has been found to
be unnecessary and also exposes a greater surface to the possibility
of infection. The more modern method is to place the virus upon the
area and to make a scarification through the virus. This may be done
by a small linear incision, by the drill method or by the multiple punc-
ture method. Wright has advised intracutaneous inoculation.

Method of Linear Incision. — After placing the virus upon the skin
a sterile needle or a small scalpel is employed for making a scarification
through the virus and sufficiently deep into the skin to permit absorption
but not to produce bleeding. The virus is then gently rubbed into the
abrasion and permitted to dry. If a dressing is desired it should be of
sterile gauze loosely applied with adhesive strips after the virus has
completely dried. Sealing with collodion should not be attempted,
since it may permit more ready growth of contaminating bacteria
and produce maceration of the skin.

The Drill Method.— A sterile drill such as is employed in the Von
Pirquet cutaneous tuberculin test is held between the thumb and middle
finger. With a twisting motion and moderately firm pressure a small
abrasion the diameter of the drill is made through the virus. This
should penetrate the epiderm, but should draw no blood.

The Multiple Puncture Method. — A sterile needle is held nearly
parallel with the skin and the point placed through a drop of virus so as

PROPHYLACTIC VACCINATION 281

to make an oblique puncture of the epidermis. This is repeated so as
to produce about six radially disposed punctures, the whole area ex-
tending not more than about 5 mm.

The Intracutaneous Method. — The virus is diluted to ten times its
volume with distilled water and injected intracutaneously by means
of a sterile tuberculin syringe and a fine needle. Two injections about
2 cm. apart are made.

All the methods indicated have given equally good results, but
convenience usually dictates the use of the linear incision or the drill
method. It is not uncommon in the use of any of these methods to
make two or three inoculations.

Vaccinia. — Following the inoculation of the virus the areas usually
remain quiescent for from two to four days when slight reddening and
itching may develop. Following this a small papule appears, rapidly
succeeded by the vesicle. It is important to note that the vesicle is
umbilicated and that its multilocular character is indicated by the
minute vesicular arrangement of the margin. The vesicle appears in
from five to six days, rapidly becomes pustular and is followed by the
formation of the crust. The crust is allowed to drop off and subse-
quent observations of the scar should show a smooth center, a somewhat
scalloped edge and more or less discrete minute marginal scars. During
the height of the local reaction the patient may complain of malaise,
headache, fever, constipation and other general symptoms. The reac-
tion of vaccinoid has been discussed in the chapter on Hypersuscep-
tibility (page 243).

Immunity as the Result of Vaccination. — The extent of immunity
has been indicated by the decrease in prevalence of smallpox since the
introduction of vaccination. It may also be measured by the success
of subsequent vaccinations. Kitasato has revaccinated a series of 931
cases with successful results as follows :

After i year 14 per cent. After 6 years,64 per cent.

After 2 years 33 per cent. After 7 years 73 per cent.

After 3 years 47 per cent. After 8 years 80 per cent.

After 4 years 57 per cent. After 9 years 85 per cent

After 5 years 51 per cent. After 10 years 89 per cent.

It will thus be seen that more than 50 per cent, of individuals are
susceptible to revaccination four years after the original vaccination.
Millard states that the Government reports of the German Confederacy
show 91 per cent, to 93 per cent, successful revaccinations in ten years
or more after the primary vaccination and concludes that " immunity
acquired through vaccination begins to disappear at about the second
year and by the tenth year it disappears almost completely." Other
investigators have obtained similar results. King reported that in
ninety-six adults who had suffered from smallpox at various ages and
showed numerous scars of the disease, vaccination was successful in
75 per cent. These figures indicate that the older conceptions of the

282 THE PRINCIPLES OF IMMUNOLOGY

durability of the immunity produced by vaccination are inaccurate.
In order to secure satisfactory immunity, vaccination should be re-
peated at intervals of a few years. In those communities where small-
pox is endemic vaccination should be repeated every year. In the
presence of epidemics, an unsuccessful vaccination should not be inter-
preted as indicating immunity and should be repeated at intervals of a
week or ten days until successful. We feel that no dependence can
be unqualifiedly placed on the signs of immunity as indicated by
Force (page 243).

Unfavorable Results of Vaccination. — If human virus be employed
the chance of inoculating syphilis must be considered, although the
danger is slight. Reports of tetanus following shortly after vaccina-
tion have not been particularly well founded and examination of a
large number of samples collected by the Hygienic Laboratory in
Washington by McCoy and Bengston failed to demonstrate the pres-
ence of the bacilli or their spores in filled capillary tubes, seed vaccine
or in bulk glycerolated vaccine. " Ivory points " were found to be
contaminated as delivered from the manufacturer of the points, as well
as after sterilization and charging. McCoy states that " the sale of
vaccine virus on or with points in interstate traffic has been prohibited
by an order of the Secretary of the Treasury.'*

The most important source of trouble is the result of vaccination in
unclean skin, the use of unclean dressings or other failures of asepsis,
more particularly those resulting from carelessness on the part of the
patient. Such infections usually remain localized but confuse the
interpretation of results and may in rare instances become gen-
eral infections.

Vaccination Against Rabies. — The cause of rabies is probably a
sporozoan parasite discovered by Negri and named by Calkins " neuro-
ryctes hydrophobise." Work with this parasite is difficult because of
failure to isolate the organism in suitable form. Therefore, the investi-
gations have been conducted with pathological material containing the
organism. It is found in greatest amounts in the nervous system and
accordingly the brain or cord is selected for experimental work. This
material is spoken o<f as the virus of rabies. Street virus is nerve
tissue obtained from an animal suffering with the natural disease. It
is extremely variable in virulence, and for this reason is not employed
for vaccination of man. Fixed virus is usually the spinal cord of
rabbits obtained after a long series of rabbit passages. By these animal
passages the virulence increases and the incubation period decreases
until a point is reached when the incubation period following inocula-
tion cannot be further shortened. All mammals are susceptible to rabies
in different degrees, but birds or reptiles are not susceptible.

The treatment of rabies in man after it has developed has been
entirely unsatisfactory by the methods of immunology. Immunization
of animals to the rabies virus produces an immune serum capable of
killing the virus. Accordingly it was hoped that such a serum could

PROPHYLACTIC VACCINATION 283

be employed for human rabies, but results attendant upon this method
of treatment have been unsuccessful. Therefore, at the present time,
efforts are directed toward producing an active immunity in those who
have been exposed to the disease. It is of interest to note that laboratory
inoculations in man rarely, if ever, lead to the development of the
disease. It is probable that in order for infection to occur the virus
must be implanted with animal sputum or some other form of con-
tamination. Bites, from rabid dogs are relatively infrequent, and it is
therefore unnecessary to immunize an entire population. Further-
more, man is somewhat resistant to infection with rabies. Statistical
evidence in regard to the frequency with which rabies follows the bites
of rabid dogs are unreliable because of uncertainty as to whether or
not the animal was rabid. Doebert found that in Prussia, where data
had been very carefully collected, there was a mortality of 14.8 per
cent, in 122 untreated persons bitten by rabid animals between the years
1902 and 1907. Other estimates conform closely to this. More recently,
however, Marx has expressed the opinion that the rate of mortality
probably does not exceed 6 per cent, to 10 per cent, of untreated
bitten persons. The mortality and morbidity rate are practically identical.
Fortunately the period of incubation of rabies is of sufficiently long
duration so that active immunization may be effected during the period
of incubation.

The period of incubation in man is variable and depends to a con-
siderable extent upon the site of the bite or scratch. According to
Bauer, the average period of incubation in 510 cases was seventy-two
days. In very rare cases the period of incubation may be less than
nineteen days and in more rare instances it may be one year or more.
Of seventy- three cases of bites about the head and neck the average
incubation was fifty-five days; of 144 cases of bites on the upper
extremities the average period was eighty-one and one-half days ;
and of seventeen cases of bites on the lower extremities the average
period of incubation was seventy-four days.

Active Immunization. Preparation of Material. — As has been
indicated above it is necessary, because of the failure of passive
immunization, to produce an active immunization. In spite of the fact
that laboratory accidents practically never lead to the development of
rabies it is considered dangerous to inoculate man with the living virus.
Ferran and subsequently Proescher have, however, employed a method
whereby the active fixed virus is employed. Both these investigators
stated that no accidents had followed the use of unmodified fixed virus.
Hogyes has successfully employed dilutions of fresh fixed virus. The
majority of investigators, however, have employed virus which has
been attenuated by a variety of methods including heat, partial diges-
tion, the action of bile, the action of glycerol, of anti-rabic serum, of
phenol and of mechanical disintegration. Nevertheless, the original
method of Pasteur is employed almost uniformly throughout the world.
For this purpose the virus is passed through rabbits until it acquires

284 THE PRINCIPLES OF IMMUNOLOGY

its minimum period of incubation. The material is introduced into the
anesthetized rabbit by subdural inoculation. The injection is made
through a small trephine opening just back of the eye and to one side
of the median line. The injected material is ground with a small
quantity of I per cent, phenol solution and 0.2 c.c. of this emulsion is
injected. After the rabbit is completely paralyzed it is killed with
chloroform and the spinal cord removed aseptically. A small ligature
is placed around one end of the cord and the cord hung in a sterile
bottle in the bottom of which has been placed sticks of potassium
hydrate. The bottle is placed in an incubator maintained at 22° to 23° C.
Pieces I. cm. in length are cut off at daily intervals and placed in glycerol
where the degree of virulence on that particular day is retained for
several weeks. In large laboratories animals may be killed on suc-
cessive days and the whole cord employed in preparing the material
for human protection. In the United States Hygienic Laboratory
pieces 0.5 cm. in length emulsified with 2.5 c.c. of salt solution serve
for one injection.

Inoculation in Man. — The determination as to who shall receive
anti-rabic treatment is often difficult, but skilled veterinarians are
able to diagnose rabies in dogs almost invariably. Knowledge of the
condition of the animal inflicting a bite is of the utmost importance.
Although cats and rats are not uncommonly victims of rabies, this is
not frequently a source of infection in man. When a dog bite is re-
ceived, the animal should be captured and observed for at least two
weeks, during which time the symptoms of rabies become manifest.
If the animal is killed the brain should be sent in glycerol to the nearest
laboratory, where it may be examined for Negri bodies. If these are
not found, material should be injected into rabbits. Negative findings
in regard to Negri bodies in the dog's brain are not to be accepted as
evidence. In our opinion it is wise to administer treatment to all
individuals who have been bitten by animals showing any signs of
rabies. The material may be supplied to the physician either in the
form of small pieces of cord to be emulsified in salt solution or in the
form of an emulsion for dilution with salt solution. The injections
are given subcutaneously under the skin of the abdomen. If a con-
siderable time has elapsed since the bite or if the bite has been inflicted
upon the head or neck the so-called intensive method of treatment is
adopted. Under other circumstances the mild treatment may be given.
When material is requested from a commercial laboratory or a state
laboratory it is necessary to indicate which form of treatment is desired.
As an example of the two methods, the scheme of treatment as shown on
page 285, adopted by the New York City Board of Health, will serve.

The Effects of Treatment.— Local reactions are frequent and are
likely to be severe about the eleventh and nineteenth days of inocula-
tion. These are urticarial in character and the more severe reactions
may be accompanied by mild constitutional symptoms. The glycerol
contained in the emulsions not infrequently produces severe pain for

PROPHYLACTIC VACCINATION 285

a few moments at the site of inoculation. The treatment, although
practically safe, is not entirely free from danger. Remlinger in a
study of 107,712 cases that had received treatment found forty cases
which developed paralysis of the extremities and two of these ter-
minated in death. The cause of this paralysis is not clear. Certain
authorities maintain that the virus contains a toxin and that this may
lead to lesions of the nerves. The mass of evidence, however, is
against rather than in favor of the conception that toxin plays any
important part in the virus of rabies. It is also possible that the
repeated injections of foreign protein may have some influence. Such
accidents are extremely rare and should not interfere with a decision
concerning administration of the treatment.

SCHEME OF TREATMENTS.
Day Mild treatment Intensive treatment

ist 14 and 13 day cord 12 and n day cord, repeat

in afternoon

2nd 12 and u day cord 10 and 9 day cord; 8 and 7

day cord in afternoon

3rd 10 and 9 day cord 6 day cord

4th 8 and 7 day cord 5 day cord

5th 6 day cord 4 day cord

6th 5 day cord 3 day cord

7th 4 day cord 2 day cord

8th 3 day cord 4 day cord

9th 2 day cord 4 day cord

loth 4 day cord i day cord

nth 3 day cord 4 day cord

I2th 2 day cord 3 day cord

I3th 4 day cord 2 day cord

I4th 5 day cord 4 day cord

I5th 2 day cord I day cord

i6th 4 day cord 4 day cord

I7th 3 day cord 3 day cord

i8th 2 day cord 2 day cord

I9th 4 day cord 4 day cord

2oth 3 day cord 3 day cord

2ist 2 day cord 2 day cord

Results of Treatment. — The benefits of this form of treatment
depend to a certain extent upon the time when the injections are begun
and also to a certain extent upon the situation of the bite. Granting
that fatalities occur in from 6 to 16 per cent, of untreated bitten indi-
viduals, the reports of fatalities in from .46 per cent, to 1.25 per cent,
of treated cases show markedly beneficial effects. More recent statistics
are highly encouraging. During the year 1916 Viala reported that 654
persons were treated at the Pasteur Institute with but one death.

VACCINATION WITH KILLED ORGANISMS

Vaccination Against Typhoid and Paratyphoid Fevers. — Al-
though various investigators had appreciated the possibility of active
immunization against typhoid fever, this subject was first placed on a
practical basis by Wright in 1896. In the subsequent year Wright
and Semple described in detail a satisfactory method for vaccination.

286 THE PRINCIPLES OF IMMUNOLOGY

They employed broth cultures of bacillus typhosus two to three weeks
old, killed by heating to 63° C. for one hour and preserved with 0.5
per cent, phenol. The vaccine was treated for sterility, standardized
and employed in doses of 750 to 1000 million organisms. In the same
year Pfeiffer and Kolle reported the demonstration of specific anti-
bodies following the immunization of man against the organism. Since
that time vaccines have been prepared in a large variety of ways and
preventative vaccination is now upon a highly satisfactory basis. Vac-
cination has been employed in military and civil life and has resulted
in a marked decrease in morbidity and mortality. The results obtained
in all civilized countries constitutes one of the greatest achievements
resulting from the study of immunology.

Preparation of Vaccines. — The organisms may be grown in broth or
upon agar. The broth culture or a salt solution suspension of an agar
culture may be killed or attenuated. The application o>f heat or chemi-
cals for the purpose of killing the organisms reduces in a certain
measure their antigenic value. If they are dried before being heated,
temperatures of 120° to 150° C. reduce the antigenic property very
little. Gay, however, points out that the measure of the antigenic value
depends upon the determination of different antibodies such as agglu-
tinins and bacteriolysins, but he notes that this offers "an indication
rather of the reaction of the animal body than a sure means of deter-
mining the degree of protection that has actually been afforded."
Numerous investigators have suggested the use of living bacteria, but
the knowledge that typhoid fever may exist as a septicemia without
intestinal lesions offers an objection, to the introduction of living or-
ganisms. It has been found extremely difficult to attenuate without
killing the bacteria, but it has been recommended that low degrees o>f
temperature, for example 53° C. (Leishman), the use of ether, alcohol,
various sugars and other chemical and physical agents may kill the
organisms without markedly reducing the antigenic properties. Certain
investigators have also suggested the employment of bacterial extracts,
but this method has not been widely employed. Gay and his collab-
orators (page 301) have claimed success in the therapeusis of typhoid
fever by the use of sensitized vaccines and have found that active
immunization progresses very satisfactorily, according to measurements
of specific antibodies, yet this method, if employed for vaccination,
is expensive and probably does not give sufficiently superior results to
justify its employment in large numbers of individuals,

It is now recognized that the typhoid bacillus may be divided into
a number of strains on the basis of cultural and immunological prop-
erties. In certain countries, including the United States, a single strain
of the organism has been employed for vaccination, but in others a poly-
valent vaccine has been employed, the French using ten strains, The
organisms are grown on large agar surfaces, emulsified in salt solution
and killed by heat. They are then standardized and a preservative,
such as phenol, lysol or formaldehyde, added. Twenty-four hours sub-

PROPHYLACTIC VACCINATION 287

sequently cultures are made to determine the sterility of the vaccine.
Standardization is usually on the basis of 1000 million organisms per
cubic centimeter. If it be desired to give a smaller number of organisms,
fractions of a cubic centimeter may be employed. It is the practice in
commercial houses to place specified doses in small ampoules so that
the physician may administer for each dose the contents of a single
ampoule. In military practice the vaccine is placed in small bottles with
a rubber cap so that a needle may be thrust through the cap and the
required amount of vaccine withdrawn into a syringe.

As the paratyphoid fevers have been studied, it has been considered
advisable to vaccinate against these at the same time as against typhoid
fever. Therefore, vaccines are now prepared containing the bacillus
typhosus, bacillus paratyphosus A and bacillus paratyphosus B. It has
been customary to introduce smaller quantities of the paratyphoid
bacilli so as not to increase to an unfavorable degree the bulk of for-
eign protein injected. Accordingly for each 1000 million typhoid bacilli
there are usually added 500 million each of paratyphoid A and B. The
actual numbers, however, vary in different countries. Castellani rec-
ommends the addition also of cholera vibrios. This transforms the
triple vaccine into a tetra vaccine. In northern latitudes this is not of
particular importance.

As has been indicated, the organisms are usually suspended in salt
solution, but recently neutral oil, such as commercial cottonseed oil,
has been employed for suspension. For such suspension the organisms
must be very carefully dried before being emulsified in the oil. These
lipovaccines have the advantage of being administered in one dose and
of producing little or no reaction. They produce immunity following
a single injection because of the slow absorption of the oil and its
contained antigen.

Method of Administration. — In the case of the lipovaccines a single
large dose of organisms may be administered. The use of the salt
solution suspensions involves several injections. As a rule, the first
dose contains 500 million typhoid bacilli and 250 million each of para-
typhosus A and B. The second and third doses contain 1000 million
typhoid bacilli and 500 million each of the paratyphoid bacilli. The
time between injections has been the subject of considerable study, but,
as a rule, a period of seven to ten days intervenes between these injec-
tions. Subcutaneous administration is practically universal. Intra-
venous injections have been recommended, but this method is not widely
practiced. Lumiere and Chevrotier have administered by mouth gela-
tine-coated pills of a dried mixed polyvalent typhoid colon vaccine. It
is probable that this method is not effectual, since the bacterial protein
must undergo at least partial digestion in the intestinal tract. Bes-
redka, however, has recently demonstrated in animals the possibility
of successful vaccination through the intestinal tract, but his animals
had previously been given bile, and it seems likely that this substance
produced sufficient lesion of the intestinal mucosa to permit of
direct absorption.

288

THE PRINCIPLES OF IMMUNOLOGY

Prophylactic Value of Vaccination. — It can readily be understood
that the control of individuals in armies offers excellent facilities for
determination of the prevalence and mortality of infectious disease.
Consequently, much of the statistical evidence favorable to typhoid
vaccination has been collected in armies. The following table, taken
from Gay, "Typhoid Fever," illustrates the prevalence of typhoid fever
in Great Britain and her colonies before vaccination was introduced :

MORBIDITY AND MORTALITY FIGURES FROM TYPHOID FEVER PER 100,000 OF ENGLISH
TROOPS IN THE YEAR 1898 IN DIFFERENT LOCALITIES.

Locality Morbidity Mortality

Great Britain 120 24

Gibraltar 420

South Africa 3290

India 3600

Egypt 8100

132

577

IOOO

2340

Gay states that even greater rates of typhoid morbidity have been en-
countered. The results of anti-typhoid vaccination are splendidly
summarized in another table from Gay's work :

RESULTS OF ANTI-TYPHOID IMMUNIZATION IN THE ENGLISH ARMY.
AND MORTALITY PER 100,000.

MORBIDITY

Locality

Vaccinated

Unvaccinated

Authority

No.

Morbid.

Mortal.

No.

Morbid.

Mortal.

India 1900

I050I

5473
58481

10378

914
380
260

539

161
36
29

40

83135
6610
10794

8936

I665
2830
1390

3040

444
390
220

500

Wright.
Leishmann.
Firth.

Report of the
anti- typhoid
committee,
London, 1913

India 1909
India 1910
Various colonies

IQI7. .

The results obtained in the United States Army under the direction
of Colonel Russell and his staff have been most impressive. In Decem-
ber of 1919 Colonel Russell summarized the results in a paper in the
Journal of the American Medical Association. He gives an analysis
of a water-borne epidemic in Hawaii as follows :

TYPHOID EPIDEMIC IN HAWAII, H. T., IN THE FALL OF 1917.

Population No. of

on Castner cases

water of

system typhoid

Vaccinated 4087 55

Unvaccinated 812 45

Cases

per
thousand

1345

55-41

Mortality

Deaths rate

Number Per cent. per

thousand

74

15-5

o.97
8.62

It is of importance to note in reading this table the large number
of vaccinated as contrasted with the Unvaccinated. It is apparent that
the vaccinated show not only a reduced morbidity percentage, but also
a diminished mortality rate. Colonel Russell gives the following table
of figures from the United States Army for nineteen years :

PROPHYLACTIC VACCINATION

289

RATE OF TYPHOID FEVER IN THE ARMY AND IN THE CORRESPONDING AGE GROUP
IN CIVIL LIFE FOR THE PAST EIGHTEEN YEARS.

Year

No. of cases

Ratio per
thousand

Deaths

Ratio per
thousand

Total

Males*

IQOO
IQOI
1902

IQOt

531

594
565

^48

575
943

8.58
5.82

60
78
69
3°

0-43
0.64
0.86
0.28

0.46
0.42
0.40
0.35

0-54

IQO4. . .

247

5.62

12

0.27

0.33

1905
IQO6. .

193
747

3-57
5.66

17

15

0.30
0.28

0.32
0.32

IQO7. .

208

3-53

16

0.19

0.28

1908
IQOQ

215

177

2.94
^..OT,

21

16

0.23
0.28

0.28
0.23

I9IO

I9nf
1912

IQIV •

142

44

18
4

2.32
0.85
0.31
0.04

IO

6

3
o

0.16
0.09
0.04
o.oo

0.27
0.23

0.18
0.18

0-34

I9H
1915
IQl6

8

2S

0.07
0.08
0.2^

3
o

7

0.03

0.00

0.03

0.15
0.18

O.I2

0.17
O.I 5

IQI7

2Q7

0.44

2^

O.OT,

O.II

O.I4

1918

768

0.30

133

0.05

O.09

O.II

* Indicates voluntary vaccination against typhoid.

t Indicates compulsory vaccination against typhoid.

t Civil deaths from typhoid fever; age group, 20 to 29 years. Rate per thousand of population.

The marked change after the introduction of compulsory vaccination
in the Army in 1911 is most striking. It is pointed out that the increase
in 1917 is in large part contributed to by delay in the vaccination and
sanitary control of National Guardsmen. As an impressive contrast
the following table illustrates the vast improvement in health condi-
tions as compared with previous wars :

RELATION OF MORTALITY IN THE WORLD WAR TO THAT OF PREVIOUS WARS.

Number of deaths

that occurred in
the World War,
Sept. I, 1917-
May 2, 1919.
Average strength
appro ximately

Number of deaths
that would have
occurred if the
Civil War death
rate had obtained

Number of deaths
that would have
occurred if the
Spanish-American
War death rate
had obtained

2,121,396

Typhoid fever

213

51,133

68,164

Ma.ls.ria

t*

17 QCI*

II ^17

Dysentery • •

xo

42

OjVO •*•

63,8o8t

* "MO^-1/

6i82t

L^t

^J\jy^y^J '

\J)£\j£t 1

* Includes malaria, remittent and congestive fevers,
t Includes dysentery and diarrhea.

During the period of the American participation in the World War
there were 1065 cases of typhoid fever in approximately 4,000,000
troops, a ratio of one case to every 3756 men. In the Spanish- American
War there was one case to every seven men. Colonel Russell's final
comment is of the greatest interest. "It is evident from these tables,
therefore, that anti-typhoid vaccination, carried out as it was by a per-
sonnel which had not been carefully trained in its administration, gave
a high degree of protection to our forces under the conditions of hur-
ried mobilization and of warfare, and reduced the rate, not only below
19

290 THE PRINCIPLES OF IMMUNOLOGY

the rates for previous wars, but also below the rate found in civil life in
some of the older states where the entire population is protected by all
the sanitary measures of modern life."

At the beginning of the World War, of the troops in Belgium only
those of the British Army were adequately protected. At the beginning
of trench warfare in 1914 an epidemic broke out, and in January and
February of 1915, 4000 cases occurred in Dunkirk. Up to May of 1915
only 827 cases were contributed from the British Army, the bulk of the
cases came from among the unvaccinated Belgian soldiers and civilians.
Vaccination was instituted in February, and the epidemic was at an
end by the middle of the summer. In the early days of the war vac-
cination had not been compulsory in the French Army, and as the
result a large number of troops were victims of typhoid fever. The
institution of vaccination completely altered the picture. Courmont
gives the following statistics for the French Army in 1916:

Deaths

Non-vaccinated cases 17.4 per cent.

Of the vaccinated cases:

Those who had one injection 6.0 per cent.

Those who had two injections 4.0 per cent

Those who had three injections 2.5 per cent.

Those who had four injections 1.9 per cent.

Duration of Protection. — When typhoid vaccination was first in-
troduced it was generally assumed that protection lasts for about two
years. Certain British troops in Mudros were found to have developed
typhoid fever within six months after inoculation. Similarly, certain
troops of the American Army developed typhoid fever a few months
after they had been vaccinated, but it was found upon investigation
that in this instance the vaccination had not been completed. On the
basis of experience, yearly vaccinations were practiced in the British
Army, although it was not considered necessary to give the three
doses at the time of revaccination ; a single maximum dose on revac-
cination apparently served to maintain immunity. Yearly revaccina-
tion, however, provides adequate protection. Knowing that infection
has occurred within a few months after proper vaccination it is no
longer advisable to state that protection lasts for more than a year.
The determination as to when revaccination must be practiced depends
in certain measure upon the degree of exposure to the disease. In
those districts where typhoid or paratyphoid fevers are endemic, we rec-
ommend that vaccination be reinforced by a single yearly inoculation of
the maximum dose. If a period of two years has elapsed since previous
vaccination, it is advisable to revaccinate with three injections.

Complications. — The reaction to any dose of typhoid vaccine is
extremely variable. Usually the second and third doses produce some-
what more severe reactions than the first dose. There are, however,
certain individuals who are apparently hypersusceptible to typhoid pro-
tein, and these may react with great severity. As a rule, reactions are
merely local and are exhibited by swelling, redness, tenderness and pain
about the site of inoculation. General reactions are much less fre-

PROPHYLACTIC VACCINATION 291

quent and are exhibited by malaise, headache, fever, constipation and
occasionally chills. Maurange investigated the general reaction fol-
lowing 39,215 inoculations and reports the following results:

T^mAc nf rpaotinn Typhoid Para bacilli

Types per cent. per cent.

None 92.23 98.59

Feeble 6.18 1.41

Moderate 1.40 o.oo

Pronounced 0.19 o.oo

Rarely the inoculations may be followed by arthritis, nephritis and
severe intestinal disturbances. Chantemesse has called attention to the
recrudescence of tuberculosis during immunization, and it has further
been suggested that other chronic diseases, including syphilis, may be
excited to renewed activity. We have observed cardiac arrhythmia in
individuals who have previously suffered from myocardial disease.

Contraindications. — The contraindications include kidney disease,
diabetes, myocardial and endocardial disease, aortitis, cachexia, gastro-
intestinal disturbances and alcoholism. The presence of acute infec-
tions is also' regarded as contraindicating vaccination. According to
Maurange, age is no contraindication, although the relative unsuscep-
tibility of old people to typhoid fever may make it seem unnecessary to
vaccinate. Menstruation is not a contraindication.

Vaccination Against Cholera. — This was first employed by Ferran
in 1884. Haffkine published results in 1888, and since then numerous
investigations have developed technical methods and have emphasized
the value of protective vaccination. Ferran injected broth cultures of
living vibrios subcutaneously, employing 0.25 c.c. as the first dose and
0.5 c.c. as the second and third doses. Haffkine employed two kinds
of vaccine, a weaker vaccine prepared from living organisms grown
on agar at 39° C. and a more virulent vaccine prepared from vibrios
which had been passed through a series of guinea-pigs. Subsequently
Kolle prepared a vaccine made from heat-killed agar cultures of viru-
lent organisms. The emulsion is made by suspending 2 mg. organisms
in saline and heating to 60° C. for one hour. Cantacuzene prepared a
vaccine by heating emulsions of the vibrios for one and one-half hours
at 55° to 56° C. The concentration of this vaccine was 500 to 1000
million organisms per cubic centimeter. Two inoculations were given,
the first of 2. c.c. and the second of 4. c.c. with a six-day interval.
Strong prepared a vaccine by incubating the emulsion in sterile water,
thereby breaking up the cells. The emulsion was then passed through
a Reichel filter and the sterile filtrate employed. At the present day
heat-killed vaccines are most commonly employed.

Results. — The earlier work of Ferran and of Haffkine was ex-
tremely encouraging, but the subsequent statistics lend even greater
support to the value of this procedure. Arnaud made a study of
108,000 men during the second Balkan War. These men were all in
infected areas. Among the unvaccinated men the morbidity was 5.75
per cent. Among those who had received insufficient vaccination it was
3.12 per cent., and among those who had received the full treatment it

292 THE PRINCIPLES OF IMMUNOLOGY

was 0.41 per cent. Kobe made an extensive study of the population
of Tokio and its suburbs. In the city of Tokio, 10.54 per cent, of the
entire population were vaccinated. The absolute number that were
vaccinated, namely, 238,936 in Tokio and 61,988 in the suburbs, as well
as the large number of controls, provides a sufficient number from
which to draw satisfactory conclusions. In Tokio cholera occurred in
1.85 per 10,000 of the unvaccinated and 0.13 per 10,000 among the vac-
cinated. In the suburbs cholera occurred in 3.09 per 10,000 of the
unvaccinated, and there were no cases reported among the vaccinated.
Cantacuzene has studied results obtained in the campaigns in the Orient
during the Balkan Wars and the World War. These were conducted
particularly during the epidemics, and by a study of the normal curve
of epidemics he finds that vaccination leads to a sharp drop in the
epidemic curve and incidence of the disease.

Vaccination Against Pneumonia. — Although vaccination against
pneumonia was practiced by Wright before the various types of pneu-
mococci had been identified, it was not until the types were carefully
studied that exact results could be obtained. Lister, after he had iden-
tified the types of organisms present in South Africa, carried out
prophylactic immunization in 11,000 workers in the Rand mines. He
employed a composite vaccine prepared from the pneumococcus types
prevalent in that region. He found that subcutaneous inoculations were
sufficient to establish an immunity, and demonstrated that the pro-
tection was effective against the particular type of pneumococcus used
in the vaccines. He emphasized the importance of using a large bulk
of organisms and considers that the minimum effective dose is at least
6000 million pneumococci of each group against which protection is
sought. The work of Cecil and Austin and o<f Cecil and Vaughan has
been of the utmost importance. Cecil and Austin employed a saline
suspension of killed pneumococci of Types I, II and III. Three or
four doses were given at intervals of five to seven days. The first dose
contained 1000 million of each of the three types ; the second contained
2000 million of each type, and the third and fourth contained 3000
million each of Types I and II and 1500 million Type III. At Camp
Upton 12,000 troops were vaccinated, and among these only seventeen
cases of pneumonia of all types developed, including those due to
Type IV as well as to the streptococcus. Among the 20,000 unvac-
cinated men, 172 cases were reported. At Camp Wheeler a lipovaccine
was employed containing 10,000 million each of Types I, II and III per
cubic centimeter, given in one dose. Eighty per cent, of the total com-
mand, or 13,460 men, were vaccinated and 363 cases of pneumonia of all
varieties developed. The study was difficult because of the prevailing
influenza epidemic. An analysis of the records shows that " there were
thirty-two cases of Types I, II and III pneumonia among the vaccinated
four-fifths of camp, and forty-two cases of pneumonia of these types
among the unvaccinated one-fifth of camp. If, however, all cases of
pneumonia that developed within one week after vaccination are ex-
cluded from the vaccinated group, there remain only eight cases of

PROPHYLACTIC VACCINATION 293

pneumonia produced by fixed types, and these were all secondary to
severe attacks of influenza. This exclusion is justified by the fact that
protective bodies do not begin to appear in the serum until the eighth
day after injection of pneumococcus lipovaccine." " The pneumonia
incidence rate per 1000 men during the period of the experiment was
twice as high for unvaccinated recruits as for vaccinated recruits, and
nearly seven times as high for unvaccinated seasoned men as for
vaccinated seasoned men." The death rate for vaccinated men, in whom
the pneumonia developed more than one week after vaccination was
12.2 per cent., whereas among the unvaccinated troops it was 22.3 per
cent. The death rate for primary pneumonias wasi only one-third as
great among vaccinated men as among unvaccinated, but the rate in
pneumonia secondary to influenza was about the same for both groups.
Among the 20 per cent, of the command which were unvaccinated 327
cases were reported. These statistics were sufficiently encouraging to
introduce vaccination into the army on a fairly large scale. Neverthe-
less, the results are not sufficiently conclusive to state positively that a
high degree of protection is obtained. Recently Cecil and Blake have
been able to produce in monkeys a characteristic pneumococcus pneu-
monia by intratracheal inoculation. These investigators have studied
the problem of vaccination with saline vaccines and lipovaccines of
killed pneumococci and in addition have investigated the value of vac-
cination with living organisms. They found that vaccination with killed
pneumococci was not effective in preventing the development of the
disease under the conditions of infection but that the vaccinated animals
showed a somewhat less severe form of the disease. The living vaccine
was considerably more satisfactory, but they state that " the method
is too dangerous for any sort of practical application." Vaccination
with living virulent pneumococcus, Type I, produces a protective im-
munity against pneumonia of homologous types. The immunity against
other types of pneumococcus following vaccination with Type I offers a
certain degree of protection against other types, but this varies con-
siderably with the individual monkey. Vaccination with " living aviru-
lent pneumococcus Type I, if administered in sufficiently large doses,
renders the monkey immune to a subsequent pneumonia of homologous
type." Cecil and Blake point out that "vaccination with attenuated
living pneumococci could probably be practiced with impunity, but the
problem of transporting and keeping alive large quantities of pneumo-
cocci in the field would be difficult to solve."

Vaccination Against Plague. — Prophylactic vaccination against
plague was first reported by Haffkine in 1897. Subsequently Pfeiffer
and also Gaffky reported experiments which support the value of this
type of vaccination. Haffkine's vaccine was prepared from a killed broth
culture of the bacillus pestis five or six weeks old. Adult males were
given 3 to 3.5 c.c. and adult females 2 to 2.5 c.c. Kolle's vaccine is
prepared from slant agar growths suspended in the proportion of 2 mg.
of bacilli to the cubic centimeter of salt solution. Kolle and Strong
also employed living organisms whose virulence had been greatly re-

294 THE PRINCIPLES OF IMMUNOLOGY

duced. Lustig and Galeotti used nucleoprotein extracted from the
organisms and Kitano and others have employed organisms grown in
Bengal isinglass medium. Kitano and Sukegawa employed sensitized
vaccines and are of the opinion that these give better results than the
usual heated vaccine. They gave in the first dose 2 mg. of the sensi-
tized organism and in the second dose 4 mg. of the sensitized organism.
If haste is essential, 6 mg. may be given at one dose.

Experimentally, it has been established that vaccinated animals dis-
play an increased resistance against the disease. The Indian Plague
Commission reported that vaccination in man diminishes the incidence
of the disease, but that it does not furnish absolute protection. Appar-
ently the duration of immunity lasts from a few weeks to a few months,
but immune bodies are not demonstrable until ten days have elapsed.
In spite of the fact that numerous investigators have reported favorably
on vaccination against plague, Flu has stated that an analysis of the
statistics fails to furnish evidence that sufficient attention has been
given in the earlier studies to the prevalence of infected rats or to
other hygienic conditions which prevailed.

Vaccination Against Typhus Fever. — Vaccination against this
disease has been attempted with the serum of convalescent patients, but
the results have not been highly satisfactory. Plotz, Olitsky and Baehr
employed a vaccine composed of fifteen strains of bacillus typhi exan-
thematici. Of a series of 5251 vaccinated individuals where typhus was
epidemic only Ihree contracted the disease, and in another series of
8420 cases only six contracted the disease. Although the work of Plotz
with this organism has been carefully done there is still doubt as to its
exact etiological relationship. In statistics concerning this disease,
the presence of infected lice should be taken carefully into considera-
tion. It cannot be stated that vaccination in typhus has any great value
until further investigations have been conducted.

Vaccination Against Pertussis (Whooping-Cough). — The dis-
covery of the bacillus of whooping-cough by Gengou almost immediately
led to investigation as to vaccination. Luttinger has summarized the
results obtained in a large whooping-cough clinic and by over 180 private
physicians and health officers. The results were sufficiently encouraging
to justify the recommendation of this procedure. Conditions of ex-
posure and the nature of surroundings, as well as the variability of the
disease, even in a single epidemic, makes the interpretation of statistics
extremely difficult.

Vaccination Against Dysentery. — Prophylactic vaccination against
dysentery has encountered great difficulties because of the extreme tox-
icity of the cultures, Shiga attempted to overcome this by employing
mixed active and passive immunization. He used a bacterial vaccine to
which was added immune serum. Experiments on 10,000 individuals
showed a definite decrease in the rate of mortality. Others have em-
ployed toxin-antitoxin mixtures with apparent success, but Hoffmann
found that this type of vaccination failed to have any effect on the control
of a dysentery epidemic which he studied. Whitmore and Fennel and

PROPHYLACTIC VACCINATION 295

also Fennel and Petersen have prepared lipovaccines. It was found pos-
sible to administer in a single dose 3000 million Shiga organisms, 3200
million Y type organisms and 2200 million Flexner organisms without
marked local or general reaction. In experiments with animals immune
sera can be prepared with much less difficulty when the organisms are
administered suspended in oil. The method ha& not as yet been given
sufficiently extensive trials in man to justify definite 'Statements as to its
efficacy, but from the experimental results obtained it appears to have
more promise than any of the other methods proposed.

Vaccination Against Influenza. — Vaccination against influenza was
practiced very extensively in the recent great epidemic. The contro-
versy over the etiological relationship of the bacillus of Pf eiffer has, in
our opinion, not been settled. The results of vaccination with this
organism might serve to settle in part the question as to the cause of
the disease, since a high degree of immunity to the disease following
vaccination, if interpreted in the sense of specificity, would indicate that
the organism employed is the exciting cause. The vaccines which have
been employed have been suspensions in salt solution, killed by heat.
In certain districts stock cultures have been employed, in others a cul-
ture of a strain or strains isolated during the epidemic has been used, and
in still others a mixed vaccine has been used composed of the bacillus
of Pf eiffer, the streptococcus, the pneumococcus, the staphylococcus and
other organisms. Reports of striking success following vaccination
have been numerous, including in particular the work of Duval and his
collaborators. In consideration of reports of this .sort the curve of the
epidemic has sometimes been overlooked. Reports of certain other
investigators have not been encouraging. McCoy states that " the gen-
eral impression gained from uncontrolled use of vaccines is that they
are of value in the prevention of influenza; but, in every case in which
vaccines have been tried under perfectly-controlled conditions, they
have failed to influence in a definite manner either the morbidity or
the mortality." At best the method must be regarded as still in the
experimental stage.

Vaccination Against Other Diseases. — Vaccines have been pre-
pared against scarlatina, cerebrospinal meningitis, tuberculosis and con-
taminated wounds. Examination of the statistics presented fails to
produce convincing evidence that vaccination against these conditions is
especially satisfactory. As time goes on, methods may be improved
and larger statistical evidence collected.

APPENDIX C
VACCINE THERAPY

INTRODUCTION.

DISEASES OF THE GENITO-URINARY TRACT.

GONORRHEA.

CYSTITIS.

PYELITIS AND SUPPURATIVE NEPHRITIS.
DISEASES OF THE SKIN.

FURUNCULOSIS.

CARBUNCLES.

ECZEMA.

RINGWORM.

OTHER SKIN DISEASES.
DISEASES OF THE RESPIRATORY TRACT.

RHINITIS.

OZENA.

ASTHMA.

PERTUSSIS.

PNEUMONIA.

OTHER DISEASES.
DISEASES OF THE EYE.
DISEASES OF THE ALIMENTARY CANAL.

TYPHOID FEVER.

PARATYPHOID FEVER.

DYSENTERY.
TUBERCULOSIS.

VACCINE THERAPY

Introduction. — A clear differentiation must be made between
prophylactic vaccination and therapeutic vaccination. The value of
various modes of prophylactic vaccination has been discussed and their
importance in protection against various diseases has been outlined.
For purposes of discussion of therapeutic vaccination it is well to con-
sider the infectious diseases as either acute or chronic and either local
or general. Acute infectious processes are for the most part self-
limited and require little in the way of specific treatment, and spon-
taneous cure is so regular as to render difficult the interpretation of
results following therapeutic vaccination. Chronic infectious diseases
tend to be progressive and finally result either directly or indirectly in
the death of the patient. Statistical reports may show instances of
amelioration of the disease, but the personal bias of the investigator
may sometimes confuse the conclusion. Generalized infections may be
treated by simple bacterial vaccination, but the results with sensitized
vaccines have been better than those with unsensitized vaccines. With
few exceptions the results of therapeutic vaccination have been best
in cases of localized infection. The vaccines employed may be in the
form of stock vaccines, but the opinion is practically universal that
wherever possible the employment of autogenous vaccines gives the
best results.

The persistence of chronic infections is, in part, due to the fact that
the chronic inflammatory fibrous tissue hinders the general absorption
of antigenic materials produced by the exciting organism. Conse-
296

VACCINE THERAPY 297

quently, immune bodies are not produced in sufficient amounts to
combat the infection. Vaccination may serve to stimulate a general
immune reaction which aids in the resistance to the local lesion. In
generalized infections the simple bacterial vaccines may add to the load
carried by the body and perhaps reduce rather than enhance immunity.
If, however, immune serum is added to the vaccine or introduced sep-
arately, the serum may operate either upon the body or upon the
bacteria so as to favor resistance.

DISEASES OF THE GENITO-URINARY TRACT

Vaccine Treatment of Gonorrhea. — If stock vaccines are to be
employed, it is desirable to use those composed of a variety of strains
of the organisms. Many of the vaccines employed are heated salt solu-
tion suspensions of the organisms. Demonchy advises the use of large
doses of unheated salt solution suspensions of stock cultures. Thomson
has prepared a so-called detoxicated vaccine. In the earlier method
Thomson dissolved the organism in N/io NaOH and precipitated with
N.HC1. The toxins remain in the supernatant fluid. Later he found that
the toxins could be removed by washing with 0.5 per cent, sodium
acid phosphate and 0.5 per cent, phenol. Haworth employed sensitized
vaccine, and recently Sezary has recommended lipovaccine. Most in-
vestigators recommend the employment of large doses of the organ-
isms, ranging from a minimum of 5000 million to a maximum of
25,000 million.

The vaccines have been employed in acute gonorrheal urethritis
but with relatively little success. They have also been employed in
vulvo-vaginitis in children, in some instances with apparent success.
Undoubtedly, the field for therapeusis of this sort is best realized in
gonorrheal arthritis. In this condition persistent vaccination has been
followed in many cases by excellent results. Somewhat similar are the
chronic infections of urethral glands, prostate, seminal vesicles and the
internal female genitalia. Results from treatment of these conditions
warrant a trial of vaccine treatment in conjunction with other modes
of treatment or in those instances where other forms of treatment have
failed or are contraindicated.

Cystitis. — The organisms which may cause cystitis are variable, but
in those cases where the disease is chronic and resistant to local treat-
ment the causative organism usually belongs in the colon typhoid group,
the bacillus coli communis being the most frequent offender. In treat-
ment of this disease it is of fundamental importance to discover the
cause. In cases due to the colon bacillus the vaccine may be given in
the form of killed salt solution suspensions of organisms isolated from
the case. Stock vaccines may be employed when necessary. The dose
is usually from 50 million to 100 million. Results have been extremely
variable, but the method is sufficiently well established to justify trial in
resistant cases. Of fundamental importance is the removal of ure-
thral stricture, prolapse of the bladder or other local conditions which
retard cure.

298 THE PRINCIPLES OF IMMUNOLOGY

Pyelitis and Suppurative 'Nephritis. — In pyelitis the causative or-
ganism should be discovered before vaccine treatment is considered.
If due to the bacillus coli communis, autogenous vaccines in doses of
from 50 million to 100 million organisms given at weekly intervals
often yield good results. Suppurative nephritis occasionally is improved
by vaccination with the causative organism, but the danger of wide-
spread infection as a result of the disease is so great that in our opinion
surgical measures are of more immediate importance unless the general
condition of the patient contraindicates operation.

DISEASES OF THE SKIN

Many of the diseases of the skin and of the subcutaneous tissues
depend upon the local action of bacteria ; a considerable number of these
is susceptible to vaccine treatment. A greater number of skin diseases
is the result of more deep seated disorders and under these circumstances
it is essential that the cause be corrected; in these instances vaccine
treatment is of little avail unless the primary disease is one susceptible to
that mode of treatment.

Furunculosis. — Furuncles are usually caused by some variety of the
staphylococcus, most frequently the staphylococcus pyogenes aureus.
Occasionally, furuncles may be the result of streptococcus infections
or of mixed infections. The single furuncle usually heals after the
pus is discharged, either naturally or surgically, and may clear up with-
out any interference whatever. Patients are seen, however, in whom
furuncles appear repeatedly. In some of these cases the underlying
cause is diabetes mellitus and in others it is apparently due to a pro-
longed decrease in the number of circulating leucocytes. Vaccination
in cases in which the boils are persistent and frequent is usually effec-
tive. Stock vaccines are frequently employed, but in this condition, as
in others, autogenous vaccines are to be preferred. Stock vaccines have
frequently failed because of failure to identify the exact organism
causing the condition. For example, staphylococcus aureus stock vac-
cines are employed on the assumption that the boils are due to this
organism, whereas if cultures were made from the boil another organism
might be isolated. It is generally recommended that the vaccine be
composed of 2000 million organisms per cubic centimeter. It is im-
portant that the first dose be relatively small and the increase in doses
gradual. At the first dose o.i c.c. is given and at the second dose 0.2
c.c. is given and the doses increase by gradations of o.i c.c. until the
maximum dose o>f i.o c.c. is reached, It is often recommended that
the doses be given eight days apart, but this period may be reduced
with advantage to three or four days. In case of diabetes the vac-
cination should proceed more slowly and with somewhat smaller
doses than in other cases. With the dosage recommended, local
reactions are slight and general reactions very rarely appear. Vac-
cination in furunculosis usually gives excellent results and is to be
highly recommended.

Carbuncles. — These are also benefited in certain instances by vac-

VACCINE THERAPY 299

cine treatment, but it must be expected that many cases will fail to
improve. On the whole, surgical treatment is more satisfactory.

Eczema. — The recent studies of this disease have shown that many
cases are the result of hypersusceptibility to proteins, usually those
contained in food. Granted that such hypersusceptibility is demon-
strable, treatment is in the form of immunization to the particular
protein concerned. Such immunization is similar to that employed
in hay fever and has been commented on in the chapter on hyper-
susceptibility (page 231). Kolmer states that the prolonged admin-
istration of an autogenous bacterial vaccine composed of staphylococci
procured from the scales or serous exudate has occasionally aided in
the treatment of obstinate cases of eczema.

Ringworm. — Strickler has recently employed a vaccine made of
several strains of the fungus and is of the opinion that the method has
some value in obstinate cases.

Other Skin Diseases. — Vaccination has been employed with a vari-
able degree of success in the different f o<rms of acne, sycosis, scrofulo-
derma, impetigo and certain forms of erythema,

DISEASES OF THE RESPIRATORY TRACT

Rhinitis. — Vaccination against acute rhinitis has been largely
prophylactic in nature, and the results of these vaccinations have been
in a general way favorable. The exact cause of this disease has not
been finally proven, but the work of Foster indicates rather strongly
that the agent is a filterable virus. The prophylactic vaccines, however,
have been mixed stock vaccines of a variety of bacteria, and it seems
probable to us that any success obtained upon this basis is probably
non-specific. Coates is of the opinion that if acute rhinitis is treated
early with vaccines there is likely to be improvement. The course of
acute rhinitis is so variable that statistical results are open to some
question. In chronic rhinitis it is maintained that autogenous vaccines
are of value. It must be understood, however, that contributory causes,
such as adenoids, enlarged tonsils, polyps and nasal deformities must
be removed. So much benefit accrues from the correcting of the
contributory causes that the beneficial effects of vaccination probably
depend in certain part upon the personal equation of the observer.

Ozena. — The cause of this disease is at present a matter of con-
siderable dispute, and the value of vaccination is undecided. The vac-
cines that have been employed are usually made from the bacillus
ozense fetidae of Perez. Horn claims that this organism is similar to
the bacillus bronchisepticus and has made polyvalent stock vaccines
which he claims are highly successful. Friel reports excellent results
from the intravenous administration of sensitized living vaccine of
Friedlander bacillus. Ersner has had disappointing results. While im-
provement may occur in a certain percentage of cases, McKenzie found
a marked tendency to relapse following the cessation of treatment.

Asthma. — As with eczema, the recent investigations of hyper-
susceptibility have placed the study of asthma upon an entirely new

300 THE PRINCIPLES OF IMMUNOLOGY

basis. According to the work of Walker and his collaborators, certain
of the cases are due to specific bacterial invasion, and probably con-
tributed to by a certain degree of hypersusceptibility to the organism.
Bacterial vaccination in these cases has been accompanied by good
results. A complete investigation of the nature of the case is essential
before any form of vaccine treatment should be attempted. Earlier
investigators have employed mixed vaccines made of organisms obtained
from the sputum.

Pertussis. — Prophylactic vaccination against this disease has been
discussed (page 294). Therapeutic vaccination has been employed by a
number of workers with, in many instances, apparently favorable re-
sults. Luttinger found that in a series of 952 cases treated by vaccina-
tion the paroxysmal stage averaged about thirty-seven days, whereas
149 cases not treated with vaccine had a duration of over fifty days.
Blum and Smith found that non-specific vaccination was practically as
effective as vaccination with the bacillus of Gengou. Barenberg also
finds that pertussis vaccine, even when given in large doses, has neither
curative nor ameliorating effect. Kraus and others have reported good
results by the use of a vaccine prepared from the sputum. The sputum
is washed, mixed with ether, shaken for three or four days, the ether
evaporated, the mixture tested for sterility and given in doses of i.o c.c.
every three or four days. If stock vaccines of the organisms are em-
ployed, it is of the utmost importance that they be fresh. Doses of
25 million organisms may safely be given.

Pneumonia. — Prophylactic vaccination (page 292) is distinctly
more promising than therapeutic vaccination. Treatment with immune
serum (page 256) is also more promising than vaccination. Coleman
is of the opinion that vaccines in pneumonia are never harmful and
may be beneficial. Teale and Embleton believe that they have obtained
good results in certain cases. Shera expresses the belief that the local
infection is too massive to permit of vaccine having any appreciable
effect in the stage of consolidation. The frequent occurrence of pneu-
mococcus septicemia as a part of the disease makes it unlikely that
vaccination will be helpful. In delayed resolution vaccines are of value
in some cases. Shera also states that empyemia when it has reached the
chronic stage may be benefited by specific vaccination.

Other Diseases. — Certain diseases of the accessory regions of the
respiratory tract, including chronic median otitis and mastoiditis, have
been treated by vaccination with the organism concerned. Results
have been variable, but inasmuch as these represent somewhat isolated
local infections it is reasonable to attempt vaccination in addition to
the usual modes of treatment.

DISEASES OF THE EYE

When conjunctivitis becomes chronic, specific vaccination sometimes
leads to improvement. The infecting agents include staphylococcus,
streptococcus, bacillus pyocyaneous, Friedlander's bacillus and others.
Autogenous vaccines may be employed in addition to other modes of

VACCINE THERAPY 301

treatment. Chronic conjunctivitis, due to the Morax-Axenfeld diplo-
bacillus, is said to respond very well to vaccine treatment. In acute
pneumococcus and gonococcus conjunctivitis, especially with ulcer,
Allen advises early and vigorous vaccine therapy and reports good
results. It is also stated that ophthalmia neonatorum sometimes im-
proves rapidly under vaccine treatment. In none of these conditions,
however, is it wise to neglect other forms of treatment.

DISEASES OF THE ALIMENTARY CANAL

Typhoid Fever. — Prophylactic vaccination has unquestioned value
in the prevention of typhoid fever (page 285). Specific therapeutic
vaccination has been the subject of experiment since the work of
Fraenkel in 1893. The vaccines employed have been usually killed
organisms either untreated or sensitized, administered either subcu-
taneously or intravenously. Certain authors have also reported the use
of living organisms, but this method has not been adopted. Gay, in his
book, " Typhoid Fever," reports the following summary of results
obtained by various methods :

SUMMARY OF RESULTS OBTAINED BY RECENT OBSERVERS (1913^-1917) IN THE TREAT-
MENT OF TYPHOID FEVER BY VACCINES ADMINISTERED IN VARIOUS WAYS.

Untreated vaccine subcutaneously . .
Sensitized vaccine subcutaneously . .
Untreated vaccine intravenously

rv. Total Estimates Bene- Mortal-
Observers cases basedon fited ity

. . 30 iooi 512 46% 14.5%
.. 14 593 239 69% 8.0%

22 501 233 62% 13 O%

Sensitized vaccine intravenouslv .

12 487 31 6 8^% 11.0%

It is usually stated that typhoid fever has a mortality of about 10 per
cent., although in the American Civil War it exceeded 35 per cent, and
in the Franco-Prussian, Spanish-American and Boer Wars it ranged
between 8 and 14 per cent. The severity of epidemics varies consid-
erably, but at the best there is little in the way of encouragement to be
found in the table given above. The basis upon which improvement
is estimated varies considerably with the different investigators and
the figures are " distinctly affected by subjective influences." Gay has
employed a sensitized vaccine administered intravenously and his re-
sults in ninety-eight cases are summarized as follows :

SUMMARY OF RESULTS IN NINETY-EIGHT CASES OF TYPHOID TREATED BY INTRA-
VENOUS INJECTION OF SENSITIZED VACCINE SEDIMENT.

No.

of

cases

Aborted 33

Benefited 32

Unaffected ... 33

The most significant figures in this table refer to those cases which
were aborted. Careful study of various epidemics fails to show any
instance where such a large percentage of the cases have aborted, and
it therefore seems probable that the vaccination had some distinct value.

Widal
titer

Blood

Treatment

No. of

Per-

Days

Age

on

culture

begun

treat-

manent

of

beginning

positive

day

ments

normal

treat-

treatment

ment

26.2

296.0

36.6%

134

1.88

20.4

7.0

24.2

156.5

70-9%

14.8

3.20

30-6

15-8

28.8

II4.8

84.8%

13-7

4-85

46.8

33-1

302 THE PRINCIPLES OF IMMUNOLOGY

This rapid improvement appeared to be somewhat more striking in the
moderate and mild cases than in those which were considered severe.

Other investigators have noted that non-specific therapy has been
quite as effective as the use of specific typhoid vaccine. Kraus found
that colon bacilli were equally effective and others have confirmed this
observation. Liidke has employed deutero-albumose, Weichardt al-
bumin solutions, Nolf pepton and still others have employed such sub-
stances as dextrose, colloidal gold and even normal salt solution. Gay
admits that the non-specific form of therapy has been as effective as
the use of sensitized typhoid vaccines, but urges the employment of
typhoid vaccine because it may be kept indefinitely in dried form under
conditions of .strict asepsis and can readily be injected in exact amounts.
He further states that " typhoid vaccine has the advantage over other
protein preparations of building up the active immunity of the patient,
and a sensitized vaccine will, in our experience, produce a higher grade
of leucocytosis."

Paratyphoid Fever. — Rathery and others have used therapeutic
vaccination in paratyphoid B fever. It was concluded that the treatment
is useful, always improves general condition, often shortens the fever
and has never led to harmful results. Others have found that typhoid
vaccine is as effective in paratyphoid as in true typhoid fever and the
non-specific therapy indicated above has also been effective.

Dysentery. — The vaccine treatment of dysentery is confined to the
bacillary form and of these varieties the cases due to the Flexner
bacillus and other related forms appear to do much better than those
caused by the Shiga bacillus. Nolf, from his observations in the Belgian
Army, concludes that vaccine therapy, when administered by the intra-
venous route is the most effective therapeutic procedure in the more
chronic forms of bacillary dysentery. His cases did not include those
caused by the Shiga bacillus. Similar results had been reported by
Baroni in the Roumanian Army. He employed either six injections of
killed organisms or four injections of living vaccine. Kountze found
that in typical cases of dysentery, vaccination produced immediate gen-
eral improvement and reduction in the number of stools. The study of
the therapy of this disease has been somewhat hampered by the failure of
investigators to identify the strains of organisms concerned. Although
the results with vaccination have been encouraging, it is by no means posi-
tively proven that this mode of treatment is superior to serum treatment.

TUBERCULOSIS

The various forms of tuberculin are vaccines and treatment by their
use is an example of vaccine therapy. The methods of preparation of
the various tuberculins have been discussed (page 238). Koch's first
work with tuberculins was stimulated by the hope that treatment with
them might be effective. The use of the material in larger amounts
than now seem necessary led to severe reactions on the part of the
patients which in some instances were disastrous. For many years
tuberculin therapy was considered extremely dangerous and was prac-
ticed by very few clinicians. Recently, however, a more thorough

VACCINE THERAPY 303

knowledge of the proper precautions in treatment has been built up and
satisfactory results are now reported. Applied to pulmonary tubercu-
losis it has been followed by improvement in many cases, particularly
in those under sanitarium treatment. It also is claimed to be an im-
portant aid in the treatment of tuberculosis of the bones and joints
and of the eye. Improvement has been reported also in cases of tuber-
culous enteritis and mesenteric lymphadenitis. Kleinberg, however,
maintains that only a small proportion of bone and joint cases improve,
that the majority show no improvement and that in some cases relapses
occurred and new abscesses appeared.

Apparently the most suitable patients for tuberculin therapy are
those with incipient tuberculosis or old cases of fibroid phthisis with
fair or good nutrition. Advanced or moderately advanced cases may
be so treated if the general condition is good. Hamman and Wolman
do not consider marked general weakness, fever, cardiac disease,
nephritis, epilepsy, syphilis of themselves contraindications but rather
unfortunate complications which may prevent specific treatment.

The injections are given subcutaneously at the lower angle of the
scapula. In order to observe whether or not reaction occurs the in-
jections are given in the afternoon after the patient's temperature has
been taken. This avoids mistaking an accidental afternoon rise of tem-
perature for a rise due to the tuberculin. Hamman and Wolman recom-
mend the following range of doses:

Tuberculin Initial dose Maximal dose

Old tuberculin 0.000,000,1 to 0.000,001 c.c. I. c.c.

New tuberculin 0.000,001 to 0.000,1 c.c. 2. c.c.

Bacillus emulsion 0.000,001 to 0.000,1 c.c. 2. c.c.

Three classes of patients are recognized: (i) children, (2) patients
who exhibit a slight fever or are not in good condition, (3) patients in
good general condition. The smaller initial doses are for patients of
the first two groups, the larger for patients in the third group. Other
forms of tuberculin are employed, but the types noted above have been
given the most extensive trial. Provided reactions are absent or very
slight, the injections may be repeated every three or four days. Tuber-
culin has been given by mouth, but is absorbed irregularly and may pro-
duce unexpected reactions. It has also been administered intrafocally
in tuberculous pleurisy, tuberculous peritonitis, lupus and tuberculosis
of the joints and of the tunica vaginalis. Results have in some instances
been encouraging. The local reactions include pain, tenderness and
swelling. General reactions are exhibited by rise in temperature,
malaise, headache, insomnia, rapid pulse, loss of weight.

Shiga has recently reported upon the use of a " serovaccine." This
is designed especially for prophylactic injection in those who by virtue
of family relations, constitution or other conditions are predisposed to
the disease and for early incipient cases. He claims to have obtained ex-
cellent results by weekly vaccination with increasing amounts of the sero-
vaccine followed after fifteen injections by two graded doses of living
avirulent tubercle bacilli. The method is prophylactic rather than curative.

INDEX

Abderhalden, building-stone theory

of, 30
Abrin, 70

Acquired immunity, 21
actively, 22
artificially, 22
naturally, 21
Agglutinability of bacteria, alterations

of, 92

Agglutination, group reactions, 85
influence of electrolytes on, 90
of heat on, 89
of hydrogen -ion concentration!

on, 91

inhibition zones in, 89
mechanism of, 91
physical basis of, 93
Agglutinins, absorption of, 86
bacterial, 79
immune, 80

production of, 80
macroscopic titration of, 83
main, 80
major, 80

microscopic titration of, 84
minor, 80
nature of, 92
normal, 80
partial, 80

preliminary titration of, 82
production of anti-typhoid, 81
specificity of, 85
Aggressins, 4

Amboceptor, activation of — by comple-
ment, 181
and complement in normal hemo-

lysins, proportions of, 135
partial, 123
Anaphylactic poisons, 218

shock, blood pressure in, 214
. coagulability of blood in, 215
distention of lungs in, 213
ferments in, 215
in guinea-pigs, 212
in man, 230
metabolism in, 214
methods of preventing, 216
Anaphylactoid phenomena, 224
Anaphylatoxin, 219
Anaphylaxis, 209

cellular theories of, 220
cross reactions in, 217
group reactions in, 217
intoxicating injection in, 211
passive, 216

period of incubation in, 211
physical theories of, 222
reaction, 212
sensitization in, 210

Anaphylaxis, specificity of, 217

theories of, 218

Anthrax, serum therapy of, 259
Anti-aggressins, 5
Anti-amboceptors, 136
Anti-anaphylaxis, 215
Antibodies, Bordet antibody, 179
leucocyte antibody, 168
production at site of injection, 35
Antibody, definition of, 22
formation, site of, 33
Anti-complementary action of cells,

tissue extracts and body-fluids, 183
Anti-complements, 137
Anticorps leucocytaire, 168
Anti-dysentery sera, therapeutic use

of, 63
Antiferment, 248

determination in blood serum, 249
Antiferment- ferment balance, 247
Antigen, definition of, 22
Anti-rabic vaccination, effects of, 284
in man, 284
results of, 285
Antitoxins, formation of, 41

influence of temperature on, 43
nature of, 43
Anti-typhoid vaccination, complications

of, 290

contraindications to, 291
duration of protection in, 290
method of, 287
prophylactic value of, 288
Asthma, cutaneous tests in, 234

vaccine treatment of, 299
Auto-serum therapy, 264
in syphilis, 265

Bacteremia, 13

Bacterial precipitins, production of, 108

products, immunization with, 24

toxins, classification of, 40

vaccine, definition of, 273
killed, 275
preparation of, 275
Bacteriolysins, 144
Bacteriolysis, bioscopic method for, 149

Buxton's method for, 148

in vitro, 146

Wright's method for, 147
Bacteriotropins, 162
Bleeding a rabbit, 83

a guinea-pig, 127
Blood antigen, 117

groups classification, Jansky, 99
Moss, 100

serum, therapeutic employment of,
252

transfusion, reactions to, 105

305

306

INDEX

Bordet-Gengou phenomenon, 173

laboratory demonstration of,

174
Botulinus antitoxin, 65

toxin, 65
Botulism, use of immune sera in, 65

Canine distemper, cutaneous reaction

in, 244

Carbuncles, vaccine treatment in, 298
Cataphylaxis, 8
Chemical agencies, anti-complementary,

182

Chemotaxis, 152
Cholera, vaccination against, 291

serum therapy of, 258
Cobra lecithid, 141
Colloid shock, 225
Complement, alterations of amount of,

127

deviation, 148
distribution of, 126
end-piece of, 133
fixation, acid-fast, 205
delicacy of, 177
group reactions in, 177
inhibition zones in, 176
tests, 206

in echinococcus cyst, 207

in glanders, 206

in gonococcus infections,

205 .

in malignant tumors, 207
in smallpox, 206
in sporotrichosis, 207
in syphilis, 186
in tuberculosis, 203
in typhoid fever, 206
in whooping-cough, 207
fixing bodies, amboceptor, nature

of, 180

relation of — to other im-
mune bodies, 178
fractions, 133
in hemolysis, influence of amount

of, 121

inhibition of — other than by fixa-
tion, 182

method of obtaining, 127
mid-piece of, 133
multiplicity of, 131
nature of, 129
origin of, 128
preservation of, 130
proportions of — in normal hemoly-

sins, 135
titration of, 119
variability of, 131
Complementary activity, influence of

lipoids on, 183
Complementoids, 132
Conglutinin, 106, 126
Crotin, 71
Curcin, 71

Cutaneous reactions, in canine dis-
temper, 244

Cutaneous reactions, in glanders, 244
in gonococcus infections, 242
in hypersusceptibility, delicacy

of, 235

technic of, 234
theories of, 236

in hyphomycetes infections, 244
in leprosy, 244
in meningococcus infections,

243

in pneumococcus infections, 243
in pregnancy, '244
in sporotrichosis, 244
in typhoid fever, 242
to vaccine virus, 243
Cystitis, vaccine treatment of, 297
Cytolysins, 115

organ specificity of, 143
Cytolysis, summary of, 149
Cytotoxins, autocytotoxins, 144
heterocytotoxins, 144
isocytotoxins, 144
lens, 144
specificity of, 142

Danysz effect (or phenomenon), 50
Dead bacteria, immunization with, 23
Defensive ferments, 245
Desensitization in hypersusceptibility,

215
Diphtheria, active immunization against,

55

antitoxin, dosage of units of, 53
injection scheme for produc-
tion of, 42

standardization of, 44
technic of producing, 42
therapeutic use of, 51
titration of, 46
carriers, anti-bacterial serum in

treatment of, 262
natural immunity to, 53
Schick test in, 53
toxin, technic of producing, 42
Drug idiosyncrasies, 237
Duck-bill platypus, 76
Dysentery antitoxin (serum therapy),

62

toxin, 62

vaccination against, 294
vaccine treatment of, 302

Eczema, vaccine treatment of, 299

Eel serum, 75

Ehrlich classification of immune bodies,

27

hypothesis, criticism of, 28
side-chain theory, 26
objections to, 49
Endotoxins, 7
Erythrocytes, chemical agglutination of,

105

fragility of, 138
iso-hemagglutinins, 98
iso-hemolysins, 136
Esterases, technic of determining, 247

INDEX

307

Exotoxins, 7, 40

Eye, diseases of, vaccines in, 300

Ferment-antiferment balance, 247
Ferments, defensive, 245

immune, 246

in blood, 246

specificity of, 245

Food adulteration, detection of, 113
Furunculosis, vaccine treatment of, 298

Gas gangrene, prophylactic use of sera

in, 68

serum treatment of, 67
use of immune sera in, 67
Glanders, cutaneous reaction in, 244
Gonococcus infections, cutaneous reac-
tions to, 242
serum therapy of, 263
vaccine treatment of, 297

Hay fever, toxins in, 23.3
Hemagglutinins, 98

auto-hemagglutinins, 98
hetero-hemagglutinins, 98
immune hetero-hemagglutinins, 98
iso-hemagglutinins, 98
normal hetero-hemagglutinins, 98
Hemolysins, 116

auto-hemolysins, 116
bacterial, 139
collection of immune, 118
immune hetero-hemolysins, 117
iso-hemolysins, 136
preparation of immune, 117, 118
titration of immune, 118
vegetable, 140
venom, 141

Hemolysis, chemical, 139
group reactions in, 123
physical, 138
Hemolytic amboceptor-antigen union,

dissociation of, 122
and antigen, quantitative rela-
tions of, 120
and complement, quantitative

relations of, 119
relative affinities of, 120
selective absorption of, 12 1
mechanism of operation of, 125
nature of, 124
rate of absorption — by blood

cells, 122
specificity of, 123
antigen, nature of, 124

quantitative relations of, 120
complement, quantitative relations

of, 119

relative affinities of, 120
selective absorption of, 121
Hemophagocytosis, 162
Hemotoxins, bacterial, 69
Hetero-hemolysins, normal, 134
Hog-cholera, serum therapy of, 270
Host and parasite, mutual relations of, I
factors favoring, 14

Hyperleucocytosis, specific, 170
Hypersusceptibility, 208

delicacy of cutaneous tests in, 235

natural, 230

occurrence of, 208

technic of cutaneous tests in, 234

tests for, 233

theories of cutaneous reactions in,

236

Hyphomycetes infections, cutaneous re-
action in, 244

Immune human serum, treatment with,
266

reactions, specificity of, 29

sera, anti-hemolytic activity of, 185
Immunity, as result of vaccination, 281

function of precipitation in, 114

relation of anaphylaxis to, 226

theories of nature of, 26

types of, 16
Immunization, active, 15

passive, 25
Infections, primary, 13

mixed or multiple, 13

secondary, 13

terminal, 13

Infection, production of, n
Infectious disease, course of, 15

non-specific therapy of, 30
Inflammation, cellular participation, 170

in resistance, influence of, 172
Influenza, vaccination against, 295
Invader, entrance of, n

factors favoring, 13

inhibiting, 14
Iso-hemagglutinins, 99

characters of, 100

incidence of, 100

in lower animals, 101

mechanism of, 101

methods for testing human blood
for, 103

Rous and Turner method, 103
with standard sera, 103

relation of — to blood transfusion,

101
Iso-hemolysins, normal, 136

Leprosy, cutaneous reaction in, 244
Leucocyte enzymes, 168

extracts, for therapeutic purposes,

169

Leucocytes, bactericidal extracts of, 167
Leucoprotease, 168
Leucotoxins, 143
Lipoyaccines, 277
Luetin reaction, 242
Lymphocyte ferments, 170

resistance to cancer, 171

Macrocytase, 173
Mass action, law of, 50
Meningococcus infection, 254

cutaneous reaction to, 243

serum therapy of, 254
Microcytase, 173

308

INDEX

Natural hemolysins, fixation of comple-
ment of, 182
immunity, 16

classification of, 18

family, 20

individual, 20

inherited, 20

racial, 18

species, 18

Neisser-Wechsberg phenomenon, 147
Normal serum, therapeutic use of, 270

Opsonins, 158

as " facultative " amboceptor, 161
experimental demonstration of, 159
for cells other than bacteria, 162
immune, 161
normal, 159

specificity and other characters of,
163

Ozena, vaccine treatment of, 299

Parasite and host, mutual relations of, I
Parasitic protozoa, poisons of, 75
Parasitism, half parasites, 2
pure parasites, 2

saprophytes, 2
Paratyphoid fever, vaccination against,

285

vaccine treatment of, 302
Pertussis, vaccination against, 294
vaccine treatment of, 300
Pfeiffer phenomenon, 144

demonstration of, 145
Phagocytic cells, influences operating

upon, 165
types of, 153
Phagocytosis, 151

analysis of mechanism of, 165
digestion of bacteria in, 153
experimental demonstration of, 155
functions of, 154
influence of phagocyte and ingested

elements on, 163
of temperature on, 157
ingestion of foreign body in, 153
mechanism of, 155
physical basis of, 156
process of, 152

relation of bacterial virulence to, 164
surface tension in, 156
Phagolysis, 155
Phasin, 71
Phytotoxins, 69
Plague, 260

serum therapy of, 260
vaccination against, 293
Plant pollens, 231
Pneumococcus infections, 256

cutaneous reaction to, 243
Pneumonia, serum treatment of, 256
vaccination against, 292
vaccine treatment of, 300
Pneumotoxin, 243
Poisonous fish, 75

substances, production of, 6

Poisons, bees, wasps and hornets, 74

centipedes, 74

duck-bill platypus, 76

of parasitic protozoa, 75

scorpion, 74

spider, 74

toads, frogs and salamanders, 75

vegetable, 69
Poliomyelitis, acute anterior, serum

therapy of, 268
Pollantin, 233
Precipitation, 106

biological relationships based on, in

delicacy of, 109

experimental demonstration of, 108

functions of — in immunity, 114

nature of reaction of, 107

organ specificity of, 112

physical basis of, 109

practical application of, no
Precipitin test in game laws, 1 14
Pregnancy, Abderhalden test, 249

cutaneous reaction in, 244
Prophylactic vaccination, 272
Proteases, technic of determining, 247
Proteins, poisonous bacterial, 7
Ptomains, 6

Pyelitis, vaccine treatment of, 298
Pyemia, 13

Rabic vaccine, preparation of, 283
Rabies, active immunization in, 283

vaccination against, 282
Resistance, factors operating against, 14
Rhinitis, vaccine treatment of, 299
Ricin, 70

Rinderpest, serum treatment of, 269
Ringworm, vaccine treatment of, 299 .
Robin, 71

Sapremia, 13

Septicemia, 13

Serum, anti-anthrax, 259

anti-bacterial, in treatment of diph-
theria carriers, 262

anti-cholera, 258

anti-gonococcus, 263

anti-hog-cholera, 270

anti-meningococcus, 254

anti-plague, 260

anti-pneumococcus, 256

anti-poliomyelitis, 268

anti-streptococcus, 253

disease, accelerated reaction, 229

delayed reaction, 228
Smallpox vaccination, 278

methods of, 280

vaccine, preparation of, 279
Sporotrichosis, cutaneous reaction in, 244
Streptococcus infections, serum therapy

of, 253

Suppurative nephritis, vaccine treat-
ment of, 298

Syphilis, auto-serum therapy in, 265
Syphilitic amboceptor, 190
nature of, 191

antigen, nature of, 189

INDEX

309

Tests, Abderhalden, 249
Dreyer, 96
forensic blood, no
tuberculin, 238
Widal, 85
Tetanolysin, 57
Tetanospasmin, 58
Tetanus, serum treatment of, 60
antitoxin, 56

prophylactic use of, 59
therapeutic use of, 58
toxin, 56

route of absorption of, 58
Thigmotropism, 171
Toxin-antitoxin union, 47

Ehrlich theory of, 47
Toxins, bacterial, 38
gas bacillus, 67
general nature of, 38
injury of, 39
L+ dose of, 46
LQ dose of, 46
minimum lethal dose of, 45
pathological effects of, 41
pollen, 71
true, 7

Tuberculin reaction, 238
conjunctival, 239
cutaneous reaction of, 239
general, 238
intracutaneous, 239
percutaneous, 239
specificity of, 241
theories of, 240
utility of, 241
Tuberculosis, complement fixation in

203

serum treatment of, 263
vaccine treatment of, 302
Typhoid fever, cutaneous reaction in,

242

serum treatment of, 263
vaccination against, 285
vaccine treatment of, 301
vaccine, preparation of, 286
Typhus fever, vaccination against, 294

Vaccination, against cholera, 291

dysentery, 294

influenza, 295

pertussis, 294

plague, 293

pneumonia, 292

rabies, 282

typhoid and paratyphoid fevers,
285

typhus fever, 294
immunity as result of, 281
unfavorable results of, 282
Vaccine therapy, 296

treatment, in asthma, 299

in carbuncles, 298

in cystitis, 297

in diseases of the eye, 300

in dysentery, 302

Vaccine treatment, in eczema, 299
in furunculosis, 298
in gonorrhea, 297
in ozena, 299
in paratyphoid fever, 302
in pertussis, 300
in pneumonia, 300
in pyelitis, 298
in rhinitis, 299
in ringworm, 299
in suppurative nephritis, 298
in tuberculosis, 302
in typhoid fever, 301
virus, cutaneous reaction to, 243
Vaccines, definition of, 273
dosage of, 277
method of counting, 276
types of, 274

autogenous, 275
killed bacterial, 275
living, 274
mixed, 275
sensitized, 274
stock, 275
tetra, 287
triple, 287
Vaccinia, 281
Vaccinoid, 243

Venom cobra, clinical tests, 142
Venoms, pathological effects of, 73
production of antisera for, 73
snake, 71
Virulence, 2

alteration of, 8
basis of, 3
decrease of, 9
demonstration of, 3
increase of, 8
Virulins, 5

Wassermann reaction, 186

antigen for, 187

complement for, 191

dependability of, 202

diagnostic value of, 201

end-piece in, 134

hemolytic system for, 194

influence of temperature upon,
196

interpretation of results of, 202

modifications of, 200

post-mortem, 203

preservation of erythrocytes
for, 195

quantitative results with, 203

reading, 199

specificity of, 200

on spinal fluid, 203

technic of, 196
test, protocol of, 199

table of methods of perform-
ing, 192
Weber-Fechner law, 152

Zootoxins, 71

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