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I

THE ACTION OF THE LIVING CELL

THE MACMILLAN COMPANY

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?i

THE

ACTION OF THE

LIVING CELL

EXPERIMENTAL RESEARCHES IN BIOLOGY

BY

FENTON B. TURCK

NEW YORK

THE MACMILLAN COMPANY

1933

COPTRIGHT, 1933,

By the MACMILLAN COMPANY.

All rights reserved — no part of this book may
be reproduced in any form without permission in
writing from the publisher, except by a reviewer
who wishes to quote brief passages in connection
with a review written for inclusion in magazine or
newspaper

Set up and printed. Published January, 1933.

PRINTED IN THE UNITED STATES OF AMERICA

NORWOOD PRESS LINOTYPE, INC.

NORWOOD, MASS., U.S.A.

To my son

FENTON B. TURCK, JR.,

my companion

in

spirit and in truth

{<'

'The intrusion of something which the
flesh engenders — not entering from outside."

IMHOTEP, 4000 B.C.

/Xi^^-5^>

<-■

LIBftAR Y r

PREFACE .^^,

While working in the Lincoln Park Greenhouse
in Chicago some forty years ago, the writer made
some curious observations concerning the growth of
plants. In attempting to find the reason for the failure
of plants to grow for a succession of years in the same
greenhouse soil, it was found that the addition of vir-
gin soil from Wisconsin to the exhausted soil proved
of little avail unless the amount of virgin soil mixed
with that from the greenhouse was equal to, or greater
than, the amount of the latter. Of itself, this consti-
tuted a matter of little interest, but in connection with
this study the surprising observation was made that the
addition of a small quantity of the exhausted soil to
the virgin soil resulted in a better growth of the fronds
than was obtained in the virgin soil alone. The reason
for this remained obscure for a number of years, but
the phenomenon excited the writer's interest to further
investigations in the field of biology.

This interest was greatly intensified by a period
spent in Europe, where, under the guidance of the
great Virchow, the writer's interest was directed
towards animal cells and the effect of environment
upon them. At that time the majority of the students
of biology were greatly influenced by the writings of
Darwin and Huxley. In consequence, many investi-
gators were enmeshed in the study of "the survival
of the fittest," as Herbert Spencer tersely stated the
doctrines of Darwin. While recognizing the impor-

vu

viii PREFACE

tance of such studies, the writer's interest was drawn
to the factors which rendered unfit those that could
not survive the rigors of an enforced environment.

This aspect of experimental biology, which has
been neglected by most biologists except in so far as
it relates to medicine, has continued to occupy the
writer's attention for many years, and the results of
numerous experimental researches have been pub-
lished from time to time over a period of four decades.
Many of these investigations have been recorded in
journals not readily accessible to biologists; hence at
the instigation of my son, Fenton B. Turck, Jr., I
have deemed it advisable to publish the main results
of my investigations in monograph form. Such a deci-
sion has proved advantageous, as it has permitted a
more logical sequence and correlation of related ideas
than was possible in the original publications. In-
deed, in reviewing some of the older data, we have
been able to deduce relationships between what at
first appeared to be unrelated facts; and in numerous
instances it has been gratifying to find that other in-
vestigators have recorded observations which coin-
cide nicely with some of our own.

In a large measure the author's work has been the
outgrowth of his initial studies on the cause of shock,
and death from this cause. Almost at the beginning
of our experiments, the conclusion was reached that
shock induced in any manner whatsoever is essentially
the expression of the action of a toxic substance liber-
ated from injured tissues. Subsequent investigation
has completely substantiated this concept, and has
shown that the same toxic substance may affect the
behavior of animal cells in a variety of ways. We shall

PREFACE Ix

therefore begin our discussion by reviewing the ini-
tial experiments concerning the toxic nature of shock.

A portion of the contents of the first few chapters
may not appear to be of immediate interest to the
general biologist. However, the investigations dis-
cussed therein really formed the basis of the author's
concepts, which have been extended to some problems
of distinct general interest. In essence, the experi-
mental evidence appears to indicate that under ap-
propriate stimulus, living cells appear to be able to
yield an endocellular component which is capable of
both exciting and depressing the activities of neigh-
boring cells. For want of a better name, this substance
has been termed cytost, and is so designated through-
out the present discussion. The postulation of the
existence of cytost and its role in the physiology of
living systems was necessitated in order to explain
and harmonize a diversified series of experimental
observations.

All that one may ask of any scientific theory is that
it satisfactorily explain known facts and if in the fu-
ture new facts are found which are not in harmony
with that theory, then the theory must be discarded.
Hence if in the future the author's cytost concept is
replaced by some other hypothesis, it matters little.
For the present the cytost concept is of service in
interpreting the meaning of performed experiments
and in guiding the imagination of the investigator in
the conduct of new experimental researches. Thus,
as will be shown later, the cytost concept has enabled
us to explain the curious stimulating effects of ex-
hausted soil upon the growth of plants in virgin soil.
To be sure, this appears to be a far cry from the cause

X PREFACE

of shock in animals, but it is the correlation of such
apparently remote topics which attests to the progress
of science and permits of a unified concept of the
activities of living systems.

It is sincerely hoped that the various aspects of the
cytost concept discussed in the succeeding pages will
be of value in this way to workers in the biological sci-
ences. Primarily the material presented in this mono-
graph has been brought together with this in mind.

The science of medicine as distinct from the art of
medicine has depended upon advances in biological
science, and it frequently happens that new biological
concepts have contributed much to medical thought.
Some of our experiments have given rise to concepts
which the writer believes to be of distinct applicability
to the problems of pathology and medicine. Since,
however, this material is not of general interest to
the professional biologist, we have segregated such
discussion in a separate chapter at the end of the book.

During the course of his career the writer has been
especially fortunate in numbering among his friends
many who have greatly aided him in the prosecution
of his experiments. It is a pleasure to acknowledge
the helpful advice and assistance of Prof. W. S. Hall,
Drs. Elsie Berwig, A. Weesner, W. A. Evans, E. L.
Heintz, Isador Diamond, L. J. Tint, M. Lincoln,
Anton Rose, Arthur Knudson, Orville McKim, the
late Edwin Banshof, J. A. Behar, and Victor Car-
rabba, who at one time or another have assisted the
writer in the laboratory.

Fenton B. Turck

New York
June 5, 1932

CONTENTS

CHAPTER

PAGE

PREFACE Vll

I INTRODUCTION 1

II SHOCK, AN INDICATION OF TISSUE DAMAGE 13

III THE RELEASE OF CYTOST FROM CELLS 47

IV THE PHYSIOLOGY OF CYTOST ACTION 74
V FURTHER EXPERIMENTS WITH CYTOST 94

VI THE ACTION OF CYTOST ON SINGLE CELLS 117

VII NATURAL RESISTANCE AND FATIGUE 139

VIII INVESTIGATIONS CONCERNING NATURAL RE-
SISTANCE 167

IX THE BREEDING AND MANAGEMENT OF ANI-
MALS 193

X RELATION OF CYTOST TO BACTERIAL INFEC-
TION 2 1 2

XI INVESTIGATIONS CONCERNING THE NATURE

OF CYTOST 230

XII CYTOST IN THE PLANT WORLD 244

XIII CYTOST IN MEDICINE 257

XIV CONCLUSIONS 271

APPENDIX: SOME REPRESENTATIVE EXPERI-
MENTS 287

BIBLIOGRAPHY 295

INDEX 307

4 2 9 27

Chapter I
INTRODUCTION

I^r ^^^ ^f^

luj LIBRARY 5r

Ue»;

One of the most striking properties of living sys-
tems is their ability to modify their activities, not in
a continuous fashion, but in response to changes in
their environment. While responsiveness to external
variations is also shown by inorganic systems, the in-
duced changes of state are by no means so complex
and varied as those shown by living matter.

A further characteristic of living matter is that in
many instances an extremely minute environmental
variation brings about a response in an organism,
which is out of all proportion to the magnitude of the
change in the environmental conditions which has
elicited the biological response.

For example, the presence of 0.0000000249%
(about 1 part in 4,016,000,000) of copper sulphate in
water was found by Kanda (1904) to have a definitely
inhibitory effect upon the growth of seedlings of peas,
broad beans, and buckwheat. Mast (1928), working
with amoeba proteus, observed that when these pro-
tozoans are transferred from a hay infusion medium
to freshly distilled water, a large proportion of them
rapidly becomes inactive and markedly radiate in
form. Furthermore, the number of individuals which
attach themselves to the vessel walls is determined by

1

2 THE ACTION OF THE LIVING CELL

the nature of the latter. Many more attach themselves
to common glass than to quartz, pyrex glass, or paraf-
fin.

If on the other hand any one of fourteen salts is
added to the water, the animals attach at once, and
become lobose in a few minutes. These experiments
show definitely that the environment determines the
activity of this unicellular organism.

Every modification of an organism's activity may
be regarded as a response to an external stimulus, —
that is, to a change in the physical or chemical nature
of the environment. While it is true that one portion
of the organism may respond to some change in an-
other portion of itself, in the last analysis this primary
change in the latter is initiated by variations in the en-
vironment.

The response to external stimuli may be very slow
indeed, or may take place with almost explosive ra-
pidity. In the latter case the initial stimulus may be
regarded as a sort of trigger which releases a rapid
series of internal changes, in much the same fashion
as the impact of a firing-pin on the primer cap of a
high explosive shell initiates a destructive explosion at
a considerable distance from the gunner. Indeed, this
somewhat crude analogy bears a close resemblance to
the chain of events which takes place in a biological
system, wherein, as we shall see, a simple physical
stimulus often initiates a chemical reaction which in
turn propagates a series of vital changes.

It has been pointed out above that the application
of a stimulus to one portion of an organism may pro-
duce a response in a distal portion of the same organ-
ism. In higher organisms, the conduction of the series

INTRODUCTION 3

of events leading to the response of an effector at a
distance from the point stimulated may take place by
way of an elaborate nervous mechanism or by means
of a humoral transmission. For example, when a tasty
food is taken into the mouth, saliva is secreted by the
salivary glands although the food stimulus is at a dis-
tance from the glands. As is well known, this effector
response is brought about by the stimulation of sensory
nerve endings in the mouth, the nervous impulse thus
started passing by way of the brain to the salivary
glands.

Bayliss and Starling's classical investigation of se-
cretin offers perhaps the most convincing evidence
of the humoral transmission of a stimulus. These
workers found that acids acting upon the intestinal
mucosa of a denervated loop of intestine caused the
pancreas to secrete. Since the injection of acids into
the portal vein did not induce a similar pancreatic ac-
tivity, it was concluded that the acid acted upon some
component of the intestinal mucosa to produce a sub-
stance, termed secretin, which was carried by way of
the circulation to the pancreas and there caused the
effector response.

The validity of this assumption was established by
the observation that when intestinal mucosa is ground
with dilute hydrochloric acid, and then boiled to
coagulate the soluble proteins, an extract results which
upon injection into an animal induces the pancreas to
secrete. Such experiments yield irrefutable proof of
the secretin hypothesis, although to date the chemical
nature of secretin remains an open problem.

The type of reasoning employed in connection with
the above mentioned experiments on secretin is com-

4 THE ACTION OF THE LIVING CELL

mon in the biological sciences. Frequently, as we shall
see, we are unable definitely to isolate and characterize
the nature of a physiologically active substance, al-
though empirical experimental facts demonstrate the
presence of such substances and the probable role
which they play in the activity of the living organism.
As the American physicist Millikan (1931) remarks,
"The distinguishing feature of modern thought lies
in the fact that it begins by discarding all a priori con-
ceptions concerning the nature of reality — and takes
as its starting point experimental facts whether they
fit into any general philosophical scheme or not. — In
a word, modern science is essentially empirical."

Galileo's simple experiment on the velocity of fall-
ing bodies of dissimilar mass sounded the death knell
of an experimentally unsupported philosophical be-
lief, and may be regarded as the birth of experimental
science. In the biological sciences, both pure and
applied, similar unsupported hypotheses exist even
today, but these are one by one being subjected to the
critical test of experiment. Indeed, at present experi-
mental biology is far in advance of any adequate the-
oretical background.

In a colony of cells we must consider the presence
of all members of the colony as environmental factors
of any single cell under consideration. In such a
colony the activities, growth and reproduction (which
may conveniently be regarded as a special aspect of
growth) of individual cells is conditioned by the
presence of other cells, both homologous and hetero-
geneous. For example, the growth of yeast cells is
more rapid in cultures heavily seeded with the inocu-
lum than in cultures started from a few cells. In 1901

INTRODUCTION 5

Wildiers advanced the hypothesis that a substance
which he termed bios was necessary for the growth
of the yeast cell. Fulmer, Nelson, and Sherwood
found that yeast will grow in a synthetic medium pre-
pared from inorganic salts and pure sugar without the
addition of bios from another source. It follows
therefore that the yeast is itself able to produce the
growth-stimulating substance. In order to explain
the difference in growth rates as a function of the
amount of inoculum used, it is assumed that the
growth of yeast is accelerated by the presence of a
sufficient amount of bios of which each cell is capable
of supplying a certain amount; if the amount of the
inoculum is insufficient to contribute the necessary
quantity of bios then the growth of the yeast is re-
tarded.

Alternatively, a pure culture of cells may, by their
metabolic activities, give rise to toxic substances
which hinder and even completely repress the normal
activities of the cells. As an illustration we may cite
the fact that dextrose broth cultures of B. typhosus
become sterile after a few days, due to the lethal ef-
fects of the end products of the bacterial metabolism.
It is worthy of note, however, that the medium may
still be utilized as a suitable environment for the
growth of bacteria of another species.

It is well known that tissue cultures in vitro suffer
death unless supplied with fresh medium from time
to time. This fact is open to two interpretations: that
death may be due to the exhaustion of some essential
food factor; or that the cells, analogously to B. ty-
phosus, produce a toxic product. That the latter
hypothesis is the correct one is shown by the observa-

6 THE ACTION OF THE LIVING CELL

tion of Carrel, that a change of the medium surround-
ing the tissue is not sufficient to keep the culture in a
healthy condition. However, he found that if the tis-
sue is carefully washed and then supplied with fresh
culture media a normal rate of growth ensues. Evi-
dently, by such a process autotoxic substances are re-
moved.

Many workers have observed that certain infusoria
when left undisturbed do not remain scattered through
their aqueous environment, but aggregate into more
or less dense groups. Having observed that Para-
moecia may aggregate about a drop of dilute acid or
carbon dioxide solution, Jennings (1902) concluded
that the carbon dioxide produced by the infusoria
themselves was responsible for their aggregation in
a culture medium. This may or may not be true, but
at any rate it appears that some product produced by
the cells themselves is responsible for the observed
effect.

Let us now consider the effect of one type of cell
upon the growth of another. Here two possibilities
exist, — that of symbiosis, and that of antibiosis, numer-
ous examples of each being recorded in the literature.
For example, Holman (1923) found that in the pres-
ence of a minute anaerobe resembling B. pneiimo-
sintes,B. influenzae exhibits a good growth upon blood
agar slants, whereas in the absence of the former little
or no growth of the latter takes place. The opposite
effect is recorded by Passini (1926), who observed
that B.putrificiis verrucosus is able to destroy 5. tuber-
culosis when present in the same culture.

That one type of cell may profoundly affect the
behavior of another type is shown by the remarkable

INTRODUCTION 7

experiment of Drew ( 1923) , who found that whereas
in tissue cultures embryonic kidney cells grow as an
undifferentiated mass, the insertion of a bit of con-
nective tissue stimulates the kidney cells to differen-
tiate.

Similar effects are to be observed in multicellular
organisms; indeed, the humoral transmission of stim-
uli already referred to may be regarded as an example
of the influence of a chemical substance produced by
one group of cells upon another tissue. Such effects
have also been observed in plants, particularly in their
heliotropic responses. As is well known, green plants
bend towards the light. Many years ago, Darwin
(1880), experimenting with the seedlings of Phalaris,
reached the conclusion that the effect of the light is
not upon that part of the plant which actually bends,
but is due to a modification of growth which is brought
about by a stimulus transmitted from the tip of the
plant. This he proved by preventing the access of
light to the tip of the seedling, whereupon, although
the stem was illuminated, no heliotropic bending took
place.

It is a reasonable assumption that the stimulus is
transmitted by means of some substance resulting
from a photochemical change induced by the light
in the cells at the surface of the tip. In agreement with
this concept, Blaauw (1908) found that the helio-
tropic response of etiolated oat seedlings conformed
to the Bunsen and Roscoe law — namely, that the
chemical effect produced by light is proportional to
the product of the intensity and the duration of the
illumination.

Further proof of the chemical nature of the helio-

8 THE ACTION OF THE LIVING CELL

tropic response is to be found in the ingenious experi-
ment of Paal (1918), who found that if the tip of a
seedling was severed from its stalk and then replaced
after the insertion of a sheet of collodion between the
stalk and the tip, a more or less normal heliotropic re-
action was obtained. When, however, tin foil was used
in place of the collodion no such response could be
obtained. Obviously the former is permeable and the
latter impermeable to the photochemical product
produced in the plant tip. According to Blaauw's
measurements, an intensity of light equal to seventeen
hundred thousands candle-meter is sufficient to elicit
a heliotropic response in the oat seedlings examined
by him. This is equivalent to saying that the seed-
lings in a darkened room would bend towards a
lighted candle some two hundred and fifty feet dis-
tant. These surprising figures well illustrate the
sensitivity of plants to changes in their physical en-
vironment.

A similar reaction is observable in the stereotropic
•response of the young roots of plants. The applica-
tion of pressure to the apex of such a root causes it
to grow away from the source of pressure. A unilateral
trauma, bruise, or application of lunar caustic elicits
a similar growth response. Hence it is highly prob-
able that each of these agencies causes the elaboration
of a substance similar to that formed in the illumi-
nated plant tips.

Such responses to changes in the physical environ-
ment are by no means limited to plants. Jacques Loeb
as well as numerous other investigators have observed
similar phototropic and stereotropic responses in ani-
mals; and in consequence he was for a long time an

INTRODUCTION 9

ardent advocate of the essentially chemical nature of
such reactions.

The foregoing illustrations well illustrate the essen-
tially chemical nature of the mechanisms for corre-
lating the activities of living systems. Any marked
change induced in an organism must of necessity inter-
fere with such a correlating mechanism and hence
give rise to abnormal responses. Hence a pathological
state results. In the root tip experiments cited above,
we have seen that simple mechanical pressure and the
application of a caustic chemical elicit the same re-
sponses. Similarly Loeb (1924), in his experiments
on the regeneration of Bryophillum colycinum, has
shown that mutilation and illumination of the leaves
both result in the liberation of various soluble sub-
stances which appear to stimulate the growth of the
plant. Such experimental results as these offer evi-
dence that divers stimulating agencies may be causa-
tive of the same response.

For some thirty years the writer has argued that
the apparent stimulating and repressive activities of
various physical and chemical agents, as well as the
pathological changes which they induce, are due to
a common cause, — the release by the cells themselves
of a substance capable of modifying cellular activity.
For purposes of convenience in discussion, the author
has termed this important substance cytost.

Although the author developed his concept of cytost
on the basis of experiments conducted in his labora-
tory, the underlying idea is by no means new. Indeed,
a similar conception dates back to early antiquity, as
recently shown by Professor Breasted (1930) in his
study of the Edwin Smith Surgical Papyrus; for it is

10 THE ACTION OF THE LIVING CELL

recorded that the early Egyptian pyramid builder
Imhotep (4,000 B.C.), who, it is believed, was deified
as Esculapius, the Greek god of medicine, believed
that injury, traumatic or otherwise, produced its ef-
fects by "not something entering from the outside,"
but by "the intrusion of something which the flesh en-
genders." Breasted remarks, "This rational distinc-
tion is unmistakable, and demonstrates the surgeon's
ability to discriminate judiciously in the world of ob-
jective phenomena and natural causes."

It will be noted that Imhotep's intuitive conclusion
is exactly the same as that which has been reached
by Loeb and others in order to explain certain tropistic
responses in plants and animals. At the present time
we hear a good deal about the therapeutic effects of
sunlight, and by analogy to the interpretation of the
phototropic response in plants, we may imagine that
in the animal external radiation stimulates the release
of some substance from the tissues which is of benefit
to the body as a whole. This conclusion was likewise
known to the early Egyptians. Together with other
aspects of Egyptian culture, this knowledge was
passed on to the Greeks, who constructed the first cele-
brated solarium, which was conducted by the great
physician Hippocrates on the island of Coz. This
solarium was dedicated to the temple of Esculapius,
and the observed beneficial effects of sunlight were
supposed to be due to its power to release the "some-
thing which the flesh engenders."

Further, it was recognized by the early Egyptians
that the process of wound healing must depend upon
a stimulating substance released by the injured tis-
sues. Consequently their physicians attempted to
stimulate the healing of wounds by binding fresh flesh

INTRODUCTION 11

over the approximated edges of the injury. After the
juices from the flesh had started the healing process,
the wound was stitched together and dressed with an
antiseptic, stimulating honey.

Lamarck, in seeking to explain the influence of such
environmental factors as light, temperature, pressure,
motion and use, imagined that these physical factors
set in motion "certain subtle existing fluids that cease-
lessly flow," bathing the body cells in a vitalizing
medium. While it is unlikely that Lamarck knew of
Imhotep's speculations, it is of some interest that he
assigned an important role to a subtle entity ever
present in the tissues, in much the same fashion as did
Imhotep.

Although at the outset such conclusions appear to
savor strongly of the metaphysical, the author has
been forced to assume a somewhat analogous hypothe-
sis in order to effect a rational explanation of the re-
sults of his experiments conducted during the last
forty years. These experiments, which have covered
a variety of biological problems, have led to results
which can be interpreted only on the assumption of*
the elaboration by tissue cells of some substance which
is capable of modifying the activities of other cells.
This substance, which has been termed cytost, appears
to be capable, depending upon its concentration, of
exerting either a stimulating or toxic action upon cells
with which it comes in contact.

In the 1880's the writer observed that slightly muti-
lated plants outgrew controls. Some years later it was
found that in animals a slight injury frequently pro-
duced a similar stimulating efifect upon the animal,
whereas a more extensive injury resulted in a depress-
ing or morbid effect. As will be shown in the succeed-

12 THE ACTION OF THE LIVING CELL

ing chapters, many experiments conducted in divers
ways have demonstrated that these results and many
other biological phenomena may be explained by the
hypothesis that when injured, cells liberate cytost into
their surrounding fluids, and depending upon the
amount of this substance liberated, other tissues in con-
tact with these fluids are affected in one manner or an-
other. Carefully controlled experiments have shown
that when the latter are brought into contact with the
cytost of the same species, the growth and activities of
tissues are stimulated if the amount of cytost in the
surrounding fluids is small. On the other hand, if the
concentration of cytost in such fluids is too great, the
activities of the cells are inhibited, often to such an
extent that death ensues.

The theories of the writer are the logical out-
growth of certain observations which were made con-
cerning the nature and cause of shock. In order that
the reader may follow the development of the writer's
concepts and thereby be prepared for the discussion
of the very interesting experiments made in the
writer's laboratory in recent years, we shall consider
in more or less chronological sequence the various ex-
perimental investigations which have led to the devel-
opment of those concepts.

At the outset it should be noted that although from
time to time the author has enjoyed the collaboration
of several competent chemists, all attempts to isolate
and characterize cytost as a definite chemical entity
have been disappointing. Its presence, like that of
the enzymes and hormones, is made manifest only by
its action upon the living cell.

Chapter II

SHOCK, AN INDICATION OF TISSUE

DAMAGE

Our ignorance concerning the mechanism of living
processes makes it exceedingly difficult to define life
explicitly. We are accustomed, therefore, to differen-
tiate animate systems from inanimate by contrasting
their inherent properties, such as growth, reproduc-
tion, and response to stimuli. In such fashion alone
may we discriminate between the two.

Our ignorance regarding the transformation of in-
organic nature into living systems — that is, the origin
of life — coincides with our knowledge of the reverse
process, death. Owing to our continual consciousness
of life, we are individually more interested in the
causation of death than in the origin of life.

Nordenskiold (1928) records that the aborigines
considered natural death to be far more wonderful
than life. While they regarded violent death at the
hands of an armed enemy or a wild beast as a matter
of course, the aborigines were so awed by so-called
natural death that they importuned an armed demon
as the causative factor. Although refusing to acknowl-
edge the existence of such a demon, we are today al-
most as blissfully ignorant as was primitive man,

13

14 THE ACTION OF THE LIVING CELL

albeit numerous theories of death have been borrowed
from physical sources, such as the spontaneous disinte-
gration of the radioactive elements.

If one considers the energy changes accompanying
such transformations as a property of the radioactive
"life" of the element, then the "life" process ceases
when the substance has been completely transformed
to elements of lower atomic weights. Analogously,
some biologists have reasoned that the death of an
organism coincides with the exhaustion of some life-
giving substance.

Other workers, notably Child, Loeb, and Pearl,
have adopted the alternative view that in the course
of life the organism suffers an accumulation of toxic
metabolites which finally attain a concentration suffi-
ciently great to interfere seriously with the normal
metabolic processes, so that death ensues.

Nowadays it is generally accepted that the chemical
transformations upon which the organism depends
are determined by the presence of enzymes. In this
connection, it is worthy of note that in vitro experi-
ments with enzymes have frequently shown that the
latter are inactivated by the end-products of the
reactions which they catalyze. (Ter Muilen, 1905;
Morgulis and Beeber, 1928.)

The careful investigations conducted by Woodruff
and his students have demonstrated unicellular organ-
isms to be immortal. The common ciliate, Para-
mecium, was found during a period of years to pass
through several thousand generations without the
intervention of natural death. Similarly, the in vitro
culture of the somatic cells of various higher animals
has indicated such cells to be potentially immortal.

SHOCK 15

That connective tissue cells from the chick heart may
be kept alive in the laboratory for years exceeding
the normal life span of the fowl, and presumably, if
the tissue culturist so desires, for an indefinite period,
was shown conclusively by Carrel and Ebeling. Nat-
ural death, it thus appears, is restricted to multicel-
lular organisms.

During the evolution of such organisms, the cells of
the various tissues have become dependent upon their
neighbors for such necessities of life as food and oxy-
gen. For example, if the blood-flow to an individual
organ is interrupted, the cells of that organ die, owing
to starvation and want of oxygen, while other tissues
continue to live unless the death of the first organ in
some fashion deprives the other tissues of their normal
environment. If, for example, the cells of the glomeru-
lus of the kidney are damaged, as in nephritis, the loss
of proteins from the blood causes water to diffuse more
easily into the other tissues of the body, which thus
become waterlogged.

Coincidentally the elimination of nitrogenous waste
products is impaired and the body cells suffer from
their accumulation. In the brain, the accumulation
of such waste products results in coma and eventually
in the death of the organism. In similar fashion, the
injury or death of any of the component tissues of a
multicellular organism will result in disturbances and
perhaps the death of other tissues in the body.

When an animal is decapitated, death ensues; yet
for some time after the decapitation the individual
tissues may be shown to be alive. The heart, if re-
moved to a suitable environment and supplied with
the necessary salts, will continue to beat for some

16 THE ACTION OF THE LIVING CELL

hours, and the muscles, upon stimulation, will con-
tract. Indeed, the major part of our knowledge of
neuro-muscular physiology is based upon experi-
ments made upon the surviving organs of "dead" ani-
mals. Eventually, of course, such isolated tissues will
die, owing to starvation or other change from the
environment in which they normally find themselves
in the intact animal.

One must, therefore, distinguish between the death
of the organism and the death of the individual com-
ponents, the tissue cells of the organism. From a prac-
tical standpoint, the former is simply a loss of the
nicely balanced and integrated activities of the indi-
vidual tissue cells, while the latter is an actual death
which may result from the former.

On the other hand, as has been noted above, if the
tissue cells are transplanted to a suitable environment,
such as that employed in the technique of tissue cul-
ture, there is no reason to believe that they should not
live forever.

The localized death of tissue cells — i.e., necrosis —
may therefore be brought about in either of two
fashions: by a localized injury — for example, the
application of a caustic chemical or a destructive
physical agent such as X-radiation; or by a change
in the internal environment induced by the disfunc-
tioning of some remote tissue or organ, such as that
of the kidney, previously mentioned. This being the
case, it is logical to assume that because of the recipro-
cal relations existing between the component tissues
of an animal, a localized injury must inevitably result
in induced changes, or perhaps even the death of other

SHOCK 17

organs and tissues at a distance from the site of the
injury.

Thus, it is a matter of common experience that an
animal may be killed by a rap on the head. Here, as
in the case of decapitation, it is clear that the death
of the individual tissues does not immediately ensue,
but follows later as a result of the inability of those
tissues to function in the correlated and reciprocal
fashion necessary to keep one another alive. It is not
so generally recognized that an apparently minor in-
jury, such as a crushed extremity or a superficial burn,
may lead to the same end. Unfortunately, however,
this is a fact which is amply attested to by the results
of experience both in practice and in the laboratory.

In such instances as those cited above, by appropri-
ate experimental procedures we may determine the
order in which the various body tissues die, and the
histological changes which either accompany or re-
sult from their death. Little more, however, may be
learned from such experiments. In order to deter-
mine the mechanism causing the death of animals
under such conditions, we must resort to other modes
of investigation which afford an insight into the dy-
namics of the principal physiological and structural
changes which precede the end result of the experi-
ment. Such methods and the findings which have re-
sulted from their application must now be considered.

It is a common and disconcerting experience that
patients who have suffered an injury involving a local-
ized mutilation of tissue frequently die although the
injury has been easily repaired by the usual pro-
cedures. Prior to death, such unfortunate individuals

18 THE ACTION OF THE LIVING CELL

exhibit a highly characteristic series of symptoms:
generally diminished reflexes, and complete loss of
the postural reflexes, together with dilated pupils.
These are accompanied by extensive circulatory dis-
turbances manifested by a lowered blood pressure,
rapid but weak pulse, frequent and shallow respira-
tions, and a subnormal temperature.

Taken collectively, these symptoms are termed the
shock syndrome, and the animal exhibiting them is
said to be in a state of shock. Since in such cases as that
just considered the state of shock results from an in-
jury, it is termed traumatic shock, to differentiate the
condition from shock induced by other means.

Shock, or perhaps more properly the series of symp-
toms enumerated above, may be induced in various
other ways. For instance, if a rabbit, particularly one
with a large abdomen, is held by its head in an upright
position for half an hour, it will exhibit the symptoms
of shock, presumably because of the stagnation of
blood in the abdomen, and an inadequate filling of the
heart during diastole. If the rabbit is held in a binder
during the course of such an experiment, shock does
not ensue, because engorgement of abdominal vessels
is prevented.

Any other condition which leads to the same circu-
latory difficulties, as for example extensive hemor-
rhage, produces a similar state of shock. The same
symptoms may be elicited by the injection of histamine
and various bacterial toxins, and an analogous state
has been frequently observed in certain pathological
conditions, such as intestinal obstruction. In such in-
stances, the condition has been designated as toxic
shock,

SHOCK 19

While the clinical aspects of shock are of vast im-
portance, and in other publications the writer has
devoted considerable attention to them, we shall here
consider shock merely as an interesting physiological
state which may be produced, as we shall show, in
experimental animals. We shall utilize the shock syn-
drome merely as an indication of a definite physiologi-
cal state which may be induced at will in a living
animal.

Because, as has been shown above, this state may be
elicited by a variety of means which obviously by their
very nature have little in common, it appears highly
probable that each of them initiates a common series
of events which lead ultimately to the same physio-
logical state. Apparently the simplest type of shock
is the postural shock, which may be elicited in the
intact animal as previously mentioned. The induction
of shock by this means is especially interesting be-
cause it involves neither the introduction of any sub-
stance into the animal, nor any manipulation tending
to injure its tissues. In this instance, the only ab-
normality involved is that of suspending an animal
in an abnormal position such that its blood under the
influence of gravity accumulates in the splanchnic
area.

From this simple experiment we may infer that in
all cases of shock, however induced, the physiological
changes observed are in great part due to engorge-
ment of the splanchnic vessels. That this does actually
occur has been demonstrated in many experiments,
performed by the writer and by others.

The fact that animals may be thrown into shock by
traumatic injuries, burns, and the injection of various

20 THE ACTION OF THE LIVING CELL

toxic substances must mean that by all of these means
one may cause a marked stasis of blood in the splanch-
nic area. It was the investigation of this point which
occupied the author's attention some thirty years ago
and led to the various experimental investigations to
be considered in this book.

Before further discussing the phenomena of shock
and considering the underlying mechanism in detail,
let us briefly recount the results of a few experiments
conducted by the author. (Turck, 1918a.)

Cats were etherized, and after the application of
an elastic ligature to a leg in such a fashion as to shut
off the circulation, the thigh muscles below the liga-
ture were bruised and the tissues mangled. After
the lapse of an hour and a half, the constriction was
released and the limb occasionally massaged towards
the body in order to reestablish the circulation.

Shortly afterwards the animals began to show
symptoms of shock, culminating in death, although
other animals which had been treated in precisely the
same fashion except that the ligature was not released,
did not exhibit signs of shock during the same inter-
val of time. Bits of muscle were removed from the
mangled limbs of the latter animals and the juice
expressed was injected intravenously into the same
animal, whereupon shock promptly ensued, although
the ligatures on the limbs had not been loosened.

A similar result was obtained by transplanting the
bruised muscle tissue from below the ligature to the
uninjured muscles above it. An even more striking
result was obtained by transplantation of the injured
tissue into the thigh of an uninjured homologous ani-
mal, the latter invariably suffering shock and death

SHOCK 21

while the injured animal was frequently saved by
amputation of its injured limb above the site of injury.

Such experiments as these show unquestionably that
so-called traumatic shock results from the absorp-
tion of a toxic substance released by the damaged tis-
sues. If it were otherwise, then we should not expect
that the mere presence of the ligature above the site
of the injury would prevent the onset of shock.
Further, as is well known, tissues may be transplanted
from one animal to another of the same species with-
out the onset of shock. Hence, in the transplantation
experiments, such as those cited above, which result
in shock, another factor must be present: i.e., a toxic
moiety derived from the injured tissues which were
transplanted to the uninjured animal. The experi-
ments above referred to are of relatively recent date,
but the essentially toxic nature of traumatic shock has
long been stressed by the author. Indeed, as long ago
as 1895 the writer pointed out that in cases of carci-
noma of the stomach the carcinoma early produced
a toxic product capable of altering the gastric, secre-
tory and motor activities of the organ, and when
brought to operation such cases frequently pass into
severe shock. A year later (Turck, 1896), the conclu-
sion was reached that such unfortunate individuals
were in such a state of autointoxication that the addi-
tional tissue products released by the operative trauma
sufficed to precipitate shock.

In a series of papers published since these initial
observations were made, the author has contributed
a considerable amount of experimental evidence
which shows definitely that the shock following injury
is due to the release of an endocellular component

22 THE ACTION OF THE LIVING CELL

which under normal conditions is held innocuously
within the cell.

Without at the moment going into details, we must
note that since this substance is endocellular, cellular
disintegration or autolysis is a necessary prerequisite
for its release into the surrounding environment. With
this in mind, we may clearly understand the above-
quoted experimental results concerning traumatic
shock. Since, as is well recognized, tissue autolysis,
in common with other protolytic digestions, is a rela-
tively slow process, we are enabled to account for the
fact that the shock syndrome does not immediately
follow an injury, but, as a rule, becomes apparent only
after a lapse of some hours.

The validity of the preceding argument is strik-
ingly proved by the following experiment. (Turck,
1918a.) Fresh muscle taken from a normal animal
was permitted to undergo aseptic autolysis until
microscopic examination revealed a loss of nuclear
staining. The autolyzed tissue was then ground in a
mortar with normal saline and the supernatant fluid
was injected intravenously into homologous animals.
Shortly after the injection of a small quantity of the
autolysate the animal passed into shock and eventu-
ally died. Increasing quantities of the autolysate
hastened the onset of shock and death. Indeed, a single
injection of a lethal quantity resulted in death within
three minutes. On the other hand when a saline ex-
tract of fresh tissue — i.e., before autolysis — is injected
in similar fashion, shock does not follow. These very
simple experiments show conclusively that autolysis
is a necessary prerequisite for the formation of the
toxic entity responsible for traumatic shock.

SHOCK 23

If the arguments advanced above are valid,
then shock should be induced by any other means
capable of producing a localized death of cells
and the subsequent release of the shock toxin by
in vivo autolysis. Further to test this hypothesis,
animals under anesthesia were superficially burned,
w^hereupon shock ensued. This result, of course,
was to be expected from our knowledge of the ef-
fect of severe burns on humans. When, how-
ever, the burned area was transplanted to the back
of a second animal, and the normal skin of the latter
grafted onto the area of the first where the burned
tissue had been removed, the second animal passed
into shock and died, whereas the donor which had
been burned recovered with but slight symptoms of
shock. In one such experiment, in which the extent
of the burn was slight, the host did not develop symp-
toms for four days. On the other hand, a third animal
upon receiving an injection of a sterile autolysate of
the burned tissue of the first animal suffered immedi-
ate shock.

Such experiments as these demonstrate that in vivo
some time is necessary for the autolysis of the in-
jured tissue, and consequent production of a concen-
tration of the toxic substance sufficiently high to elicit
the response which we term shock. It is because of
this that the prompt removal of damaged tissues fol-
lowing an injury is efficacious in preventing the onset
of shock, whereas cases in which surgical interven-
tion is unduly delayed often show some degree of it,
all too frequently resulting in death. From the experi-
mental results cited above, it is clear that in such in-
stances in order to be efficacious in preventing shock,

24 THE ACTION OF THE LIVING CELL

removal of the damaged tissues must be accomplished
before autolysis has progressed to any appreciable ex-
tent.

Objectively, but without understanding the under-
lying reason for the procedure, this fact was recog-
nized by Duboys, who served with the French troops
in this country during the Revolution. He states that
"American surgeons amputated at once and lost but
few, but the French delayed and lost many." The
recognition of the value of such treatment was per-
haps due to Benjamin Rush, the surgeon-general of
our army during that period. Similarly, Larrey, a
French surgeon who early advocated the "debride-
ment" of wounds and thereby saved numerous lives
during the Napoleonic Wars, remarked, "The effects
of commotion (shock), far from being aggravated,
diminish and disappear insensibly after the opera-
tion."

The question may arise in the reader's mind: Why
does not the tissue damage which inevitably results
from simple cuts, burns, and bruises cause shock?
Simply because such injuries are not sufficiently ex-
tensive. Obviously when the area of tissue damaged
is but small, the toxins liberated are inadequate in
quantity to elicit serious consequences. Nevertheless
since the state of shock is but the culmination of a
series of physiological changes, we may well expect
the liberation of the shock toxin even in small amounts
to manifest some physiological activity.

This indeed is the case, and the writer's experiments
have, he feels, placed this substance in a unique posi-
tion in the internal environment of the animal : that is,
as a substance capable of modifying to a considerable

SHOCK • 25

degree the activities of the component body cells. For
this reason, the name shock-toxin has been abandoned,
and the name cytost applied to the substance liberated
by cells which initiates the series of physiological
changes which may result in shock. It must be remem-
bered, however, that for such a state to ensue, a rela-
tively large amount of cytost must be liberated.

From the standpoint of general biology, the effects
of small quantities of cytost upon the organism are of
far more importance than the effects of quantities suf-
ficiently great to cause shock. Indeed, as the reader
will see as our discussion progresses, cytost apparently
plays a unique role in the physiological behavior of
the animal. As the investigations concerning this as-
pect of our subject have been carried out almost ex-
clusively in the writer's laboratory, the primary ob-
ject of the succeeding chapters will be to disclose the
methods used in our experiments, the results obtained,
and the conclusions which may logically be deduced
from them.

However, before passing to this admittedly more
interesting portion of the author's work, we shall
further discuss the earlier experiments relative to
shock. Such discussion is necessary for two reasons:
first that the reader may gain an insight into the finer
details of the action of cytost; and second that he may
follow without difficulty the gradual development
of the writer's thesis as it has taken place over a period
of some years.

Let us now return to our discussion of shock par-
ticularly as regards the modes of experimentation
other than those previously considered.

Years ago (Turck, 1897), the writer found that

26 THE ACTION OF THE LIVING CELL

shock could be induced in animals by exposing the
viscera to a draught of cold air. Further, the onset
of symptoms was found to be accelerated by frequent
manipulation of the exposed viscera. After dogs had
been caused to pass into a profound state of shock by
such means, they were bled and their separated blood-
serum was injected into normal dogs. The effects so
elicited in the latter were found to be governed by
the amount of serum injected. For example, 5 cc. of
such serum produced little effect; SO cc. caused the
animal to show decided evidence of fatigue; while
larger quantities brought about collapse. (Turck,
1901.) At that time the analogy was drawn between
these results and those obtained by Mosso with fatigue-
sttiff. The latter author found that if the blood of a
fatigued animal were injected into a normal animal,
the unfatigued one showed signs of fatigue. In other
words, the production of shock in an animal brings
about the presence in the blood of some substance
capable of causing the onset of the signs of fatigue in
normal animals. As will be shown later, it appears
that this substance is cytost, the substance released
from injured tissues which, as we have seen, is the
primary cause of shock.

Further, the analogy between the shock-toxin and
the fatigue stuff of Mosso is especially interesting for,
as will be shown in a later chapter, there exists a close
correlation between an animal's resistance to fatigue
and to cytost.

The evidence so far presented refers only to the
primary cause of shock, — the release, by injury, of
the endocellular substance cytost, which is capable,
if present in sufficient concentration, of causing the

SHOCK 27

unique train of physiological events which finally
lead to the condition termed shock. While recogniz-
ing that an adequate understanding of the cause of
such a condition is of paramount importance to the
surgeon, our interest in the subject is to be rather
closely confined to the underlying physiology of the
reaction and the relationship of such physiological
behavior to the problems of general physiology.

That the reader may properly understand the
underlying mechanism by which such reactions are
produced, we must revert to some experiments which
antedate by some years the majority of those pre-
sented above.

Since our problem is to determine the effects of a
localized tissue damage upon the organism as a whole,
it is obvious that development of a technique for pro-
ducing such a localized injury in various parts of the
body is highly desirable. By means of an apparatus
termed the gyromele, the author (Turck, 1896b,
1903a) has been able both to apply various reagents
to specific areas of the stomach mucosa and to with-
draw at will material from any portion of the organ.
The instrument consists essentially of a hollow rotat-
ing sound which may be introduced into the stomach
by way of the esophagus. By means of the rotating
mechanism, the distal portion of the sound may be
approximated to any desired area of the gastric mu-
cosa, the exact position of the tip being easily located
by palpatation or fluoroscopy. Once the tip of the
instrument has been placed in the desired position, a
small sponge heretofore sheathed in the hollow sound
may be extruded, thus permitting the localized appli-
cation of any desired reagent to the mucosa. Similarly

28 THE ACTION OF THE LIVING CELL

the sponge may be utilized for the extraction of mucus,
bacteria, etc., from such foci. After the sponge has
been utilized for the desired purpose, it is resheathed,
and the instrument withdrawn from the stomach.

The course of action of reagents applied in this
manner may be followed cytologically by the micro-
scopic examination of samples of the stomach mucosa
removed by means of intragastric nippers designed
for this purpose. In some instances the course of ac-
tion of gastric irritants was observed by means of a
permanent fistula established by making a valvular
opening in the anterior wall of the stomach. This
was accomplished by folding the mucous membrane
in such a fashion as to make a valve similar to the
ileocecal. (Turck, 1896a, 1898.) Upon introducing
a speculum and a small electric lamp through such a
fistula, the entire interior of the stomach could be
viewed; at other times, the valvular fold prevented
the escape of the stomach contents.

Let us now consider the results obtained by the
application of such experimental methods. By means
of a needle douche introduced through the esophagus,
a 0.2% emulsion of mustard in water was douched over
the stomach mucosa of dogs in which gastric fistulae
had been established. The changes so induced were
observed through the fistula as described above.
Within five minutes a local hyperemia of the mucous
membrane was observed ; and after twenty minutes
the tissues were markedly congested. A similar local-
ized hyperemia of the mucosa was induced by the
introduction of water at a temperature of 40° to 45° C.,-
and by the mechanical stimulation of the membranes

SHOCK 29

by means of the gyromele, which was revolved within
the stomach for three minutes.

The important point which we must here stress is
that a similar localized response — namely hyperemia
— is elicited by means of three decidedly dififerent
agents: a chemical irritant, a temperature rise, and
mechanical irritation. (Turck, 1900.) Repetition of
these experiments in animals whose abdomens had
been opened in such fashion as to permit observation
of the splanchnic circulation showed that a moderate
stimulation of the gastric mucosa by any of the three
agents discussed above did not produce any marked
changes in the splanchnic circulation. When, how-
ever, the chemical or mechanical irritation was pro-
longed, or the temperature of the water introduced
into the stomach was sufficiently high, there was evi-
denced a marked change in the splanchnic circulation.
There first appears a marked hyperemia, which is
followed by an intense venous congestion, the veins
becoming distended, and as a result the intestines as-
sume a dark purplish shade. Finally such splanchnic
congestion causes a pronounced peripheral anemia.
It is worthy of note that this picture is identical with
that observed in shock produced by any other means.
(Turck, 1897.)

A similar state of affairs was induced by inflating the
stomach with air after ligation of the pylorus. Within
five minutes, venous engorgement of the gastric ves-
sels ensued, followed in another five minutes by
splanchnic congestion, and subsequently by cardiac
dyspnea, respiratory failure, and finally complete col-
lapse of the animal. Comparable results were obtained

30 THE ACTION OF THE LIVING CELL

by inflating the intestines with air. When a stream of
compressed air was allowed to impinge upon the ex-
posed viscera of rabbits, the viscera became bluish-
black, due to engorgement, and the animals developed
the general symptoms of shock.

Lastly the writer has induced shock symptoms in
intact animals by placing them for a minute or less
close to the arm of a centrifuge revolving at ten thou-
sand revolutions per minute. (Turck, 1918a, 1918b.)
The same result was also obtained by discharging
a 38-calibre blank cartridge in a box containing the
experimental animals. In the case of aquatic animals
such as fishes, turtles, and frogs, a similar state ensued
when the cartridge was fired just beneath the surface
of the water in the aquarium. (Turck, 1918b.) At
autopsy, the animals used in such experiments were
found to have suffered a splanchnic congestion identi-
cal with that observed in experimental shock pro-
duced by other means.

Although these experiments were originally de-
signed to duplicate in the laboratory a condition analo-
gous to the "shell-shock" often experienced by sol-
diers in combat, they are here presented merely as an
illustration of a means of producing a profound state
of shock in intact animals. While by such experi-
mental means no externally obvious injury is pro-
duced, it seems clear that the pressure waves en-
gendered alike by the centrifuge and the explosion
cause an injury which leads to a series of in vivo
changes finally resulting in a state of shock.

Since the same final state may be induced by such
a variety of markedly different experimental methods,
the question arises : What is the common factor which

SHOCK 31

eventually elicits the same end? It is quite apparent
that the experimental methods, such as the applica-
tion of caustic chemicals, burns, trauma, and air pres-
sure, have, from a purely physical standpoint, noth-
ing in common. We must therefore seek the common
factor within the animals themselves, or more prop-
erly within the living cells whose individual activities,
as we have seen, determine to a large extent the be-
havior of one another.

In order to understand the cellular changes which
follow the application of such agents as those enumer-
ated above, let us return to the author's experiments
on the stomach mucosa. In these experiments, by the
methods previously mentioned, we were enabled to
establish a localized injury by mechanical means, or
by the application of irritating chemicals. After ex-
perimentation with various irritants, mustard, because
of its relatively mild, yet prolonged, irritating quali-
ties, was utilized for the production of an acute gas-
tritis. (Turck, 1902.) That we may follow the course
of the physiological changes induced by the mustard,
we shall first consider the local effects.

After the washing out of the stomachs of healthy
dogs which had been without food for twenty-four
hours, a ten per cent aqueous suspension of ground
mustard was introduced into the organ by means of a
stomach tube. After the mustard had remained in
the stomach for an allotted time, the abdomen of the
animal was opened under anesthesia and specimens
of tissue were excised from the oardia, fundus, body
pylorus, duodenum, and gall ducts, and prepared by
fixation and staining for cytological study. Such ma-
terial was obtained at various times up to forty-eight

32 THE ACTION OF THE LIVING CELL

hours after the introduction of the mustard. (Turck,
1902.) The salient cytological changes observed may
be summed up as follows:

First an exudate, consisting of cells, granular
debris, fibrillar masses, leucocytes, and erythrocytes,
appeared on the surface of the mucosa within an hour
or two after introduction of the mustard emulsion. At
this time the cells, including the nuclei, stained
readily. The capillaries within the interglandular
connective tissue beneath the epithelium were mark-
edly engorged, and the blood vessels in the submucosa
were likewise distended. After about two hours, the
mucosa was found to be covered with a thick layer of
broken down, desquamated cylindrical epithelial
cells. Some cells in the ducts appeared to be degener-
ated acid cells, while some proliferation of the inter-
stitial tissue was apparent. The blood vessels of
the outer coats, at this time, were found to be some-
what distended and filled with blood.

At the expiration of six hours, mitosis was visible
everywhere, the nuclear figures being plainly dis-
cernible. The interglandular connective tissue, espe-
cially at the upper portions of the mucosa and beneath
the surface lining, was markedly infiltrated with leu-
cocytes and round cells. The blood vessels of the sub-
mucosa and serous coats were enormously distended
and filled with blood. The cardia fundus showed the
most marked inflammation, the epithelial cells being
disarranged and exhibiting various degrees of degen-
eration ranging from a simple granular stage to com-
plete necrosis. The cytoplasm of such cells stained
comparatively easily with eosin, the intensity of the
staining being conditioned by the degree of degenera-

SHOCK 33

tion, while the nuclei took the nuclear stain lightly
or not at all.

Stomach tissue which had been exposed to the
action of the mustard for longer periods exhibited
much the same picture, but with further separative
changes and extension of the inflammatory process to
the duodenum and bile ducts. The surface epithelium,
curiously enough, was not destroyed in proportion to
the very marked changes occurring in the deeper tis-
sues of the mucosa. Apparently, then, the mustard
emulsion produced a localized tissue injury, which
in turn was responsible for the more extensive damage
observed in the deeper tissues. This observation is
of especial importance because it shows clearly that
when one tissue is damaged by experimental means,
adjacent tissues not directly in contact with the injuri-
ous agent may suffer from the primary injury. This
argues for the transmission of an injury from one tis-
sue to another and presents us with the problem of
finding the mechanism of such a transmission.

Previously, we have called attention to the fact
that the introduction into the stomach of a dilute mus-
tard emulsion brings about a congestion of the vis-
ceral circulation analogous to that caused by me-
chanical irritation of the stomach lining. When more
concentrated mustard emulsions are utilized for this
purpose, the general symptoms of shock are elicited.
In such an experiment, within five minutes after the
introduction of the irritant, the splanchnic vessels
were found to be hyperemic; within ten minutes the
splanchnic veins evidenced marked congestion and
distention; and after the lapse of twenty minutes, the
splanchnic congestion had become so extensive as to

34 THE ACTION OF THE LIVING CELL

cause peripheral anemia. (Turck, 1900.) Clearly, a
condition of shock had been induced by the localized
action of mustard in the stomach.

That the action of the irritant per se was limited to
the stomach and not due to its absorption was easily
demonstrated as follows: After the mustard had re-
mained in situ long enough to induce hyperemia of the
splanchnic vessels, it was removed by lavage and the
administration of slippery elm water, whereupon
the splanchnic congestion was speedily reduced.

If, as these experiments indicate, the action of the
mustard is purely local, then the general physiological
changes which are induced by its application to the
gastric mucosa must be brought about by some sub-
stance liberated by the tissue cells under the action
of the mustard. Such an hypothesis must necessarily
be advanced to explain the production of shock by
the mechanical irritation of the stomach, for obviously
the mechanical instrument utilized to effect the pri-
mary injury can by no stretch of the imagination be
endowed with any other than a purely local action.

We are now in a position to understand why shock
may be induced by such diverse means as trauma, me-
chanical irritation, burns, and chemical irritants.
Each of these means results in cell destruction with
the liberation of the endocellular substance, cytost,
which is absorbed, and causes a splanchnic congestion,
in turn giving rise to the external symptoms collec-
tively termed shock.

If this hypothesis is correct, then any agent capable
of effecting the liberation of cytost in sufficient quan-
tity will cause splanchnic congestion, stagnation, and
shock. For example, toxins formed in the stomach

SHOCK 35

as a result of bacterial growth may act as local irri-
tants and thus initiate a series of events similar to those
observed in the experiments with mustard. This was
pointed out by the author thirty-five years ago.
(Turck, 1896.)

The accumulation of such toxins in the stomach
may result in atony, splanchnic congestion, and even-
tually in collapse. This was shown experimentally
as follows:

The stomach contents were withdrawn from a pa-
tient suffering from gastritis with marked distention,
who had fasted for fourteen hours. The fluid was
filtered through paper and then passed through a
Pasteur filter, the sterility of the filtrate being checked
by a control culture. A portion of the filtrate was then
injected subcutaneously into rabbits. Within half an
hour the animals showed paralysis of the hind legs,
the toxic ef^fect resembling that observed in rabbits
bitten by venomous snakes. Injection of a further
quantity of the filtrate caused the death of the animal
within three hours. Injection of the stomach contents
of healthy individuals does not elicit such marked
symptoms. (Turck, 1896.)

The normal healthy stomach is not a satisfactory
environment for the growth of microorganisms.
However, any factor which causes a marked irritation
of the stomach mucosa results in an exudation of a
sticky material which accumulates upon the stomach
wall. The author found this to be an excellent cul-
ture medium for various bacteria known to occur
in the stomach. Apparently in situ the mucin deposit
protects the organisms from the action of the hydro-
chloric acid of the gastric juice. Cultures from such

36 THE ACTION OF THE LIVING CELL

material are readily obtained by means of the gyro-
mele.

The introduction of mild irritants into the stomach
of an animal results in pathological changes which
render the mucosa a satisfactory culture medium for
the growth of microorganisms. In his early experi-
ments the author found that if a dog's stomach is
washed out daily with tannic acid for a period of
two weeks and then inoculated with material taken
from the stomachs of patients suffering with gastritis,
the bacteria grow readily and by their growth induce
a series of tissue changes resulting in a pathological
picture identical with that found in cases of gastritis
in humans. (Turck, 1896.)

First the gland cells become enlarged and cloudy,
numerous leucocytes appear on the surface and
marked hyperemia is evident. After the lapse of a
few months the cellular changes appear more marked,
cystic degeneration of some glands occurring while
others undergo a metamorphosis into mucoid glands.
Eventually there is an extensive round cell infiltra-
tion and connective tissue formation, while the sur-
face becomes coated with leucocytes. This pathologi-
cal picture is distinctly analogous to that obtained by
the action of mustard upon the stomach mucosa.
Hence again we may reasonably postulate the inter-
vention of a common factor resulting from tissue
injury.

It was easily supposed that the general toxic effect
which accompanies gastritis was due to the absorption
of toxins generated by the bacteria growing in the
stomach. This was of course a natural supposition.
Since, however, much the same state of affairs may

SHOCK 37

be induced by the action of mustard in the stomach,
and, as the author has shown (Turck, 1902) that in
such experiments the exudate which first forms is not
a good medium for the growth of bacteria, it seems
more reasonable to postulate that in gastritis the ap-
parent general toxemia is due to endocellular sub-
stances released from the cells injured by the meta-
bolic activities of the microorganisms.

Such a concept is in agreement with the well known
fact that bacterial toxins, with the exception of that
formed by B. botulinus, are not absorbed through
the gastrointestinal tract.

Early in the history of anesthesia, it was recognized
that a state of profound shock may be induced by the
anesthetic. In 1866, Sabaarth reported 119 cases of
death during chloroform anesthesia, less than half
of which appeared to be due to a direct toxic effect
of the anesthetic. In the same year Nothnagel reported
that inhalation, or injection of chloroform and ether,
produced a fatty degeneration of various organs. So
far as we know, the experiments of the latter author
were the first to demonstrate the specific toxic action
of these anesthetics upon the body cells.

Early in the author's career, he was impressed by
the fact that some experimental animals showed evi-
dence of shock more readily than others of the same
species, even comparatively trivial manipulation
under anesthesia resulting in shock and subsequent
death a day or two later. As it seemed likely that such
results were due largely to the effect of the anesthetic
per se, the latter were investigated in the absence of ex-
perimental surgery. (Turck, 1903b.)

A series of healthy dogs were kept under deep

38 THE ACTION OF THE LIVING CELL

chloroform or ether anesthesia for periods of from
three to six hours. From twelve to twenty-four hours
after the removal of the anesthetic, the animals uni-
formly exhibited the symptoms of shock, such as sub-
normal temperature and blood pressure, and pale
tongue and cold surface; frequently death ensued. At
the time these experiments were published the author
commented that: "The conclusions that can be drawn
from the details of the experiments made on animals
under chloroform or ether are that where shock is
produced it does not materially differ from the shock
that results from trauma. The most constant patho-
logic factor is failure of circulation, and this is espe-
cially expressed in splanchnic congestion." (Turck,
1903b.)

When blood serum obtained from dogs which had
been subjected to anesthesia the day previous was in-
jected intravenously into normal dogs, the latter de-
veloped symptoms of shock. This result shows that
in shock produced by anesthesia cytost is liberated in
the blood in much the same fashion as in shock induced
by other means.

The following protocols taken from the paper
above referred to clearly show the nature of this re-
action.

•

Experiment 16. Mongrel, placed under ether anesthesia; pres-
sure 140; kymographic tracings. Ether. Temperature, 41. In-
jected 90 cc. serum from dog which had been under anesthesia for
5 hrs. The serum had been removed the following day. Blood pres-
sure fell 80-70 ; forty-five minutes, temperature 36° C. Coagulation
marked, interfering with further blood pressure records. Anes-
thesia removed ; animal placed in warm location ; temperature fol-
lowing day 37° C. After three days, animal still showed symptoms

SHOCK 39

of shock. Died on third day. Postmortem revealed no evidence of
infection. Cultures negative.

Experiment 17. Small black and tan under chloroform. Blood
pressure, 130. Serum used from a dog which had been under anes-
thesia five hours, and bled the following day; 25 cc. of serum in-
jected. Fifteen minutes, pressure fell to 115; twenty minutes, 110;
thirty minutes, 95 ; 25 cc. serum injected. Blood pressure, 15
minutes following the last injection, 65-70. Wide excursion. Anes-
thesia removed. Animal died the following day.

Experiment 18. Mongrel bull. Ether anesthesia. Blood pres-
sure 150. Serum from an animal that had been under ether for
four hours. The animal had been bled immediately after removal
of the anesthetic. Injection of 22 cc. followed by another injection
of 22 cc. No effect on blood pressure, 20 cc. more no effect on blood
pressure; 20 cc. pressure 100. In one hour pressure 85. Anesthetic
withdrawn and animal recovered.

Experiment 22. Dog, yellow; temperature 38.8°. Injected serum
from animal six hours under anesthesia. Temperature, 38.3°.
Amount injected, 25 cc. and 20 cc. Temperature went down to
37.2°. The following day rectal temperature 38.3°. Animal list-
less ; cannot be around, and lips appear pale ; marked loss of sensa-
tion. Second day, temperature 39.5°. Animal depressed; marked
loss of appetite. Fourth day, animal bled ; shows rapid coagulation
and taking.

At this point it should be noted that when animals
are anesthetized for a period sufficiently long to in-
duce shock, invariably it is found that the coagulation
time of the blood is shortened. We shall reserve dis-
cussion of the significance of this observation for a
later chapter, but it may not be amiss here to note that
an increased ease of coagulation of the blood will
facilitate clogging of the capillaries and hence con-
gestion in the larger vessels — a characteristic phe-
nomenon in all cases of shock regardless of the means
by which this unique physiological state is induced.

For a long time it has been common knowledge that

40 THE ACTION OF THE LIVING CELL

when subjected to surgical procedures, animals or
patients are more apt to pass into a state of shock if
the anesthesia is unduly prolonged. This is readily
understood when we recall that our experiments have
shown that both anesthetic and trauma are able to
effect a liberation of the shock-toxin, cytost. Hence,
when operated upon, an anesthetized animal is sub-
jected to the action of a higher concentration of cytost
than in the case of subjection to either of the experi-
mental methods alone. This state of afifairs, then, is
closely analogous to that previously postulated to
explain the marked susceptibility to surgical shock
of patients with carcinoma of the stomach.

In this connection it is of interest to note that such
cases when subjected to operation are subjected to
the action of cytost liberated in three ways: by the
carcinoma, by the anesthetic, and by the operative pro-
cedure — a truly formidable combination of factors
calculated to liberate cytost.

At this point it is perhaps proper to stress the fact
that in general the subjection of animals to two or more
harmful agencies, as in the examples just considered,
may lead to a synergism, or apparent potentiation of
one another. Thus, in 1900, the writer found that ani-
mals which had been in shock were easily susceptible
alike to the action of Staphylococcus albus, Staphylo-
coccus Pyogenes aureus, and Bacillus coli, whereas
injection of cultures of the first and third of these
organisms into normal animals yielded only negative
results.

It follows therefore that in the presence of a high
concentration of cytost resulting from the action of
the agency utilized to produce shock, the virulence of

SHOCK 41

these saprophytic organisms was apparently in-
creased. When, however, we remember that by the
virulence of a microorganism we mean little more
than a quantity which is inversely proportional to the
resistance of the host, it is seen that in the presence
of a relatively high concentration of cytost the ability
of the host to overcome a comparatively weak invader
is nullified. This interesting aspect of the subject will
be treated more fully in a subsequent chapter.

Let us now return again to the effects of anesthesia,
for we must consider yet another source of toxemia
resulting from the action of the anesthetic upon the
gastrointestinal tract. (Turck, 1903b.) Animals in
which gastric fistulae had been previously established
were anesthetized with chloroform or ether in such
a fashion as to preclude swallowing of these sub-
stances. This was accomplished either by tracheot-
omy or effective plugging of the esophagus. After
brief periods of anesthesia, the stomach and its con-
tents were examined by means of the fistula.

An initial hyperchlorhydria was observed, this be-
ing followed by a diminished secretion of acid and
ferments. Subsequently large quantities of mucus
were formed. These results harmonize with those
previously found when other irritants such as tannin
and mustard were applied to the gastric mucosa.
(Turck, 1895, 1896.) Incidentally, these results were
subsequently confirmed by Pawlow (1902) .

The presence of anesthetics in the stomach was
proved by distilling washings and applying the usual
chemical tests to the distillate. Early in this chapter
we have summarized the author's work concerning
the production of shock by gastric irritants. From

42 THE ACTION OF THE LIVING CELL

what has been said, it should be apparent to the reader
that since, following inhalation, the anesthetics actu-
ally reach the stomach and elicit therein effects analo-
gous to those produced by other gastric irritants, we
may well expect shock to result from the action of
ether and chloroform on the stomach mucosa. This
is in agreement with the observations of Brunton
(1895), who found that the ingestion of chloroform
produced vomiting and irritation of the gastrointes-
tinal tract, leading to circulatory depression similar
to that observed in shock.

For many years, clinical observers have noticed that
following anesthesia there may be a marked atony
of the stomach and intestines. In animals after liga-
tion of the pylorus and cardia the writer has observed
through a fistula that such atony results in an accumu-
lation of gases which distend the stomach. When the
gases are removed, gas formation is found to continue
at a rate more rapid than could be accounted for by
fermentation. The gas, therefore, must have resulted
from diffusion of the blood gases. As a result of the
distention of the organ by these gases, the splanchnic
circulation must be affected in much the same manner
as was found to be the case when the stomach was in-
flated with air.

That toxins are actually produced in the stomach
as a consequence of atony was easily demonstrated by
the injection of filtered stomach contents into normal
animals. When stomach contents withdrawn from
normal animals was injected into dogs, quantities as
great as 40 cc. did not produce toxic effects, whereas
smaller quantities of such material taken from patients
with atony of the stomach — particularly that result-

SHOCK 43

ing from chloroform and ether anesthesia — upon in-
jection caused marked symptoms of toxemia.

As a result of the experimental research presented
in this chapter we may safely conclude that the pri-
mary cause of shock, regardless of the technique util-
ized to elicit the reaction, is the liberation of an endo-
cellular toxin which, by its action upon the circula-
tory system, results in an engorgement of the vessels
in the splanchnic area.

In subsequent chapters we shall consider the con-
ditions under which cytost is liberated, its probable
nature, and the mechanism of its action on tissues.
We shall have little more to say regarding the various
aspects of shock; nevertheless it may interest the
reader to note that the writer's early experiments upon
the toxic nature of shock laid the groundwork for
more general investigations which are to be con-
sidered in this book.

In the interests of historical accuracy, the follow-
ing may prove worthy of note:

In 1896 and subsequently the writer published a
series of papers in which it was maintained that trau-
matic shock was essentially a "shock by toxin" from
damaged tissues. In discussing the results of experi-
ments with anesthetics it was stated (1903b) : "The
explanations that are usually given for the complex
symptoms found in shock from anesthesia cannot be
explained by the simple failure of blood pressure and
respiration, but there appear to be some direct toxic
bodies formed that are found in the blood serum.
The injection of serum from animals that have shown
evidence of shock produces many of the symptoms
manifested in shock, which include loss of sensation,

44 THE ACTION OF THE LIVING CELL

general malaise, marked fall in blood pressure, ster-
torous respiration, and, what is generally more sig-
nificant, rapid fall in blood pressure."

And, "The conclusions that can be drawn from the
details of the experiments made on animals under
chloroform or ether are that where shock is produced
(by the anaesthetic) it does not materially differ from
the shock that results from trauma."

While in papers published previously to that from
which the foregoing is taken, the author stressed the
concept that shock was induced by toxins liberated
from damaged tissues, the above statements show
clearly that the author was cognizant of the very gen-
eral nature of the underlying mechanism of shock
brought about by divers experimental means. This
was early recognized by Munch, who published a re-
view of the writer's early experiments in 1903.

In general, however, it must be admitted that the
writer's long-standing thesis did not until recently
receive either general recognition or experimental
confirmation by others. However, with the advent
of the Great War, and the appalling incidence of
shock amongst the wounded, a concentrated effort was
made by many workers to discover the underlying
physiology of shock. Until almost the end of the War,
nevertheless, a number of prominent research workers
were committed to the theory that shock is of nervous
origin. In 1918, Quenu, on the basis of his experience
with the wounded, was led to postulate the existence
of a shock toxin. Shortly afterward, Dale and Laid-
law (1919) found that the intravenous injection of
histamine would produce symptoms identical with
those of shock, and in consequence it has been postu-

SHOCK 45

lated that this substance, or a similar substance, is
liberated when tissues are injured. As will be shown
in our subsequent discussion, it is improbable that
cytost and histamine are identical.

Bayliss, Cannon and their associates have shown in
a fashion analogous to that employed by the author
that crushing the thigh muscles of anesthetized cats
will induce shock in the animals. Cannon (1923)
showed further that if circulation in the animal's legs
is arrested by clamping the abdominal aorta, no symp-
toms of shock appear until the release of the aortic
clamp.

These results are in agreement with those of the
author's experiments, wherein he utilized a tourniquet
to prevent circulation in the animal's limbs. Again,
by means of a parabiotic union of the circulation of
two animals. Cannon has shown that the production
of an injury in one animal leads to a state of shock
in both animals.

This type of experiment has also been conducted by
the writer (unpublished) , but in view of the fact that
much the same result is achieved by the injection of
the blood serum or tissue juices of the injured animal
into a normal animal, the writer has felt it unneces-
sary to adopt the elaborate technique of parabiotic
experiments. Furthermore, in such an experiment,
the "normal" animal may well be rendered hyper-
sensitive to the shock reaction because of the tissue
damage incident to the establishing of the parabiosis.

The validity of the writer's contention concerning
the toxic nature of shock has been recognized by Jean-
neney (1921), Hartmann (1922), and Limousin
(1923).

46 THE ACTION OF THE LIVING CELL

The reader interested in the history of the author's
views on the toxic nature of shock is referred to these
authors, and especially to two papers by the writer
published in 1922 and 1929, entitled respectively
"Shock and Fatigue" and "Researches on the Shock
Reaction before the Great War, Confirmed by Ac-
tual Experience in War." The latter paper contains
a review of our investigations from the time of their
inception, and the former contains a number of de-
tailed protocols of the experiments on shock.

In a recent publication, Zschau (1931) has pointed
out that the so-called electrosurgery has an advantage
over the ordinary methods of surgical procedure, in
that the incidence of operative shock is unusually
slight. This he attributes to the fact that the resorp-
tion of the products of tissue disintegration is con-
siderably lessened by the lymph coagulation which
attends the use of the electric knife, but does not take
place when ordinary surgical methods are employed.

Chapter III

THE RELEASE OF CYTOST FROM CELLS

In the previous chapters we have called attention
to the fact that under the proper conditions, living
cells appear to be potentially immortal. The "proper
conditions" consist in an adequate food supply and
a satisfactory mechanism for the removal of non-
utilizable and toxic metabolites. By its growth and
ever-changing activities the cell expends energy, and,
not being endowed with an inexhaustible supply of
the latter, obtains it by the oxidative degradation of
foodstuffs.

While the greater part of ingested foods is actu-
ally utilized as a source of energy a smaller quantity
is transformed by suitable chemical reactions into the
proteins, carbohydrates and fats which constitute the
basis of the morphological structure. As a conse-
quence of the chemical changes taking place within
the cells, numerous end products which are not
utilizable either as a source of energy or for struc-
tural purposes are excreted. Many of these end
products, if not removed from the immediate environ-
ment of the cell, exert a toxic action which ultimately
results in the death of the cell.

It is now generally accepted that the chemical

47

48 THE ACTION OF THE LIVING CELL

changes incident to the life process are conditioned
as regards both their velocity and specificity by bio-
chemical catalysts or enzymes. The action of these
remarkable substances is as yet but imperfectly under-
stood; nevertheless the evidence for their existence
is irrefutable. For example, starches may be hydro-
lyzed to glucose by means of strong mineral acid or
by sprouted barley. In 1832 Payen and Persoz ob-
served that an extract of barley which was free from
living cells would, when added to starch, efiPect its
hydrolysis with great ease.

For many years it was believed that the action of
such extracts differed materially from the enzymatic
activities of living cells. It remained for Buchner
in 1897 to establish beyond a doubt that the chemical
activities of cells were actually controlled by endocel-
lular enzymes. This worker, in his now classic ex-
periment, subjected ground yeast cells to a tremen-
dous pressure, thus obtaining a yeast juice free of
living cells. This expressed fluid was found to possess
all the fermentative properties of the original yeast
cells.

Since Buchner's time the presence of a great many
enzymes has been demonstrated in somewhat analo-
gous fashion. Indeed enzyme preparations have been
obtained from various cells, thus enabling us to ac-
count for the majority of the chemical transformations
effected by living matter. Nevertheless the chemical
structure of the individual enzymes still remains one
of Nature's secrets.

Now these enzymes for the most part exist within
the cells and if an insufficient quantity of proper food-
stuffs is available to the cell, they readily attack the

RELEASE OF CYTOST 49

structural elements; hence, if such action is unduly
prolonged the cell structure becomes disorganized
and death ultimately results. This process of self-
digestion was first pointed out by Hoppe-Seyler, who
noted that the tissues of dead animals underwent lique-
faction even though there was no apparent evidence
of bacterial action. Some twenty years later Salkow-
ski (1890) found the end products resulting from
such liquefaction to be nearly identical with the
end products produced by the action of pancreatic
trypsin on proteins. While today it is known that the
intracellular proteases differ in their properties from
the intestinal proteases, their actions are nevertheless
analogous as first surmised by Salkowski. The essen-
tial difference between these two classes of enzymes
appears principally in the optimal hydrogen ion con-
centration for their activities — the intracellular pro-
teases exhibiting their maximal activity in faintly acid
media whereas trypsin is inactivated in the presence
of acids.

In 1900 Jacoby observed that when a portion of
liver was ligated in situ it underwent changes analo-
gous to those previously found by Hofmeister and
Salkowski in dead tissues. Jacoby coined the word
autolysis to describe the self-digestion of tissues both
in vivo and in vitro. It has been pointed out above
that starving tissues undergo autolysis. In this con-
nection it is worthy of note that Cusa-Bianche states
that tissues taken from starving animals and tissues of
normal animals which have undergone in vitro autol-
ysis present much the same histological picture.

Analogous conditions are found in unicellular
organisms; for Condradi (1903), Rettger (1904)

50 THE ACTION OF THE LIVING CELL

and Effront (1905) have drawn attention to the fact
that while many bacteria and yeast do not suffer self-
digestion in culture medium, autolysis becomes rapid
when such cells are transferred to either distilled
water or saline solutions. Obviously under such con-
ditions the microorganisms are deprived of adequate
nutriment and hence are placed in a position com-
parable in some respects to the tissues of a starving
animal.

Since these early investigations, a very considerable
literature has accumulated concerning the nature of
autolysis. In general it has been found, in agreement
with the fact that tissue proteases are active only in
acid media that the addition of acid substances in-
creases the rate of autolysis of excised tissues, whereas
the converse is true of alkalis. Indeed if tissues are
made sufficiently alkaline, autolysis ceases. In the case
of mammalian tissue no evidence of autolysis either
in vivo or in vitro can be detected as long as the tissues
are held at a hydrogen ion concentration equal to or
less than that characteristic of the animal's blood. It
has long been known that excised tissues develop a
post mortem acidity; and potentiometric measure-
ments by Dernby (1918) and by Kochler, Seving-
haus and Bradley (1921) have shown the velocity of
this acid production to be extremely rapid. While the
mechanism by which injured or dying tissues produce
the acid is not known, it is nevertheless real, as various
workers have actually isolated lactic acid from such
tissues. As a consequence any pathological change
which causes an increase in the acidity of a cell will re-
sult in an autolytic process, and diffusible substances
are produced which diffuse out of the cells ; hence the

RELEASE OF CYTOST 51

mass of the latter decreases. The mechanism by which
the intracellular acidity is raised is immaterial for
atrophic changes will always follow. Halogenated
organic compounds such as phosgene, mustard gas
and chloropicrin, readily penetrate cells undergoing
hydrolysis with the liberation of hydrochloric acid.
As a result the cells die and autolyze rapidly.

However, the mechanism utilized to produce the
primary acidity need not in itself be a chemical cap-
able of liberating an acid, for as we have seen excised
tissues themselves produce sufficient acid to initiate
autolysis. We now know as a result of Warburg's
(1928) experiments that the majority of tissues are
capable of producing lactic acid, particularly if de-
prived of an adequate oxygen supply. In other words,
when the metabolism of the tissues is altered, acids
accumulate.

That such an altered metabolism may be induced by
relatively slight changes in the physical environment
is shown by the experiments of Kubowitz (1929).
Utilizing Warburg's technique, this worker found
that upon exposure to temperatures slightly higher
than 25° C. the frog's retina suffered an inhibition of
the Pasteur reaction with a consequent production
of lactic acid by glycolysis. While at present evidence
is lacking that other types of injury may cause a simi-
lar alteration in metabolism in other tissues of higher
animals, it is a reasonable supposition that such is the
case, because various types of injury are known to
cause the onset of autolysis. Further, it has long been
recognized that trauma results in an increased acidity
of tissues; and Chambers and Pollack (1927) have
recently observed this in single cells — the starfish egg.

1 n B & R \'

4

52 THE ACTION OF THE LIVING CELL

By means of a micro-pipette these authors injected
the indicator, brom thymol blue, into the eggs. Upon
injury of the egg by repeated thrusts of a micro-
needle the indicator underwent an immediate change
in color from blue to yellow, indicating an increase
in the hydrogen ion concentration. Previously
Chambers (1924) had noted that tearing of an egg by
a micro-needle leads to cytolysis which spreads from
the site of injury; and the experimental results of
Chambers and Pollack show that the increase in
acidity apparently precedes the cytolysis, for they
state that upon injury of an egg suspended in sea
water colored with brom thymol blue, the water ad-
jacent to the egg transiently became yellow prior to
cytolysis.

These beautiful experiments are especially perti-
nent to the present discussion because they unquestion-
ably demonstrate the production of an acid by a single
cell when injured. As yet the precise mechanism
underlying this curious behavior is unknown. How-
ever, it is clear that once the acid is formed, the intra-
cellular proteolytic enzymes will be enabled to hydro-
lyze the cell proteins and hence the degradation of
morphological structure will ensue. Incident to this,
various substances of endocellular origin will be re-
leased into the surrounding medium — the sea water
in the case of the starfish egg and the body fluids in
the case of the cells of higher animals and plants. If
the foregoing is accepted, we have a rational explana-
tion of the mechanism by which cytost is released from
the injured cells.

Various observations of the author have suggested
that a tissue toxin or cytost may be liberated under

RELEASE OF CYTOST 53

a variety of conditions which we may briefly con-
sider. In 1893 the writer had under his care a patient
suffering from alcoholism. Curiously enough, al-
though the use of alcohol had been discontinued for
some months, a marked toxemia persisted. Hence the
conclusion was reached that the toxic symptoms evi-
dent in the individual in question arose from the ab-
sorption of toxic products resulting from the necrosed
cells which had been produced by the injuries in-
flicted by the previous prolonged ingestion of alco-
hol (Turck, 1893). In view of the preceding dis-
cussion, we may now assume that in this instance the
prolonged use of alcohol had resulted in an autolysis
of the tissue cells of the gastrointestinal tract and that
the observed symptoms of toxemia were due to the
continued absorption of cytost from the injured tis-
sues.

Two years later, while investigating the bacteri-
ology of the stomachs of patients with gastric carci-
noma, it was found that in such cases the carcinoma
created a favorable soil for the microorganisms, par-
ticularly lactic acid-producing organisms. Fresh sec-
tions of the gastric mucosa which were examined im-
mediately after removal showed typical pathological
changes incident to inflammation : cells loosened from
the membrane, cystic degeneration, mucoid meta-
morphosis, marked interstitial changes, leucocytic in-
filtration, and engorgement of the blood vessels. The
writer stated, "If it is not alone the product formed
in the growth of carcinoma of the stomach that causes
this inflammatory and rapid destructive process, then
the infection from growing pathologic and non-
pathologic microorganisms colonizing upon the sur-

54 THE ACTION OF THE LIVING CELL

face of the mucous membrane offers a possible ex-
planation of these changes." (Turck, 1895.) When
cultures of microorganisms obtained from patients'
stomachs were introduced into the stomachs of normal
dogs, the microorganisms rapidly disappeared. This
result indicates that the bacteria alone were incapable
of causing the degenerative changes observed in the
stomach, and that the real cause of the alterations
in the stomach cells was the liberation of cellular tox-
ins as a result of cellular autolysis, primarily caused
by the carcinoma.

In agreement with this it was found that if prior to
the introduction of the bacteria the animal's stomach
was daily swabbed with tannic acid for a week, the
organisms grew rapidly and the animal showed loss
of appetite, refused food and grew thin. Apparently
the tannic acid had in a fashion similar to that of
carcinoma so altered the gastric mucosa that the
microorganisms could easily multiply.

Shortly after these experiments, other caustic irri-
tants, such as mustard, were applied to the gastric
mucosa, whereby a similar result was obtained, the
sticky exudate resulting from the action of the irri-
tant forming an excellent culture medium for the
rapid proliferation of bacteria. (Turck, 1902.)
Furthermore the histological picture of the stomach
tissues in cases of carcinoma was found to be closely
akin to that obtained by the prolonged action of mus-
tard emulsions on the gastric mucosa.

As a result of his observations of toxemias in pa-
tients suffering from carcinoma, alcohol poisoning and
various gastric disturbances, the writer came to the
conclusion that cell necrosis played an important role

RELEASE OF CYTOST 55

in the various pathological processes. In the case of
stomach ailments, various experiments were devised
to ascertain the presence and the nature of toxins
formed in the stomach. In the previous chapter we
have referred to experiments which showed that the
injection of the gastric contents of patients suffering
from gastritis produced signs of toxemia and shock.
Clinical observers have repeatedly noted symptoms
of so-called auto-intoxication in patients with gastric
atony and dilation. This suggested a study of the
fatigue of gastric muscle. Experimentally it was
found that the gastric muscle could conveniently be
fatigued in either of two ways: by rapidly revolving
the gyromele with the stomach; and by alternately
inflating and collapsing a rubber bag inserted into
the stomach. (Turck, 1903a.) By either of these
means the muscles may be so completely fatigued that
they fail to respond to stimuli, and show marked
elongation and lack of tone. After such treatment por-
tions of the stomach tissues were subjected to histologi-
cal examination whereby the muscle fibers of the py-
lorus were found to have suffered the most marked
changes, the nuclei staining poorly with methylene
blue while the cytoplasm appeared to be less granu-
lar than normally. These latter observations are indic-
ative of autolysis and show that injury of the stomach
tissues is followed by morphological changes analo-
gous to those found in other tissues under similar con-
ditions. Further congestion and dilation of the capil-
laries was evident. These histological findings are
in agreement with Oilman's (1903) observations on
the effect of fatigue on the nuclei of voluntary muscle
cells.

56 THE ACTION OF THE LIVING CELL

In order to determine the presence of "fatigue
toxins" in the muscles, the muscularis was removed,
minced and extracted with saline. Upon injection of
such extracts into the muscle walls of the unf atigued
stomachs of other animals, the abdomen being opened,
it was found that a contraction occurred, followed in
half an hour by a marked dilation, the stomach ulti-
mately becoming distended by gas to three or four
times its normal size. Rhythmical contractions van-
ished and the musculature became flabby and would
not respond to stimuli. As a control, a volume of
saline equal to that of the extract employed was in-
jected into the stomach walls of other animals. In
such control experiments the organ showed spontane-
ous movements within twenty minutes after injection
and the muscles readily responded to stimulation.

The extract of fatigued muscle was injected into
the peritoneal cavity of a dog. Some eighteen hours
later, the animal showed an intense thirst but immedi-
ately vomited water after drinking. This behavior is
noteworthy since it is identical with that frequently
seen in shock. Twenty-seven hours after the injection
the animal's abdomen was opened and the stomach
found to be dilated and flabby, and to contain fluid.
Rhythmical movements were scarcely discernible and
the response to stimulation was poor. A similar effect
was obtained by the injection of the fatigue extract
into the jugular vein. A normal dog was fed a mix-
ture of bread, milk and bismuth. Radiographic
examination showed peristalsis to be proceeding nor-
mally. Injection into the jugular vein of the extract
of fatigued gastric muscle completely inhibited the
peristaltic movements.

RELEASE OF CYTOST 57

These experiments show that distention of the stom-
ach results in the production of a toxic substance and
this is in agreement with the clinical findings. It will
be remembered that in the preceding chapter experi-
ments were presented which showed that a state of
shock could be induced by distending the stomach
with air. In the latter case a sufficiently high con-
centration of toxins was produced to yield general-
ized effects. In both instances, however, the libera-
tion of the toxic substance is due to the same cause —
dilation of the stomach — but in the shock experiments
the forced dilation was of considerably greater dura-
tion.

How, we may now ask, is the toxin produced?
When the stomach is dilated by any means blood is
forced out of the vessels in the wall of the organ. In
using the method of intermittent dilation which has
been discussed above, it was noted that "at the rest
pause, the peritoneal walls became bluish-purple, and
the vessels engorged. As each distention occurred, the
outer wall showed an anemic appearance from stretch-
ing, when upon releasing the air it would become rosy
in tint and hyperemic. After a repetition of this
dilation, the engorgement of the vessels disap-
peared. (Turck, 1903a.) Further, in an experiment
in which the femoral vein was exposed it was noted
that distention of the stomach by air caused a dis-
tention of the vein, which reverted to its normal
size when the air was permitted to escape from the
stomach.

Such circulatory disturbances must of necessity
interfere with the normal metabolic processes of the
cells of the stomach tissues; hence, as has been dis-

58 THE ACTION OF THE LIVING CELL

cussed at the beginning of this chapter, autolysis must
result. In the case of the simple distention experi-
ments this autolysis in all likelihood does not proceed
very far ; nevertheless protein decomposition products
are liberated in sufficient quantity to affect the muscle
cells locally, resulting in a state of fatigue. On the
other hand when the stomach is distended by air for
a considerable period, as in the shock experiments,
the blood supply to the tissues is interfered with for
a sufficiently long time to permit marked autolysis to
progress; hence a much larger amount of protein
split products is liberated into the general circulation
and shock results. These inferences are in agreement
with the histological evidence which, as previously
stated, showed a decrease in the cell granules and the
affinity of the nucleus for stains — a result always en-
countered in autolyzing cells. Further, it is now com-
mon knowledge that impairment of the circulation
leads to autolysis and atrophy.

In 1897 the author reported the results of various
ligation experiments. It was found that ligation of the
portal vein resulted in the death of the animals within
three days, and the liver tissues whose blood supply
had been thus interfered with showed evidence of ex-
tensive autolysis. After ligation of the veins along the
lesser curvature of the stomach, microscopic examina-
tion revealed evidence of local autolysis and promi-
nent erosions in the gastric mucosa. Similar results
obtained by the local ligation of various portions of
the stomach lead to the conclusion then expressed that
"Erosions of the stomach occurred wherever the
venous outlet was obstructed."

In 1914, incidental to a study of the diffusion of
bacteria into the intestinal wall, various portions of

RELEASE OF CYTOST 59

the intestine were ligated. In a series of one hundred
and fifty-five animals including monkeys, rats, rab-
bits and guinea pigs, it was observed that "the higher
the ligation the more quickly was the issue fatal." In
some experiments, bits of intestine were removed some
six hours after the operation for ligation. Micro-
scopic examination revealed necrosis of the entire
coat, including the muscular coat. This was evidenced
by the poor staining ability of the cytoplasm and
nuclei of the cells. The animals all showed the shock
syndrome a few hours after the tying ofif of the intesti-
nal loops; in conformity with the conclusions ex-
pressed in the previous chapter, it seemed likely that
the observed symptoms and death were due to a toxic
proteose fraction resulting from the tissue autolysis.
Further experiments proved this to be the case, for it
was found (Turck, 1922) that when the submucous
juice obtained from such animals was diluted with an
equal volume of normal saline and injected either into
the same animal or into another of the same species, a
fatality always resulted.

Some of the results of the experiments just men-
tioned are summarized in the tables on pages 60
and 61.

Reference to the results shown in the tables shows
that in general the higher the ligation of the intestine,
the more rapid was the onset of symptoms and subse-
quent death. This is readily understood when it is
remembered that the autolytic process is an enzymatic
one. In general, tissue autolysis, as we have seen, is
the result of changes in the internal environment of
the cell which in turn so alters conditions that the
endocellular protolytic enzymes are able to effect
hydrolysis of the cell proteins. Consequently when

60 THE ACTION OF THE LIVING CELL

TABLE I

Animal

Position of
Ligature

Time of
Appear-
ance of
Shock

Time of
Death

Time of
Examina-
tion

Findings

No. 2
No. 3

Duodenum
Jejunum

4 hrs.

5 hrs.

24 hrs.

24 hrs.

S'^^'^"' ) fluid
Mucosa J

Hemorrhage

■ Peritoneal

coat

No. 4
No.s

Jejunum
(lower)
Ileum

S hrs.
8 hrs.

48 hrs.

24 hrs.

Effusion of
blood in
coats

No. 6

Ileum

7 hrs.

24 hrs.

Effusion of
blood in

No. 7

Ileum

9 hrs.

24 hrs.

coats
Effusion of
blood in
coats

No. 8
No. 9

Ileum

Section tied
(small and
large intes-

5 hrs.
12 hrs.

24 hrs.
24 hrs.

Hemorrhagic
points near
ligature

No. 10
No. II

tines)

5 loops at dif-
ferent levels

8 loops

2 hrs.
I hr.

4 hrs.
3i-hrs.

Shock syn-
drome

No. 12

Colon and
ileum loops

S hrs.

24 hrs.

The intestines were tied off in positions indicated in the second column.
The figures in the third column indicate the time which elapsed before the
symptoms of shock became apparent, while the figures in the fifth column
indicate the time which elapsed between the ligation and examination under
anesthesia. The principal findings of interest are indicated in the sixth column.

cellular necrosis is permitted in organs which them-
selves secrete proteolytic enzymes, the digestion of
tissue proteins is markedly facilitated since the latter
are subjected to attack not only by the endocellular

RELEASE OF CYTOST

61

TABLE II

Dogs

Dog

Loops

Time

Intravenous Inj.

Death

No. I

Jejunum

3i hrs.

5 cc.

3 minutes.

No. 2

Upper ileum

5 hrs.

I cc.

2 hours.

No. 3

Lower ileum

6 hrs.

2 CC.

24 hours.

No. 4

Duodenal
jejunum loops

3i hrs.

0.5 CC.

3 minutes.

No. s

Middle
jejunum loops

4 hrs.

I CC.

2 hours.

No. 6

Upper ileum

8 hrs.

2 CC.

24 hours.

No. 7

Colon

8 hrs.

3 cc.

24 hours.

No. 8

Colon

10 hrs.

5 cc.

24 hours.

In these experiments loops of intestine were tied off as indicated in the second
column. Submucous juice was withdrawn from the loops after the periods
noted in the third column. This was diluted with an equal volume of normal
salt solution and the quantities of the extract noted in the fourth column were
injected intravenously into the animals. This always caused death within
from three minutes to twenty-four hours, as shown in the fifth column.

enzymes but by those exterior to the cells as well. Now
enzymatic action is known to be greater in the upper
alimentary tract than at lower levels. It follows that
for a given injury, the cells lining the upper portion
of the tract must be more rapidly autolyzed than those
at lower levels. As a net result the concentration of
cytost necessary to cause generalized symptoms is more
rapidly released than is the case in lower levels of the
intestine. Thus we arrive at an adequate theoretical
basis for the results observed in these experiments.

The results of these experiments are in harmony
with the author's early contention that a tissue tox-
emia frequently caused death from shock during ob-
dominal operations. (Turck, 1901.) Further, they
of]fer a rational explanation for the statement that "in

62 THE ACTION OF THE LIVING CELL

operations in the upper abdominal area there is a
greater danger from shock than in operations on the
lower abdominal area. Even traction on the stomach
(i.e, interference with circulation) greatly increases
the liability to shock, while exposure to air adds
greatly to the danger. If death does not follow, still
the toxins formed are detrimental to convalescence,
and the future welfare of the patients." (Turck,
1902b.) This conclusion, expressed some thirty years
ago, has since been confirmed by both clinical obser-
vation and laboratory experiments. Indeed the last
sentence of the above quotation suggests the investi-
gation of the action of small quantities of cytost upon
the well-being of the animal. During the last decade
this aspect of the problem has been carefully investi-
gated in our laboratory and the results obtained are
of very considerable interest and will constitute the
major portion of the discussion in previous chapters.
Individuals suffering from intestinal obstruction
frequently suffer from an intense toxemia. At one
time it was assumed that this was due to the absorp-
tion of bacterial toxins produced by the activity of
microorganisms in the stagnant contents of the in-
testinal tract. More recently such toxemias have been
ascribed to the liberation of toxic products from the
cells of the intestine. Roger was forced to this con-
clusion by his observation that extracts of the intestinal
mucosa were more toxic to animals than extracts of
the intestinal contents. This, it should be noted, is in
perfect harmony with the results of the writer's ex-
periments — in which the presence of bacteria could
not have played any significant role. These conclu-
sions have furthermore been substantiated in recent

RELEASE OF CYTOST 63

years by the experiments of Draper and his co-
workers. (Draper and Johnson, 1930.)

In the first chapter attention has been called to the
fact that local application to the gastric mucosa of
caustic chemicals leads to cell necrosis and subse-
quent shock. The experiments upon shock induced
by general anesthesia suggested the mechanism of the
reaction to be the same as in other cases of experimental
shock ; i.e., an autolysis of tissue giving rise to the shock
toxin. If this hypothesis is correct, then one should
be able to induce a localized tissue autolysis at any
desired site by the injection of a substance such as
chloroform. If the extent of the autolysis induced in
this manner is sufficiently great, then the experimental
animals should exhibit symptoms of shock.

Experiments of this type have been reported by the
v^riter (Turck, 1922b). Cats w^ere injected intramus-
cularly twice weekly with 0.25 cc. of absolute alcohol,
ether or chloroform. The animals died after five or six
months of such treatment. At autopsy these animals
all showed a chronic interstitial nephritis with stag-
nation of blood and autolysis of the tubules. In these
experiments it was of course recognized that the ob-
served pathology might have been due to the direct
action of the drugs upon the tissues and not caused by
cytost released from cells at the site of the injection.
In order to settle this point experiments of the fol-
lowing nature were conducted. Chloroform was in-
jected deeply into the tissues of cats. After a lapse of
time sufficient to permit a complete local necrosis, the
tissues were removed from the site of the injection.
A sterile saline extract was prepared and injected two
or three times a week into normal animals. At autopsy.

64 THE ACTION OF THE LIVING CELL

the kidneys of the latter were found to have suffered
degenerative changes similar to that produced in ani-
mals by the direct injection of chloroform. Only one
conclusion may be drawn from these results; that the
observed pathology was due not to the direct action
of the drug on the kidney but was the result of endo-
cellular substances released from those tissues directly
injured by the chloroform, — i.e., those tissues at the
site of injection.

Further evidence substantiating these conclusions
is to be found in the following experimental results:

An isotonic saline solution was injected into the
muscles of the thigh. After a brief period the fluid
was aspirated from the site of injection and injected
intravenously into another animal of the same species.
No evidence of discomfort of physiological change
was observed in the second animal. When however
either chloroform, ether or alcohol was substituted for
the saline solution quite different results were ob-
tained. The drugs were allowed to remain in situ
for one or two hours ; the fluid was then aspirated from
the resulting sterile abscess and injected intraven-
ously into homologous animals, whereupon the latter
quickly showed the symptoms of shock and subse-
quently died.

The results of such experiments are summarized in
Table III, page 65.

As shown in the table, the rapidity with which death
may follow the injection of tissue break-down prod-
ucts is very striking. At autopsy, all of the animals
were found to have suffered a marked congestion of
the splanchnic viscera, identical with that observed in
shock induced by other means. Kidney lesions were

RELEASE OF CYTOST

6S

TABLE III

Quantity

Quantity As-

Time of

Animal

Irritant

Injected in

pirated AND
Injected into
Second Animal

Death after

First Animal

Injection

I. Rabbit

Chloroform

2 cc.

i.o cc.

5 Min.

2. Cat

Chloroform

2 cc.

1.5 cc.

3 Min.

3. Dog

Chloroform

4 cc.

2.5 cc.

10 Days

4. Cat

Chloroform

4 cc.

2.5 cc.

I Day

5. Rabbit

Ether

4cc.

1.5 cc.

I Min.

6. Cat

Ether

4 cc.

1.5 cc.

2 Min.

7- Dog

Ether

6 cc.

2.0 cc.

4 Min.

8. Cat

Ether

4 cc.

1.0 cc.

2 Hours

9. Cat

Ether

6 cc.

1.5 cc.

10 Hours

10. Rabbit

Alcohol

10 cc.

1.0 cc.

2 Min.

II. Rabbit

Alcohol

IS cc.

2.0 cc.

8 Hours

12. Cat

Alcohol

15 cc.

2.0 cc.

24 Hours

13. Dog

Alcohol

20 cc.

1.5 cc.

15 Hours

14. Cat

Alcohol

15 cc.

1.5 cc.

S Min.

15. Cat

Alcohol

15 cc.

2.0 cc.

18 Hours

found in those animals which lived for some hours
after the injection of the aspirate. It is of interest to
note that this is a common finding in animals which
have suffered extensive tissue destruction by burns or
various infections.

These experiments demonstrate conclusively that
the anesthetics so far examined are actually able to
effect tissue destruction with the consequent liberation
of the toxic cytost — that is they cause an in vivo autoly-
sis.

A second series of experiments were made with cats
in order to compare the ease with which chloroform,
ether and alcohol effected tissue autolysis. For this
purpose the animals received two intramuscular in-
jections of the chemicals, the second injection being
administered two hours after the first. A few hours

66 THE ACTION OF THE LIVING CELL

after the second injection all of the animals developed
the typical symptoms of shock and later died. The
results are summarized in Table IV.

TABLE IV

Quantity

Onset of

Time of

Reagent

Shock after
Injection

Death after

ist. Injection

2nd. Injection

Injection

I. Chloroform

I cc.

1.5 cc.

4 Hours

24 Hours

2. Chloroform

2 cc.

I cc.

3 Hours

36 Hours

3. Chloroform

1.5 cc.

1.5 cc.

5 Hours

48 Hours

4. Chloroform

2 cc.

2 cc.

4 Hours

72 Hours

5. Ether

2 cc.

2 cc.

6 Hours

24 Hours

6. Ether

2 cc.

2 CC.

3.5 Hours

36 Hours

7. Ether

3 cc.

3 cc.

4 Hours

40 Hours

8. Alcohol

5 cc.

10 cc.

6 Hours

72 Hours

9. Alcohol

5 cc.

lo cc.

5 Hours

112 Hours

10. Alcohol

2 cc.

10 cc.

4.5 Hours

80 Hours

II. Alcohol

10 cc.

10 cc.

4 Hours

24 Hours

From these results it is apparent that chloroform is
the most active reagent of the three. Since shock is
produced by cytost liberated from the tissues it fol-
lows that chloroform is able to liberate the cell con-
tents more easily than does ether or alcohol. This re-
sult is of interest in view of the fact that animals
anesthetized for long periods with chloroform pass
into a state of shock more frequently than is the case
with the other narcotics. The results of the experi-
ments just discussed offer ample proof of the state-
ment previously made, that shock produced by
general anesthesia is due to the same causative factor
as is shock caused by other injuries. (Turck, 1903b.)

Under general anesthesia the excretory functions
of the organism is impaired, hence the animal is un-
able to rid itself of toxins as easily as is the case when

RELEASE OF CYTOST 67

other means are employed to induce shock. Thus, al-
though the concentration of anesthetic in the tissues
may be insufficient to cause an extensive necrosis, the
added factor of diminished excretory power perhaps
permits a greater accumulation of cytost in the tissues
than might be the case in unanesthetized animals in-
jured by other means.

The foregoing may be summarized as follows: in-
juries regardless of their nature stimulate the autol-
ysis of cells. Such autolysis results from the activity
of endocellular enzymes. Now it should be obvious
that cellular autolysis may be complete or partial,
for it is conceivable that if the injury be slight, the
damage may be easily repaired and consequently the
autolytic process will not proceed far enough to seri-
ously disorganize the cell.

This fact is attested to by the observations of Cham-
bers and other micro-dissectionists who have fre-
quently injured cells of various sorts by their manipu-
lations. If the injury be slight and non-extensive, the
cells show no apparent ill effects and continue to live
normally. On the other hand, as has been stated previ-
ously, if the injury is marked, as for example in
Chambers' experiments with the starfish egg, then
cytolysis, death and presumably autolysis ensue.

For some years it has been recognized that whereas
the normal living cell exhibits a selective perme-
ability, the latter ceases upon death (Jacobs, 1924).
So far as is known today, all normal cells are imper-
meable to colloids. Hence they are probably im-
permeable to enzymes since all enzymes which have
been carefully studied appear to be colloidal (Wald-
schmidt-Leitz, 1926; Gortner, 1929). It follows

68 THE ACTION OF THE LIVING CELL

therefore that any endocellular enzymes found in the
medium surrounding cells must have been liberated
by necrosis. This concept finds confirmation in the
fact that active cytolytic agents or mechanical dis-
integration may be employed to achieve that extrac-
tion of enzymes from suspensions of cells.

When, as a result of natural processes or injury,
autolysis of cells takes place, the contents of the cell
including the endocellular proteases responsible for
the autolysis may diffuse out into the surrounding
fluids. In the case of higher animals these substances
find their vs^ay into the blood and are eventually ex-
creted in the urine. This has been proven by Pfeiffer
( 1914) v^ho w^as able to detect the presence of proteo-
lytic enzymes in the blood and urine of individuals
suffering from extensive burns. Similarly in infec-
tuous diseases accompanied by tissue destruction ab-
normal quantities of such enzymes have frequently
been found in the blood and urine.

Just as beginning students of physiology frequently
ask "Why do not the stomach and intestines digest
themselves?" we may ask "Why do not the autolytic
enzymes destroy normal cells?" While the first ques-
tion has never been satisfactorily answered, it was
pointed out at the beginning of this chapter that the
hydrogen ion concentration of the normal cell is not
sufficiently great to permit activity of the endocellular
proteases.

Prior to the recognition of the importance of the
intracellular hydrogen ion concentration in control-
ling the activities of the endocellular enzymes, it was
believed that autolysis was inhibited solely by the
presence of specific antienzymes. Some years ago

RELEASE OF CYTOST 69

Hildebrandt (1893) found that blood serum inhibited
the action of trypsin. Since that time many other
workers have studied this effect and it is now known
with certainty that some substance present in the
serum inactivates all proteolytic enzymes which may
find their way into the blood. For our purpose it is
not necessary to consider the various hypotheses which
have been advanced to explain this fact.

As we have seen endocellular proteases may diffuse
from injured cells into the blood. Under normal cir-
cumstances, the inhibitory substances present in the
serum may diffuse into injured cells and perhaps there
inhibit the activities of the proteolytic enzymes. Any
factor which prevents such an inhibition will there-
fore enhance the rapidity of the autolytic process.
Simniezki (1902) found that depending upon the
concentration used, alcohol either weakened or com-
pletely inhibited the antienzymatic activity of blood.
In connection with the experiments on shock pro-
duced by general anesthesia the author (Turck, 1903)
compared the antiferment action of serum from nor-
mal animals with that obtained from animals that
had shown symptoms of shock induced by anesthesia.
For this purpose the well known Mett method of meas-
uring proteolysis was adapted as follows: egg albu-
min was aspirated into glass capillary tubes of 1 to
2 mm. diameter and coagulated by heating to 95° C.
for a short time. The tube containing the coagulated
protein was broken into short lengths and these were
added along with proteolytic enzymes to the sera
under investigation and to normal serum. These
preparations were then held at 37° C. for ten hours,
after which the proteolytic activity of the mixtures

70 THE ACTION OF THE LIVING CELL

was ascertained by measuring the difference between
the length of the capillaries and the length of the
column of undigested egg white. The differences so
determined are summarized in Table V. The weights
reported in the table are the amounts of dried enzyme
preparation added to the tabulated volumes of serum.
In the case of anesthetized animals similar quantities
of serum were utilized in each case.

TABLE V

Antiferment Activity of Blood Serum as Measured by the Mett

Technique

Serum from normal dogs

+PANCREATIN

+CARICA PAPAYA

3.2 mm.

2.4 mm.

3.1 mm.

1.5 mm.

Serum from anesthetized dogs

Anesthesia lasted for 6 hr.

9.0 mm.

8.8 mm.

6hr.

8.4 mm.

8.6 mm.

4 hr.

7.4 mm.

8.7 mm.

ihr.

3.4 mm.

4.7 mm.

ihr.

4.3 mm.

4.1 mm.

The figures in this table represent the lengths of egg albumin in the Mett
tubes which was digested by the various preparations during an interval of
12 hours at 37° C. In each instance 1.5 gram of the dry enzyme was added
to 5 cc. of serum. Note that the serum from the anesthetized animals per-
mitted digestion to take place much more rapidly than was the case with normal
serum. (Turck, 1903.)

From the tabulated results it is apparent that nor-
mal serum inhibits the action of the added enzymes
to a greater extent than does serum from anesthetized
animals. Further, the longer the period of anesthesia,
the less is the extent of the inhibition.

In order to ascertain whether or not the anesthetics
employed were capable of lowering the antiferment
activity of normal serum the following procedure was

RELEASE OF CYTOST 71

followed. Chloroform or ether was added to serum
and after the lapse of two or three hours, the chemi-
cals were removed by warming the serum to 45° C.
in vacuo. When sera treated in this fashion were ex-
amined by the Mett method it was found that they
had lost their ability to inhibit the action of prote-
olytic enzymes.

From these results it follows that by lowering anti-
ferment action of serum, anesthetics may aid the auto-
lytic process. Confirmation of this hypothesis is found
in the experiments of Yamakawa (1918) who found
that in the presence of various surface active sub-
stances such as alcohol and chloroform, the serum
proteins undergo auto digestion. The experiments
of this investigator are of importance for they demon-
strate the fact that autolysis may take place in the
serum providing that the activities of the ever present
antienzymes are inhibited. It follows therefore that
during anesthesia toxic protein fractions may possibly
be derived from serum proteins as well as from intra-
cellular proteins.

It therefore seems reasonable to postulate that the
shock observed during anesthesia may in part be due
to the action of such hydrolytic products of the serum
proteins.

While on the subject of anesthetics it may be well
to mention some other observations made at the time
(1903) these experiments were conducted. It was
noted that after prolonged narcosis the animal's blood
coagulated more rapidly than is normally the case.
This may be interpreted as indicating the presence
within the serum of some substance which hastened
coagulation. The various theories of blood coagula-

72 THE ACTION OF THE LIVING CELL

tion uniformly assume that incident to an injury some
substance is liberated from the tissues which initiates
the series of changes which finally lead to the forma-
tion of the fibrin clot. While the nature of this tissue
substance and its mode of action are still in dispute, its
importance in initiating the clotting process is quite
generally accepted. For our present purposes, how-
ever, the exact details of the clotting process are im-
material. The fact that the blood of anesthetized
animals coagulates more rapidly than that of normal
animals is indicative of the fact that prolonged anes-
thesia causes the liberation of tissue components in
much the same fashion as does a traumatic injury.

When serum drawn from an animal under anes-
thesia for three hours was added to a drop of normal
blood, agglutinization of the corpuscles occurred im-
mediately. Serum from animals anesthetized for
longer periods exhibited this agglutination action even
after a fivefold dilution.

Frequently the immediate coagulation of blood has
been noted after prolonged anesthesia. However, it
is not generally realized that this is due to some sub-
stance formed in the circulating blood as a result of
the action of the anesthetic. The author in 1902-3
observed that the addition of "shock serum" to an
equal quantity of normal blood resulted in the im-
mediate coagulation of the latter. In general it was
found that the intensity of the agglutinizing and clot
forming reactions depended upon the duration of the
anesthesia. Little such action was noted with the sera
of animals anesthetized for an hour or two, whereas
longer periods of narcosis invariably led to the for-
mation of such agglutinins and precititins. For the

RELEASE OF CYTOST 73

moment we wish merely to note that such bodies ap-
pear to be formed in shock produced by other means.
Their formation may be assumed to arise as a result
of tissue autolysis; hence they may be identical with
cytost, may be formed concomitantly or they may re-
sult from the action of the liberated cytost. These con-
cepts will be discussed in a later chapter.

Chapter IV
THE PHYSIOLOGY OF CYTOST ACTION

It will be remembered that our purpose in intro-
ducing the subject of shock was not to present an
exhaustive account of the various theories and their
relation to surgical practice, but rather to present evi-
dence of the physiological relations between various
cells. We shall not therefore offer an extensive re-
view of this interesting subject, but shall limit the
following to the author's own experiments. The
reader desirous of pursuing further the clinical as-
pects of the subject is referred to the reviews of
Cannon (1923), MacLeod ( 1930), and Turck (1922,
1929).

In previous chapters, experiments have been pre-
sented which show the primary cause of shock to be
the liberation of cytost from injured tissue cells. So
far, however, the discussion has been restricted to the
evidence concerning the release of cytost and the ter-
minal results of its action. We may now briefly con-
sider the manner in which the released cytost affects
the animal as a whole — that is, the physiological
changes which ultimately lead to a state of shock.

At the time the author began his investigation it
was generally held that shock was of purely nervous

74

PHYSIOLOGY OF CYTOST ACTION 75

origin. In support of this contention were the experi-
ments of Goltz (1872), who found that, upon per-
cussing the mesentery of the frog, there followed a
reflex vagal inhibition of the heart and subsequent
dilation of the splanchnic vessels. Beginning in 1899,
Crile recorded observations showing that long con-
tinued stimulation of the sensory nerves elicited a
fall in blood pressure. Since in severe operations or
injury to the peritoneum a large number of sensory
nerves are subjected to excessive stimulation, Crile
concluded that shock under such conditions was due
to exhaustion of the vasomotor center. It was there-
fore natural that at the outset the author's contention
that shock was the result of tissue toxins should meet
with disapproval, as, for example, the statement of
Dr. James G. Kiernan (1897) : "I do not want unduly
to swell the neurologic trend of the discussion at the
present time, yet I cannot but take issue with the posi-
tion that local tissue change irrespective of nerve ac-
tion underlies the results as Dr. Turck has claimed.
This seems to be turning back the page of pathology
to the days of Broussaid. . . ." Curiously enough,
however, this attitude persisted up till the time of the
World War, despite the fact that in the interim the
author had published the results of numerous experi-
ments, showing the toxic nature of shock. The high
incidence of shock in wounded soldiers stimulated
others to investigate the underlying physiology. As
a consequence, various investigators, notably Bayliss,
Cannon, and their associates (1919), arrived at con-
clusions essentially the same as those expressed by
the author some twenty years earlier.

In 1897 (Turck, 1897), the writer published ob-

76 THE ACTION OF THE LIVING CELL

servations made upon dogs which had been brought
into a state of shock by various experimental means,
such as exposure of the abdominal viscera to a draught
of air, and the injection of stomach contents. In these
experiments it was noted that coincident with the on-
set of shock, the stomach and intestines assumed a dark
blue color due to stagnation of blood in the vessels.
In experiments in which both arterial and venous pres-
sures were recorded, it was noted that during shock
the veins and viscera showed an increased pressure,
whereas the arterial pressure dropped below the nor-
mal value. As the depth of shock increased, stagnation
in the visceral vessels was found to increase, the intes-
tinal vessels becoming engorged more rapidly than
those of the stomach. When means were taken to re-
vive the animals, the reverse of the above changes took
place, the congestion being reduced and the arterial
circulation reestablished. On the basis of such obser-
vations the conclusion was drawn "That we cannot
apparently experimentally produce profound shock
without the occurrence of congestion and that the re-
duction of such shock does not occur until we reduce
the congestion and distribute the blood over the sur-
face." In all subsequent experiments in which shock
was produced, this stasis of blood in the visceral ves-
sels was always found. It is this visceral congestion
which is responsible for the various external symp-
toms of shock, such as weakened pulse, diminished
respiration, and lowered temperature.

The splanchnic circulation, physiologically con-
sidered, is distinct from the somatic or peripheral cir-
culation, there being an inverse relation between the
volume of blood circulating in the vessels of the

PHYSIOLOGY OF CYTOST ACTION 77

somatic and splanchnic areas. Because of this, dila-
tion of the peripheral vessels is normally accom-
panied by contraction of the vessels of the splanchnic
area. The latter, in fact, may be regarded as a reser-
voir or regulatory apparatus for the entire circula-
tion, blood passing from this area to the peripheral
vessels when these are adequately stimulated. Con-
versely, stimulation of the abdominal vessels, by func-
tional activity or any other means, results in an in-
creased flow of blood through the splanchnic vessels.
We know that upon passing from a cool into a warm
atmosphere, the peripheral vessels dilate; upon re-
turning to the warm atmosphere, the peripheral ves-
sels rapidly contract, forcing the blood back to the
splanchnic area. In consequence, the vessels of the
latter dilate to accommodate the increased influx of
blood. Such facts as these illustrate the extreme deli-
cacy of circulatory regulation.

In a previous chapter, there has been presented ex-
perimental evidence showing that direct stimulation
of the gastrointestinal mucosa promptly causes a lo-
cal hyperemia. If the stimulation be excessive, as by
the prolonged action of mustard in the stomach, or the
air blast, then this hyperemia becomes pathological
and eventually results in a marked venous congestion.

The pronounced sensitiveness of the splanchnic ves-
sels to stimuli is a necessary function, the mild hyper-
emia following such stimulation being necessary for
the secretory and absorptive processes of the stomach
and intestines. Such mild dilation of the capillaries
of this area does not result in any obvious outward
symptoms. When, however, such dilation becomes
excessive, the diminished blood flow in the peripheral

78 THE ACTION OF THE LIVING CELL

vessels results in a lowered blood pressure, weak pulse
and lowered surface temperature, the magnitude of
such changes being conditioned by the extent of the
splanchnic dilation.

The author's (Turck, 1900) observations that
splanchnic congestion might be brought about by
changes in temperature or irritation of the gastric
mucosa are not surprising when viewed from our pres-
ent knowledge of the physiology of the splanchnic
circulation. Very striking, however, was the observa-
tion that upon subcutaneous injection, into rabbits, the
gastric contents from a patient with a dilated stomach
caused dilation of the splanchnic vessels, and collapse.
A more rapid response was obtained by injecting such
material into the mesenteric vein of a rabbit. When a
similar quantity of water was injected in like manner,
the animal exhibited no change other than a slight
hyperemia, which always follows exposure of the
viscera. At the time these experiments were pub-
lished (1900), attention was called to the essential
similarity between these results and the effects pro-
duced by the entry of peptones, primary and second-
ary albumoses, into the circulation. Further, the
analogy was drawn between splanchnic congestion
and severe hemorrhage, which produces much the
same outward signs. This was summed up in the state-
ment "that splanchnic congestion simulates hemor-
rhage ; it might be expressed as a bleeding within one-
self." (Turck, 1900.)

We are thus in a position to explain in part the
occurrence of so-called postural shock, which was
mentioned in the first chapter. When a rabbit is held
by the head in a vertical position, the force of gravity

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PHYSIOLOGY OF CYTOST ACTION 79

suffices to cause an accumulation of blood in the
splanchnic area and consequently the animal mani-
fests the outward symptoms of shock. Such an animal,
however, will usually recover when returned to its
natural position. In this respect, postural shock dif-
fers from the profound state of shock induced by tis-
sue damage. It follows, therefore, that the production
of true shock entails something more than a mere re-
versible accumulation of blood in the splanchnic area,
as is the case in postural shock.

At this point we may recall the author's observa-
tions, mentioned previously, that in experimental
shock the blood was found to clot more readily than
is normally the case, and the serum from animals in
profound shock contained substances capable of ef-
fecting hemagglutination and coagulation of blood
drawn from intact animals. Such being the case, it
follows that such processes may take place within the
blood vessels. Thus, the blood which accumulates in
dilated vessels may suffer a partial or complete coagu-
lation, the extent of this process depending upon the
amount of coagulins present in the blood. In any
case, even though the extent of agglutination and co-
agulation be slight, the net effect will be to raise the
viscosity of the blood and thus hinder its passage
through the vessels and thence mechanically to dis-
tend the vessels even more.

Leo Loeb ( 1904) has shown that coagulins may be
extracted from tissues and this fact is in harmony
with the author's concept that shock is produced pri-
marily by the products of cellular disintegration.

As a result of the agglutination and coagulation of
the blood, the capillaries may be easily blocked, thus

80 THE ACTION OF THE LIVING CELL

causing a stagnation of blood in the smaller and then
in larger vessels. In order to follow such changes it
is necessary to resort to histological methods. For
this purpose, bits of tissue were removed from animals
in a state of shock and examined by the usual histo-
logical procedures. Before passing to a discussion of
the actual observations, we may summarize the essen-
tial results of such an examination. In all cases of
acute shock, regardless of the experimental means
used to produce the reaction, general examination dis-
closed that marked stasis originated in the capillary
bed of the visceral organs, the stomach, intestines,
liver and lungs. "This stasis occurs in the venules
and backs down into the larger veins in the portal zone
of the liver, pulmonary and arterial zone (venous)
of the lung, and submucous zone of the stomach and
intestines." These conclusions are based upon a study
of many reactions such as those shown in the follow-
ing photomicrographs. The principal points of inter-
est are noted in the legends accompanying the photo-
graphs.

It is noteworthy, as shown in figure 6, that in
shock the brain tissues exhibit no evidences of conges-
tion, the cells appearing normal. In all experiments
with shock, wherein the brain has been examined, the
same result has always been found. This constitutes
additional evidence that shock is not the result of a
central nervous disfunction.

A study of the accompanying photomicrographs,
which are representative of hundreds prepared by the
author, has shown conclusively that the first reaction
which occurs in the liver appears to be confined to the
finer capillaries, which are in direct contact with

PHYSIOLOGY OF CYTOST ACTION 81

the columnar liver cells. Apparently it is here that
coagulation of the blood takes place, for, as is clearly
shown in the pictures, the central vein (hepatic) is
entirely empty. The coagulation, or gelation, in these
fine capillaries prevents the free flow of blood. In
consequence, as more blood enters from the portal
vessels, a considerable pressure is exerted upon the
capillary walls.

This pressure it is which brings about the distention
of the capillaries, and their subsequent engorgement
with blood. This series of events is the primary change
induced in the liver and is always found in fresh speci-
mens taken from animals in shock. A similar state of
affairs is found in the lung (Fig. 3) . In cases of pro-
found shock or in animals whose death has been in-
duced by the action of cytost, further changes are to
be noted. For example, in the section shown in fig-
ure 5, a definite clear area containing blood corpuscles
is observed surrounding the distended capillary. This
clear area, which has not taken the stain, is due to
exeresis of fluid from the capillaries. This outflow of
fluid, as well as the presence of blood corpuscles in
the clear area, is evidence of the increased perme-
ability of the capillaries.

Such increased permeability has been recognized
by many workers in this field. Following the initial
work of Dale and Laidlaw ( 1919) , who observed that
the administration of histamine to cats caused a rapid
concentration of the blood cells, due to increased per-
meability of the capillaries, it has been assumed by
some that traumatic shock is due to the liberation of
histamine or a histamine-like substance. This point
will not now be elaborated upon, for as will be shown

82 THE ACTION OF THE LIVING CELL

in the next chapter, the properties of cytost are quite
at variance with those of histamine. For the present,
the point we desire to stress is that in shock the in-
creased diffusion of fluid from the blood occurs sec-
ondarily to the congestion of the capillaries.

With the increased permeability of the capillaries,
cytost present in the blood may be expected to diffuse
along with other blood constituents into the fluid
(lymph) bathing the tissue cells. This consideration
is of importance since, as will be shown subsequently,
cytost in sufficiently high concentration may exert a
distinctly deleterious action upon the various cells
with which it comes in contact.

In the experiments so far considered, cytost has ap-
peared to act selectively upon the abdominal viscera
and lungs. As will be seen presently, however, this
selectivity of action is more apparent than real, for
other tissues, such as muscle and brain, are affected by
cytost. Such effects, nevertheless, become evident in
such tissues only when they are exposed to the direct
action of cytost in a concentration considerably greater
than is necessary to bring about similar changes in the
viscera.

The extreme sensitivity of the viscera to such a
tissue toxin is perhaps analogous to the fact that among
humans, at any rate, these organs are the most sus-
ceptible to disease. Statistical studies of the causes
of death have shown that, in the great majority of
cases, death is actually due to various derangements
of these particular tissues. (Pearl, 1922.)

This is perhaps not surprising, for these organs
really represent the actual metabolizing center of the
mammal. The skeletal tissues are merely a support-

PHYSIOLOGY OF CYTOST ACTION 83

ing structure endowed with facilities for locomotion,
while the nervous system serves mainly as a coordi-
nating mechanism. The latter tissues are, of course,
dependent upon the normal functioning of the viscera
for their continued existence.

What have the various viscera in common that can
account for their ready susceptibility to the action of
cytost? While this question cannot be completely an-
swered, a valuable suggestion is found in their com-
mon histogenesis. As the reader is aware, in the early
stages of embryonic development three distinct germ
layers are formed. These are termed respectively the
ectoderm, the mesoderm, and the endoderm (ento-
derm).

Embryologists have been able to follow the subse-
quent development of these primitive layers into the
various tissues of the body. During the course of
embryonic development, the innermost layer, or en-
doderm of the vertebrate, has been found largely to
retain its original epithelial character. From it are
formed the epithelial lining of the enteron, its auxil-
iary organs and glands — the stomach, liver, pancreas,
lungs, and urinary bladder.

Those organs which are particularly sensitive to
cytost have then a common origin, and it is perhaps
justifiable to conclude that their peculiar sensitivity
is simply that of a primitive germ layer, the endo-
derm. This conclusion seems further justified when
it is remembered that of the three germ layers, the
endoderm suffers the least differentiation during de-
velopment.

The marked sensitiveness of the tissues of endo-
dermal origin was first stressed by the author in con-

84 THE ACTION OF THE LIVING CELL

nection with his experiments on inflammation of the
stomach. At that time, while considering the patho-
logical changes induced by the introduction of caustics
and bacteria into the stomach, it was concluded that
"it seems that the entoderm is far more sensitive to
irritation than the ectoderm."

In the foregoing discussion it has been shown that
cytost appears to act selectively on the endodermal
cells and thus affects the adjacent blood in the capil-
laries of the various viscera. When autolyzed tissue
is injected into an animal, the pathological lesions pro-
duced in the lungs resemble those of other visceral tis-
sues (Fig. 3). Since the capillary network of the air
sacs is the most dense capillary plexus of the body, and
material may be introduced into the lung without
recourse to experimental surgery, the lung appeared
to offer an excellent site for the investigation of the
localized stasis induced by cytost. To this end, auto-
lyzed tissue was introduced into cats' lungs. The re-
sults of these experiments, which are summarized in
Table VI (Turck, 1919), were far more striking
than could possibly have been imagined.

The experiments were conducted as follows : Fresh
lung tissue from healthy cats was autolyzed in a
sterile chamber of chloroform vapor for forty-eight
hours, when the mixture was found to yield an in-
tense biuret reaction. By means of a tube attached
to a respiratory indicator, 0.5 to 1 cc. of such auto-
lyzed tissue suspension was insufflated into the
tracheae of normal cats. The animals' increased in-
spiration aspirated a portion of the material into the
finer bronchii, and in many instances, as shown in
Table VI, caused the animal's death within four or

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P9

P 10

P II

P 12

P 13
Pi4

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P i8

TABLE VI
Cats Insufflated with Autolyzed Cat Lung Tissue

Time of Death

AFTER

Insufflation

4 mm.

3 mm.

5 mm.

10 hours

24 hours
Shock

15 min.
S hrs.

10 days
8 days.

3 min.
2 hrs.

36 hrs.

4 min.

5 min.
Shock

Time of Exam-
ination Made

UNDER

Anaesthesia

45 hours

25 mm.

3 days.

5 mm.

Pneumonic Find-
ings ON Imme-
diate Post-
mortem

Bronchial
Type

+

+

+
+
+

+
+

+

+
+
+

+
+

Lobar
Type

+

+

+

+

+

Remarks

Intense con-
gestion of
both lungs.

Lungs appeared
soaked in
blood.

Liver lobes
involved and
lungs con-
gested.

Sneezing. Rales

Rales.

Coughing.

Resection of
lungs.

Profound symp-
toms from the
beginning.

Pleuritic effu-
sion.

Coughing and
sneezing.

Coughing.
Rales.

Intense engorge-
ment.

Reaction in
both lungs.

Reaction in
both lungs,
with hemor-
rhages.

85

86 THE ACTION OF THE LIVING CELL

five minutes. In such instances an immediate autopsy
showed the tissue changes to be focal in type, with
disseminated hemorrhagic areas, while the entire
lung was hyperemic, occasionally being soaked with
blood.

In those experimental animals which survived for
several hours or longer, there was found classical
pneumonitis of the bronchial type, with numerous
focal lesions, becoming hard and resistant.

The rapidity with which small quantities of cy-
tost insufflated in the lung cause death is most prob-
ably due to the fact that the thin respiratory epi-
thelium allows the toxin to come into immediate
contact with the endodermal cells without the neces-
sity of passing through the capillaries. This results
in a prompt stasis of blood in the vessels, and conse-
quent death, due to stopping of the heart. That auto-
lyzed lung tissue may actually effect such a stasis
was shown by the fact that the intravenous injection
of such material caused an instantaneous engorge-
ment of all the lung capillaries.

This is in agreement with the observations previ-
ously reported, that autolyzed tissue products formed
either in vitro or in vivo, when introduced into the
circulation, cause capillary stasis in the lungs and
other viscera. It is of considerable interest that, as
revealed at necropsy, the insufflated autolysate caused
no such reaction in the trachea or bronchioles, the
reaction always appearing to take place in the distal
portion of the bronchial alveolar system connected
with the surrounding dense capillary plexus. Only
when death is delayed does the reaction extend up to

Figure 7.

Lung removed fnjm a cat which died immediately following
tracheal insufflation of homologous cytost. This photograph presents
a picture typical of that always found in such experiments. The lung
is virtually soaked in blood and assumes the blood-red color in place of
the normal gray color.

PHYSIOLOGY OF CYTOST ACTION 87

the bronchial mucosa, giving rise to classical pneu-
monitis of the bronchial type. It follows, therefore,
that such an extension is secondary to the primary
reaction in the capillary plexus. It is interesting to
compare this action with that in the abdominal vis-
cera, where, it will be remembered, the primary stasis
was found to occur first in the capillaries, then ex-
tending to the larger vessels. These observations lead
one to believe that the primary reaction to "shock-
toxin," or cytost, is always in the endodermal cells and
then extends to the capillary endothelium.

As, conceivably, one might consider the insuffla-
tion technique to involve the complicating factor of
introducing a liquid into the lung, experiments of
another type were conducted. (Turck, 1919.) "A
group of ten cats was selected and examined to ex-
clude any respiratory disturbance and then placed
in a large cage. The front paws of five of these ani-
mals were coated with a thin paste of autolyzed cat
lung tissue. The animals were tagged, and placed
back in the cage with the other five cats that had not
had the application of 'lung paste.' Within two hours,
several of the cats began to sniffle and sneeze. Within
twelve hours all the cats, those that had had their
paws coated and the others in the same cage that had
not had the application of paste, had symptoms of
involvement of the lungs. Eight of these cats died
from pneumonia within 48 hours to one week after the
application of the lung paste. Some of the animals
showed extensive respiratory pathological conditions.
They also showed nasal discharge, eyes reddened, con-
junctive discharging and extensive rales over both

88 THE ACTION OF THE LIVING CELL

lungs. The temperature at first dropped and later
rose, but the temperature records were no guide to
the degree of lung involvement."

At autopsy, the findings were of a nature similar to
those observed in the preceding series of experiments,
wherein the autolysate was introduced by insufflation.
In agreement with the observations on shock pro-
duced by various means, the blood was early found
to have a high viscosity and diminished clotting time.
This, as we have seen, may be an important factor in
the development of capillary stasis.

Results identical with those discussed above were
obtained many times with different series of animals
and were not therefore due to a chance infection in
one particular group. As a control upon these obser-
vations, pneumococcus cultures, types I, II, III, and
IV, were insufflated into the trachea of cats. No pneu-
monia was produced, provided "flooding" of the lung
was prevented.

In the "cats' paw" series of experiments, the amount
of cytost which found its way to the animals' lungs
must have been exceedingly small. Indeed, that the
amount necessary to elicit a typical lung involve-
ment is minute to a degree was found in another series
of experiments wherein the autolyzed tissue was not
applied directly to the animals, but was merely
sprayed about their cages by means of a nebulizer.
Cats kept in such cages were found to develop respira-
tory involvements in much the same manner as the
animals whose paws had been smeared with a paste
of autolyzed lung tissue. At autopsy the findings
were identical with those recorded in the latter case.
(Turck, 1919.)

PHYSIOLOGY OF CYTOST ACTION 89

Incidental to the experiments just discussed, an
observation of considerable interest was made. When
autolyzed lung tissue from dogs, sheep, beef, rab-
bits and horses was applied to cats' paws or adminis-
tered by insufflation, the cats usually failed to show
any signs of respiratory involvement. Similarly,
intravenous injection of such autolysates failed to
provoke the symptoms of shock such as result when
a tissue autolysate is injected into an animal of the
same species.

These results make it appear that cytost possesses
a certain species specificity. This aspect of the prob-
lem will be discussed further in a subsequent chapter.

As a result of these experiments we may conclude
that the zone of attack of cytost lies in the endodermal
cells of the lungs, liver and upper alimentary tract
which are in intimate contact with the endothelium
of the capillaries. The injection of cytost extracts in
such concentration that immediate shock and death
do not ensue leads to a more generalized involvement
of the body tissues — particularly of the kidney. It
has been known for years that a severe nephritis may
follow a superficial burn. As has been shown in our
previous discussion, burned tissues liberate cytost. If
the burned area is extensive, then shock and death
may follow promptly. On the other hand, if the tis-
sue damage is restricted the animal may suffer a slow
absorption of the toxic cytost. Under such conditions
the concentration of cytost in the circulating blood is
usually too small to cause coagulation and consequent
marked interference with the circulation. Under such
conditions, therefore, the cytost is enabled to exert its
toxic action on more remote tissues. The kidney dam-

90 THE ACTION OF THE LIVING CELL

age which becomes evident following severe burns
belongs in this category.

In order to study such conditions, areas on the backs
or buttocks of animals were anesthetized by injections
of novocaine. The muscle and skin were then grasped
by clamps and burned to char by the application of
a white-hot branding iron for a few moments. A few
hours after such treatment the animals passed into
shock (Turck, 1918) . In Table VII are recorded the
time of the onset of shock following such burns.

TABLE VII

No.

A>flMAL

Onset of Shock
AFTER Burn

Death, Time after
Burn

I

cat

2 hrs.

8 hrs.

2

cat

li

lo hrs.

3

cat

4

24 hrs.

4

cat

3

63 hrs.

5

cat

5

recovered

6

dog

4

24 hrs.

7

dog

2

10 hrs.

8

dog

2

22 hrs.

9

dog

li

8 days

lO

rabbit

2

6 hrs.

II

rabbit

4

7 hrs.

12

rabbit

2

5 days

13

rabbit

3

recovered

H

rabbit

5 days

The crisp charred tissue was dissected from the
burned areas of such animals and pulverized. One-
gram portions of this material were then extracted
with 10 cc. of water and filtered. This extract was then
injected intravenously into normal homologous ani-
mals, 1 cc. portions being used for cats and rabbits,
and 2 cc. portions for the dogs. By this means a much

PHYSIOLOGY OF CYTOST ACTION 91

higher concentration of cytost was introduced into
the circulation than is the case when natural absorp-
tion of the burned tissue is permitted to take place.
Consequently the onset of symptoms in this series of
animals is much more rapid, as shown in Table VIII.

TABLE VIII

No.

Animal

Onset of Symptoms
Time after Injection

Death in

IS
i6

17
18

19

20
21
22
23
24

25

26

27
28

cat

cat

cat

cat

cat

dog

dog

dog

dog

rabbit

rabbit

rabbit

rabbit

rabbit

immediately
immediately

5 minutes
10 minutes
30 minutes
immediately
10 minutes
30 minutes
immediately
immediately

5 minutes

4 minutes

5 minutes
10 minutes

2 hours
recovery
24 hours

5 hours

30 minutes

6 hours
recovery
12 hours
recovery

At autopsy, all the animals listed in Tables VII and
VIII showed the same general pathology as that
previously described in the earlier experiments with
cytost. However, in this series of experiments special
attention was directed to the involvement of the kid-
neys. Quite striking was the observation that within
five minutes after the intravenous injection of the ex-
tract of charred tissue, kidney lesions, usually in the
form of a glomerulitis, could be detected. This was
determined by removal and sectioning of the organ.

It was further observed, as might be expected, that
in general the type of kidney involvement varied

92 THE ACTION OF THE LIVING CELL

with the degree of the burn and the interval of time
between the burning and removal of this kidney for
examination. While for our present purposes it is
not desirable to stress the pathology of kidney lesions,
a few additional facts are pertinent to the general
problem of the mode of action of cytost on the kidney.

Animals which had been burned and apparently
had recovered from such maltreatment were found
upon subsequent examination to be suffering from
kidney lesions.

The kidneys of such animals showed changes of
both the degenerative and inflammatory proliferative
type in the glomeruli and approximal tubules.

Since glomerulitis may be observed within five
minutes following the injection of cytost, it seems that
this is the primary change in the kidney induced by
stasis of the blood in the glomerular capillaries.
Those animals which survived the injection of the
burned tissue extract likewise were found to have
suffered degenerative changes in the kidney similar
to those induced by direct burns. It seems likely,
therefore, that degeneration and secondary prolifera-
tion in the areas under consideration are induced by
the primary response to cytost. The blood stasis in-
duced by the latter interrupts the normal metabolism
of the kidney cells and permits an accumulation of
toxic metabolites, which in turn results in further
tissue destruction and cellular proliferation. If the
latter becomes excessive, then the damage is irrepar-
able.

It is interesting to compare these results with the
changes induced in the stomach by the application to
the mucosa of emulsions of mustard. In the latter

(8
U

o

t-

o

u
a
cr

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u

.s „•
^ "a

-^ a

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1-1 "-M

O 3

13

r-i

U

g o

o '^

>- r,
<u p

- . -i"? '".V ^'v

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a,

PHYSIOLOGY OF CYTOST ACTION 93

experiments, it will be recalled, the mustard first in-
duced merely a hyperemia. This was followed later
by degenerative changes and subsequent active pro-
liferation of the cells, as evidenced by the occurrence
in sections of many karyokinetic figures. Further,
these changes continued to take place even after re-
moval of the mustard, the latter merely acting as a
trigger to release the cytost from the mucosal cells
in the same fashion that trauma or burning releases
it from epithelial and muscle cells.

While burns and other injuries always produce
acute lesions of the lungs and liver, these, if not ex-
tensive, may clear up rapidly, leaving little or no
evidence of any tissue damage. This difference be-
tween the other organs and the kidney, wherein, as
stated above, cytost may produce a chronic injury,
may in part be attributed to the single functional
blood supply of the latter, which depends almost en-
tirely on the renal vessels and a few auxiliary anas-
tomoses. During blood stasis, therefore, the kidney
is more severely taxed than are the other viscera, which
have a double vascular blood supply. Again, the kid-
ney cells are known to be very sensitive to toxic sub-
stances, such as the salts of mercury and uranium and
various organic compounds. In this respect they re-
semble the endodermal cells of the liver, lung,
stomach, and intestines. Hence it seems probable that
the kidney cells may be more easily injured by cytost
than are other tissues. Photomicrographs showing
congestion of the glomeruli, venous stasis, and clump-
ing of the corpuscles in the region of the straight
tubules are shown in the accompanying illustrations.

Chapter V
FURTHER EXPERIMENTS WITH CYTOST

The investigations presented in the preceding
chapters have shown that cytost, a product of tissue
autolysis, when introduced into the circulation
promptly induces a splanchnic stasis which is fol-
lowed by shock and death. Now such effects are in-
duced only by relatively large quantities of cytost.
This is evident from the fact that shock does not fol-
low minor trauma and burns, although from the
preceding discussion it should be apparent that such
injuries must provoke the liberation of cytost in pre-
cisely the same manner as does more extensive tissue
damage. Such being the case, it is of interest to fol-
low the reaction of animals to injections of sublethal
quantities of cytost prepared by the in vitro autolysis
of homologous tissue.

The cytost extracts used in the following experi-
ments were prepared from cat's muscle, which was
allowed to autolyze in chloroform vapor for 24 hours.
The autolyzed tissue was triturated with an equal
weight of sterile water, heated to boiling, and centri-
fuged to remove the insoluble matter. The super-
natant fluid was then sterilized by passing it through
a Berkefeld filter and used directly for injection.

94

EXPERIMENTS WITH CYTOST 95

The toxicity of such preparations was ascertained
by injection into the femoral vein of anesthetized cats.
When administered in this fashion, 1.5 cc. of such a
preparation caused death of the animal within 1 or
2 minutes. As in the other acute experiments which
we have considered, immediate autopsy always dis-
closed marked congestion of the lungs and abdominal
viscera. When injected intraperitoneally, intramus-
cularly, or subcutaneously, such cytost extracts do not
cause immediate congestion and death, but, depend-
ing upon the dosage, bring about different pathologi-
cal conditions, as shown in the following experiments
selected from many recorded in the author's labora-
tory notebooks.

No. 73

Male cat, weight 343 gms.

March 29, 1920. Temperature 101.4°. 1 cc. injected intraperi-
toneally and 1 cc. subcutaneously.
March 31. Similar injections. Temperature 102°.
April 4. Similar injections. Temperature 102.2°.
April 6. Animal died.

Autopsy : stomach congested, dilated ; duodenum, congested ;
intestines hemorrhagic ; spleen enlarged ; kidneys congested.

Castrated male cat. Weight 2980 gms.

March 31, 1920. 1 cc. injected intraperitoneally and 1 cc. subcu-
taneously. Temperature 101°.

April 4. Similar injections. Temperature 101.6°.

April 8. Sick, temperature 103.8°. Weight 2210 gms.

April 10. Weak, does not eat, temperature 102.5°. Weight 2000
gms.

April 11. Temperature 99° ; exit.

96 THE ACTION OF THE LIVING CELL

Autopsy : stomach, few hemorrhagic spots. Duodenum, con-
gested and atrophied ; spleen, enlarged, infracts ; liver, very
hemorrhagic; lungs, characteristic lesions; kidneys, congested.

No. 86
Female cat. JVeight 2900 gms.

April 8, 1920. 1 cc. injected intraperitoneally and 1 cc. subcu-
taneously. Temperature 101°.

April 13. Similar injections. Temperature 101°. Animal appears
to be weak.

April 18. Similar injections. Temperature 100°.

April 19. Died.

Autopsy: Stomach and duodenum show erosions and what
appear to be breaking doAvn ulcers; liver, very hemorrhagic;
spleen, enlarged ; kidneys, congested ; lungs, characteristic ap-
pearance.

Results similar to the above have been obtained a
great many times. Occasionally, however, a cat was
encountered which was apparently capable of toler-
ating such injections of cytost for a much longer pe-
riod of time. Such animals however eventually suc-
cumb to the long-continued action of cytost, and when
autopsied are found to exhibit degenerative tissue
changes similar to those recorded in the preceding
protocols. For example, consider cat No. 89.

No. 89
Castrated male cat. Weight 3250 grams.

April 26, 1920. Injected 1 cc. intraperitoneally and 1 cc. subcu-
taneously. This was repeated on each of the following dates
unless otherwise noted.

May

2,

1920.

Weight

3240.

9.

Weight

3190.

18,

Weight

3300.

EXPERIMENTS WITH CYTOST

97

May 25,

June

17,

30,

July

13,

27,

Aug.

11,

25,

Sept.

14,

29,

Oct.

18,

Nov.

9.

Dec.

7,

Jan.

7,

23,

Feb.

6.

15,

Mar.

5,

April

9.

Aug.

6,

Nov.

19,

May

7,

13,

27,

Aug.

22,

Sept.

7,

22,

Oct.

1,

10,

Nov.

9,

16,

Dec.

11,

1921.

1922.

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Weight

Animal

3300.

3490.

2950.

3070.

3130.

3060.

3500.

3500.

3570.

3690.

3910.

3940.

3960.

4010.

3890.

3890.

3770.

3920.

3240.

3630.

3510.

3640.

3650.

3480.

3350.

3480.

3450.

3490.

3000.

2990.

died.

No injections. Cat appears dull.
Y2 cc. used for each injection.
Yz cc. used for each injection.
No injections.
Y2 cc. used for each injection.

No injections.
No injections.
No injections.

Autopsy: Lungs, chronic lobular pneumonia; heart, peri-
carditis and dilatation hepatitis; stomach, gastrointestinal
catarrh, ulcer all over and in pyloric region ; kidneys, chronic
glomerulitis.

It will be noted that this animal was markedly re-
sistant to the action of cytost.

98 THE ACTION OF THE LIVING CELL

As shown in the protocol, this animal (cat No. 89)
withstood periodic injections of cytost for a period of
more than two years. During this time several dif-
ferent cytost preparations were used, each of which
was definitely toxic when injected into the femoral
vein of other cats. It follows, therefore, that this
animal possessed to a marked degree a natural im-
munity to the action of cytost. As this was also en-
countered in several other animals the author was led
to attempt the production of an artificial immunity to
cytost. This interesting aspect of the problem will be
discussed later.

It has been shown previously (Chap. Ill) that cy-
tost may be liberated in vivo by agents which bring
about cellular destruction and autolysis. If such
processes are induced continuously for a protracted
period of time, we should expect to obtain results
similar to those following the injection of tissue
autolysates. Now in vivo autolysis may be accom-
plished by a variety of means, such as trauma, burns,
and the application of caustic chemicals such as mus-
tard oil, ether, chloroform, and alcohol. Of these the
last three are most adaptable for prolonged experi-
ments, since by regulating the amount injected one
may to some degree control the extent of autolysis.
Animals which died as a result of such treatment,
or were killed after a number of such injections,
quite uniformly were found to have suffered the
same organic lesions as those injected with ex-
tracts of autolyzed tissues. Such observations con-
firm the hypothesis that the tissue damage caused
by these reagents is responsible for the observed
pathology.

EXPERIMENTS WITH CYTOST 99

The protocols following are representative of many
of the author's observations.

Cat No. XII. Male.

March 1,1919. 2 P.M. Temperature 40°. 5 cc. of ether injected
subcutaneously on right side. No immediate effects although
animal appears stupid.

3 P.M. Temperature 37°. Lying down, moaning. Remained
this way for ^ hour, then recovered.

March 4. 5 cc. ether injected on left side. In 10 minutes com-
menced to stagger.

March 8. Thin and weak. 5 cc. ether injected on right side.

March 13. 5 cc. ether injected on left side. Stupor, then coma.

March 14. Found dead.

Autopsy: Liver and spleen, enlarged and congested; omen-
tum, sclerosed, hardened; stomach and duodenum, congested,
stained with bile ; lungs, red, solid, pneumonic ; brain, normal.

Cat No. XVII. Male

Feb. 28, 1919. 2:50 P.M. Temperature 40°. Injected 10 cc. al-
cohol in each side.
3:00 P.M. Temperature 38°.

March 4. Injected 10 cc. of alcohol on each side.

April 17. Injected 10 cc. of alcohol on each side. Staggered about
for ten minutes, then became comatose, remaining so for 8 hours.

April 18. Found dead.

Autopsy: Pneumonic spots, marked congestion; stomach
and duodenum, congested, hemorrhagic; liver, very congested,
omentum hardened ; spleen enlarged, infarct ; kidneys, marked
congestion; brain, normal.

In such experiments, objection may be raised
against the relatively large quantities of the reagents
injected into the animals. In order to obviate this, the
following procedure was devised. The animal's leg

100 THE ACTION OF THE LIVING CELL

was ligated and a small quantity of the chemical to
be studied was injected below the site of the ligature.
By such means, free diffusion of the reagent was pre-
vented and the tissues adjacent to the site of the in-
jection received the brunt of the action. By this means,
a greater tissue damage and consequent increase in
the amount of cytost liberated was achieved, for the
oxidation or elimination of the injected substance was
prevented. Upon removal of the ligature, the accu-
mulated cytost was released into the circulation and
carried to the viscera, which reacted in the manner
previously described.

Cat No. XIX.

March 1, 1919. Leg tied off. 3 cc. alcohol injected below ligature.

Two hours later ligature released. Leg swollen.
March 7. Swelling of leg has disappeared, animal becoming thin.
March 10. Animal failing rapidly. Skin excoriating over site of

injection.
March 11. Found dead.

Autopsy: Lungs, partially consolidated ; stomach, duodenum,
and liver, markedly congested; brain, meninges congested;
heart, ventricles normal.

Cat No. XX

March 1, 1919. Leg tied and 4 cc. ether injected below ligature.

Latter removed after 2 hours.
March 4. Leg swollen, animal quiet and stupid.
March 10. Does not eat, thin, weak.
March 13. Found dead, body still warm.

Autopsy: very thin, fat and muscles gone. Lungs, congested,
hemorrhagic, red, solid ; stomach and duodenum, congested ;
liver, congested, fatty degeneration; kidneys, congested, fatty;
spleen enlarged; brain, normal.

EXPERIMENTS WITH CYTOST 101

Cat No. LI I

June 4, 1919. Leg tied off. Fifteen minutes later injected 1 cc.
chloroform. After 1 hour ligature removed.

June 5, 9:15. Respiration rapid and weak. Rales in lungs.
Mucous membranes pale. Taken from cage and laid on desk,
is relaxed, does not move. Raises head for an instant but can-
not hold it up.

10:15. Gasps for breath. Temperature 38.5°. Respiration
and heart stopped.

10:20. Autopsy: Lungs, lower lobes engorged, hemorrhagic;
liver, stomach and upper intestines congested.

When fluid was withdrawn from the site of the in-
jection and injected into other animals, the latter
showed a typical response, as demonstrated in the
following protocol.

Cat No. XXII

March 1, 1919. 2:00 P.M. Temperature 40°. Four cc. of auto-
lytic products, resulting from the injection of ether into the
leg of another cat, were injected subcutaneously.
2:30. Temperature 38°.

March 2. Eats. Temperature normal.

April 14. Animal very thin.

April 20. Thin, coughing.

April 26. Extremely thin. Constantly coughing and sneezing.

May 6. Does not eat. Staggers.

May 11. Found dead.

Autopsy: Lungs, pneumonic spots, and spots resembling tu-
bercules; liver, congested; stomach and intestines, congested,
hemorrhagic areas and apparently ulcers in intestines; kidneys,
congested.

Such experiments as these show definitely that cy-
tost liberated in vivo by the action of chloroform,
ether, and alcohol leads to the same pathological end

102 THE ACTION OF THE LIVING CELL

as does cytost from autolyzed tissues. In both in-
stances the primary congestion results in damage of
the tissues of endodermal origin.

When somewhat smaller quantities of cytost are in-
jected or liberated in vivo much the same results may
be obtained, but a much longer period of time is neces-
sary before the pathological changes become appar-
ent. In this connection the reader may recall the pro-
duction of kidney lesions by superficial burns and by
the repeated injection of small quantities of chloro-
form and ether (page 92). Similarly, repeated in-
jections of small quantities of tissue autolysates re-
sult in the production of chronic lesions. The results
of some experiments of this nature are summarized in
Table IX. The cytost preparations were prepared as
stated in the preceding protocols.

Aside from the resulting pathology, the marked
loss in weight of the experimental animals is interest-
ing. This may be taken as indicative of a lowered
metabolism. On the basis of these experiments alone
one cannot decide whether this is due primarily to
the action of the cytost on the body cells, or to im-
paired circulation resulting from blood stasis induced
by the cytost.

In the writer's experience, gastrointestinal ulcers
are rare in cats; hence he is forced to conclude that
those found in the animals injected with cytost were
due to the prolonged action of the latter on the gastro-
intestinal mucosa, which, like the other tissues sensi-
tive to cytost, arises from the endoderm of the early
embryo.

As is now well known, in the adult animal ingested
foods are for the most part utilized as a source of

EXPERIMENTS WITH CYTOST

103

energy for the organism, a relatively small fraction
being used for the repair or replacement of tissues.
While for the latter purpose the nature of the ingested
fats and carbohydrates appears to be immaterial as

TABLE IX (TURCK, 192 1)

Amount
Injected

Mode of
Injection

Interval

Period

Original
Weight

Weight
AT Death

Loss

Pathology

I or 2 cc.

Subcut.

2-5 days

6 months

2970

1420

1550

arterial
sclerosis

I cc.

Subcut.

daily

2 months

2335

I180

"55

arterial
sclerosis

2 cc.

Intra-
peri-
toneal.

2-3 wks.

I year

3960

2550

1410

arterial
sclerosis

2 cc.

Subcut.

2 days.

2 months

3980

3090

890

chronic
lobar
pneu-
monia,
ulcers,
nephtitis.

I or 2 cc.

Subcut.

4 days.

I month

3130

1950

1 180

pneu-
monitis,
hepatitis,
nephritis,
dudodenal
ulcer.

well as interchangeable, there is a definite protein
requirement. This is necessary, as shown in the re-
searches of Osburn and Mendel (see Mendel, 1924),
to supply certain essential amino acids which the or-
ganism is incapable of synthesizing from other com-
ponents of its diet.

During complete starvation, such essential amino
acids are not available; hence the animal is forced to
draw upon the proteins of some of its body cells in
order to satisfy the nitrogen requirements of other

104 THE ACTION OF THE LIVING CELL

cells. That the body proteins are actually utilized
during starvation is shown by the fact that a more or
less constant excretion of nitrogen takes place during
the period of starvation. Such being the case, it seems
likely that during inanition the essential amino acids
are liberated by autolysis of the tissue cells. This hy-
pothesis is substantiated by the observations of Casa-
Bianchi (1912), vs^ho found the histological changes
incident to starvation to be similar to those observed
during the autolysis of tissues. As we have seen, tissue
autolysis liberates the toxic substance, cytost; hence
we should expect to find some evidences of the latter's
action in starving animals. In agreement with this
conclusion, Asada (1919) found the capillaries of the
lungs and other organs of fasting rabbits to be in-
tensely congested — a typical "cytost reaction."

When animals are caused to suffer partial inanition
(malnutrition), the changes have frequently been
found to be more extensive than during total starva-
tion. The reason for this is not clear, although au-
tolysis made necessary by the deficient diet is seem-
ingly an important factor. Some years ago the writer
(1907) observed degenerative changes in all the or-
gans of rats which had been kept for a month upon
a diet of meat extractives, although control animals
fed meat or extract-free meat for the same period
showed no harmful effects. When dogs were fed in
similar fashion with beef extract containing B. coli
for from four to six months, the animals died from
perforated ulcers of the stomach, or hemorrhage. At
the time these experiments were performed the au-
thor's interest was directed towards other ends than
those now under discussion; hence cultures of the

EXPERIMENTS WITH CYTOST 105

colon bacillus were added to the diet. However, since
this organism is normally found in the alimentary
tract and the heart's blood of the experimental animals
was found to be sterile, we may for the moment at-
tribute little significance to the presence of the bacilli
in the food. As a result of such feeding the dogs were
found at autopsy to have suffered multiple peptic and
duodenal ulcers, and venous congestion of the liver
and kidneys.

Upon histological examination, the liver cells, par-
ticularly in the central portions of the lobules, were
found to have undergone degenerative changes, while
in the peripheral portions numerous small vacuoles
were observed in them. Similarly the stomach and in-
testines showed glandular and vascular changes, ap-
pearing edematous, with the blood vessels more or
less engorged. The kidneys were also involved ; the
glomeruli were separated from the capsule, the capil-
laries were engorged, and the peripheral cells lining
the tubules were swollen ; in some instances, their out-
lines had disappeared. In some cases in which the pe-
riod of feeding lasted for several months, arterio-
sclerotic changes were found. Judging by the failure
of the nuclei to stain properly, all the organs showed
evidence of autolysis. In none of the experimental
animals could any evidence be found for the existence
of a bacteriemia or inflammatory reaction. In conse-
quence it appears that the colon bacillus played a
minor role in these experiments and that the observed
pathology was due to the products of tissue autolysis
resulting from partial inanition. If the reader will
compare the tissue changes induced by such treatment
with those produced by cytost, either injected as such

106 THE ACTION OF THE LIVING CELL

or produced in vivo by the other means previously dis-
cussed, he will see that the effects are the same. These
experiments therefore support the contention that cy-
tost may be liberated in vivo by starvation.

Much the same end may be achieved by feeding
animals a complete although unsatisfactory diet. For
example, six monkeys were fed small squares of bread
fried in cottonseed oil in addition to the usual daily
vegetable rations. (Turck, 1917.) All six of the ani-
mals kept on such a diet died within twenty-eight to
sixty-one days, although other monkeys kept in the
laboratory and fed vegetables alone remained in a
healthy condition. At autopsy all six of the fat-
fed animals presented much the same picture. The
splanchnic vessels were markedly congested, and the
tissues, especially the liver, were found to have suf-
fered fatty infiltration. Peptic ulcers were found in
the pyloric region of the stomach.

Although these changes were induced by a high
fat diet, it does not seem likely that the fats per se
were responsible for the observed pathology. The
latter suggest the action of cytost, whose liberation
may be accounted for in the following way.

The normal diet of the monkey consists largely of
carbohydrate. Consequently when forced to ingest
relatively large amounts of fats, these animals, one
may well imagine, are subjected to a severe acidosis
and, as we have seen, an acidosis in the tissues leads
to rapid autolysis and the subsequent liberation of
cytost. Further, in these experiments, it seems likely
that the fats may have interfered with the digestion
and absorption of food; hence the animals may have
suffered partial inanition which, as pointed out above,

EXPERIMENTS WITH CYTOST 107

leads to autolysis. Whichever explanation is correct,
the fact remains that the histological examination of
the body tissues disclosed marked autolysis of the
tissue cells.

This fact is but the expression of a general rule that
nutritive disturbances which hinder the normal course
of metabolism always result in the degeneration of an
animal's tissues. This is seen in the various forms of
illness, which are of common occurrence in captive
wild animals, and will be discussed more fully in
Chapter IX.

The reader is probably familiar with the well
known fact that disuse of a tissue, such as a muscle,
leads to atrophy and cellular degeneration. With this
fact in mind, the writer conducted the following ex-
periments. (Turck, 1906.) The experimental ani-
mals were placed in small sterilized cages which pre-
vented them from moving about. Ninety-six guinea
pigs and thirty-six rabbits were caged in this fashion
and given a normal diet. During the first few months
the animals gained in weight, due to deficient exercise.
Later, although supplied with sufficient food, they
became emaciated. At autopsy, general tissue de-
generation and autolysis were found, as well as peptic
ulcers in some cases. During the course of these ex-
periments, routine blood examinations were made and
those animals showing any evidence of infection were
immediately discarded. At the expiration of nine
months but six guinea pigs remained alive. At au-
topsy general tissue degeneration and autolysis were
found in all cases, as well as several instances of pep-
tic ulcers.

More than three hundred years ago Borelli demon-

108 THE ACTION OF THE LIVING CELL

strated that the movements of the body fluids in the
capillaries is entirely dependent upon the contraction
of the surrounding muscles, and today physiologists
are generally agreed that the venous return to the
heart is dependent upon this factor. Hence, during
enforced quiescence there must be some degree of
stagnation of blood in the finer vessels, which prevents
both an adequate nutritive supply and the removal of
toxic waste products. From the preceding discussion
it should be obvious that the former will cause an
increased liberation of cytost, while the impairment
of circulation must necessarily bring about the ex-
posure of the tissue cells to higher concentrations of
the toxin than would otherwise be the case.

In consequence, it is not surprising that the tissue
degenerations and pathological findings in animals
whose quiescence has been enforced should closely
parallel those induced by the frequent injection of
sublethal quantities of cytost.

Of all the results obtained in the writer's experi-
ments, the following are the most interesting (Turck,
1921b) : A pair of two months old kittens from the
same litter were injected subcutaneously with like
quantities of the same cytost extract. One, which we
may call "A," was injected once a week; while the
other, "B," received the injections daily or every other
day. Much to our amazement, "A" gained in size
and weight more rapidly than the other, untreated kit-
tens of the same litter, whereas "B" ceased to grow,
and suffered a progressive decline in weight. This
was accompanied by senile changes, until, after two
months of such treatment, the kitten (then four months
old) appeared as a decrepit old animal,

"Wip

Figure 10.

Kitten B, age 4 months. Premature senescence and apparent rachitis
induced bv cytost injections as described in the text.

EXPERIMENTS WITH CYTOST 109

The kitten's coat remained undeveloped and it ac-
quired a curiously dejected facial expression. The
spine assumed an inward curvature and the bones of
the paws did not develop properly. These malforma-
tions are clearly shown in the accompanying photo-
graphs, taken shortly before the animal's death, which
occurred five months after the injections of cytost
commenced.

At autopsy arteriosclerosis, general atrophy, ar-
thritis and fibrosis of the kidney, liver, and lungs were
apparent. Such tissue changes as these are commonly
found in aged animals, and because the factor respon-
sible for these changes is unknown, are usually classi-
fied as senile changes. (See Fox, 1923.)

Since in kitten "B" these pathological changes were
induced by the injection of cytost, it seems possible
that senescence in the metazoans may in part result
from the accumulation of cytost. As stated previously,
tissue culturists have found that cells appear to be
immortal if provided with adequate nourishment and
means for the removal of waste products. However,
even though the former is provided, cells in culture
rapidly degenerate and die if their metabolites are
not removed. (See Lewis, 1924.)

While the writer has not encountered another case
in which senile changes were induced as rapidly as in
kitten "B," he has by repeated injections of cytost in
many cats accelerated the onset of senescent changes,
as shown by the high incidence of arteriosclerosis in
animals so treated. (See Table IX.) In such cases, this
results long before death in external changes such as
we normally associate with old age. For example,
cat 114 (see protocol below) showed graying of the

110 THE ACTION OF THE LIVING CELL

hair, sclerotic changes in the marginal vessels of the
ears, and hemorrhagic zones at the junction of the
gums and teeth.

Cat No. 114 (Abstract of Protocol)

Young female, white with black spots

Nov. 9, 1920. Weight, 1750 gms. Injected intraperitoneally 1 cc.
of cytost extract prepared by boiling 10 gms. of autolyzed cat
heart muscle with 50 cc. of water and evaporating to 25 cc.

Nov. 11, 12, 13. Similar injection. Sneezes, discharge from eyes.
These symptoms subsided by the 20th.

Nov. 13-21. Similar daily injections.

Dec. 7. Weight 2015 gms., good appetite but has appearance and
actions of animal about to break down. Hence allowed a
month for recovery.

Dec. 13. Weight, 1835 gms.

Jan. 9, 1921. Weight, 2270 gms. Appeared normal hence in-
jections commenced again. Used 1 :25 extract of autolyzed cat
muscle. Injected 1 cc. subcutaneously each day from Jan. 9,
1921, until Feb. 17. During this time the cat progressively
gained in weight.

Feb. 17. Weight, 2640 gms.

Feb. 18. In heat, assaulted by tomcat.

Feb. 19-26. Daily subcutaneous injections of 1 cc.

Feb. 27. Weight, 2520 gms. Blood vessels around margin of ear
appeared sclerotic. Hemorrhagic zone at margin of gums. In-
dicates vascular and nephritic changes. Daily injections of 1 cc.
continued until

April 7. Aborted two kittens about 4 weeks gone. No injection.

April 8. Aborted another kitten. No injection.

April 10-May 26. Daily injections but no noteworthy changes.
Weight, 2420 gms. Apparently the cytost extracts used were
too weak. Hence changed to muscle extracts made by ex-
tracting 10 gm. portions of autolyzed muscle with 10 cc. of
water. 1 cc. of the extract injected daily until

July 7. Unable to climb or jump, very sensitive, resents handling,
poor appetite, receding gums and extensive dental caries. Daily
injections continued till

EXPERIMENTS WITH CYTOST 111

Dec. 10. Gray hair becoming more and more pronounced in the
patches of once dense black hair. Most marked in tail. Daily
injections continued.

Feb. 4, 1922. Weight, 2270 gms. Black hair on head replaced by
white hair. Injections continued.

Feb. 8. Weight, 2170 gms. Cat weak, anterior incoordination, eye
discharge. Daily injections continued until March 15, 1923.

March 17, 1923. Cat died. Weight, 2050 gms. Autopsy per-
formed five minutes after death.

Autopsy: Lungs, chronic fibrous pneumonia; heart, hyper-
trophied, dilated; liver, fatty degeneration; stomach, full of
erosions, dilated, fibrosis; intestines, fibrosis, erosion, begin-
ning ulcers; kidneys, chronic nephritis; adrenals, enlarged;
bones, extremely brittle; brain and cord, congested; aorta and
arteries, hard.

In no instance has the writer been able to elicit such
degenerative tissue changes by the injection of similar
quantities of heterologous cytost — that is, cytost pre-
pared from the tissues of animals of another species.
Even cytost prepared from a lion's muscle, when in-
jected in similar fashion into cats, does not lead to any
pronounced pathology such as induced by that pre-
pared from cats' tissue.

In various experiments in which parallel groups
of animals have been injected with homologous and
heterologous cytost, only those treated with the former
exhibit such tissue changes as have been described
above.

It will be recalled that in the experiments with the
kittens mentioned a few pages back, kitten "A," which
received cytost injections spaced a week apart, gained
in weight more rapidly than its litter mates. This sug-
gests that, in small quantity, cytost may actually be
capable of accelerating the metabolism of the tissue
cells. In order to test this conclusion a series of mature

112 THE ACTION OF THE LIVING CELL

cats were periodically injected subcutaneously with a
cytost extract prepared from autolyzed muscle as de-
scribed on page 94. The injections differed from
those used in previous experiments only in that the
dosage was carefully regulated to 0.25 cc. per kilogram
of body weight. The animals were injected for a pe-
riod of from one to eight months. In each instance the
cats gained in weight, while a series of control animals
of approximately the same age and weights, kept un-
der identical conditions, did not exhibit any marked
increase in weight. Data concerning ten cats treated in
this way are summarized in Table X. (Turck, 1921 ) .

TABLE X

Number of
Injections

Interval

BETWEEN

Injections

Period m
Months

Original

Weight in

Grams

Weight at
End of
Period

Net Gain
IN Weight

lO

2 weeks

2i

4230

S35S

II2S

7

2 to s days

I

2880

3140

260

S

2 to 3 weeks

3

2950

3650

700

21

2 to 5 days

4

2180

3090

910

5

2 to 3 weeks

2

2840

3260

420

13

2 to 3 weeks

6

2830

3780

950

IS

2 to 4 weeks

8

3000

4020

1020

12

2 to 4 weeks

6

3S00

4520

1020

H

2 to 4 weeks

6

3020

4010

990

13

2 to 4 weeks

7

4250

5030

780

Aside from the increase in weight, these animals
had a general appearance equalled by but few of the
non-treated animals. Their coats were sleek and their
muscles firm and free from fat. Likewise, they showed
a playfulness and courage which are quite uncommon
in old cats. When injured, their wounds healed rap-
idly without suppuration. Two years after these ex-

EXPERIMENTS WITH CYTOST 113

periments, seven of the ten cats which had been kept
in the laboratory were still healthy and vigorous.

In order to follow more closely the rate of increase
in weight of animals so treated a series of cats was in-
jected every four days with 0.25 cc. of homologous
cytost extract and the weights of the animals were de-
termined at the time of each injection. In figure 11
data from three representative experiments have been
plotted. In each instance it will be noted that the
cytost injections led to a marked increase in weight up
until the time of the tenth or twelfth injection, after
which the animal's weight fell, only to rise again to
a value which was maintained despite further injec-
tions of cytost.

In these experiments, the small quantity of cytost
injected first causes an increase in weight, due pre-
sumably to increased metabolic activity; then a re-
action sets in which causes a loss in weight although
the original level is not reached. This is apparently
due to tissue damage caused by an excess of cytost,
for post-mortem examination of some of these animals
disclosed evidences of chronic involvement of the
lungs, and gastritis. However, such damage is slight
and appears to be readily overcome by the animal,
which then regains its lost weight. The final rise in
weight may perhaps be attributed to the production
of antibodies against cytost. It seems probable that it
is the presence in these animals of the latter, which we
may call anticytost, which prevents the development
of pronounced pathological changes such as those
found in other experiments previously cited. It is in-
teresting to contrast this series of experiments with
those summarized in Table IX on page 1 03. In the lat-

114 THE ACTION OF THE LIVING CELL

ter, larger quantities of cytost were injected and these

led to a rapid decline in weight, and subsequent death.

During the course of the writer's experiments it was

! 1 1 1 1 I 1 M ' 1 M I

> 111 1 1 1 1

I 1 1 1

4-0 JO . . . . . i

, - -M i- -f- — ,- K- -| - -4-| - ^-L.

_,_ i_Ti;feis; 41 iirt _i_ir r

±1 -1 X tJ?SlS4l 41 ZLLTt: > i- ^ ^

y V\ i^ 1 ■

J^^^ ^^ tJ-1- L-

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X ^ "" ' *^V \ " "* r^ ^ t

•'^ ^ftA .... ^'^ ^ 1 J , . y ., '^ *'' . ., . i . , , , .,..,,. J.

3500 p /^ -* f s; -- /- -- -4-

..:fe'^ ^ ," — ^ ^v ^.^

^ — " ^ ^ - - ^ ^ 'r

^ S _ 1— '

^^' ± II _.

7 -^ 1 r 1

/

/

•anon jf'.. , ._ _

Z it it IT "I. "^ ■ l_

. . . , 1

-f- -^ -1-zt: ii "^ J- "• — ^

r 1

1

2500 ,..-L..--...

I 1

"It ""it

J , ,1

1

-hi x " - i ill .- -Ill J

?.000 ! ; 1 1 1:1 1 1 !

I c 3 4 5 o 7 3 9 10 II 12 13 14- 15 16 17 18 19 20 21 22 23 24

Figure 11. Graphs showing the effects of injections of homol-
ogous cytost upon the weights of three cats. The weights of the
animals are shown as ordinates and the number of injections of
0.25 cc. of a 10% solution of homologous cytost as abscissae. These
injections were spaced four days apart. (From a paper read by the
author before Section D of the Brit. Assn. Adv. Sci., Sept. 9, 1930.)

noted that when pregnant cats happened to receive
sublethal injections of cytost, they frequently aborted
or gave birth to dead or weakly kittens. This led to a
brief study of the effects of cytost upon pregnant cats
and their offspring.

had

and

Section through a pregnant guinea pig which
experienced severe shock. Clearly shows congestion
small hemorrhages in the entire visceral area excepting the

brain and spinal cord.

EXPERIMENTS WITH CYTOST

lis

Eight healthy pregnant cats were daily injected in-
traperitoneally with 1 cc. of cytost. As might be ex-
pected from what has been said above, the cats devel-
oped various pathological conditions of the viscera
which caused the death of two of the mothers. Five of
them gave birth to dead kittens, while all but one of
the kittens born alive died shortly after birth. Data rel-
ative to these experiments is recorded in Table XI.

TABLE XI
Effects of Large Doses of Cytost on Mothers and Offspring

Mothers

Kittens

SURVIVED

DIED

DEAD IN
UTERO

BORN

died"

LATER

SURVIVED

TOTAL

Alive

Dead

I

I
I

4

3

5
3

I

5
I

3

2

3

5

2

O
O
O
O
O
O
I

o

4
4

5

I

5
3
3

2

6

2

4

II

12

ID

I

27

From these results it follows that the injected cytost
affected the kittens as well as the mothers. That this
is entirely possible is shown by the fact that when
shock was induced in a pregnant animal, the embryo
was found to suffer marked stasis of the blood in much
the same manner as did the mother. In figure 12 is
reproduced a photomicrograph of a guinea pig em-
bryo in utero of a mother which had been subjected
to fatal shock. Examination of this section discloses

116 THE ACTION OF THE LIVING CELL

marked congestion with small hemorrhages through-
out the visceral area, although there is no evidence of
congestion in the spinal cord. (Turck, 1918b.)

This result indicates that if for any reason a preg-
nant animal suffers an accumulation of cytost we may
expect the fetus to be affected in some adverse way.
Experiments which will be discussed in Chapter VIII
have shown that such is the case.

Chapter VI

THE ACTION OF CYTOST
ON SINGLE CELLS

Some years ago Leo Loeb (1897) observed that
excised somatic cells and tissues could be grown in
test tubes upon solid media such as blood agar, a
method which presented a serious drawback in that
the processes of growth and differentiation could not
be continuously observed. This difficulty was sur-
mounted by Harrison ( 1907) , who found that isolated
bits of nervous tissue taken from frog embryos could
be kept alive for many days in a hanging drop of frog
lymph. Such a technique permits the observation of
the cells by means of a high power microscope, and
present day methods of tissue culture are an elabora-
tion of Harrison's method.

In general the technique is quite simple. Under
aseptic conditions tissues are excised from an animal
and immediately placed in a balanced salt medium,
such as Ringer's or Locke's solution. While so im-
mersed, the tissue is cut into pieces a millimeter square,
or less. These are then transferred to a sterile cover
slip, covered with a drop of suitable medium, and the
cover slip is then inverted over a depression in a sterile
slide and sealed in position.

117

118 THE ACTION OF THE LIVING CELL

After incubation, if the culture medium is suitable,
the cells are found to migrate from the periphery of
the tissue fragment or explant. This migration takes
place only along solid supporting substances; hence
blood plasma is usually added to the medium, since
after coagulation of the drop, the fibrin threads form
a suitable pathway for movement of the cells.

That the plasma per se is not essential to the cellular
migration but simply offers a convenient mechanical
support is shovv^n by the fact that glass wool or spider's
web immersed in a fluid medium offers a substitute
satisfactory for this purpose. (Harrison, 1914.)

This migration of cells from tissue explants will
take place in simple inorganic media such as Locke's
solution,^ although in such instances, because of the
absence of suitable organic foodstuffs, the cells do not
undergo appreciable proliferation and growth. Such
experiments, however, demonstrate that the cells of
recently killed animals are alive, and are capable of
remaining alive in Locke's solution for some time.
The duration of life under such conditions varies with
the type of tissue and the temperature. For example,
at 37° C. kidney epithelium and smooth muscle may
live for ten days or longer, while nerve, and skeletal
muscle may be kept alive but a few hours. (Lewis,
W. H., and Lewis, M. R., 1924.)

Although migration of cells such as is referred to
above increases the area of the explant, no growth as
measured by an increased mass of living tissue takes
place unless suitable foodstuffs are added to the saline
medium. When, however, the composition of the cul-

'^ Locke's solution contains 0.9% sodium chloride, 0.025% calcium
chloride, 0.42% potassium chloride, and 0.02% sodium bicarbonate.

ACTION OF CYTOST 119

ture medium is properly adjusted, a number of the
cells which have migrated to the periphery of the ex-
plant soon show mitotic division.

As stated above, the presence of blood plasma ap-
pears to be of value principally because of its ability to
form a solid matrix suitable for the migratory activity
of the cells. So far as is known, it will not sustain un-
limited growth, and if taken from an old animal may
exhibit definitely toxic effects (Carrel and Ebeling,
1923), although when taken from young animals
heterologous plasma appears to be almost as satisfac-
tory as homologous plasma for migratory purposes
(Burrows, 1910, 1911).

While a large number of substances have been ex-
amined with reference to their suitability as a medium
for cell growth in tissue cultures, but one substance
seems to be completely suitable for this purpose:
namely, an aqueous extract of young embryos. (Ebe-
ling, 1913; Carrel, 1913.) By the use of such an ex-
tract Ebeling (1922) has succeeded in keeping a cul-
ture of chicken fibroblasts more or less indefinitely —
for fifteen years at the time these experiments were
reported. In the course of this period the original
chick embryo cells had passed through some two thou-
sand generations, and no apparent diminution in
growth rate was observed. The duration of this ex-
periment was well beyond the average life span of
the hen and in consequence offers excellent evidence
of the potential immortality of somatic cells, to which
we have referred previously.

Just how embryo juice keeps cells in culture alive
is unknown. It is obvious that the growing cells de-
mand a source of nitrogen for the development of

120 THE ACTION OF THE LIVING CELL

their unique proteins ; yet when amino acids are added
to non-dialyzable portions of embryonic extract, the
mixture does not exhibit growth-stimulating proper-
ties analogous to that of the undialyzed extract
(Baker and Carrel, 1926) . On the other hand, Wright
(1925) has obtained from extracts of chick embryos
an active dialysate which markedly stimulates the
growth of cells in cultures.

It follows, therefore, that the growth-promoting
principle must be a substance of relatively low molec-
ular weight; otherwise one should not expect it to
pass through the collodion membranes used for dial-
ysis. Such being the case, this substance cannot be a
proteose, as suggested by Baker and Carrel (1928),
who found that peptic digests of albumin and fibrin,
in contradistinction to the pure proteins, augmented
the growth of tissue cultures. Unlike the embryo ex-
tracts, such digests will not allow the tissues to live
indefinitely; hence their growth-stimulating proper-
ties are more apparent than real.

Presumably such preparations offer the tissues ni-
trogen in a readily assimilable form, but do not pos-
sess some as yet unknown factor which is essential to
the continual growth of the tissue. This factor con-
tained in Wright's dialysates may be a stimulant which
permits the cells to utilize the foodstuffs available in
the media. Carrel (1928) , Fischer ( 1925) , and others
have observed that embryo extracts lose their growth-
stimulating activity if heated above 56° C. and in con-
sequence it has been suggested that the mysterious
growth factor is of enzymatic nature, since, as is well
known, many enzymes are rapidly inactivated at this
temperature.

ACTION OF CYTOST 121

An alternative explanation may, however, be of-
fered, for at this temperature some of the water-
soluble proteins in the extract are coagulated, and it is
conceivable that the active constituent of the embryo
juice may be absorbed upon the coagulum and thus
become unavailable to the growing tissue. This con-
cept is in harmony with Carrel's (1913) observation
that the growth-promoting stimulant is removed from
the embryo extracts when the latter are passed through
a Chamberland filter, which presumably absorbs the
active substance.

The conclusion to be drawn from these investiga-
tions is simply that cells in tissue culture require, aside
from adequate food, some substance which stimulates
in some fashion their metabolism and growth. We
shall now pass to some related experiments conducted
by the author (Turck, 1921).

The reader may recall the author's experiments, in
which mustard infusions were introduced into the
stomachs of dogs, and that such treatment, if pro-
longed, results, as stated previously, in severe inflam-
mation of the gastric mucosa and eventual destruction
of the mucous membrane and gland cells, although the
surface epithelium is not so seriously damaged as are
the deeper tissues. This fact, it may be remembered,
led to the conclusion that the latter were affected, not
by the mustard, but by substances released from the
surface epithelium under the destructive action of the
mustard. Further histological examination disclosed
distinct evidence of mitosis in the affected tissues;
hence we are led to conclude that cytost, aside from
its toxic action, may actually be a stimulant of cellular
metabolism and growth.

122 THE ACTION OF THE LIVING CELL

Again, when cytost extracts such as those frequently
referred to in our previous discussion are injected into
animals, the cells at the site of the injection are to a
certain extent stimulated to multiplication, as shown
by the presence of mitotic figures. This was observed
in the following way: A homologous cytost prepara-
tion was injected intramuscularly into one limb of a
cat, and a like quantity of sterile water was injected
similarly into the parallel leg. After the lapse of six
hours the tissues surrounding the sites of the two in-
jections were excised, fixed, and sectioned. Upon mi-
croscopic examination the tissues surrounding the cy-
tost injections showed distinct evidence of mitosis,
such as is seen in a healing wound, while the tissues
about the water injection showed no such evidence of
proliferation.

Similarly when the heterologous cytost prepared by
a rat or rabbit was injected in this fashion, localized
mitosis did not take place. Hence these simple ex-
periments offer further evidence of the specificity of
cytost. It is interesting to compare these results with
the growth-stimulating factor present in embryo ex-
tracts where such marked specificity is not shown, for
Fischer (1925) has found that duck fibroblasts grow
well in chicken embryo extract, while Carrel and
Ebeling (1922) report that the tissues of the rat may
likewise be grown in the same medium.

In view of these results, it became of interest to
examine the effects of cytost upon cells in tissue cul-
ture. Quite unexpectedly, cytost added to hanging-
drop cultures of various tissues was found to exhibit
two effects : in very low concentration it stimulates the
cells to growth in a manner similar to that of chick

ACTION OF CYTOST 123

embryo extract; in higher concentration, it exerts a
definitely toxic action, in some instances completely
inhibiting growth. (Turck, 1921.)

For the purposes of these experiments, the tissues
were first grown in the usual blood plasma chick em-
bryo extracts. In this fashion 34 cultures of chick cells
and 25 cultures from a human fetus (about 3 weeks
old) were prepared. As needed, transplants were made
from these stock cultures to hanging drops of the
media under examination. The cytost extract used in
the following experiments was prepared by auto-
claving 10 grams of autolyzed tissue with 10 cubic
centimeters of water.

Bits of tissue were cut from the outgrowth of the
stock cultures and placed momentarily in Ringer's so-
lution, which served to wash away the media, and then
transferred to a drop of homologous plasma on a cover
slip. In the first series of experiments a minute quan-
tity of cytost was added to the medium. This was ac-
complished by touching the plasma drop with the
tip of a platinum needle which had been immersed
in the cytost extract. After the cover slips had been
sealed onto hollow ground slides, the cultures were in-
cubated at 37.5° and transplants to similar media were
made every 48 hours. After several days the cultures
made in pure plasma showed very little growth, while
the same tissues in plasma plus homologous cytost grew
very nicely.

This was not an isolated result, but was duplicated
in 308 successive transplants of both human and chick
tissues. Hence we may safely conclude that cytost
is capable of stimulating cell growth in vitro. The
tissue fragments which did not grow in pure plasma

124 THE ACTION OF THE LIVING CELL

were not dead, however, for when transplanted to
fresh plasma containing a trace of cytost they com-
menced to show growth, and after a few transplants
in such media they underwent active proliferation and
apparently could be carried along in such a medium
indefinitely.

In one instance after the tissues had been grown in
plasma plus a trace of cytost for eighteen successive
transplants, bits of the tissue were implanted in pure
plasma and in the pure cytost extract. In this case the
tissues in the latter medium died within two days,
while those in the pure plasma remained alive and
grew slightly although not so well as cultures made
with plasma and a trace of cytost.

This experience led to an investigation of the ef-
fects of higher concentrations of cytost upon tissue
cultures. To this end transplants were made from the
stock cultures to a medium consisting of two drops of
homologous plasma mixed with one drop of homolo-
gous cytost. After incubation for 48 hours these trans-
plants were found to be dead. This result was obtained
a number of times and definitely shows the distinct
toxic action of cytost when present in sufficient quan-
tity. The lethal effect cannot be attributed to simple
dilution of the plasma, for Carrel and Burrows (1911)
have shown that a threefold dilution of plasma ac-
tually accelerates the growth of tissue cultures.

Now the interesting question arises : Will heterolo-
gous cytost behave in the same manner as the homolo-
gous extract? To answer this, the above experiments
were repeated, using chicken cytost with human tis-
sues and human cytost with chick tissues. Transplants
from the stock cultures, originally obtained from the

ACTION OF CYTOST 125

human fetus, were made to human plasma containing
a trace of chicken cytost, and after 48 hours such
preparations were compared with control cultures
containing human cytost. In each instance growth
w^as apparent, but the cultures made with chick cytost
did not show so much growth as the controls, from
which it may be concluded that heterologous cytost is
not so efifective a growth stimulant as a similar prep-
aration from homologous tissues.

This conclusion was substantiated by experiments
made with chick tissues in a medium of chicken
plasma plus traces of human cytost. These differences
between homologous and heterologous cytost are much
more clearly shown by the fact that when chick tis-
sues were transplanted to a medium consisting of two
drops of chicken plasma and one drop of human cy-
tost, the resulting cultures underwent growth, al-
though the latter was slight compared to growth ob-
tained in control cultures made simultaneously. This
result is in marked contrast with that obtained with
chicken cytost in equivalent concentration. In the
latter case, as stated above, the cultures were killed.

Thus we arrive at the interesting conclusion that
whereas heterologous cytost has not the growth-
stimulating potentialities of homologous cytost, it also
does not exert the toxic effects characteristic of the
latter in high concentrations.

The stimulating effects of tissue extracts upon tis-
sue cultures have been in part confirmed by Drew
(1922, 1923), who found autolyzed tissues to possess
a growth-stimulating principle analogous to that
found in embryo extracts. It will be noted, however,
that the growth-stimulating substances present in the

126 THE ACTION OF THE LIVING CELL

cytost preparation differ from those in the embryo
extracts in that they are not thermolabile, for, as stated
at the beginning of this section, the extracts used by
the author were autoclaved, a procedure which in-
activates embryo juice.

This difference was made more strikingly apparent
in the following way. It will be recalled that the au-
thor was able to obtain the typical systemic effects of
cytost when extracts of charred tissues were injected
into cats. Such being the case, it became of interest
to investigate the action of such extracts upon tissue
cultures. To this end 10 gram portions of tissue were
heated in a crucible at 300° C. until a black ash was
obtained. This ash was extracted with 1 cc. of water
and filtered to remove the insoluble material, the re-
sulting solution being used as "ash cytost" in the ex-
periments described below.

As in the previous experiments, transplants of chick
tissues were made from the stock cultures to a drop
of homologous plasma and a trace of the "ash cytost"
was added on the tip of a platinum needle. So far as
could be judged, the cultures thus prepared grew as
well as control cultures made with plasma and un-
ashed cytost. Again, when transplants were made in
a mixture of two parts of plasma with one part of the
"ash cytost," the cultures were found to be dead at
the end of 48 hours. When similar experiments were
conducted with chick tissues and ash cytost, prepared
from human tissues, decidedly different results were
obtained. The heterologous ash cytost did not appear
to augment growth appreciably, but it did not kill the
culture, as was the case with the extract of homolo-
gous ash.

ACTION OF CYTOST 127

These rather startling results tempt one to specu-
late as to the possible nature of the growth-stimulating
agent present in the ashed tissues. At first one might
be led to believe that simple inorganic salts were re-
sponsible for the observed effects. This seems some-
what attractive, in view of the experiments of Loeb
and Blanchard or amoebocyte tissue. They found that
various inorganic salts exerted a marked influence
upon the migration of cells from experimental amoeb-
ocyte tissue. The concentrations used by these authors,
however, was considerably greater than could pos-
sibly exist in the cytost-plasma media used in our ex-
periments. Further, in the present state of our knowl-
edge it is difficult to conceive of any mechanism
whereby inorganic salts would exert such a specific
action as that observed in the tissue culture experi-
ments. One is therefore forced to an alternative hy-
pothesis: that the ash cytost contains some markedly
heat-resistant organic compound which is not com-
pletely destroyed during the ashing procedure.

Admittedly this concept seems a wild stretch of the
imagination, but no other postulation seems to fit the
empirical results of the experiments. As far as the
writer is aware, no organic compounds of biological
importance can withstand the temperature attained
during ashing, although my friend Dr. K. C. Blan-
chard informs me that he has encountered some syn-
thetic organic compounds such as tetra substituted
ureas and aromatic silicon derivatives which do not
suffer appreciable decomposition at such tempera-
tures. Of course these substances are of no biochemical
interest, and are simply referred to here to illustrate
the possibility of the existence of organic compounds

128 THE ACTION OF THE LIVING CELL

which are markedly thermostable. Be that as it may,
we are at present in complete ignorance of the nature
of the growth-promoting substance in cytost, and ash
cytost, and must therefore content ourselves with the
results of the experiments.

As was stated towards the beginning of this chapter,
various tissues will remain alive for some time but
will not grow in Locke's solution. In consequence it
became of interest to ascertain whether or not the
addition of cytost to such a medium would permit
growth to take place. To this end bits of muscle from
the thigh and heart of a chick embryo were care-
fully washed in Ringer's solution and then implanted
in a hanging drop of Locke's solution with the ad-
dition of a minute quantity of chicken cytost on the
tip of a platinum needle.

After two days in the incubator these cultures
showed very little growth. They were, however,
transplanted again to a similar medium, and controls
were made in Locke's solution without the addition of
cytost. After 48 hours the latter transplants showed
practically no growth, while the cultures made in the
Locke-cytost medium exhibited a small but distinct
growth.

These cultures were then divided in two, one set
being carefully washed in Ringer's solution to remove
cytost, and then implanted in Locke's solution; while
the second half of the cultures was transferred to the
Locke-cytost medium. After two days' incubation,
the latter were found to be growing nicely, whereas
the former, washed free of cytost, showed but a very
meagre growth. Upon again transplanting these cul-
tures to their respective media, the cultures made with-

ACTION OF CYTOST 129

out cytost showed no signs of growth, whereas those
made with the addition of cytost showed a satisfac-
tory growth, although, because of the lack of suitable
supporting structure, it was not so good as in controls
made with blood plasma.

These experiments make it clear that even in such
an unfavorable environment as Locke's solution, cytost
may exert its growth-accelerating properties. Simi-
larly in such a medium cytost may, if its concentra-
tion be too high, exert a definite toxic action com-
parable to that found in the plasma cytost mixtures.
This was demonstrated experimentally by implant-
ing bits of actively growing cultures in a mixture of
one drop of Locke's solution and one drop of chick
cytost. Such cultures failed to grow, and unlike those
made in Locke's solution alone were found to be dead
after 48 hours' incubation.

The lethal action upon tissue cultures of cytost in
high concentration is perhaps not of any especial sig-
nificance other than that it shows that a product of
cellular disintegration may be toxic to contiguous
normal cells — a fact in harmony with the numerous
experimental results quoted in preceding chapters,
which have shown that cytost introduced into the in-
tact animal or liberated in vivo by a localized injury
results in damage of the body cells, particularly those
of endodermal origin. On the other hand the stimula-
tion of cell growth by cytost is of very considerable
interest because a number of statements found in the
literature confirm the idea that cells influence the
growth of one another by the liberation of some sol-
uble substance. Thus, although many workers have
attempted to culture single cells in vitro they have

130 THE ACTION OF THE LIVING CELL

quite uniformly met with failure. Witness for ex-
ample the remarks of Harrison (1928) : "It is a very
interesting and at present inexplicable fact that single
somatic cells isolated in culture media do not prolifer-
ate. Experiments to this end in my own laboratory
some years ago but not published did not succeed and
other workers have reported similar experience. As
Fischer put it,"A colony of fibroblasts cannot arise
from a single cell even when the nutrient conditions
are most favorable. Likewise small groups of cells
if isolated do not undergo division and their growth
remains at a standstill. On the other hand certain
tumor cells (Rous chicken sarcoma) are capable of
multiplying and producing colonies when isolated
singly."

It seems, therefore, that in order that a somatic cell
may undergo division it must obtain suitable stimu-
lants from its neighbors. In this connection, the most
careful observations seem to be those of Haberlandt
(1919-1922) , who, working with plant tissues, found
a direct correlation between the size of the transplant
or the number of cells within it, and the frequency of
mitoses. In consequence this investigator concluded
that some substance arising from the injured cells,
which he termed "division hormones," incited the
intact cells to division. This is in direct confirmation
of the writer's hypothesis — Haberlandt's division hor-
mones being synonymous with cytost.

Similarly, other names have been applied to the
growth-stimulating substances present in tissue ex-
tracts. They have been termed "trephones" by Carrel
(1924), "desmones" by Fischer (1925), and "archu-
sia" by Burrows and Johnson (1925). Since, how-

ACTION OF CYTOST 131

ever, the exact nature of these substances remains un-
known, these various names are of no descriptive value.
Therefore the writer prefers to retain the name "cy-
tost" simply because it was the first one coined to de-
scribe this mysterious substance.

In a large mass of cells such as the transplants used
in tissue culture it is inevitable that some suffer injury
from one cause or another. In consequence, by the
mechanism previously discussed cytost will be liber-
ated from such cells. It seems likely, therefore, that
the growth observed in cultures made in pure Locke's
solution may be due to the stimulating action of the
cytost so derived.

In agreement with this concept is the fact pre-
viously mentioned on page 130, that transplants of
single cells, even in apparently suitable media, do not
undergo division and growth. Further, an enormous
number of papers have been published which present
evidence that the crowding of tissues in cultures ex-
erts a decidedly beneficial effect upon their out-
growth. (See Fischer, 1923.) This again may be at-
tributed to the action of cytost derived from some
cells upon other members of the colony.

In view of the experimental results obtained with
vertebrate tissues, it became of interest to investigate
the effects of cytost upon unicellular organisms such
as protozoans and bacteria. A survey of the literature
concerning cultures of such organisms discloses a num-
ber of facts which may be interpreted in a manner
similar to that used to explain the empirical observa-
tions on tissue cultures. Thus, Robertson (1921),
working with Enchelys farcimen, found that the di-
vision rate of this organism in a hanging drop is sig-

132 THE ACTION OF THE LIVING CELL

nificantly increased when two animals are present in
the same drop. Similarly, Calkins (1926), working
with Uroleptus, found the division rate to be inversely
proportional to the number of protozoans introduced
into a given volume of culture medium.

From these experiments it seems likely that these
microscopic animals release a substance which ac-
celerates the growth of the same species. With Pare-
mecia, variable results have been obtained by different
workers (reviewed by Allee, 1931). In part, this
seems to be due to differences in experimental tech-
nique, although the recent work of Petersen (1929)
has thrown considerable light upon the factors which
influence the growth of this ciliate.

By alterations in experimental procedure Petersen
has been able at will to obtain results which confirm
or negate Robertson's conclusions, for she has shown
that the volume of the medium employed is a decisive
factor in the final results obtained. For example, she
found that when the volume of media was slightly less
than 1 cc, the division rate of individual Paramecia
was markedly accelerated by the presence of other
animals of the same species, whereas in larger volumes
of fluid, the organisms were without apparent influ-
ence upon one another. This fact, of course, may be
due simply to a dilution effect, for if a given concen-
tration of the growth stimulant derived from a few
animals is necessary in order to accelerate the division
rate of others present, it is evident that simple dilu-
tion of the pericellular fluid will lower this concen-
tration below the necessary minimum.

It has long been recognized that in mass cultures of
protozoans, some metabolites are excreted into the

ACTION OF CYTOST 133

medium, rendering the latter unfavorable for the con-
tinued growth of the organism. In the case of Para-
mecium, this has been carefully investigated by Wood-
ruff (1911, 1914), who has also demonstrated (1913)
that the toxins which are elaborated in mass cultures
are more or less species specific, for he finds that
media from such cultures, which is incapable of sup-
porting further growth of Paramecia, is satisfactory
iov tht c\i\\.iY2i\.ioii oi Stylonychia. Robertson (1924),
in discussing his experiments with Enchelys, states
that "the ultimate cessation of reproduction in old cul-
tures is attributable to the accumulation of a product
of growth, and possibly of the same product that was
originally responsible for the acceleration." This con-
clusion of Robertson's is similar to that advanced by
the writer (Turck, 1921), to explain the action of
cytost on cells of all types.

In order to obtain cytost from Paramecium, the or-
ganisms were grown in large numbers in small dishes
for 10 days. The growths were then centrifuged,
washed, and dried on a steam bath. The resulting
mass was autoclaved with 10 times its weight of water
and filtered in precisely the same fashion as that
utilized for the extraction of cytost from other tis-
sues. In order to ascertain the effects of such cytost
upon the growth of Paramecia, six or eight of the
latter were transferred from the stock cultures to each
of several small tubes containing 1 cc. of water. Some
of these tubes were then inoculated with paremecium
cytost by adding as much of the dried organisms as
could be held on the tip of a platinum needle.

After twenty-four hours at room temperature, the
animals in the tubes containing the cytost were found

134 THE ACTION OF THE LIVING CELL

to have undergone an increase in number greater than
that found in the control tubes to which no cytost had
been added.

When this result had been confirmed several times,
the experiment was repeated but the ash obtained by
ignition of the dried animals was substituted for the
cytost. In water containing traces of such ash the
Paremecia were found to multiply more rapidly than
in the control tubes, but the increment in number of
the control cultures was nowhere nearly so marked as
in the previous experiments with the dried animals.

Since, as is well known, traces of various mineral
salts may markedly afifect the behavior of protozoan
cultures, it is impossible to state definitely that the ash
cytost possesses any specific ability to accelerate the
division rate of such organisms. On the other hand,
the unashed material, when present in traces, exerts
a distinct stimulation of growth as measured by in-
creased numbers of organisms. Further, this effect
is more or less specific, for when cytost prepared from
chicken, rat, or human tissue was substituted for the
Paramecium cytost, no such increase in division rate
was noted.

As was found to be the case with tissue cultures, the
growth-stimulating principle in the dried ciliates is
water-soluble. This was shown in a series of experi-
ments in which the 10% extract referred to above was
used. Four or five Paramecla were transferred to
tubes containing 1 cc. of tap water. A loopful of the
cytost solution was added to several of these tubes and
the remainder were kept as controls.

After 48 hours at room temperature, the number of
organisms in each of the tubes con\.2i\nmg Paramecium

ACTION OF CYTOST 135

cytost was found to be significantly greater than in the
controls. Incidental to these observations, it was noted
that the protozoans in the water containing cytost were
considerably more active than those in plain tap water.

In order to examine this further, hanging drops
containing a single Paramecium were touched on the
periphery with a platinum needle which had pre-
viously been immersed in cytost solution. Under such
conditions the single animals became very active, dart-
ing back and forth across the field. After approxi-
mately ten minutes, the magnitude of these movements
diminished, shortly assuming a circular path whose
radius became progressively smaller until finally the
animals simply rotated on their own axis. Controls in
drops of tap water did not exhibit such behavior.

It follows, therefore, that some component of the
cytost influences the movement as well as the repro-
duction of Paramecium.

When a loopful of the aqueous cytost extract was
added to a few drops of water containing several Para-
mecia, 2l similar behavior was noted. Following the
onset of the circular movements, the axial rotation en-
sued. This became progressively slower, and, after
the lapse of an hour and a half, the organisms assumed
a spherical form resembling a cyst. It seems likely
that this behavior, perhaps the beginning of of en-
cystment, represents a response to the unfavorable en-
vironment created by too high a concentration of
cytost in the aqueous medium. Similar results were
obtained when the ash of Paramecia was substituted
for the cytost extract, but not when heterologous cy-
tost was employed in similar concentration.

From these experiments, each of which was re-

136 THE ACTION OF THE LIVING CELL

peatedly confirmed, we may conclude that homologous
cytost afifects protozoans in a manner similar to that
found in tissue cultures: i.e., in very low concentra-
tion, it appears to exert a definite stimulation of ac-
tivity and growth, whereas in higher concentrations
it becomes distinctly toxic. The latter conclusion is
further substantiated by the observation that the ad-
dition of large quantities of homologous cytost to hay
infusion (a loopful per cubic centimeter) renders the
medium unsuitable for the cultivation of Paramecium.

When we turn to the growth of bacteria, we find that
an enormous literature has accumulated concerning
the requirements for and the nature of bacterial
growth. Owing to the non-uniform nature of the
media employed in experiments concerned with such
studies, and to numerous conflicting statements as to
the results obtained, it is virtually impossible to draw
any definite conclusions concerning the general na-
ture of bacterial growth.

Two undisputed facts, however, are worthy of com-
ment in connection with our discussion. It is now quite
generally accepted that, following the inoculation of
a bacterial culture, a definite time interval (the lag
phase) may elapse before active proliferation begins.

Various hypotheses have been advanced to explain
this phenomenon : some believe that during the lag pe-
riod some substance essential for growth is liberated
into the medium from the cells of the inoculum ; others
have imagined that the lag phase represents a period
during which the organisms recover from injury
caused by the accumulation of toxic metabolites in
the parent culture. Penfold (1914) has suggested that
a time period is necessary for the synthesis of certain

ACTION OF CYTOST 137

substances which must attain a definite concentration
in the cells before active growth ensues. These and
other explanations have been reviewed by Buchanan
and Fulmer (1928), and we shall not therefore elab-
orate upon these ideas.

If, as suggested above, some accelerating substance
is necessary for the rapid growth of bacteria, they can,
if supplied with adequate food and kept in an optimal
physical environment, synthesize this substance for
themselves. This becomes self-evident when one re-
calls that cultures of bacteria may be obtained from a
single bacterium isolated by the micro-pipette method
first introduced by Barber (1904). In this respect,
then, the bacteria differ from the somatic tissues of
higher animals, which, as found in tissue culture ex-
periments, demand a source of preformed cytost,
trephone, archusia, or whatever one prefers to call
the growth-accelerating substance.

This is not surprising, considering the relatively
undifferentiated nature of bacteria and their lowly
position in the scale of life. Nevertheless, it is of in-
terest to ascertain if cytost prepared from bacteria
is capable of accelerating the growth of such or-
ganisms.

For this purpose the author selected Bacillus coli
communis, since, in the course of another investiga-
tion in 1912, he had accumulated some 80 grams of
the dried organisms, which had been grown in mass
culture on solid synthetic media. As the colonies had
been removed from the surface of the latter by scrap-
ing, the mass of organisms was undoubtedly contami-
nated with some of the media. This unfortunately
could not be avoided with the facilities at the author's

138 THE ACTION OF THE LIVING CELL

disposal. In consequence, in the experiments to be
cited presently, the possibility exists that the observed
effects were in part due to an enrichment of the media
by this means. This objection, however, seems of
doubtful significance, in vievv^ of the fact that the
amount of additional nutriment introduced into the
medium along with the cytost must have been of a
negligible order.

Cytost was prepared from the dried B. colt men-
tioned above by autoclaving 1 gram of the dried or-
ganisms with 10 cc. of water. Plates were poured with
5 cc. of nutrient agar to which had been added 0.1 cc.
of the aqueous extract of dried B. colt. These plates
and controls were then inoculated with a standard
loopful of a 24 hour broth culture of B. coli. After
24 hours, the plates made with the bacterial cytost
showed a much better growth than the controls. Simi-
lar results were obtained using 0.15 cc. of the B. coli
cytost per 5 cc. of medium. It seems therefore that
although cytost is not essential to the growth of these
organisms, its presence in the medium accelerates the
growth of the latter.

Similar concentrations of B. coli cytost added to
cultures of staphylococcus and streptococcus in nutri-
ent media failed to cause any observable increase in
growth; hence the specificity of cytost is apparent in
even such lowly forms as the bacteria.

Chapter VII
NATURAL RESISTANCE AND FATIGUE

All forms of life from the lowest bacteria to man
are so constituted that they will live only within cer-
tain definite limits of environmental changes. While
it is true in many instances that the interval between
such limits may be rather wide, it is well known that
organisms live normally and longest only under opti-
mal environmental conditions. This is a necessary con-
sequence of the operation of the mechanism of natu-
ral selection, which has permitted the survival of only
those individuals which are more or less adapted to
the particular environment wherein the development
of a given species has taken place. This has been due
to the fact that natural selection operates according
to the laws of chance, rather than purposefully, to
produce and maintain a race of organisms best fitted
to survive and reproduce its kind.

The various races of animals and plants resulting
from such a process are therefore not ideally adapted
to their environment, but only better adapted to it
than their ancestors. By a similar process the consti-
tuent body cells of the metazoans and higher plants
have become adapted to a definite internal environ-
ment which is comparatively constant for a given

139

140 THE ACTION OF THE LIVING CELL

species when the external environment of the organ-
ism is not permitted to undergo marked changes. Con-
versely, external changes v^ill result in internal varia-
tions which tend to compensate for the former and
thus to a degree prevent serious impairment of the
body cells. This type of compensation is well il-
lustrated in the warm blooded animals, which have
developed during the course of evolution a tempera-
ture-regulating mechanism which permits them to
maintain a body temperature very nearly constant
throughout a wide range of external temperature vari-
ations.

Similarly all living organisms have developed to a
certain degree various mechanisms which enable them
to sufifer environmental changes which do not vary
too far from the norm to which they are best adapted,
although in some few instances this inherent adaptive
ability is exceedingly elastic. In general, individual
cells such as protozoans and the cells of higher ani-
mals in tissue culture demand for their normal ac-
tivities a rigorously constant environment, while
higher animals, due to the compensating and inter-
related activities of their component tissue and organs,
are much better able to withstand marked environ-
mental changes such as those of temperature, radia-
tion, and oxygen pressure.

In many instances such ability is sufficiently pro-
nounced to permit an actual acclimatization to a sud-
den shift in environment. To this end, living beings
are provided with certain defense mechanisms which
as a rule do not become apparent until called upon
to defend the organism against an environmental
change or a sudden new environmental factor which

NATURAL RESISTANCE AND FATIGUE 141

the individual has not been previously forced to con-
tend with. As a rule such defense mechanisms do not
appear instantly when needed, but are developed as
necessary from some latent source when the organ-
ism is appropriately stimulated by an environmental
factor. As a simple instance of this in man, let us
briefly consider the phenomena incident to sunburn.
We are all familiar with the fact that after a winter
indoors the exposure of our skin to the bright summer
sun for a sufficiently long period usually results in a
painful sunburn. If, however, we submit to a series
of such exposures, of lesser duration, the skin becomes
"tanned," after which prolonged exposure to the sun
does not result in a burn. This is due to the fact that
while our skin contains more or less pigment which
hinders the absorption of solar radiation, the concen-
tration of this pigment ordinarily is not sufficiently
great to prevent serious consequences from a single
prolonged exposure. On the other hand, the series of
short exposures necessary to elicit a good tan stimu-
lates the formation of an increased amount of the
protective pigment within the skin. In this way, then,
the human body can protect itself against highly in-
tense solar radiation. Due to the slight amount of pig-
ment normally present within the skin, all humans
possess to some degree a natural resistance to sunlight,
but, as the phenomena of tanning shows, this natural
resistance may be intensified by the suitable stimula-
tion of pigment formation — i.e., of a latent natural
resistance.

The development of callus of the skin as a pro-
tection against a mechanical injury may be analyzed
in a similar fashion. Our skin affords us a certain

142 THE ACTION OF THE LIVING CELL

degree of protection from the elements. Although this
is by no means a perfect protection, nature has en-
dowed the dermal cells with the ability to fortify
themselves, and consequently the more delicate under-
lying tissues, when properly stimulated. To this end
continual exposure of the skin to mechanical friction
results in the formation of callus. The reader will
readily recall other obvious examples of such de-
vices, by which an animal or plant is enabled to cope
with limited variations in an environment to which
it is not perfectly adapted.

It will be noted that whereas the mechanism of the
increased development of resistance to sunburn is
readily understood on the basis of the increased for-
mation of pigment, no such simple explanation is
forthcoming to account for the formation of callus.
In the majority of cases, the mechanism underlying an
observed increase in natural resistance is not in the
least obvious. Similarly, while individual variations
in natural resistance are apparent at all times, we have
little exact knowledge concerning the underlying
causes of such variation. To illustrate : When a series
of animals receive identical wounds inflicted by the
same means and in the same manner, the various in-
dividuals respond dififerently. In some, the wounds
heal uneventfully; in others, they may heal slowly;
and in some individuals shock and other untoward
disturbances are experienced. To what may such dif-
ferences in reaction be attributed? We may answer,
to individual differences in natural resistance. Quite
obviously, we cannot logically postulate a natural re-
sistance to the particular type of wound inflicted, or
the particular device employed to produce the injury.

NATURAL RESISTANCE AND FATIGUE 143

In consequence, the observed variations in the re-
sponse to the wounds must be due to variations in the
natural resistance of the animals to wounds and the
sequelae thereof.

In this connection it is interesting to note that, as
the author and others have observed, battered street
cats are usually better subjects than pampered house
cats for laboratory experiments which involve surgical
procedures. May we not conclude from this simple
observation that because of his fighting existence and
frequent bloody encounters, the alley cat's natural re-
sistance to traumatic injury has been significantly
raised above that of his brother, the protected house
cat?

As has been shown in the previous chapters, the au-
thor's experiments upon shock produced in various
ways have all led to the conclusion that cytost, a prod-
uct of cellular disintegration, is the causative factor
primarily responsible for the train of physiological
changes which follow severe injuries. It seems, there-
fore, that individual differences in susceptibility to
shock — i.e., natural resistance to shock, must be in-
herently due to differences in susceptibility to cytost
— a concept in harmony with our observations relative
to the differences in response which normal animals
show upon injection of extracts of autolyzed tissue.

As has been stated above, an animal's natural re-
sistance to a given environmental factor may be en-
hanced by appropriate stimulation by that factor at
an intensity lower than that required to inflict severe
injury. In the case of sunlight, the development of
colored pigments in the skin is a tangible explanation
of the increased resistance of the animal. On the other

144 THE ACTION OF THE LIVING CELL

hand, if the pigments so developed were colorless,
but absorbed light of the near ultra-violet portion of
the spectrum, v^e might be equally well protected from
the sun; yet in all probability we should be in a quan-
dary as to the mechanism by which increased resistance
to sunlight is accomplished. Similarly, one is mysti-
fied by the vast majority of changes to which many
animals, and man, are resistant or may become ac-
climatized. The observed resistance or acclimatiza-
tion is usually attributed to natural resistance or adap-
tation — admittedly and necessarily vague terms.

However, although we may not completely com-
prehend the underlying mechanism by which in-
creased natural resistance takes place, it seems neces-
sary to postulate that such a physiological mechanism
is absolutely essential to life.

While the tissue culture experiments previously re-
ferred to have shown life to be potentially immortal
if provided with an adequately controlled environ-
ment, the existence of animals and plants in their natu-
ral environment is sooner or later terminated by death,
natural or otherwise. This is necessarily so, because
an organism's ability to increase its natural resistance
towards all sorts of changes is not unlimited — if it
were, we might reasonably expect all animals to be
immortal.

The study of disease has disclosed a number of im-
portant facts concerning natural resistance, or im-
munity, as this is termed by medical men.

Following Pasteur's classic experiments on immu-
nization to the virus of rabies, and Behring's discovery
of diphtheria antitoxin, it became apparent that some
organisms, notably the bacteria, when introduced into

NATURAL RESISTANCE AND FATIGUE 145

the circulation of higher animals, were capable of
eliciting the production of specific antibodies. The
latter by one means or another afford the host some
degree of protection against the invading parasites.
In general, the antibodies which are formed in re-
sponse to an infection are specific towards the species
of microorganism which caused their formation.
Thus, it is common knowledge that an attack of mea-
sles during childhood usually renders an individual
immune to further infection by the causative organ-
ism; yet no immunity towards other infectious dis-
eases results. In this instance the antibodies formed
appear to last throughout the remainder of the host's
life. If one wishes, this may be regarded as an adap-
tation of the host to a new environment — i.e., to one
wherein the chances of infection with measles is ever
present.

In many instances, antibodies produced during the
course of disease are either negligible in amount or
rapidly disappear from the body of the patient follow-
ing his recovery. Witness, for example, the all too
frequent recurrence of colds, grippe, and so on, in
some individuals. Despite the fact that in such infec-
tions specific antibodies are not demonstrable, we
nevertheless find many individuals who seldom are so
afflicted even although they have been exposed to the
same predisposing causes as their less fortunate broth-
ers. Classical immunology ofifers no explanation other
than natural resistance or natural immunity for this
commonly observed fact. While such terms may suf-
fice to indicate the observed differences, they denote
no definite causative factor.

In order to effect a satisfactory explanation of such

146 THE ACTION OF THE LIVING CELL

observed instances of natural resistance, we must seek
further than the antibodies of classical immunology.
It is hoped that the reader will not misunderstand the
above statement. We do not wish to deride the results
of the brilliant investigators who have contributed so
much to the conquest of disease and the advancement
of our knowledge concerning the manner in which
higher animals protect themselves against their inva-
sion by more lowly forms of life. It is our desire rather
to stress the fact that while the formation of specific
antibodies towards invading organisms affords an ani-
mal some measure of protection against infection,
other physiological factors are perhaps of equal im-
portance in this respect.

As is well known, many animals are resistant to-
wards diseases which ordinarily affect man; and con-
versely, man appears to be immune to some diseases of
animals, although careful study has failed to disclose
the existence of specific antibodies in the immune in-
dividuals. In consequence, the conclusion is forced
that factors other than specific antibodies must be in-
voked to explain such differences in susceptibility.
Judging from the remarks of such prominent inves-
tigators as Manwaring (1929) and Zinsser (1928),
the immunologists themselves have recently staged an
aboutface in the interpretation of such confusing
facts.

According to the tenets of classical immunology,
only specific antibodies should be capable of combat-
ing the invasion of higher animals by bacteria or
other toxins. In recent years, however, there has been
accumulated a considerable volume of evidence which
shows that some infections may be overcome, at least

NATURAL RESISTANCE AND FATIGUE 147

in part, by the injection of non-specific (i.e., heterolo-
gous) proteins which, so far as is known, bear no re-
lation whatsoever to the bacterial products (see re-
view by Petersen, 1928).

For example, numerous clinical investigators have
reported that they have obtained encouraging results
in the treatment of arthritis by the intravenous injec-
tion of divers substances, such as dead typhoid bacilli,
or sterile milk. Such substances obviously bear no re-
lationship to the causative factor in arthritis; yet in
some mysterious way they are able to stimulate the
animal's resistance towards the primary factor respon-
sible for the pathological condition. The very nature
of the treatment indicates that this increase in resist-
ance is brought about in a non-specific fashion: that
is, in some manner other than by the elaboration of
antibodies specific for the invading organism causative
of arthritis.

With these facts before us, it is of interest to exam-
ine their possible relationship to some observations of
the writer concerning the nature of natural resistance.

Many years ago, in connection with the experiments
previously mentioned wherein emulsions of mustard
were introduced into the stomachs of dogs, at fre-
quent intervals for a period of some months, the ani-
mals gradually developed a tolerance for the markedly
irritating substance. This was evidenced by the fact
that after several weeks of such treatment the animals
failed to expel the stomach contents following the in-
troduction of the mustard. While this observed fact
may appear quite remarkable, it is of interest to note
that it is analogous to the distinct tolerance which cer-
tain Oriental and tropical peoples have developed to-

148 THE ACTION OF THE LIVING CELL

wards the highly spiced condiments which they con-
sume daily.

The development of this tolerance towards the in-
troduction of mustard into the stomach naturally
raised the question as to whether the animal's increased
resistance towards this substance was localized in the
gastric mucosa or was a general resistance to be found
in all the body tissues. This problem was solved in part
by the simple expedient of applying ordinary mustard
plasters to the skin of the animal. By this procedure
it was observed that coincident with the tolerance to
mustard developed by the stomach tissues, the exter-
nal application of mustard in the form of a plaster
did not result in the usual vesicant efifect. From this
we may conclude that the tolerance developed towards
the introduction of mustard into the stomach is due to
a generalized increase in resistance towards this sub-
stance (Turck, 1905). Although the exact nature of
this resistance has not been elucidated, it seems clear
that no specific antibodies were produced, since all
the evidence accumulated by immunologists shows
that only proteins are capable of acting as antigens for
the production of specific antibodies (Wells, 1925) ;
while the irritant principle present in mustard is
known to be a volatile oil of non-protein nature. Such
being the case, the observed results must be interpreted
on other grounds.

In an earlier chapter, evidence has been presented
which indicates that the general toxic effects produced
by the introduction of mustard into the stomach may
be interpreted as due to the liberation of cytost from
the mucosa cells injured by direct contact with the ir-
ritant. The tolerance to mustard developed by the

NATURAL RESISTANCE AND FATIGUE 149

stomach might be accounted for by postulating the
development of an increased resistance of the mucosal
cells to the action of mustard. While such a concept
would suffice to explain the development of an in-
creased tolerance towards the introduction of mustard
into the stomach, it would in no manner account for
the development of a generalized resistance towards
mustard, as evidenced by the experiments with the
plasters. Consequently, we must seek further for an
adequate explanation of the facts.

If it be accepted, as appears reasonable, that the
mustard liberates cytost from the cells of the gastric
mucosa, we may develop an hypothesis along the fol-
lowing lines. Let us assume first that the vesicant ac-
tion of mustard when applied locally to a given tissue
takes place in the following manner. At the site of
contact the mustard injures a number of tissue cells
which in turn liberate cytost. The latter, upon dif-
fusing to the underlying cells, subjects them to its
action, and so on. Eventually the capillaries are
reached, and, as has been shown previously, their per-
meability is increased by the action of cytost. Because
of this increased permeability, there is an exosmosis
of fluid into the surrounding tissues, and bleb forma-
tion takes place. Now it is to be noted that in this
chain of events the factor leading to vesication is the
peripheral liberation of cytost and its subsequent ac-
tion upon the underlying tissues and capillaries.

Since divers chemical substances can behave as vesi-
cants, it would appear that in general their actions are
similar to that postulated above for mustard. Indeed,
that vesication of the skin is due to a product liberated
by the tissue cells themselves is shown by the beautiful

ISO THE ACTION OF THE LIVING CELL

experiments of Sir Thomas Lewis (1924), who has
demonstrated that in young subjects wheals may be
formed on the skin by firmly stroking it with a blunt
instrument. The wheal formation is preceded by a
red tache which indicates a dilation of the capillaries
of the skin. These observations of Lewis of¥er irre-
futable evidence that some product liberated from the
injured tissues is responsible for the observed increase
in capillary permeability and the subsequent localized
edema.

Lewis and Grant (1924) believe the substance
which is liberated from the tissues to be of a histamine-
like nature because they found that a similar wheal
formation follows the introduction of histamine into
the skin. It may be remembered that some investi-
gators have reached the conclusion that traumatic
shock is the result of the liberation of histamine by
the injured tissues; hence, on this basis, there exists
a distinct relationship between shock and vesication.
In this respect, then, these results are in agreement
with the author's cytost theor}^, the only essential dif-
ference being the names applied to the tissue break-
down product responsible for the observed physio-
logical changes. For the reasons given previously, the
writer prefers to adhere to the name cytost until the
exact chemical nature of the substance is known.

To return to the problem of the immunity to mus-
tard found in the animals studied by the writer, it
seems obvious that the resistance developed is not
really an increased tolerance for this substance, but
is rather the expression of an increase in resistance of
the body cells to the substances liberated from the tis-
sues by the action of mustard. That is, during the

NATURAL RESISTANCE AND FATIGUE 151

course of the experiments the animal's resistance to
cytost must have been raised.

These experiments, made many years ago, demon-
strate that it is possible to increase the resistance of an
animal to the breakdown products of its own tissues
and open the way to a fascinating field of investiga-
tion, — the production of such an increased resistance
by various experimental methods. This problem is of
importance because in the battle between animals and
their environment, the former are subjected to many
conditions which tend to cause tissue damage and the
consequent liberation of cytost. If the animal is not
more or less resistant to the action of the latter, he
will be subjected in a greater or lesser degree to the
various ailments which, as has been shown in previous
chapters, may be induced by the parenteral introduc-
tion of cytost. If such be the case, the animal will be
less suited than his more resistant brothers to cope with
the environment which nature presents to him. It fol-
lows, therefore, that an animal's resistance to cytost
is a factor of considerable importance in determining
his so-called natural resistance. In consequence, if one
is enabled to raise an animal's resistance to cytost, it
follows logically that we shall in some degree be able
to raise his natural resistance. In other words, we may
be able to increase the adaptation of a given animal to
his environment and thus in some measure cause his
earthly struggle for existence to be easier and, per-
haps, more pleasant.

Resistance to fatigue may be regarded as one im-
portant aspect of natural resistance, for obviously the
more easily an animal is fatigued by his daily routine,
the less is his ability to cope with his environment.

152 THE ACTION OF THE LIVING CELL

Similarly, when a given organ is fatigued, it is ren-
dered incapable of performing its allotted physiologi-
cal functions in a manner prerequisite for maximal
efficiency. Many years ago the writer reached the con-
clusion that numerous gastric disturbances might be
attributed to fatigue of the digestive organs. In par-
ticular, let us consider the digestive disturbances and
accompanying pain due to failure of the stomach to
undergo the usual cycle of contractions and relaxa-
tions which are necessary for the complete mixing of
ingested foods with the digestive enzymes of the gas-
tric juice secreted by this organ.

Protein foodstuffs are digested in the stomach by
the enzyme pepsin, and such digestion is only possible
when the enzyme is brought into intimate contact with
its substrate. In the normal animal this is accom-
plished by the churning of the stomach contents, which
is brought about by the more or less rhythmic move-
ment of the stomach walls. The lack of such move-
ment also fails to cause ready movement of the stom-
ach contents through the pyloric valve into the small
intestine. Such being the case, the afflicted individual
suffers from both faulty nutrition and physical dis-
comfort varying from an uncomfortable sense of full-
ness to distinct pain. If long continued, these distress-
ing sensations prevent the individual from living a
normal physical life, and conceivably the continued
discomfort may lead to a mental state which prevents
the individual from having normal relations with his
neighbors — an important factor in one's environment.

Every clinician has encountered patients whose
principal source of complaint may be traced to gastric
disorders similar to that outlined above, a condition

NATURAL RESISTANCE AND FATIGUE 153

not difficult of diagnosis, for the salient characteristic
is the atonicity of the stomach. Believing such ato-
nicity to be due to the fatigue of the gastric muscula-
ture, the author injected intravenously into dogs
extracts of material taken from the stomachs of fast-
ing patients suffering from atony of the stomach. The
animals receiving such injections exhibited a slight
distress, and the results suggested the presence of a
fatigue toxin in such stomach contents, although it
must be admitted experiments of this type are not very
convincing. Because of this, the stomach muscula-
ture of normal dogs v^as fatigued by means of a rapidly
revolving cable introduced into the stomach by means
of the gyromele, or by recurrent tension brought about
by the alternate inflation and exhaustion of a rubber
bag inserted in the stomach (Turck, 1903). Both
these methods result in a stretching and relaxation
of the muscles, treatment v^hich von Uexkiill found
to be the most efifective method of stimulating muscle
by mechanical means. In the intact animal such treat-
ment does not result in a rapid fatigue of the muscle,
because the firm external abdominal muscles must be
fatigued before the gastric muscles can be stretched
sufficiently to cause a distinct state of fatigue. If, how-
ever, as in the experiments cited belov^, the animal's
abdomen is opened, the continued intermittent ten-
sion which is caused by either of the two methods men-
tioned above causes the gastric muscles to show signs
of fatigue, such as marked dilation, and failure to re-
spond to stimuli which ordinarily cause rhythmical
contractions of the organ.

The muscles of such stomachs were removed by dis-
section, minced, and extracted with normal saline so-

154 THE ACTION OF THE LIVING CELL

lution. Upon injection of such an extract into the gas-
tric musculature of animals whose stomachs had not
been fatigued, they rapidly developed signs of fa-
tigue dilations, beginning within about half an hour
following the injection, and progressing until the
stomach had undergone distention to a volume some
three or four times its normal size. This was accom-
panied by a loss of tone, and complete loss of irrita-
bility. These efifects were not due to the volume of
saline injected but must have been caused by some
product extracted from the fatigue muscle, because,
upon injecting a like quantity of saline into the stom-
ach musculature of other animals, similar results were
not obtained.

Further evidence for the presence of a "fatigue
toxin" in the tissue extracts was found by injecting
such extracts into the peritoneal cavity of normal dogs.
Animals so treated developed an intense thirst some
eighteen hours following the injection, but vomited
water or milk as soon as taken into the stomach. Upon
opening the abdomen two days after the injection of
the toxin-containing extract, the stomach was found
to be dilated and flabby. In another similar experi-
ment, following the injection of the muscle extract
the animal was fed bread, milk and bismuth, and ex-
amined by the X-ray, as described by Cannon (1902) .
In such experiments it was found that inhibition of
gastric peristalsis began shortly after the injection;
and three or four hours later it was found that the
movements would cease entirely for a time, and then
continue sluggishly. Such results were not obtained
when an extract of unfatigued muscle was substituted
for the extract of the fatigued muscle.

NATURAL RESISTANCE AND FATIGUE 155

These experiments, the details of which were pub-
lished almost thirty years ago (Turck, 1903), demon-
strate that fatigue of the gastric muscles gives rise to
the formation of a toxic entity capable of causing
changes similar to those associated with fatigue in the
muscles of other animals. Since such a fatigue syn-
drome may be produced by the parenteral introduc-
tion of the toxin into other animals, it follows that if
this same toxic substance, or another substance of simi-
lar nature, is produced elsewhere in the body in suffi-
cient quantity, then a similar reaction of the stomach
is to be expected.

The reader will recall, perhaps, that in our dis-
cussion of shock produced by anesthesia it was shown
that under prolonged anesthesia the stomach was
found to become distended, flabby, and insensitive to
stimuli. This was shown to be due to the action of
cytost liberated by the action of the anesthetic upon
various cells of the body. At the time the experi-
ments upon the stomach, cited above, were made, the
author's cytost theory had not been developed to the
extent presented in our previous discussion. In view
of the latter, however, it now appears that the "fatigue
toxin" extracted from the stomach muscles is to be
regarded as synonymous with the cytost liberated
from tissues by the various means detailed in the pre-
ceding pages. If it be remembered that, as shown pre-
viously, the visceral organs of endodermal origin are
peculiarly sensitive to the action of cytost, one can
readily account for the behavior of the stomach under
the conditions of these early experiments.

If the fatigue toxin formed in the musculature of
the stomach by the experimental means cited above

156 THE ACTION OF THE LIVING CELL

is identical with cytost, we should expect to find histo-
logical changes similar to those observed in other
tissues when induced to discharge cytost by any means.
Such is actually the case, for microscopic examina-
tion of bits of muscle taken from the cardiac and py-
loric ends of the fatigued stomachs showed some stasis,
congestion, and distention of the capillaries. These
changes appeared much more marked in the pyloric
end of the stomach than in the cardiac end, a diflfer-
ence of some interest, inasmuch, as demonstrated by
numerous observers, only the pyloric end of the stom-
ach shows definite peristaltic movements.

Under higher magnification it was observed that
the cytoplasm of the cells stained poorly and appeared
to be less granular than that of normal muscle cells.
Similarly, the nuclei did not stain well with methylene
blue, as is the case in normal tissues. (Turck, 1903.)
These observed histological changes are distinctly
analogous to those observed in other instances of
beginning cellular autolysis, which we have shown to
be prerequisite for the liberation of cytost.

These observations are substantiated by the obser-
vations of Oilman (1903) on the nuclear changes
incident to the fatigue of striated muscle.

From the above discussion it will be seen that mus-
cular fatigue involves the production of cytost which
in turn tends to inhibit the action of the muscle. This
conclusion must not be interpreted as meaning that
muscular fatigue is caused solely by the accumulation
of cytost resulting from muscular activity. The re-
cent remarkable advance in our knowledge of the
chemistry of muscular contraction would nullify any
such interpretation of the facts; for quite obviously

NATURAL RESISTANCE AND FATIGUE 157

the exhaustion of available phosphagen, or any other
substance essential to the activity of muscle, would
likewise lead to fatigue. Similarly, the accumulation
within the muscle of any toxic metabolite such as lac-
tic or carbonic acids would likewise lead to fatigue.
Fatigue due to the latter substances has long been
recognized, but, as Bainbridge (1919, page 79) has
recently pointed out, neither of these substances "ever
attains during exercise such a concentration in the
blood as to be directly toxic."

Vincent and Sheen (1903), and Bayliss (1918),
have found that when injected into an animal a neu-
tral extract of muscle may cause a loss of tone of the
arterioles. This action, as has been shown previously,
is a typical cytost reaction, and is of interest in con-
nection with the earlier investigations of the author.

During violent muscular exertion such as rowing
or running, the circulatory and respiratory increases
necessitated by the increased muscular activity usually
cease shortly after the muscular exertion has stopped,
although other evidences of fatigue may persist for
some time afterwards. This is perhaps due to the
accumulation of cytost during the period of exercise.
In this connection it is of interest to note that upon
reviewing the available literature Bainbridge (1919,
page 186) has been unable to find any evidence that
metabolites formed as a result of muscular activity
have any part in causing a fatigue of the central nerv-
ous system. This is of interest in the present discus-
sion; for, as has been shown in earlier chapters, the
parenteral introduction of even large quantities of
cytost does not lead to any involvement of the central
nervous system.

158 THE ACTION OF THE LIVING CELL

Nowadays it is universally recognized that regular
exercise is necessary to insure a normal functioning
of an animal's organs and their efficient coordination
in bodily activities. Further, college and professional
athletes after proper training engage almost daily in
the most violent forms of exercise, and not only do not
suffer any serious consequences, but seem to be actu-
ally benefited by their exhausting performances.
Indeed, there is a prevalent impression that the
mortality rate among athletes is lower than among
non-athletic individuals. While a few statistical
studies of college men have appeared to indicate the
correctness of this opinion, we cannot seriously con-
sider such data, because as a rule the college athlete
is at the outset a selected individual — selected for his
healthy and vigorous constitution. Admitting this
source of error in such studies does not in any way
invalidate the common knowledge that properly regu-
lated exercise is distinctly beneficial to any animal.

At first glance this appears to be paradoxical in
view of what has already been said concerning cytost
and its possible relation to muscular fatigue. In the
past much mystery has surrounded the mechanism by
which exercise results in beneficial effects to the organ-
ism. While the increased blood flow through the vari-
ous organs results in a more rapid removal of metab-
olites and a better nutritive supply, this cannot be
the sole beneficial factor; otherwise violent anger
with its concomitant increase in circulation should be
as stimulating to the organism as is exercise.

During exercise an increased metabolism takes
place, the energy liberated in excess of the body re-
quirements being utilized to do the external work

NATURAL RESISTANCE AND FATIGUE 159

demanded by the nature of the exercise, such as row-
ing a boat, or throwing a ball. Studies directed toward
this end have demonstrated that with cessation of the
physical activities, the excess metabolic activity is
utilized in connection with the repair and synthesis
of tissue; and the latter may and usually does take
place to a greater extent than is warranted by the
tissue substance loss during the period of exercise.
This is attested to by the fact that an individual's mus-
cular development is conditioned by the extent to
which his muscles have been used in past perform-
ances.

Conversely, the disuse of muscles leads to atrophy.

The manner in which functional activity causes
an increase in growth and incidentally in natural re-
sistance has up to the present been shrouded in mys-
tery, although the writer feels that at least a partial
explanation of the observed facts may be efifected as
follows. Let us recall the facts demonstrated previ-
ously: that (a) cytost in low concentration stimulates
the proliferation of cells in tissue culture; (b) cytost
in too high a concentration brings about the death of
cells exposed to its action ; (c) severe muscular fa-
tigue is accompanied by an accumulation of cytost.

From the last it follows that cytost is liberated by
the muscle cells as a consequence of their activities
incident to contraction. If it be imagined that the
amount of cytost liberated during muscular activity
is determined by the extent of such activity, it be-
comes apparent that if formed in the proper amount
the cytost so liberated should by analogy to the tissue
culture experiments stimulate the growth of the cells
of the muscle. In this manner we may account for

160 THE ACTION OF THE LIVING CELL

the growth stimulus induced by functional activity.
Continuation of this line of thought seems to indi-
cate logically that aside from mere muscular develop-
ment, the general benefits of exercise upon the body as
a whole may be traced to the action of cytost ; for even
though exercise brings about the liberation of cytost
in the muscles alone, the substance may be carried to
the other tissues of the body by means of the circula-
tion; and if, as postulated above, the concentration of
cytost in the body fluids is of such an order of mag-
nitude as to cause an increase of metabolism of the
cells bathed by these fluids, there will result a general
increase of the animal's metabolism and growth, for
each is an integration of the chemical activities of
the constituent body cells.

If there is any truth in this concept, one would ex-
pect that the introduction of small quantities of cytost
into the circulation of animals must result in an in-
creased metabolic activity and general salutary ef-
fects akin to those resulting from regular exercise.
Now this is precisely what has been observed in the
author's laboratory when very low concentrations of
cytost in the form of extracts of autolyzed tissue are
injected into cats for a protracted period of time. If
the reader will refer to the results tabulated on page
1 12 and summarized in the graph on page 1 14, he will
note that as the result of such a series of injections
the experimental animals underwent an increase in
weight considerably greater than did the control ani-
mals of the same age group on a similar diet. When,
as shown in the experiments just referred to, the cytost
injections were continued for a much longer period
of time, the animals suffered a decline in weight, and

NATURAL RESISTANCE AND FATIGUE 161

eventually pathological changes more or less similar
to those found in animals receiving large doses of
cytost over a shorter period of time.

This result is of importance because it demonstrates
that the tissue cells of the intact animal behave towards
cytost as they do towards other alterations in their
immediate environment. As is well known, tissues in
general demand the presence of certain essential ele-
ments and compounds in their surrounding fluids.
In general, the mere presence of these essential sub-
stances does not fulfil the needs of living cells. In
order that the cells may live a normal life, the con-
centration of the essential elements and compounds
in the surrounding fluids must be within certain opti-
mal limits. When present in a concentration less than
or greater than the optimal, such essential substances
exert definitely toxic actions. This has been definitely
shown by the exhaustive investigations of Jacques
Loeb and others on the phenomena of salt antagonism.

Similarly the writer's investigations concerning the
effects of cytost upon cells in tissue culture have shown
that while the presence of this substance appears neces-
sary for the growth of cells under such conditions, an
excess of cytost rapidly inhibits growth and may actu-
ally lead to the death of the cultures.

From these results it may be reasoned that in the
intact animal the normal functioning of the body
cells is in part conditioned by the concentration of
cytost. Such being the case, it follows that as a result
of proper exercise the tissue growth which results
from the liberation of cytost must be due to the fact
either that cytost does not accumulate in the body
fluids to such an extent as to manifest toxic actions;

162 THE ACTION OF THE LIVING CELL

or, if it does, its actions are counterbalanced by the
development of factors antagonistic to cytost — i.e.,
by the development of an anticytost.

The name "anticytost" implies an antibody capable
of overcoming the toxic actions of cytost. While it
is true that we have been unable to demonstrate the
presence of such an antibody by the usual methods of
serology, a considerable mass of experimental evi-
dence has been accumulated which substantiates such
a concept. This evidence will be considered in detail
in the succeeding chapter.

If for the moment the reader will accept the concept
of anticytost, we may proceed a little farther with
the discussion of some of the aspects of fatigue. If, as
suggested at the beginning of this chapter, muscular
fatigue is in part due to the accumulation of cytost in
or about the muscles, it follows that, other things being
equal, an animal whose body fluids contain an appre-
ciable amount of anticytost will fatigue less easily
than will an individual less well equipped to com-
bat the toxic effects of cytost, — that is, having avail-
able less of the specific antibody necessary to counter-
act the toxic efifects of cytost. Such being the case, it
follows that since regular exercise leads to the devel-
opment of a certain degree of resistance to fatigue, it
must likewise lead to an increased production of anti-
cytost; for only by this means could a resistance to
the fatigue-causing cytost be developed.

As a corollary it follows that one reason a relatively
inactive animal is readily fatigued by a moderate
physical exertion is that his body cells have not devel-
oped a sufficiently marked resistance to cytost to en-
able them to withstand the sudden onslaught of this

NATURAL RESISTANCE AND FATIGUE 163

substance from the active muscles. By proper train-
ing, however, such a resistance is developed; for the
gradual liberation of small quantities of cytost by ap-
propriate exercise may be considered to stimulate the
formation of anticytost and thus endow the individual
with an appropriate mechanism for combating fa-
tigue.

On this basis one is enabled to account for the fre-
quently remarked fact that oftentimes individuals of
perfect muscular development are less able to with-
stand certain types of physical exertion than are those
of distinctly less prowess. On the basis of what has
been said above, distinct muscular development as
seen in ex-athletes and captive wild animals depends
upon past performances which have stimulated the
development of the muscles, while resistance to fa-
tigue depends upon the immediate presence of anti-
cytost in sufficient quantity. Thus, an ex-athlete or
captive animal may, due to forced inaction, fail to
stimulate the formation of sufficient anticytost to over-
come the fatiguing effects of what otherwise might be
regarded as a relatively simple physical task. From
these considerations it is seen that there is not neces-
sarily a correlation between physical development
and resistance to fatigue.

Before leaving this discussion, one more factor
must be considered: the results attending complete
physical inaction. Such results are well seen in indi-
viduals afflicted with some pathology which has
caused a degeneration of some portion of the motor
nerve paths — such, for example, as infantile paralysis,
or, in experimental animals, section of the motor
nerves passing to a limb. In consequence of such in-

164 THE ACTION OF THE LIVING CELL

volvement, nervous impulses are not carried to the
affected limb and the muscles receive no stimuli from
the central nervous system. Therefore, unless moved
by external manipulation, the muscles of the injured
member participate in little or no movement, and
after a time they undergo degeneration or atrophy.
Consequently the injured limb may dwindle to a size,
measured in terms of tissue mass, considerably less
than that of the opposite limb. The essential point to
be observed here is that inaction leads to atrophy. No
factor other than enforced inaction is necessary to
bring about atrophy; it may be accomplished simply
by splinting a limb in one fixed position for a few
months.

Just why the degenerative changes described by the
word atrophy should be caused by muscular inaction
has never been entirely clear in the past, although it
usually has been attributed to the impairment of circu-
lation consequent to inaction. This is due to the fact,
as first suggested by Borelli in the seventeenth cen-
tury, that the venous return of blood to the heart is
owing not to the heartbeat, but to the extrinsic circu-
latory mechanism of the veins; for their muscle con-
tractions, by exerting a pressure upon the underlying
veins and capillaries, force the blood by virtue of the
unidirectional valves towards the abdomen and trunk.
When the muscles are completely inactive this proc-
ess cannot proceed at all; or when partially inactive,
the magnitude of the venous return to the heart is
seriously diminished. There follows then, as the case
may be, either a complete or a partial stagnation of
blood in the tissues of the immobilized part. Such
being the case, the tissues suffer a lack of foodstuffs and

NATURAL RESISTANCE AND FATIGUE 165

oxygen, and an accumulation of metabolites, includ-
ing any cytost which may have been formed by their
activities.

Since under the conditions specified above the
activities of the tissues have been minimized as a re-
sult of inaction, the cytost arising from this source
cannot in all likelihood attain a sufficient concentra-
tion in the body fluids to be of serious consequence.
Nevertheless, the faulty nutrition of the tissues and
their prolonged exposure to acidic metabolites must
result in some degree of autolysis, as explained in an
earlier chapter; and, as show^n previously, the auto-
lytic process results in the liberation of cytost in con-
siderable quantities. In consequence, there will be
an accumulation of this substance in and about the tis-
sues of the inactive limb, and these tissues will there-
fore suffer. By this line of reasoning we reach the con-
clusion that the cause of atrophy in immobilized
tissues is twofold, being due in part to impaired
nutrition and in part to the accumulation of cytost.

If an animal is completely immobilized, then his
entire system will be exposed to the toxic action of
large quantities of cytost arising in the manner de-
scribed above; for the general impairment of the cir-
culation will also inhibit the excretion of any toxic
products formed in the tissues. The writer has per-
formed experiments in this connection by bandaging
rabbits in such a fashion that all movements other than
those incident to respiration were inhibited. In the
writer's experience, all the animals so treated died
within thirty-six hours following the application of
the bandages. At autopsy the blood was found to have
undergone coagulation and stasis in the viscera simi-

166 THE ACTION OF THE LIVING CELL

lar to that observed in other instances of acute death
by cytost. These experiments represent an extreme
case of cytost intoxication due solely to inaction.

Mention has previously been made of the experi-
ments conducted by the writer in 1901, in which the
less stringent method of confinement in close-fitting,
sterile cages was used to cause inactivity. As previ-
ously recorded, it was found that under such condi-
tions the vast majority of the animals so treated died
within a few months, while the majority of those sur-
viving this treatment were found at autopsy to have
developed various lesions similar to those produced
in later years by the repeated injection of homologous
cytost.

The foregoing discussion offers a partial explana-
tion for the numerous ill effects, such as malaise, ease
of fatigue, and general lowering of the so-called
natural resistance, which follow periods of enforced
or willful inactivity in both man and animals.

Chapter VIII

INVESTIGATIONS CONCERNING
NATURAL RESISTANCE

In the last chapter we were led to certain specula-
tions regarding natural resistance and the role played
by cytost in this important aspect of normal physi-
ology. Let us now consider the results of various ex-
periments which have been made by the writer in an
attempt to test the validity of his conclusions con-
cerning natural resistance. For convenience, such
experiments may be divided into three groups:
(a) those dealing with the susceptibility to cytost as
evidenced by the ease of the induction of shock by
the injection of that substance; (b) wound healing;
and (c) experiments concerning the breeding of cap-
tive animals. In each of these groups we shall for the
present limit our discussion to animals whose natural
resistance has been raised by a preliminary series of
cytost injections.

It will be remembered that when small graded
doses of cytost are injected every few days for some
weeks into laboratory animals, these animals undergo
a progressive increase in weight and apparent well-
being. If such injections are discontinued before the
animals suffer a decline as shown in the graph on

167

168 THE ACTION OF THE LIVING CELL

page 1 14, we may suppose that their natural resistance
to cytost has been increased beyond that of similar un-
treated animals which have been kept under other-
wise identical conditions. As will be shown pres-
ently, this supposition is justified by the experimental
results. Such being the case, we may reasonably speak
of such animals as having been actively immunized
to cytost by the treatment they have undergone. It
must be stressed, however, that the term immunity is
here used in a relative manner, for we cannot by any
treatment render an animal absolutely immune to cy-
tost. All we can do is to raise his degree of tolerance
to cytost above that of his untreated mates.

If a series of cytost injections, which leads to in-
creased weight and general health, really is capable
of raising an animal's resistance to cytost, then ani-
mals so treated should be more resistant to shock than
are animals whose resistance has not been so raised.
This point is easily settled by experiment. A potent
cytost preparation prepared as previously described
from autolyzed cat's muscle was first assayed by injec-
tion into normal cats in order to find the dosage neces-
sary to cause a severe shock. This dosage was then
injected under parallel conditions into normal cats
and into cats actively immunized to cytost, as above
described. The untreated animals always passed into
severe shock frequently resulting in death, whereas the
actively immunized animals developed a shock of con-
siderably lesser degree and but few fatalities resulted.

When the quantity of cytost injected was reduced
to such an amount that it caused a severe but non-fatal
shock in normal animals, the actively immunized ani-
mals were found upon injection to suffer shock in but

NATURAL RESISTANCE 169

a slight degree, and to recover from it rapidly. In
some instances, the immunized animals were able to
withstand the intravenous injection of a quantity of
cytost from 100 to 500 times as great as the amount
found to cause the death of normal animals within
three minutes. Furthermore, the immunized animals
proved to be refractory to the usual procedures which
are known to induce shock. (Turck, 1918, 1921.)

These striking results prove that the preliminary
series of cytost injections cause the elaboration of
some defense mechanism within the body of the
treated animals. These facts, taken in conjunction
with our previous observation that cytost is to a
marked degree species-specific, suggest that the in-
creased tolerance to cytost shown by the treated ani-
mals is due to the elaboration of a specific antibody or
anticytost.

Two different sets of experiments tend to substan-
tiate this concept. First, it was found that cats which
had been subjected to a series of injections of heterol-
ogous cytost prepared from beef or rat tissues did not
develop an increased resistance to cytost. This was
demonstrated by the fact that animals so treated, when
injected with homologous cytost passed into shock
and died as rapidly as did untreated normal animals.

These results offer further evidence that the proc-
ess of active immunization to cytost depends upon the
elaboration of a specific anticytost analogous in some
respects to the specific antibodies of classical im-
munology. By analogy, then, it appeared likely that
such antibodies might be produced in the body of
another animal, such as the horse or goat, and that pos-
sibly by injection of the serum of such an immunized

170 THE ACTION OF THE LIVING CELL

animal cats might be given a passive immunity to
cytost. To this end horses and goats were given a
series of injections of autolyzed cat tissue, and after
some months blood was withdrawn and the serum
separated as in the usual methods for the preparation
of antisera, and preserved by the addition of tri-
cresol (Turck, 1919).

The existence of anticytost in horse or goat serum
prepared in this fashion was demonstrated as follows.
A quantity of the antiserum was mixed with an
amount of cat cytost known to be fatal when injected
into cats. The mixture was then injected into normal
cats. As a rule the animals receiving the cytost-anti-
cytost mixture developed severe shock but did not
die as did the animals receiving cytost alone. Alter-
natively, when injected first with the antiserum and
then with a potent cytost preparation, it appeared
that the antisera afforded the animals some measure
of protection against cytost although the degree of
immunity obtained in this manner was in no way as
striking as that resulting from active immunization,
as described above. While this result proved disap-
pointing, it is in harmony with common experience
with many bacterial endotoxins, where it has repeat-
edly been found impossible to convey a reliable pas-
sive immunity by the injection of antisera.

However, since some degree of success attended
these experiments, the writer feels that the results
mentioned above favor the concept of an anticytost
as a defense mechanism of the organism. In this con-
nection it is of interest to recall the investigations of
Weichardt (1912), who claimed to have obtained
an antitoxin against the toxin of muscular fatigue.

NATURAL RESISTANCE 171

Since, as postulated in the previous chapter, it ap-
pears that cytost is concerned in muscular fatigue,
Weichardt's fatigue antiserum may be closely re-
lated to or identical with anticytost. In view of our
present knowledge, however, it appears more likely
that more convincing experimental results can be ob-
tained by active immunization with homologous cy-
tost than by the transmission of antibodies elaborated
in another animal. In consequence, in the majority of
the experiments to be discussed subsequently, the
various animals used were immunized actively rather
than passively.

From the standpoint of general biology, the im-
munization to shock discussed above is of importance
only in so far as it demonstrates our ability to raise
an animal's resistance to the effects of high concentra-
tions of cytost. From a general standpoint this is of
interest because, as we have shown, various bodily
activities as well as injuries lead to a liberation of
this endogenous tissue product.

If, as postulated previously, an Increased tolerance
to cytost is a factor in an animal's natural resistance,
then we should expect that actively immunized ani-
mals would recover from minor injuries more rapidly
than non-immunized. In order to test this concept,
the rate of healing of standardized wounds was com-
pared in immunized and non-immunized cats. For
this purpose an area back of the heads of the animals
was shaved, and, under ether anesthesia, a triangular
flap of skin was removed between the occipital tuber-
osity and withers. As far as possible, these injuries
were made alike, the dimensions of the triangles being
held to a standard size. The rate of wound healing

172 THE ACTION OF THE LIVING CELL

was then estimated by measuring the size of the
wound and area of granulation from day to day. With-
out exception it was found that the wound healed much
more rapidly in the immunized than in the non-
immunized cats. These results are summarized in
Table XII. (Turck, 1921.)

TABLE XII
Rate of Wound Healing in Immunized Cats (Turck, 1921)

Group

First
Week

SECO^fD
Week

Third

Week

Fourth
Week

Fifth
Week

Sixth
Week

I

2

3

3X3X4

2.7X2.7X3.7

3.5X3X4.5

3X2.5X4
2X2.5X3
2.7X2.5X3

2.5X2X3
2X2X2.5
2X2X2.5

1.7X1.5X2

1X1XI.5

1X1XI.5

0.5X0.7X1
Healed
Healed

Healed

The figures given above represent the average measurements in centimeters
of the unhealed portions of the standardized wounds (3X3X4 cm.) in three
different groups of cats. The first group were untreated in any way. The
members of the second group received injections of a few drops of cytost around
the margin of the wounds, while all members of the third group had been
actively immunized to cytost as described in the text.

These results indicate that the immunized animals
can more readily cope with minor injuries than can
the non-immunized. Since all animals during their
existence are subjected to various minor injuries of
one sort or another, it follows that an increased tol-
erance to cytost must be of considerable service to
the animal in overcoming adverse disturbances in his
environment.

It has long been recognized that, other things being
equal, the rate of healing of a localized injury is in a
large measure determined by systemic conditions of
a so-called "constitutional" nature. While the term
constitutional is admittedly vague, it nevertheless

NATURAL RESISTANCE 173

conveys the idea with which we are all familiar, — that
individual internal factors over which we have little
control determine the physiological activity of an
organism.

Thus, it is well known that captive wild animals
do not breed well. Commonly this fact has been at-
tributed to constitutional alterations arising from
changed feeding habits and inaction enforced by cag-
ing which play an important role in their altered
breeding activities and the viability of their ofifspring.
We have seen previously that these same factors lead
to a liberation of cytost and various pathological con-
ditions which may be attributed to the action of this
substance. Consequently, it became of interest to ascer-
tain whether or not immunization to cytost would re-
sult in an increased fertility and possibly in a greater
viability of the ofifspring. As captive wild animals
were not available to the writer, he was forced to con-
duct his experiments upon domesticated and labora-
tory animals.

A series of pullets which had not begun to lay eggs
were divided into three flocks. The first flock (A) of
ten received a course of four Immunizing injections
of chicken cytost, spaced one week apart. The sec-
ond flock (B) of twenty-five received similar in-
jections throughout the period of observation, while
the third flock (C) of ten were held as controls. The
daily egg production of each flock was noted over a
period of seven months, from January through July.
During the first three months the number of eggs pro-
duced was about the same, although the members of
flock A (the immunized birds) yielded a slightly
greater number of eggs. At the end of the fourth

174 THE ACTION OF THE LIVING CELL

month, however, distinct differences in egg produc-
tivity became apparent in the various flocks. While
the control birds reached and maintained an egg pro-
duction of about 30%, the immunized birds showed
a marked increase in the rate of egg production, attain-
ing 60% at the end of the seventh month ; whereas the
members of flock B which had received the cytost in-
jections every week throughout the period of observa-
tion, after the fourth month suffered a rapid decline
in egg production, which fell almost to zero after the
seventh month. (Turck, 1923.) The results of these
experiments are represented graphically in figure 13.

These experiments with chickens recall those made
with the cats, wherein it was found that although a few
doses of cytost appeared to be distinctly beneficial to
the animals, a long continued series of injections ulti-
mately led to retrogression and pathological changes.
It may reasonably be assumed that the diminished egg
production observed in flock B is due to the produc-
tion of circulatory disturbances and consequent tis-
sue damage similar to that found in cats when treated
in the same manner.

When attempts are made to breed cats under labora-
tory conditions it has been frequently observed that
the kittens born are sickly and survive but a short time.
Further, as we have shown previously (page 87),
when apparently healthy cats are placed in cages con-
taminated with cytost, the animals rapidly develop
respiratory disturbances, involvement of the lungs,
and frequently pneumonia. Hence when one allows
pregnant cats to give birth to kittens in cages con-
taminated with cytost, or places the newly born kit-
tens in such cages, the young animals are subjected to

NATURAL RESISTANCE

175

a combination of factors which early brings about
their death. Obviously, then, such conditions may be
utilized as a rather severe test of our ability to raise an
animal's natural resistance by injections of cytost.

6C.

Chickens

ECGSX

JAN. Feb. Mar. Apr. May Ju.n. Jul.

Figure 13. Graphs showing the effects of cytost injections upon
the monthly egg production of hens. The ordinates represent the
percentage of the hens which laid eggs each day and the abscissae
represent the time after the beginning of the experiment. Curve A
refers to the hens stimulated by cytost injections; curve B represents
the hens in which a decline was caused by cytost injections; and
curve C the control flock. (From Turck, 1923.)

Kittens born of normal mothers were divided into
two groups. The members of one group were in-
jected with a cat anticytost serum which had been
prepared as above described, while no attempt was
made to immunize the individuals of the other group.
All the kittens were then placed in cages which had
been sprayed with a potent cytost preparation. All

Untreated kittens

AGE

DEATH IN

8 weeks

12 days

4 weeks

10 days

4 weeks

12 days

4 weeks

1 1 days

176 THE ACTION OF THE LIVING CELL

the kittens died within a month, although all the ani-
mals treated with the anticytost lived approximately
twice as long as the untreated ones. (Turck, 1923.)
The longevity of the members of both groups is sum-
marized below:

Passively immunized kittens

AGE DEATH IN

8 weeks 17 days

4 weeks 25 days

4 weeks 32 days

These experiments show that specific anticytost
sera afford the animals a slight degree of protection
from the cytost infected cages. A large series of ex-
periments has shown that the average life of healthy
kittens exposed to cytost contact is from ten to twenty
days, whereas if injected with anticytost serum be-
fore being brought into contact with cytost, they
usually live for a longer period, although they ulti-
mately succumb if not removed from such contact.

These experiments were continued in slightly dif-
ferent fashion, as follows. Female cats were actively
immunized to cytost as above described, and allowed
to give birth to their offspring in cages sprayed with
cytost. In this case the kittens, although in contact
with cytost, were found to live for a considerably
greater period than did those born of non-immunized
mothers. A number of the former received injections
of anticytost as well, and the kittens so treated with-
stood the contact with cytost slightly better than did
those born of actively immunized mothers but re-
ceiving no anticytost serum. Since, however, in these
two groups of animals the length of life in the cages

NATURAL RESISTANCE 177

did not differ very greatly, it seems likely that the
tolerance to cytost exhibited by both groups of kit-
tens is directly traceable to the fact that their mothers
had been actively immunized. The length of life of
the kittens born of immunized mothers is summarized
below. (Turck, 1923.)

Untreated kittens born of

Kittens from activ

ely immunized

actively immunized

mothers and injected with

mothi

?rs

anticytost

serum

DEATH IN

DEATH IN

Born in cage

8 weeks

Born in cage

6 months

Born in cage

57 days

Born in cage

4 months,

Born in cage

18 days

11 days

Born in cage

32 days

Born in cage

1 month

Born in cage

11 weeks

Born in cage

8 weeks

The fact that all the kittens in these two groups
lived for a comparatively long period under condi-
tions known to be rapidly fatal to kittens implies that
the resistance of these animals was greater than is
usually the case with young kittens, and from the very
nature of the experiment can only be attributed to the
fact that the mothers had been actively immunized to
cytost. This fact suggests that the immunized mothers
were able to transmit their acquired immunity to their
offspring. This perhaps is simply another way of
stating the well-known fact that vigorous healthy ani-
mals usually give birth to young which as a rule have
a greater natural resistance than do the offspring of
normally inactive animals.

Although the latter statement by itself conveys noth-
ing new, it is of singular interest that immunization
of the mothers to cytost enables us to insure the pro-

178 THE ACTION OF THE LIVING CELL

duction of offspring having a higher natural resistance
than is ordinarily the case. This conclusion is not
based upon the experiments just cited alone, for re-
sults of a similar and even more striking nature have
been obtained with mice, rats, and guinea pigs as well.
Before considering these in detail, it is worth while
to outline the probable relation between the health
of the mother and that of her offspring.

It is now generally accepted by biologists that ac-
quired characteristics of the parents cannot be in-
herited by their offspring. This conclusion is based
upon the results of thousands of experiments in this
direction which have been made since the beginnings
of scientific biology. While from time to time appar-
ent evidence to the contrary has been recorded in the
literature, subsequent investigation has in each case
proved such evidence to be the result of either faulty
experimentation or observation. Further, the modern
theory of heredity, which has withstood innumerable
onslaughts, furnishes an adequate basis for the non-
inheritance of acquired characters. According to pres-
ent day concepts, an animal's inheritance is uniquely
determined by the genes passed on through the germ
plasm from one generation to another. So far as is now
known, an organism's inheritance can be modified
only by factors capable of altering such genes. To
date, the only known method of accomplishing this is
by means of the X-ray which, as Muller (1925) has
shown, may cause such alterations in the genes as to
lead to the formation of mutants. Such being the case,
it appears obvious that ordinary factors affecting the
activities and health of the parents can in no wise influ-
ence the inheritance of the offspring.

NATURAL RESISTANCE 179

At first glance these conclusions may appear to be
at variance with the results of the author's experiments
concerning the breeding of animals. Such, however, is
not the case, as the following considerations will show.
Supposing that, given an adult man who by right of
inheritance is an excellent physical being capable of
withstanding without difficulty the rigors of present
day civilization, we suddenly transfer him to the Arc-
tic regions and fail to equip him with proper clothing
or housing to withstand the extreme cold. It is quite
obvious that under such conditions the man would
rapidly succumb to the intense cold, regardless of the
genes he had inherited from his forbears. Again, if
after a brief exposure to the intense cold and before
death, we were to transfer him back to a more suitable
environment, we know, from experience, that regard-
less of his genes, the individual would show the results
of his unfortunate exposure, and would for a time be
susceptible to various maladies, particularly respira-
tory infections. In other words, while the genes de-
termine one's inheritance, environment determines
largely the value of this inheritance to the individual
concerned. Further, as shown in the simple example
cited above, a relatively brief exposure to a distinctly
unfavorable environment may so alter one's natural
resistance that for a longer or shorter time he is unable
to cope with simple variations in an environment to
which he is normally relatively resistant.

There is no reason why these simple concepts should
not be applied to the developing fetus in utero. In
other words, regardless of the genes which have been
passed on to the fetus by its parents, the immediate
environment of the fetus must, to a great extent, de-

180 THE ACTION OF THE LIVING CELL

termine the resistance of the fetus both before and
after birth. Similarly the development of the fetus in
the body of the mother must alter her internal environ-
ment. The latter fact is easily made apparent in the
various anatomical and physiological changes which
accompany pregnancy, and by the vomiting and other
evidences of toxemia frequently to be observed.

Such unpleasant effects, which are commonly ob-
served in women and higher animals during preg-
nancy, may in part be ascribed to the action of cytost
liberated from the uterine tissues as a result of the
activities of the developing fetus. For the first week
following insemination the developing egg is a free
body, but later attaches to the uterine wall. In the case
of the monkey, the changes incident to this attachment
have recently been described by Hartman (1931).
According to this author the embryo of the monkey
begins to implant itself in the tissues of the mother
about eight or nine days after fertilization, and even-
tually becomes half buried in the uterine wall. Hart-
man's experiments show that this implantation is ac-
companied by an erosive action which for a time
causes the maternal tissues literally to "melt away"
before the egg and thus affords a mechanism for the
attachment of the egg.

The "melting away" of the mother's tissues, as Hart-
man terms the process, is undoubtedly autolysis — the
usual procedure by which cells disintegrate. It seems,
therefore, that some secretion of the egg must act as a
trigger which initiates such autolysis and thus permits
implantation to take place. Such an autolysis, in com-
mon with any other, must liberate cytost into the circu-
lation of the mother and cause her to show the effects

NATURAL RESISTANCE 181

of cytost intoxication — in this case the so-called tox-
emia of pregnancy. On a similar basis we may explain
the occurrence of such toxemias in pregnant women;
for in their case it has long been known that the human
embryo becomes completely embedded in the uterine
tissue, and this must take place by a mechanism similar
to that in the monkey. For obvious reasons, embryolo-
gists have not been able to follow this process in the
woman as Hartman has done in the monkey; but in
this instance it does not appear fallacious to argue by
analogy.

The concept of toxemia outlined above is easily sub-
jected to experimental verification; for symptoms
similar to those accompanying the toxemia of preg-
nancy should be elicited by the parenteral introduc-
tion of cytost into pregnant animals. This has been
shown by the writer (1921, 1922) as follows: When
homologous cytost preparations were injected intra-
peritoneally into healthy pregnant cats, the animals
showed signs of acute nausea within from thirty sec-
onds to two minutes after the injection, and violent
retching continued for thirty to fifty minutes, fre-
quently accompanied by symptoms of eclampsia. As
has been stated previously, such injections, if con-
tinued, may induce abortion. On the other hand, injec-
tions of heterologous cytost into pregnant cats does
not result in the onset of any of these difficulties.
These results seem to prove unquestionably that the
toxemia of pregnancy is actually due to the release
of cytost brought about by the activities of the devel-
oping embryo in utero.

In a normal pregnancy the majority of the symp-
toms of toxemia disappear long before the end of

182 THE ACTION OF THE LIVING CELL

gestation. This fact signifies one of two things : either
the mother develops an immunity towards cytost, or
the production of cytost by the fetus ceases. In order
to analyze these concepts, let us return to Hartman's
observations on the monkey, where it was found that
the egg digs into the maternal tissues until half buried.
Since, as we have seen, this implantation is accom-
panied by autolysis, we may inquire why this autoly-
sis does not continue until all the uterine tissues have
disappeared. Two answers to this perplexing ques-
tion suggest themselves. Either the stimulus of autoly-
sis, the fetus, has undergone a change, or the mother's
tissues have become resistant towards the invasive ac-
tion of their offspring. Each of these answers con-
tains an element of truth, as is evidenced by the fol-
lowing considerations.

In the previous discussion we have shown that the
repeated injection of small quantities of cytost leads
to the development of an active resistance towards
this substance, presumably through the development
of an anticytost. With this in mind we may postulate
that because of the slow and continual absorption of
cytost from the disintegrating uterine tissues, the
mother's resistance to this substance is raised in a
manner analogous to that resulting from the regular
injection of small quantities of homologous cytost.
That such a resistance is developed during preg-
nancy is shown by the following considerations.

Injection of cytost in suitable quantity into preg-
nant cats causes nausea and retching which usually
pass off within an hour. When like quantities of cy-
tost are injected into male cats kept under the same
conditions in the laboratory, vomiting and retching

NATURAL RESISTANCE 183

are likewise induced, but as a rule the onset of such
unpleasant symptoms is found to be more rapid and
their duration more prolonged than in the case of the
pregnant females. (Turck, 1921b.) This observation
appears to substantiate the hypothesis that the females,
as a result of their pregnancy, develop a certain re-
sistance towards cytost.

It may be remembered that in our experiments con-
cerning the action of cytost on the abdominal viscera
it was shown that once autolysis is started the process
appears to pass from cell to cell, gradually involving
greater areas of tissue. With this in mind, it seems evi-
dent that the autolysis of the uterine tissues due to the
activities of the embryo would spread not only to
other tissues but to the embryo itself, unless checked
in some way. It follows from the foregoing discus-
sion that such a check is afforded by the anticytost
developed by the mother. In those all too numerous
instances where toxic symptoms are maintained
throughout pregnancy, especially in eclampsia, it ap-
pears that for some reason or other the mother is
incapable of elaborating sufficient anticytost to com-
bat the activities of cytost in her body. This unfor-
tunate circumstance may lead to two difficulties, one
affecting the mother and the other the offspring. The
first leads to various pathologies such as kidney in-
volvement, and the second to ill health and perhaps
faulty development of the young. Indeed, with regard
to the latter, Holland (1920) has concluded— "that
the toxemia of pregnancy is the principal cause of
antefetal death."

This conclusion, which is the result of empirical
observation, cannot be questioned ; hence it is of inter-

184 THE ACTION OF THE LIVING CELL

est to seek the cause underlying the observed facts. To
this end let us attempt an analysis of the facts in the
light of the foregoing discussion.

In an earlier chapter attention has been drawn to
the fact that when shock is induced in a pregnant
guinea pig the embryonic tissues suffer congestion in
much the same manner as do the viscera of the mother
(see Fig. 16). Since shock, as we have shown, is due
to the action of cytost, such an observation may be
taken as evidence that the presence of an excess of
cytost in the circulation of the mother will be re-
flected in the developing fetus. This may well lead
to faulty development of the latter, and such faulty
development in utero may seriously affect the animal's
antefetal life. Thus we are able to see a connection
between the toxemia of pregnancy and the life of the
young from a rational point of view.

It follows, therefore, that anything we can do to
lessen the toxemia of pregnancy, that is, to combat the
action of cytost in such instances, will enable us to
offer a measure of protection to young animals. The
importance of this possibility lies not solely in pro-
tecting the young against cytost during their compara-
tively brief period in utero, but in the fact that by so
doing we may, barring accident, insure the develop-
ment of normal young which will better be able to
live in accordance with the heritage of their germ
plasm. To accomplish such a result, we must take
advantage of all available means to combat the action
of cytost in utero before as well as after birth.

One means available toward this end is the im-
munization of mothers against cytost so far as is pos-
sible. This will have a dual effect, for not only will it

i

NATURAL RESISTANCE 185

decrease the tendency towards toxemia in the mother,
and hence the possibility of attendant damage to the
developing fetus, but, as shown in the experiments
with kittens in cytost-sprayed cages, the offspring will
be relatively more resistant to cytost than are the
young born of non-immunized mothers. That a course
of immunizing injections actually does decrease the
liability towards the toxemia of pregnancy follows
from the fact that the nausea and retching which fol-
low the intraperitoneal injection of small quantities
of cytost is not observed when a similar injection of
cytost is administered to pregnant female cats which
have been actively immunized by the procedure pre-
viously described. (Turck, 1921, 1923.)

From a laboratory standpoint cats are not conven-
ient animals for investigations concerning breeding
activities, and it was exceptionally fortunate for the
writer that through the kindness of the late Dr. Edwin
J. Banzhof we were able to conduct such experiments
on a large number of guinea pigs used for breeding
purposes in the laboratories of the New York City
Department of Health. For a period of years it had
been observed by members of the Health Department
Laboratories that a large proportion of the breeding
stock died in pregnancy, and that the young, when
born, were often feeble and did not survive for long.
This situation occurred time and time again during
the winter months, although the animals were kept in
a warm, well-ventilated room. At first it was thought
that the unusually high mortality was due to some
infection, and attempts were made to combat the situ-
ation by sterilizing the cages and by transferring the
animals to other rooms. Such means, however, proved

186 THE ACTION OF THE LIVING CELL

of no avail; hence the conclusion was drawn that the
breeding stock through generations of domestication
had become unusually susceptible to the action of cy-
tost liberated during pregnancy. In order to test this
concept, it was decided to attempt immunization of
the guinea pigs to cytost.

To this end, a cytost solution was prepared by ex-
tracting autolyzed guinea pig muscle with ten times
its weight of water, and after removal of the insoluble
material by centrifugation, the extract was passed
through a Berkefeld filter in order to insure sterility.
This preparation was then used directly for the im-
munization experiments reported below.

In the first experiment twenty old females of the
same age and weight were taken from the breeding
stock which had been doing poorly. Breeding was
started on December 2, 1923. The animals were
divided into two groups of ten each. Beginning Feb-
ruary 4, 1924, each of the members of one group re-
ceived a series of seven subcutaneous injections of the
cytost extract, spaced one week apart, while the mem-
bers of the other group were held as controls. The
amount of the cytost extract used for each injection
was two or three minims. In this experiment it was
desired to ascertain the effect of the injections on the
mothers at the time of parturition and shortly there-
after. All the animals in both groups gave birth to
young atvarious times between February 3 and March
10. By the end of April all the members of the in-
jected series were still alive, whereas four of the mem-
bers of the controlled group had died on the follow-
ing dates: April 4, 12, 14, and 18. Subsequently all

NATURAL RESISTANCE 187

these surviving animals, including the young, v^ere
sent to the writer's laboratory, w^here they vs^ere kept
under continued observation. By December, 1924, all
the mothers of the control series had died, w^hile all
the members of the immunized group were still alive.

This result leaves no doubt but that the attempted
immunization was actually efiFective in protecting the
mothers against cytost liberated during pregnancy.
Further, this result is of interest in that it shows again
that animals may actually be immunized to cytost by
such a series of injections — perhaps because of the
elaboration of a specific anticytost, as previously sug-
gested.

It may be suggested that these rather striking re-
sults are due to some factor other than the cytost in-
jections. Such a possibility would demand serious
consideration were it not for the fact that essentially
the same result was obtained time and time again on
repetition of the experiment.

In these early experiments the cytost injections
were begun too late during the course of the preg-
nancies to warrant any conclusions as to the effect
upon the offspring; hence other experiments must be
considered. Before passing to these, we may interpo-
late an experiment made under the writer's direction
by Dr. Banzhof and his associate, Mr. A. M. Klein.
A number of guinea pigs was given a series of three
cytost injections spaced a week apart, and was then
set aside in the animal house for a period of fifteen
months. At the end of that time, they were large and
healthy, while, according to Dr. Banzhof, animals of
the same age and size usually show a mortality rate

188 THE ACTION OF THE LIVING CELL

of fifty per cent within a year when kept under the
same conditions in his animal house. These results
of Dr. Banzhof offer striking confirmation of the
writer's results with cats (page 112), and again
demonstrate that it is possible to raise the natural
resistance of animals by a properly graded series of
cytost injections. Unfortunately Dr. Banzhof did
not record the gain in weight of his animals; there-
fore we cannot compare this aspect of their growth
with that of controls, as was done in the writer's experi-
ments with cats.

Old guinea pigs were found to be less resistant to
injections of large quantities of cytost than younger
animals. This fact may be taken as indicative of the
fact that the older animals suffer from a greater ac-
cumulation of cytost than do the younger ones; con-
sequently the former are more apt to die as a result
of the toxemia of pregnancy than the latter, a conclu-
sion in agreement with observations from experience.
That older animals actually are more sensitive to cy-
tost is evidenced in that they succumb more rapidly to
injections of large quantities of cytost than do younger
animals.

Turning now to the influence of the immunization
of the mothers on the offspring, we have found the
following: When female guinea pigs receive a series
of immunizing cytost injections, as above described,
but given prior to pregnancy, it is found that the off-
spring are more vigorous, have a healthier appetite,
grow more rapidly, and present a healthier appear-
ance than do the offspring of non-immunized animals.
A series of young females was divided into two groups,
one being immunized and the other held as control.

NATURAL RESISTANCE 189

During the course of the experiment both groups of
animals and their offspring were kept under identical
conditions in the laboratory and were given the same

rations.

Following the birth of the young, the following dif-
ferences were noted: The litters of the immunized
mothers were larger than those of the controls, and
the young were more active and took to the breast
more rapidly than did those of the non-immunized
mothers. In a few instances, some of the latter died
shortly after birth from what appeared to be acute
inanition. This difference in appetite became much
more marked a month after birth, when the animals
born of immunized mothers showed the voracious
appetite common to healthy young animals, while the
members of the control group showed much less inter-
est in food.

The difference in food intake was shown by the fact
that one month after birth the average weight of the
young of the immunized mothers was 60 grams greater
than the average weight of the young born of the con-
trol group, whereas after the lapse of two months the
difference in weight averaged 80 grams. Again, a dis-
tinct difference was noticeable in the appearance of
the fur, that of the control group appearing rough,
while that of the members of the other group was
sleek and glossy, an indication of better health. Dur-
ing this period a distinct difference in natural re-
sistance was evidenced by the fact that several of the
young in the control group succumbed to respiratory
disturbances which caused their death, whereas none
of the animals born of the immunized mothers suf-
fered from this cause or from any other. In other

190 THE ACTION OF THE LIVING CELL

words, the young born of immunized mothers ap-
peared superior in every way to the controls.

These differences recorded above cannot be at-
tributed to differences in the germ plasm, since all the
animals used in these experiments were derived from
the same breeding stock. Further, the observed dif-
ferences between the offspring of non-immunized and
of immunized mothers were sufficiently consistent to
justify the conclusion that the immunization of the
mother to cytost is decidedly beneficial to her off-
spring.

When we attempt to interpret this result, we are
confronted with two possibilities, for as a result of
the immunization we may consider that the observed
results evidenced by increased resistance of the young
are due solely to the lack of toxic effects upon the
embryo during intrauterine life, thus permitting a
normal development; or on the other hand, it may be
supposed that the increased resistance developed by
the mother is passed on to her offspring. From an
experimental standpoint the latter conclusion appears
the more likely for several reasons.

In the first place, it has been pointed out above that
by immunization cats may be rendered less susceptible
to the respiratory disturbances which are induced by
keeping the animals in cytost-sprayed cages. Further,
it was found that in such cages the longevity of kittens
born of immunized mothers was greater than that of
those whose mothers had not been immunized. This
result can only be explained on the assumption that
the kittens during their intrauterine existence had ac-
quired, in a degree, the immunity induced in their
mothers.

NATURAL RESISTANCE 191

Bearing this in mind, it seems likely that the in-
creased natural resistance of the young guinea pigs
discussed above must likewise be due to the passage
of the mothers' increased resistance to the offspring.
This conclusion does not controvert the generally ac-
cepted doctrine that acquired characters are non-in-
heritable, for it may be effectively explained on the
basis of the concept of anticytost. It will be recalled
that some evidence has been offered that the immuni-
zation procedure accomplishes its desired end by caus-
ing the elaboration of a specific anticytost. If the pla-
centa is permeable to anticytost, than the fetus may
acquire this substance by diffusion from the blood of
the mother. Consequently at birth the young animals
are endowed with a certain supply of anticytost which
offers them protection until such time as they become
able to develop anticytost of their own.

Such a concept is in complete accord with the re-
sults of the experiments discussed above, and is in
agreement with the findings of Ehrlich (1891, 1892),
who found that when female mice were immunized
to the toxins ricin and abrin, their progeny possessed
a resistance to these substances which was greater than
that normally found in the offspring of non-
immunized mothers.

Ehrlich further observed that the increased re-
sistance to ricin and abrin was transmitted to the off-
spring when only the mother was immunized to the
toxin, whereas immunization of the father alone did
not result in an increased resistance of the young to
these toxins. A similar finding is recorded by Behring
(1889) in the case of immunization to diphtheria
toxin.

192 THE ACTION OF THE LIVING CELL

These results argue in favor of the placental trans-
mission of antibodies, and it seems logical to conclude
that when a pregnant animal is immunized to cytost,
the anticytost formed passes in similar fashion to the
fetus.

Chapter IX

THE BREEDING AND MANAGEMENT

OF ANIMALS

In the foregoing chapters we have advanced a num-
ber of concepts and experiments relative to the action
of cytost in the animal body. It has been shown that,
depending upon its concentration, this substance may
exert both stimulating and toxic effects, so far as an
animal's health and well-being are concerned. Let
us now consider the application of the information
so far deduced to the problem of animal breeding.

The object of the latter, whether for laboratory or
farm purposes, is to produce under more or less fixed
conditions uniformly healthy and fertile animals
which are as resistant as may be expected to unavoid-
able alterations in their environment. It is obvious
that in order to achieve this end as economically as
possible the breeding stock must likewise be kept in
a healthy resistant state. If the surroundings natural
to a given species cannot be completely duplicated the
breeder must devise various ways of compensating for
this enforced change in the animal's mode of living.

To this end intelligent breeders have always tried,
in so far as possible, to duplicate climatic conditions,
provide exercise, and give adequate attention to

193

194 THE ACTION OF THE LIVING CELL

dietary considerations. Nevertheless such attempts
have frequently met with lack of success, even in the
hands of experienced husbandrymen. It is of inter-
est, therefore, to inquire into the relationship betw^een
such factors as those mentioned above and the various
aspects of the cytost problem that have been con-
sidered in the preceding pages.

First consider the question of diet. As is well
known, a given diet may be entirely adequate for an
animal when regarded solely from the standpoint of
energetics, and yet be inadequate in so far as it is
lacking in essential components which the animal is
incapable of synthesizing from other components of
its food. It is evident, then, that before attempting
to breed a given species on a large scale one should
carefully study the dietary requirements of the ani-
mals at hand. Information as to the types and quan-
tities of food demanded by the ordinary domesticated
animals is usually obtainable, although it must be ad-
mitted that in most instances such needs have been
determined as a result of empirical experience rather
than by carefully planned investigations such as those
Osborne and Mendel have made upon the rat.

The accumulated experience of years has taught
husbandrymen that aside from an adequate diet, cap-
tive wild animals and domestic animals must be pro-
vided with adequate facilities for exercise, and kept
under conditions which preclude crowding. The
necessity of considering such factors in the care of
animals is easily understood in the light of the vari-
ous experiments conducted with cytost, and we may
profitably consider them from the standpoint of the
cytost theory.

ANIMAL BREEDING 195

As regards the question of diet, the reader should
recall the experiments on partial inanition which have
been discussed in Chapter V, where it was shown that
as a result of malnutrition an animal is forced to call
upon some of its body cells for essential foodstuffs
omitted from its diet. In consequence, along with the
nitrogenous products liberated as a result of cellular
autolysis, it may be expected that an excessive amount
of cytost may find its way into the circulation, and,
as has been shown previously, this may result in vari-
ous pathologies.

Besides containing an adequate quantity of essen-
tial foodstuffs, an animal's diet must be so adjusted that
it cannot interfere in any way with the normal proc-
esses of digestion and absorption, and thereby prevent
the proper utilization of the various dietary com-
ponents. Such a necessity is attested to by the results
of the writer's experiments with monkeys, which were
fed bread fried in oil, together with the other foods
commonly given to such animals. As stated on page
106, the monkeys on the high fat diet died within a
few months, although other monkeys kept under sim-
ilar conditions but not fed any of the bread fried in
oil continued to live an apparently normal existence.
In other words, although the diet was adequate from
a nutritional standpoint, the addition of the heated
oil brought about an end result similar to that induced
by partial inanition. Indeed, we may reasonably take
the view that the oil so inhibited the normal proc-
esses of digestion and absorption that the experimental
animals were in the predicament of "starving to death
at a banquet," and hence suffered an accumulation of
cytost in much the same manner as occurs on an incom-

196 THE ACTION OF THE LIVING CELL

plete diet. Similarly any other means which promotes
faulty absorption in one way or another leads to the
same ends.

Some years ago it occurred to the writer that such
faulty absorption of essential foods might be brought
about by the admixture of various substances with an
otherwise satisfactory diet. Various irritants such as
mustard and silver nitrate were tried without any
especially gratifying results. Shortly thereafter, it was
found that the desired result could be achieved by add-
ing large quantities of beef extractives and bouillon
cultures of B. coli to an otherwise satisfactory diet.
Rats kept on such a diet died within a month. As the
blood was found to be sterile, death was not due to a
bacteriemia. In agreement with this, it was found
that the same end resulted when the bacteria were
omitted but the feeding of large quantities of beef ex-
tract was continued. (Turck, 1906, 1908.)

Dogs treated similarly usually died in about three
months. At autopsy the blood was found sterile, but
marked evidences of cytost action were apparent. The
viscera were found to be markedly congested, and in
many instances multiple peptic ulcers were found.
These latter were indicative of the fact that autolysis
had taken place. At the time it was noted that "there
was no bacteriemia, no inflammatory reaction in the
form of round cell infiltration such as one would ex-
pect in a reactionary inflammation induced by pyo-
genic microorganisms or toxins. It was not the pic-
ture of reaction to an infection, not the picture of a
local acting agent, but rather of a systematic condition,
and of an induced cellular change." (Turck, 1906.)

The question of exercise has been fully discussed

ANIMAL BREEDING 197

in Chapter IV; therefore it will not be necessary to
consider this factor again.

Turning now to the question of crowding, we find
that as a result of experience it has long been known
that such a condition acts adversely on caged animals,
although in the case of higher animals little actual ex-
perimentation has been directed towards a study of
this factor. What literature exists on this topic has
recently been reviewed by Allee (1931), and the
reader is referred to his book and the excellent mono-
graph on the rat by Greenman and Duhring ( 1923) ,
for the details of experiments made by others.

At first it is rather difficult to understand why the
overcrowding of cages should lead to the ill health
of animals except in so far as it limits their freedom
of motion, thus preventing a normal amount of exer-
cise. Undoubtedly this is an important factor in the
effects of overcrowding, but, as will be shown below,
it is not the only one.

The reader may recall that it has been shown that
the toxic effects of cytost and the respiratory involve-
ment attendant thereto may be induced in cats simply
by spraying their cages with an extract of homologous
cytost, or by the application of a paste of autolyzed
tissue to the paws of the animals. This finding demon-
strates among other things that when present in the
air surrounding the animals cytost may enter the re-
spiratory tract and there set up a congestion.

Now other things being equal, the extent and
severity of such a congestion will be dependent upon
the amount of cytost inspired and this in turn must
bear some relation to the amount present in the air. On
this basis, it follows that as a result of inhaling air con-

198 THE ACTION OF THE LIVING CELL

taining cytost an animal may suffer an involvement
of the lungs varying in severity from nothing to a true
pneumonia. This is especially interesting because re-
spiratory involvements of one sort or another are fre-
quently responsible for a high mortality amongst small
mammals, particularly rabbits and guinea pigs. Such
troubles are usually attributed to bacterial infections,
although as the writer has pointed out elsewhere
(Turck, 1919), it appears that the bacterial invasion
usually follows the congestion of the lungs.

The fact that animals may suffer from the toxic ef-
fects of homologous cytost in the surrounding air sug-
gests that this is an important factor in producing the
effects of overcrowding. Various experiments which
we shall now consider substantiate this conclusion.

When the dust which accumulates about animal
pens or cages was collected, converted into a sterile ex-
tract, and injected into homologous animals, it was
found that the animals died rapidly, in some instances
almost immediately, the time of death depending upon
the quantity of the extract injected. A similar finding
was obtained when concentrated extracts of the hair
and epithelial tissues were substituted for the dust ex-
tract. Since all of the substances used as a source of
cytost in these experiments will commonly occur in
animal cages, pens, and runs, it is obvious that such
places constitute a potential source of trouble unless
frequently cleaned. Obviously in crowded cages this
potential source of cytost is increased, due to the
greater number of animals per unit area. In conse-
quence we may conclude that the ill effects attendant
upon overcrowding are in part due to the greater
chances of exposure to air containing a relatively

ANIMAL BREEDING 199

higher concentration of cytost than would be the case
if fewer animals were confined within the same area.
Further, it is possible that cytost finds its way into the
air of the cage by way of the effluvia of the breath.

Further evidence that in crowded cages cytost may
be present in sufficient quantity to elicit various respi-
ratory disturbances was obtained as follows : Pregnant
animals were allowed to give birth to their young in
cages which had previously been inhabited by a num-
ber of adult animals. Under such conditions it was
found that the newly born offspring usually died
within two weeks, from respiratory disturbances.

The results of experiments of this type confirm the
hypothesis that the ill effects of overcrowding are due
in part to the resultant relatively high concentration
of cytost which accumulates in the cages or pens.

In raising small animals in the laboratory, it almost
inevitably results that a certain degree of crowding
is necessitated by the requirements of space. This con-
dition should not present any serious difficulties if the
cages are cleaned thoroughly at regular intervals,
preferably daily. Such a cleaning should involve not
only the removal of excreta and the like, but the com-
plete removal of hair and dust. As a further means of
minimizing the dangers of contact with cytost, the
rooms in which the animals are kept in their cages
should be well ventilated, as this will prevent the ac-
cumulation of cytost-laden air.

This fact has been appreciated by the laboratory
workers engaged in researches on the white rat, and
a visit to laboratories where this animal is raised in
large numbers will show that the conditions stated
above are scrupulously observed. For some curious

200 THE ACTION OF THE LIVING CELL

reason breeders of other species of animals have not
seen fit to employ the same care in the treatment of
their charges. Indeed, such breeders are inclined to
scoff at the pampering which the white rat receives in
the larger laboratories. They argue that the rat is a
hardy animal naturally acclimated to life under con-
ditions usually regarded as unhygienic for other spe-
cies. While this is true, the scoffers forget that the rat
breeders would not indulge in the care and attendant
effort which they expend on their animals unless it
had been found to pay, not only financially, but also
in insuring the production of a constant supply of
hardy animals suitable for purposes of experimenta-
tion.

It may not be amiss to suggest that the ill health all
too common amongst humans in congested districts
may in part be due to the same cause as that found
in the case of caged animals. Everyone has heard the
virtues of "country air" extolled, and I believe that
most of us are ready to admit that there is something
invigorating about country air that is lacking in the
city. By analogy with the animal experiments dis-
cussed above, it seems probable that in densely popu-
lated areas the atmosphere must contain a certain
amount of human cytost which is lacking in the coun-
try air. On this assumption the invigorating nature
of extra-urban atmosphere lies in its comparative free-
dom from cytost. This inference, of course, is only
speculation which cannot be proved by direct experi-
ment, but several facts of human experience appear
to testify to its correctness. Thus, it has long been
recognized that patients suffering from respiratory
infections recover more rapidly outside the larger

ANIMAL BREEDING 201

cities. Again, most individuals have at one time or
another experienced a feeling of discomfort when
together with a number of people in a poorly venti-
lated room or hall. This discomfort may in part be
due to the accumulation of small quantities of cytost
in the air of the room ; and if this be true, it is obvious
that the greater the number of people in the room the
more rapidly will such an accumulation take place.

We have no direct evidence that humans actually
discharge cytost into the air, but that they must give
off something which is characteristically human is
evidenced by the fact that bloodhounds can trail a
man; and that many other animals, such as the deer,
can scent a human at some distance, providing the
wind is in their direction. Indeed, many animals ex-
hibit a remarkable sense of smell, which enables them
to seek their own kind. Since, as has been shown previ-
ously, cytost is distinctly species specific in its action,
it is conceivable that such species specific scent as is
shown by the dog is in part due to a special sensitivity
to cytost rather than to a sense of smell as we ordi-
narily understand it.

The foregoing discussion, although admittedly
somewhat speculative, suggests that the animal
breeder should utilize every means at his disposal to
combat the action of cytost upon his animals. Aside
from the selection of an adequate diet, the prevention
of overcrowding, the preservation of cleanliness, and
the provision for adequate exercise, it is conceivable
that the professional breeder might profitably con-
sider the possibility of immunizing his animals to
homologous cytost. By so doing he might be able to
lower the mortality rate of his breeding stock and of

202 THE ACTION OF THE LIVING CELL

their offspring. This would seem particularly im-
portant when the animals are of necessity kept in
highly artificial surroundings, such as is usually the
case with laboratory animals.

In the preceding chapter, we have presented ex-
perimental evidence that immunization to cytost is
of considerable value in the maintenance of a guinea
pig colony, since it appears to insure the birth of young
animals having a higher natural resistance than is
normally the case under similar conditions of breed-
ing.

Similar experiments have been conducted with
white mice. These experiments are especially in-
structive, since the mouse is an animal with a span
of life shorter than that of most other laboratory ani-
mals; and since they reach maturity more rapidly
than do other species, it is comparatively easy to fol-
low through several generations under fairly constant
environmental conditions. Further, it is well known
that white mice show a very high mortality even under
apparently ideal conditions of temperature, feeding,
etc. This fact, which frequently proves troublesome
to the breeder, is ideal for our purposes, since a com-
parison of the mortality rates of mice immunized to
cytost and those of non-immune animals offers a ready
means of testing the validity of our conclusions.

In the experiments to be discussed presently, the
mice were kept in clean, spacious cages in a well venti-
lated, sunny room, and all of them were given the
same food. They were in charge of an experienced
breeder of mice who for a period of seven years had
kept careful records of the food requirements, mor-
bidity, and mortality. At times the latter had reached

ANIMAL BREEDING 203

the discouraging proportion of 50%. During the
period post mortems, bacterial cultures, and similar
data had yielded no information concerning the high
death rate, and the conclusion was drawn that death
was due to "natural causes" incident to breeding under
domesticated conditions.

As ordinarily used, the term "death from natural
causes" signifies little short of a confession of igno-
rance. As it seemed reasonable to suppose that the
high death rate was due to cytost intoxication, experi-
ments were begun with this in view.

For this purpose mouse cytost was prepared from
autolyzed mouse muscle in a manner identical with
that used for the preparation of guinea pig cytost, as
described in the preceding chapter. Initially the ex-
tract was prepared by the extraction of the autolyzed
muscle with an equal weight of water. Such prepara-
tions proved too toxic for the experiments, for upon
the injection of 0.5 cc. of such an extract subcutane-
ously or intraperitoneally, the mice always died
within 12 to 48 hours. When the dosage was halved
(0.25 cc), the mice were found to withstand a series
of five or six such injections spaced a week or ten days
apart. In this instance, the usual birth rate prevailed,
but 95% of the young died within two months after
birth.

After considerable experimentation of this sort it
was finally discovered that it was necessary to employ
a considerably more dilute tissue extract in order to
achieve any distinctly beneficial effects upon the
breeding of such animals. In our final series of ex-
periments, which we wish to discuss here, it was found
that a cytost preparation obtained by extracting the

204 THE ACTION OF THE LIVING CELL

autolyzed mouse tissue with forty times its weight of
water proved the most serviceable. Mice were able
to withstand a series of several injections of 0.25 cc. of
such an extract without any apparent harm.

A series of mice were immunized by means of six in-
jections of 0.25 cc. of such a cytost preparation, spaced
10 days apart. The second series received eight injec-
tions of 0.06 cc. of the cytost preparations, spaced one
week apart. These two groups of mice, together with
untreated controls, were kept in spacious cages under
identical conditions, and allowed to breed. The effi-
cacy of the immunizing treatment was judged by the
mortality rate of the offspring of the three groups.
During the period of observation, which extended
over several months, 30% of the young born of non-
immunized mothers died; whereas in the two im-
munized groups the mortality rate of the young was
5% and respectively.

The lower mortality rate found in the cytost-
injected animals indicates that the immunization pro-
cedure adopted was actually effective. As it had been
observed that during the winter months many mice
died of pneumonia when their cages were placed on
the floor of the breeding room so as to be exposed to
draughts of cold air, cages containing immunized and
non-immunized mice were kept on the floor through-
out the winter and spring. Under these conditions
many of the non-immunized animals died, although
all the immunized lived through this period. This
difference was especially noticeable in the case of the
newly born, for the mortality of the offspring of the
non-immunized mothers amounted to 50%.

On comparing the young born of the two groups

ANIMAL BREEDING 205

of mice, distinct differences were noticed. Those born
of immunized mothers exhibited a thick, sleek fur, in
marked contrast with the rougher coats of the young
members of the non-immunized colony. Similarly
the former surpassed the latter in activity and virility.
Indeed, in these respects the young born of the immun-
ized mothers approached the behavior of young wild
mice. This is especially interesting, since Donald-
son has pointed out that in the case of the albino rat,
the young behave similarly to wild rats if they are
born of hardy stock under ideal conditions. The young
born of the immunized mothers also gained in weight
more rapidly than did those of the other group. This
is in accord with the findings in the experiments with
guinea pigs referred to previously.

Greenman and Duhring (1932) in their discussion
of the breeding of albino rats state that in addition to
the observance of the various environmental factors
noted in the preceding discussion, one should "select
animals for breeding which shall have been reared
for one or more generations in an exercising cage dur-
ing the period of rapid growth from 25 to 130 days
of age." This advice is based upon the empirical
observation that by so doing one obtains a breeding
stock having a higher natural resistance than is other-
wise the case. In view of the discussion in Chapter
VII, and the experiments with guinea pigs and mice,
it should be clear that the utility of exercise in im-
proving a breeding stock must depend upon the re-
lease of cytost by this means. This, as we have seen,
results in the production of anticytost, and a conse-
quent increase in natural resistance.

Greenman and Duhring, as well as King (1919),

206 THE ACTION OF THE LIVING CELL

have pointed out that the popular conception that in-
breeding results in the degeneration of a stock is er-
roneous. The latter author calls attention to the fact
that the dire effects usually associated with inbreed-
ing are due to environmental rather than genetic fac-
tors. In other words, when animals are inbred in
breeding establishments, sufficient attention is not
given to the prerequisites for successful breeding,
which have been discussed in the beginning of this
chapter. Indeed, provided sufficient attention is paid
to such factors as diet, cleanliness and provision for
exercise, it is entirely possible to breed from poor stock
a strain showing a high natural resistance. In this
connection Greenman and Duhring remark, 'Tf for
any special reason it is necessary to breed from a poor
stock, it is by no means a hopeless task to rebuild
lost vigor." And again, "Brothers and sisters may be
mated with no unfavorable results when breeders are
carefully selected." This means simply that provided
the brothers and sisters are in a high state of natural
resistance we may expect their offspring to exhibit
a similar natural resistance.

The conclusions of the investigators just cited are
in harmony with the experimental results of the
author. In the case of the latter, however, the in-
creased resistance of the breeding stock was achieved
by means of cytost injections rather than by the more
natural methods employed by Greenman and Duh-
ring. The fact that both methods of stock improve-
ment lead to the same end argues well for the validity
of the author's hypothesis that so-called natural re-
sistance, however developed, is due to an increased
tolerance towards cytost.

ANIMAL BREEDING 207

In the case of the colonies of mice investigated by
the author, no special facilities for exercise were pro-
vided. Injections of cytost, however, proved an effec-
tive substitute, since our colonies v^ere carried on for
a period of three years without the introduction of
any new stock. Prior to the application of cytost to
these colonies it had been found necessary to clear
out all the stock, old and young, every year or two
and begin again with fresh breeding stock in order to
insure the production of healthy, vigorous young.

It has been stated previously that experiments indi-
cated that in both cats and guinea pigs the immunity
to cytost developed in the mothers was transmitted to
the offspring. This was also observed in the case of
mice, as noted above. With the latter animals we have
been able to follow the effects of cytost immunization
through several generations. This was made pos-
sible by the fact that young mice mature rapidly. Thus
by observing the mortality, appearance, activity, and
fecundity of succeeding generations of mice, it was
found that the beneficial effects of cytost immuniza-
tion appear to be transmitted through at least three
generations.

Perhaps it is erroneous to speak of the transmis-
sion of cytost immunity, for this seems to imply the
alteration of a genetic factor. What we mean to con-
vey is simply the experimental fact that succeeding
generations of animals having an increased natural
resistance result from the immunization of mothers.
This may be due to the fact that, as stated above, the
first generation born of immunized mothers shows
a greater activity than do the offspring of those not
immunized. Such an increased activity must of neces-

208 THE ACTION OF THE LIVING CELL

sity result in effects akin to those resulting from any
other form of exercise. In consequence we may as-
sume that the increased activity of such young animals
suffices to stimulate anticytost production to a degree
comparable to that which results from active immuni-
zation.

Some exceedingly interesting results have been ob-
tained by the immunization of old guinea pigs. As is
well known, with the onset of age females tend to
become non-fertile; or, if fertile, they frequently
abort, or give birth to young which die shortly after
birth. When such animals, which are normally dis-
carded by breeders, were subjected to a series of cytost
injections identical with those used to immunize
younger animals against the toxemia of pregnancy, it
was found that they became fertile and proved useful
as breeding stock for another year. This apparent
rejuvenation suggests that their lack of fertility prior
to immunization was due to the toxic effects of cytost
which had accumulated in their bodies during the
course of their lives. On the other hand, if such an
assumption is true, it is difficult to see why the addi-
tional cytost administered in the immunizing pro-
cedure should not result in a distinct toxicity.

This difficulty may be circumvented by assuming
that the apparent poor fecundity of such animals is
due not to a persistent accumulation of cytost, but
rather to an anticytost deficiency. Indeed, this is made
manifest by the extreme susceptibility of such animals
to the toxemia of pregnancy and abortion. In other
words, prior to insemination they do not suffer from
an accumulation of cytost, but when the embryo be-
gins to attack the uterine tissues, the resulting libera-

ANIMAL BREEDING 209

tion of cytost proves too much for their powers of
resistance. On this basis it is readily seen that when
such animals become pregnant their abilities to pro-
duce normal, healthy young must be seriously im-
paired.

In captivity the guinea pig at best is not a very active
animal; hence it is easy to imagine that, due to its
limited muscular activity, it fails to stimulate anti-
cytost formation in the natural manner we have postu-
lated. From this brief discussion it should be clear
that the cytost injections employed in immunizing
such aged animals of poor fecundity must function
solely by stimulating the formation of anticytost in
quantity sufficient to overcome the effects of any cytost
liberated during the course of a pregnancy. In con-
sequence such animals are permitted to carry the de-
veloping fetus to term in a normal fashion.

From the preceding discussion it appears likely
that much the same results could be achieved by sub-
jecting old, apparently sterile females to a course of
exercises of gradually increasing severity. So far as
the writer is aware this type of experiment has not
been investigated, but the results should prove of inter-
est.

The conclusions reached above may be summarized
as follows: In order that domesticated and captive
wild animals may live a normal, healthy existence in
accordance with their birthright, every precaution
should be taken to inhibit the formation of cytost or
contact with cytost in excessive quantities. Oppor-
tunity must be offered to develop anticytost in a nor-
mal fashion, since this will protect the animals against
cytost intoxication. These ends may be achieved by

210 THE ACTION OF THE LIVING CELL

supplying the animal with an adequate quantity of its
natural foods, with proper housing conditions, and
with facilities for exercise. If for any reason such
conditions cannot be adequately fulfilled, then this
lack may in part be remedied by active immunization
with homologous cytost.

The writer makes no claim that such immunization
offers a panacea for all animal ills, but suggests, rather,
that the results of his experiments indicate that the
stimulation of anticytost formation offers a powerful
weapon for the prevention of such ills. Of course, like
all preventive measures, it is not infallible; but the
experimental results enumerated in this chapter sug-
gest that it is worthy of a trial in otherwise obscure
cases, and in connection with the breeding of animals
under the conditions enforced by captivity.

It would be exceedingly interesting to ascertain
whether or not immunization to homologous cytost
would stimulate breeding in those wild animals
which are known to breed poorly or not at all when
held in captivity. In this connection it is opportune
to note the methods being used in rearing the baby
gorilla at the New York Zoological Park. Accord-
ing to current press accounts (March, 1932), those
in charge of this young animal have departed from
the time-honored methods of raising gorillas in cap-
tivity and have permitted their charge to romp in
the snow during the winter time. According to some,
this should prove suicidal, since the gorilla is natu-
rally acclimated to a warm environment. With the
animal in question, however, now five years old, this
procedure has proved actually beneficial, and he seems
to be withstanding the rigors of New York weather

ANIMAL BREEDING 211

better than any of his predecessors at the Zoo. In
common parlance we should state that this young
gorilla has become acclimatized. This is undoubtedly
true — by his continual exercise his natural resistance
has been raised to such a level that he can withstand
our climate without succumbing to the ills which
afifect gorillas in captivity.

Such animals frequently contract pneumonia and
die. As we have shown elsewhere, a low resistance
to cytost is a predisposing cause of respiratory in-
fections. It will be interesting to watch the New York
gorilla during the next five or ten years. If during
this period he escapes the various respiratory infec-
tions, may we not attribute it in part to an increased
resistance to cytost developed as a result of his un-
orthodox unbringing at the New York Zoo?

Chapter X

THE RELATION OF CYTOST TO
BACTERIAL INFECTION

The relation between bacterial invasion and cytost
may be viewed from three different aspects: the re-
lease of cytost as a result of injury caused by bacterial
infection ; the cumulative effects of bacteria and cytost
acting together; and the relationship between cytost
immunity and bacterial immunity. At the outset we
should expect that any microorganism capable of ef-
fecting the destruction of the tissues of a higher ani-
mal would bring about the production of cytost in
much the same fashion as would any other means of
injury. In consequence, the infected animal should
exhibit symptoms of cytost intoxication as well as
those due to the bacterial toxins per se.

When subtlethal quantities of exocellular bacterial
toxins, such as diphtheria toxin, are injected into the
skin of an animal, a local reaction sets in after the
lapse of several hours. The reaction usually com-
mences as a slight reddening at the site of injection,
indicating a localized capillary dilatation. This is fol-
lowed by a localized edema, which is due to an in-
creased permeability of the capillaries. Since, as we
have seen, both these effects are caused by cytost, and

212

BACTERIAL INVASION 213

since they appear only some hours after the injection
of the toxin, it seems likely that they are not caused
directly by the toxin, but by the action of the toxin on
the surrounding tissues, in the same manner as was
found to be the case when irritating chemicals, such
as chloroform, were injected. Such reactions are by
no means limited to the bacterial exotoxins, but fol-
low the intradermal injection of living bacteria, dead
bacteria, and bacterial extracts. (Simmonds, 1928.)

In agreement with this concept is the fact that the
proteolytic organisms produce such localized reac-
tions much more rapidly than do the non-proteolytic.
This is especially interesting because, as has been
shown earlier, the autolytic process which leads to
the liberation of cytost is essentially proteolytic in
nature. In agreement with this is the well known fact
that infection with highly proteolytic organisms such
as the Welch bacillus frequently leads to shock, ap-
parently identical with that which results from the
liberation of cytost by any other means.

The shock which sometimes follows such infection
is really due to cytost liberated as a result of the activi-
ties of the microorganisms upon the tissues of the host;
for if the infected tissue is dissected out, extracted with
water, sterilized, filtered, and injected into another
animal, shock frequently ensues. This result must be
due to cytost present in the extract, and not to the
bacteria or their exotoxins, since the latter are re-
spectively removed and inactivated by the processes
of filtration and the temperature incident to steriliza-
tion. When a similar experiment is conducted with
tissues infected with weakly proteolytic or non-

214 THE ACTION OF THE LIVING CELL

proteolytic organisms, shock does not usually follow
the injection of the tissue extract.

Such experiments show conclusively that bacterial
infection may lead to cytost intoxication, provided the
infecting organism is capable of causing the libera-
tion of cytost from the infected tissue of the host. In
such a case, then, the host may sufifer from the sum-
mation of two harmful factors: the cytost liberated
by the activities of the organism on the tissues, and
the specific toxins elaborated by the bacteria them-
selves. On this basis it should be clear that if prior
to infection the animal has a high degree of resistance
towards cytost, it may suffer less from the action of
the infecting organism.

In connection with some experiments upon the dif-
fusion of bacteria through the intestinal wall, the
writer (Turck, 1914) has made some interesting ob-
servations which indicate that the joint action of bac-
teria and cytost leads to a distinct potentiation of the
latter. Before discussing the reason for this, let us
consider the experimental findings.

Dogs were opened under anesthesia, and a culture
was taken from the lumen of the intestines. The con-
tents (secretions) were removed from the duodenum
and jejunum, diluted with four volumes of normal
saline solution, and placed on ice until needed for
subsequent stages of the experiment. This extract
will be referred to below as S. The intestines were
then removed, and divided into two sections, the first
comprising the duodenum and upper jejunum and
the second the lower jejunum and ileum. The mucous
membranes were removed from these by scraping and
then ground with four volumes of saline. These ex-

BACTERIAL INVASION 215

tracts will be referred to below as Mi and M2 re-
spectively. Lastly, the muscular coat of the intestines
was ground in a meat chopper and extracted with four
volumes of saline for a few hours and then centrif uged
to remove insoluble material. This extract of the
muscle coat will be designated as C,

The B. coli obtained from the intestinal lumen was
grown in broth culture for 24 hours ; 10 cc. of the cul-
ture was mixed with 0.2 cc. of extract S and incubated
for 30 minutes ; then 0.1 cc. of Mi was added, and incu-
bation continued for another 20 minutes.

One tenth of a cubic centimeter of this mixture was
injected into the marginal vein of a rabbit's ear, where-
upon the animal developed symptoms identical with
those of anaphylactic shock and died in three minutes.
Injection of a similar preparation containing M2 in
place of Ml led to the same end, although the onset of
the anaphylactic reaction was considerably delayed.
Similar injection of an equivalent quantity of the coli
culture without the addition of the intestinal extracts
did not cause the appearance of shock or death. In-
jection of a mixture of S and Mi or M2 in quantity
equivalent to that employed in the first experiment led
to a mild shock but death did not follow. Injection
of a like quantity of a mixture of 10 cc. of the extract
of the muscular coat (C) and 0.2 cc. of S caused
anaphylaxis and death, although the onset of symp-
toms was not so rapid as in the experiments with the
mixtures containing B. coli.

Essentially the same findings were obtained in the
case of dogs, provided the amount of the injected ma-
terial was increased proportionately to the difference
in weight of these animals.

216 THE ACTION OF THE LIVING CELL

In another series of experiments the technique was
modified to permit of a quantitative evaluation of
some of these findings. Cultures of B. coli were ob-
tained from the feces of the dogs used in this experi-
ment. Samples of blood were taken from the femoral
vein and from the branches of the mesenteric vein
near the junction of the jejunum and duodenum. The
jejunum was ligated at the duodenal junction and at
a point 10 cm. below this. Sterile broth was placed in
the jejunal loop so obtained and allowed to remain
sufficiently long to obtain a rich culture of the micro-
organisms present on the intestinal wall. This broth
culture was then removed by aspiration and replaced
by a 48-hour broth culture of the B. coli previously
obtained from the animals' feces and the abdomen
was closed. After ten hours the abdomen was re-
opened and portions of tissue were removed from
the duodenum near the pyloric junction and from por-
tions above and below the ligatures, for histological
examination. After washing, the mucosa of the duo-
denum, jejunum, and the upper part of the ileum were
scraped from the muscle walls.

The blood samples, the mucosa and submucosa were
frozen in carbon dioxide to prevent decomposition,
and then dehydrated in vacuo over sulphuric acid.
The dry residues so obtained were weighed and then
brought into 2% suspension in 8% saline. These stock
suspensions were then diluted from twenty to two
hundred fold for subsequent experiments. One-cc.
portions of these various dilutions were mixed with
0.5 cc. of a forty-eight hour broth culture of B. coli
and incubated for 30 minutes. One-tenth cc. of these
mixtures was then injected into the marginal ear vein

BACTERIAL INVASION

217

of young rabbits. When the anaphylaxis-like symp-
toms occurred within three minutes, shortly followed
by death, the results were considered positive, whereas
very slight convulsions, followed by recovery, or
death after 24 hours or a week, were regarded as nega-
tive.

This decision appears to be legitimate, since very
slight shock followed by rapid recovery frequently
follows the injection of many substances; and the de-
layed death was usually found to be due to a gener-
alized infection which obviously could not take place
within the brief time necessary for attainment of the
positive results following the injections.

The results obtained in this experiment are sum-
marized in Table XIII below. The column headed
Dilution refers to the dilution of the stock suspen-
sions of the various extracts which were mixed with
the B. colt culture as described above.

TABLE

XIII

Dilution

1:20

1:60

1:100

1:200

Mucosa and submucosa

Submucosa

Serum from mesenteric vein

Serum from femoral vein

+
+
+

+
+
+

+
+

The plus sign denotes the appearance of distinct anaphylactic like symp-
toms within 3 minutes followed by death while the minus sign d^-notes that
the animals showed but slight convulsions and subsequent recovery.

As a control upon these experiments it was found
that double the amount of B. colt culture or of the
various extracts employed could be injected singly
without the onset of any untoward symptoms. Since,

218 THE ACTION OF THE LIVING CELL

however, B. coli and the mucosal extract together
form a highly toxic combination, we must admit that
a definite potentiation of some sort is brought about by
the mere mixing together of these components. Ap-
parently the presence of the microorganisms increases
markedly the toxicity of the cytost initially derived
from the mucosa.

That this is true is indicated by the fact that in the
more concentrated dilutions of the extract of the
serum taken from the mesenteric vein a toxic action
was observed when it was used in conjunction with
B. coli, whereas the serum from the femoral vein ex-
hibited no such toxicity. This result indicates that
the blood leaving the intestine contains a toxic moiety,
presumably cytost, which is either excreted or altered
in some fashion before it reaches the general circu-
lation.

That the actual toxic agent in these experiments is
cytost is evidenced from the fact that immediate au-
topsy of the rabbits which died a few minutes fol-
lowing the injection of the various toxic mixtures
disclosed an acute venous dilation of the splanchnic
vessels. Presumably, as in other cases of shock, this was
the cause of the sudden collapse and death.

Just why the presence of the colon organism should
so markedly intensify the action of the cytost is not
entirely clear, although it seems highly probable that
such intensification is due to a surface adsorption of
the cytost by the microorganisms. As is well known,
colloid particles are in general stabilized by an ad-
sorbed ionic layer and the mutual precipitation of
oppositely charged colloids involves the interaction
of such surface layers. Since, as pointed out elsewhere

BACTERIAL INVASION 219

in the text, the splanchnic stasis which is so character-
istic of cytost action appears to involve the agglutina-
tion of blood cells, it may be that the agglutinating
efifect of minute quantities of cytost is greatly magni-
fied by the adsorption of the latter on various blood
colloids or upon bacteria, vs^hich in many respects be-
have as colloids.

In agreement with this concept, it was found that
a similar potentiation of tissue extracts containing cy-
tost could be achieved by admixture with a dilute
agar-agar suspension. That is, if, by preliminary as-
say one ascertains the maximal quantity of a potent
cytost preparation which may be injected into an ani-
mal without the production of any noticeable evi-
dence of toxicity, and then injects this quantity of cy-
tost in admixture with a small quantity of a dilute
solution of agar-agar, the symptoms of shock are
found to follow immediately.

This finding strengthens our contention that the
role of the colon organism in the experiments cited
above is essentially that of a colloid which, by adsorp-
tion of the cytost, markedly potentiates the latter. This
is of considerable interest, since it opens the way to a
possible explanation of the interesting mysteries of
the well known phenomena of anaphylaxis and ana-
phylactic shock. As is well established, these promptly
follow the injection of a foreign protein into the cir-
culation of any animal which, some weeks previously,
has received a sensitizing dose of the same protein.
Various ingenious hypotheses have been advanced
to explain this puzzling phenomenon, but as yet no
single theory has been found capable of accounting
for all the known facts of anaphylaxis.

220 THE ACTION OF THE LIVING CELL

Whereas we cannot offer a comprehensive theory
of anaphylactic reactions, the experiments cited above
indicate that such a theory should include a recogni-
tion of the possible role of cytost in such reactions. In
this connection it may be imagined that the initial or
sensitizing dose of protein, bacterial or otherwise,
brings about in some fashion the liberation of cytost in
an amount sufficient to cause shock when adsorbed
upon an appropriate colloid. Then the parenteral
introduction of the second or intoxicating dose of the
protein may furnish the necessary surface for the ad-
sorption of and consequent potentiation of the cytost.
This hypothesis does not account for the well known
specificity of anaphylactic reactions, is necessarily
vague, and is offered merely as a suggestion for the
consideration of those interested in this field of inves-
tigation.

The essential point is that regardless of the mechan-
ism of anaphylactic shock, it seems to be ordinary
shock, such as we have discussed at some length, which
is brought about in an indirect fashion involving the
foreign protein introduced into the circulation of an
animal. This fact has been recognized by Zinsser
(1931) in his recent discussion of anaphylaxis. He
calls attention to the fact that the typical symptoms
of anaphylactic shock may be elicited by a variety of
injuries which can have nothing in common with a
true anaphylactic reaction. Zinsser suggests that the
reason for this may be that the union of the antigen
and its specific antibody on the surface of the body
cells causes a surface injury similar to that brought
about by trauma or other means.

From the above discussion it should be clear that

BACTERIAL INVASION 221

when any highly invasive microorganism gains ac-
cess to the body v^e may expect an analogous behavior
— that is, a cytost effect in some respects comparable
to that obtained in the experiments cited. Obviously
the severity of such an effect vsrill depend upon both
the cytost available for adsorption upon the bacteria
and the invasiveness of the infecting organism.
Further, if, as we have shown, the latter is capable
of causing the disintegration of tissue and thence lib-
erating cytost, the host is forced to cope with a three-
fold difficulty.

In previous chapters it has been shown that vari-
ous chronic disturbances, especially of the organs of
endodermal origin, may be brought about by the con-
tinued injection of sublethal quantities of homolo-
gous cytost. From this, it may be deduced that, by
the combined action of bacteria and small quantities
of homologous cytost for a protracted period of time,
similar chronic conditions may be induced. This is a
concept rather difficult to subject to experimental test,
since animals vary so in the rapidity of their response
to sublethal quantities of cytost. Nevertheless the
conclusion seems logical.

It is relatively easy to understand the importance
of all these considerations in the case of animals actu-
ally suffering from an acute infection, although at
first sight they do not appear to be of special impor-
tance in the life of an animal not suffering from infec-
tious disease. There are, however, a number of dis-
eases known to science which have all the earmarks
of being infectious, but for which up to the present
time no specific causative factor has been found. In
some instances such ailments may be traced to a non-

222 THE ACTION OF THE LIVING CELL

specific microorganism, while in others no micro-
organism appears to be concerned. In the first instance,
it is known that a number of unrelated microorgan-
isms may give rise to the same type of pathology.
This of course indicates that the particular patholo-
gies observed in such instances are due, not to the
specific activities of the microorganisms concerned,
but to a common factor resident within the host.

Such considerations suggested to the writer that
cytost might be this common factor. With this in
mind, experiments have been conducted towards this
end, and it has been found that several so-called non-
specific diseases may be simulated in animals by the
injection of cytost. These experiments, which have
been discussed elsewhere (see Chapter XIII), will
not be treated of here, since they are not of general in-
terest. In this connection, however, it should be noted
that the role played by the non-specific organisms fre-
quently associated with such diseases is probably akin
to that of B. coli in the experiments described earlier
in the chapter: that of an adsorbant which intensifies
the action of cytost.

So far we have considered the relation of bacterial
infection to cytost only from the standpoint of cytost
formation and intoxication. Passing now to a con-
sideration of the possible relationship between im-
munity to cytost and to disease, it is at once apparent
that in the case of infection by proteolytic micro-
organisms which can liberate cytost, the degree of
an animal's immunity to cytost will be a determining
factor of some importance in his offensive against the
invading bacteria. If, then, we consider all members
of a given population to have equal chances of infec-

BACTERIAL INVASION 223

tion, it is obvious that those individuals having the
greatest resistance towards cytost will be the best
equipped to offset the ravages of the microorganisms.
Consequently, the employment of any of the methods
discussed in the chapter on natural resistance, which
may raise an individual's resistance to cytost, may be
expected to result in an increased resistance towards
bacterial invasion.

It must be noted that such an increased resistance
is non-specific, and would not offer an immunity
towards specific toxins, such as diphtheria toxin.
Nevertheless, that cytost may play a role in the devel-
opment of specific immunity is indicated by some re-
cent experiments of Bresredka (1931), who found
that the application of friction to the shaved skin of
a rabbit was sufficient to endow the animal with a local
non-specific immunity towards bacterial toxins, which
lasted for from twelve to fourteen hours. If during
this period an ointment^ containing diphtheria toxin
is applied to the skin, the animals are found to develop
a specific immunity to the toxin. This is at first purely
local, and later becomes general.

In a previous chapter we have called attention to
the experiments of Lewis and his associates, which
demonstrated that prolonged gentle friction on the
unbroken skin leads to a localized reaction identical
with that which follows the intradermal injection of
tissue extracts. As previously stated, this indicates
that such gentle friction is sufficient to cause the liber-
ation of cytost.

Such being the case, it seems clear that the immune
reactions observed by Bresredka were due to the re-
lease of cytost, which was brought about by the rub-

224 THE ACTION OF THE LIVING CELL

bing of the animal's skin. This seems all the more
probable since he records that the immunity devel-
oped in this way is non-specific. Perhaps the specific
immunity which follows the inunction of the diph-
theria toxin is due to the combination of the toxin
with the non-specific anticytost or cytost whose forma-
tion is brought about by the mild injury attendant
upon the friction. Be this as it may, the experiments
of Bresredka make it appear highly probable that a
close relationship exists between cytost immunity and
bacterial immunity.

These considerations clearly justify the statements
of Zinsser (1931) that "Antibody production is after
all only the expression of underlying cellular activi-
ties," and, "The forces of natural resistance are normal
reactions." The writer feels that his investigations on
cytost and anticytost reactions lend considerable
weight to such views.

In a paper read before the Pan-American Congress
in 1901 and published in 1904, the writer directed
attention to this fact. To quote: "In another series of
experiments, I found that when an animal is stimu-
lated by heat applied within the splanchnic area by
methods previously described, immunity or resistance
against infection was thus produced. When the serum
of such an animal was injected into another animal
there was obtained an increased resistance or partial
immunity to infection. Animals so protected could
not always be saved but death from infection was re-
tarded, sometimes for one or two weeks." And again:
"These facts establish a most important point in the
pathology of shock, namely the alteration of the tissue
cells and blood in shock."

BACTERIAL INVASION 225

As shown early in the text, excessive heat applied
to the splanchnic area results in varying degrees of
shock which, as we have seen, is due to the liberation
of cytost from the tissues. The increased resistance
to infection which was observed in the animals so
treated must have been due to the cytost released into
the circulation by the thermal injury of the splanch-
nic tissues. In this respect then, these early experi-
ments of the writer are in harmony with the more
recent findings of Bresredka cited above. Indeed, as
one examines the recent writings of prominent im-
munologists, one becomes impressed by the fact that
the experts in this field are beginning to realize that
cellular products liberated as a result of tissue damage
are of paramount importance in the development of
immunity, both specific and non-specific. In this con-
nection, the reader will find much of interest in the
reviews of Manwaring (1930), Zinsser (1931), and
Irons (1931).

The localized formation of cytost due to the action
of bacteria in the tissues of a higher animal may lead
to the operation of other defense mechanisms. As is
well known, when a local infection resulting in abscess
formation takes place, a migration of phagocytes to
the site of infection occurs, and the body attempts to
protect itself against a generalized infection by wall-
ing off the infected tissues by the development of a
barrier of connective tissue.

The importance of both these types of defense
mechanism has been recognized for a long time, but
the manner in which they are brought into play is
obscure. The migration of leucocytes to a site of in-
fection is usually classed as a chemotactic response,

226 THE ACTION OF THE LIVING CELL

although in general little definite commitment has
been made as to the exact nature of this response. Such
a migration of leucocytes is found to take place fol-
lowing any kind of tissue injury, such as mechanical
mutilation or the application of irritating chemicals
such as chloroform or croton oil, as well as after bac-
terial infection. It would seem, therefore, that the
specific nature of the cause of injury is of no impor-
tance in determining the migration of the white cells,
but that an endocellular substance liberated from the
tissues is responsible for the chemotaxis.

It is possible that cytost may be such a substance,
for we have frequently observed an increased leu-
cocyte count in animals at short intervals following
the injection of extracts of autolyzed tissue. This ob-
servation suggests that the migration of leucocytes to
the site of infection is brought about by the cytost
liberated from the tissues of the host by the activities of
the invading organisms.

We have shown elsewhere in the text that when
present in appropriate concentration cytost stimulates
the rate of proliferation of cells. With this in mind,
it seems likely that the connective tissue barrier which
is thrown about a localized infection may result from
the stimulus afiforded by the cytost liberated by bac-
terial action.

From the above it should be clear that as a result
of bacterial infection cytost may be liberated, and in
consequence the animal may sufifer from a cytost in-
toxication as well as from the specific disease en-
gendered by the bacteria. The liberation of cytost in
this manner may cause serious consequences, or it may
stimulate the defense mechanisms of the host in such

BACTERIAL INVASION 227

a fashion that an effective barrier to generalized infec-
tion results.

This conclusion may appear paradoxical, but it is
no more so than the well established relationship be-
tween toxins and the formation of their specific anti-
toxins.

The colon of higher animals always contains large
numbers of various bacteria; indeed, it has been esti-
mated that from 5 to 20 per cent of the dry weight of
the fecal mass consists of bacteria. Under normal
conditions the bacteria present in the colon do not
appear to cause the host difficulties of any sort. Oc-
casionally, however, these organisms do gain access
to the body and infection results. Many years ago the
writer had occasion to investigate this matter. It was
found, by the microscopic examination of appropri-
ately stained sections of the intestinal tract of animals
which had been fed cultures of B. colt, that the bac-
teria may diffuse into the intestinal wall, passing be-
tween the epithelial cells and between the muscle cells
of the muscular mucosa into the areola tissue. (Turck,
1909, 1910.) Later it was found that this process could
be followed very easily in fetal animals after injection
of bacterial cultures into the intestine. (Turck, 1908,
1914.)

In all instances it was observed that as the diffusing
bacteria entered the submucous zone rapid bacterioly-
sis took place. In consequence, this zone of bacterial
destruction was named the "Zona Transformans."
Presumably it is the zone which normally protects an
animal from infection by the organisms present in the
intestinal tract. If for any reason the Zona Trans-
formans ceases to function in its normal capacity, then

228 THE ACTION OF THE LIVING CELL

it ceases to exercise its protective function and the
varied intestinal flora may gain access to the body.

When shock is induced in animals it is found that
the nature of the intestinal wall is so altered that it be-
comes readily pervious to bacteria. Thus the abdomen
of animals under ether anesthesia was exposed to an
air blast until marked venous stasis and shock were
produced. After recovery from the latter, some of
the animals were fed cultures of B. colt. A day later
sections were obtained from the intestine. Both in
animals that had been fed the cultures of bacteria and
those which had not, it was found that bacterial inva-
sion had taken place.

A similar result was obtained with animals which
had been brought into shock by prolonged anesthesia.
(Turck, 1914, 1917.) Curiously enough, the bacterial
invasion was not accompanied by leucocytic infiltra-
tion or any other evidences of an inflammatory proc-
ess.

The essential point to be noted here is that inter-
ference with the splanchnic circulation which always
occurs in shock lessens the ability of the Zona Trans-
formans to cause the bacteriolysis of the invading
organisms. It follows from this, and the fact that
cytost intoxication always leads to a greater or lesser
degree of splanchnic stasis, that such intoxication must
be regarded as a predisposing cause of infection by
way of the intestinal route.

The bacteriolysis which we have found to take place
in the Zona Transf ormans opens the way to an under-
standing of the diffusion or non-dififusion of other
substances through the walls of the intestine. As is
well known, protein molecules do not pass through

BACTERIAL INVASION 229

the intestines in most individuals. Since, however,
the size of the protein molecule or micelle is very con-
siderably less than that of the bacteria used in the
author's experiments, one might expect them to dif-
fuse even more readily. When it is remembered that
the process of bacteriolysis may involve the action of
proteolytic enzymes, it seems likely that the non-
absorption of unhydrolyzed protein is possibly due
to the proteolytic activity of the Zona Transformans.

Some individuals are sensitive to certain foodstuffs
w^hich upon ingestion give rise to anaphylactic reac-
tions of one sort or another. It would seem that this
effect must be due to the absorption of specific un-
hydrolyzed protein which for some reason or other
succeeds in passing the Zona Transformans in the same
fashion in which we have found bacterial invasion
to take place in animals wherein the shock was pro-
duced.

At this time we do not wish to stress the medical as-
pects of these concepts, but it may not be amiss to
point out that the treatmentof infectious diseases might
be more intelligently prosecuted if the ideas devel-
oped in this chapter were kept in mind by the practi-
tioner. By this we do not wish to imply that the use
of specific antisera and medication should be super-
seded, but rather that such methods of proven value
might reasonably be supplemented by such methods
as lead to a lessened susceptibility to cytost. Those
interested in the writer's conclusions in this regard
should consult Chapter XIII, wherein this topic is
more fully discussed.

Chapter XI

INVESTIGATIONS CONCERNING THE
NATURE OF CYTOST

It has been pointed out in previous discussion that
extracts of autolyzed tissue, if boiled for some time,
maintain unimpaired their toxicity for animals. This
indicates that the substance in such extracts which is
responsible for the various effects described in pre-
ceding chapters is quite stable. Furthermore, if solu-
tions of cytost are made either alkaline or acid and
then boiled, the toxic substance is unaffected. Conse-
quently its molecule cannot be readily hydrolyzable,
as is that of a simple peptide.

Sterile solutions of cytost have been kept in sealed
ampules for years without any apparent loss in ac-
tivity, and dry samples of tissues and tissue extracts
likewise maintain their toxic nature.

Recently confirmatory evidence of the extraordi-
nary stability of cytost has come from a quite unex-
pected source. The noted archeologist, Flinders
Petrie (1932), in his book Seventy Years in Arche-
ology, records that contact with the mummy dust
caused the onset of various respiratory disturbances,
headaches, and colds. Two statements of Petrie are
illuminating (page 32) : "All work in the pyramid

230

NATURE OF CYTOST 231

was unwholesome, owing to the dust raised which al-
ways produced feverish headaches after a few hours
of it." And (page 94) "The efifect of being surrounded
with so much organic dust from the mummies was to
give me a bad infection of the breathing passages."

Now it may be recalled that we have found that
the endodermal cells of the lung are peculiarly sensi-
tive to cytost and that various respiratory disturbances
varying from sneezing to a true pneumonia could be
induced by the introduction of minute quantities of
cytost into the respiratory tract of animals. This re-
sult was accomplished by the simple expedient of
spraying the cages of animals with a potent extract
of autolyzed tissue as well as by coating the paws of
cats with a paste containing cytost.

It would seem that the discomfort experienced by
Petrie and his associates when in contact with the
mummy dust was essentially the same as that experi-
enced by the cats whose cages had been sprayed with
cytost. One may raise the argument that exposure
to any atmosphere containing finely particulate mat-
ter will cause some respiratory distress. This of course
is true, but in general unless the dust contains some
toxic substance its effects are purely mechanical and
pass away very shortly after the exposure.

Such, however, is not the case with cytost, for, as
our experiments have shown, cytost-laden air induces
pathological conditions which last for some time. In
this connection, it is of interest to note that Petrie com-
plains that the mummy dust caused headaches and
"colds" which prevented further work for a few days,
and this is what we should expect from exposure to
cytost, but not from exposure to a dry, non-toxic dust.

232 THE ACTION OF THE LIVING CELL

There have been a number of deaths among those
engaged in the excavation of ancient tombs, and it is
said that the Arabs consider this as the vengeance of
the dead upon the excavators. Be this as it may, the
observations just mentioned indicate that the dead
may retain their cytost intact for centuries, and one
might reasonably imagine that some of the deaths
among the archeological explorers have been due to
overexposure to the cytost-laden dust of which Petrie
speaks.

To return to the experimental aspect of the prob-
lem, we have noted in our discussion of the induction
of shock by severe burns that extracts of the charred
tissue, when injected into other animals, caused shock.
This observation suggested that the tissue might be
charred in vitro with the same results, and this was
subsequently confirmed by experiment. Naturally
this finding evoked an interest in the determination
of the degree to which charring could be carried with-
out loss of cytost activity. To our amazement, it was
found that excised tissue could be burnt to a gray
ash without greatly diminishing its potency.

Such an ash when dissolved in water to form a 10%
solution and injected intravenously into animals
caused shock and death, provided a sufficient quantity
was injected. Thus, 1 cc. of a 10% solution of the ash
obtained from cats' muscles, when injected in this
manner into cats weighing approximately 2500 grams,
will always cause the immediate onset of shock and
usually produce death within a very brief time after
the injection.

In order to obtain tissue ash of such toxicity, it is
necessary that the tissue be fired at relatively low tern-

NATURE OF CYTOST 233

peratures — in the neighborhood of 300° C, for at
higher temperatures the ash loses its toxicity. It be-
comes practically inactive if fired at 700° C. in an
electric furnace. In the experiments discussed below,
the various tissues were always ashed at a temperature
approximating 300° C.

In order that we might compare the toxicity of
various cytost preparations, we have adopted as a
convenient reference standard a solution containing
0. 11 2 gm. of ash in 1 cc. This was prepared by extract-
ing a weighed quantity of a given ash with sufficient
water to yield a solution of this concentration, boiling
for a few minutes, and then removing the slight quan-
tity of insoluble material by filtration.

Various quantities of a solution of the ash of guinea
pig muscle prepared in this way were injected sub-
cutaneously into a large number of guinea pigs. As
much as 1.5 cc. of the solution per 100 grams was in-
jected in this manner without the occurrence of a
single death.

Animals which received such large injections were
ill for a day or two following the injection, but made
uneventful recoveries within a few days, although the
skin sloughed off at the site of injection.

Much more striking results were obtained by In-
jecting the solution of the ash intraperitoneally.
When injected in this manner, %6 cc. of the solution
proved lethal to all of 10 guinea pigs which received
this quantity. A few minutes after the injection the
coats of these animals appeared ruffled, and their res-
piration rate increased. Many of them vomited, and
all died within from IS minutes to 2 hours following
the injection of the cytost. Slightly larger doses — i.e.,

234 THE ACTION OF THE LIVING CELL

/

% cc. — always brought about death within fifteen
minutes, and in many instances within several seconds
following the injection. At autopsy, immediately fol-
lowing death, all the animals killed in this way ex-
hibited the typical pathology of "death by shock" ; the
lungs and abdominal viscera were always found to
be markedly congested.

This extreme rapidity of the onset of symptoms,
and death, shows that the ash itself must be toxic, and
that the typical cytost efifect obtained is due to the ash
and not to the liberation of cytost from the animal's
own tissues by the action of the ash thereon. Further
evidence for this argument is found in the marked dif-
ference in the response of the animals to the ash solu-
tion when injected subcutaneously. In the latter case
the sloughing of the skin at the site of injection shows
that the ash may act locally, causing the death of tis-
sues. The rate of absorption, however, is consider-
ably less than in the case of the intraperitoneal injec-
tions; hence the viscera are not suddenly exposed to
a sufficiently great concentration of cytost to cause
congestion, stagnation, and death.

Similar experiments were carried out upon rats
and cats with the ash of homologous tissues, and quali-
tatively the experimental findings were essentially the
same.

We have stated earlier that saline extracts of auto-
lyzed tissue appear to have a certain species specifi-
city when the power to elicit shock is determined.
Naturally, this raised the question as to whether or
not the tissue ash would show a similar specificity in
this regard. To this end experiments, of which the
following are representative, were conducted:

NATURE OF CYTOST 235

A solution of the ash obtained from cats' muscles
was prepared, containing 0.112 gm. of the ash per
1 cc. of solution. This was injected intraperitoneally
into guinea pigs in order to determine the minimal
lethal dose of the solution. Proceeding in this fashion,
it was found that the minimal lethal dose of the solu-
tion when injected intraperitoneally was from ^ to
Yi cc. per 100 gms. The lesser quantity caused the
death of 50% of the injected guinea pigs within 48
hours, and 1^% within 72 hours, whereas the higher
quantity always caused death within 15 minutes. In
consequence, it must be concluded that qualitatively
the guinea pig and cat tissue ash both contain some-
thing toxic to guinea pigs. When, however, one com-
pares the minimal lethal doses of the two solutions, it
is noticed that on a weight for weight basis the solu-
tion of guinea pig tissue ash is more toxic for guinea
pigs than is the solution of cat tissue ash.

This is made evident by comparing the quantities
of the two solutions which are necessary to cause the
death of guinea pigs within fifteen minutes. In this
respect, Yi cc. of the cat tissue ash solution contain-
ing 0.056 gm. of ash is equivalent to ^ cc. of the solu-
tion of guinea pig ash containing 0.028 gm. of ash.
Considering the weights of the ash involved, it is ap-
parent that the homologous ash is twice as potent as
is the heterologous.

The same result was obtained when the ash of beef,
dog, and rat tissue was substituted for the cat tissue
ash used in the above experiments. In each instance
it was found that solutions of those ashes were all capa-
ble of causing the death of guinea pigs when injected
intraperitoneally, but that the amounts necessary to

236 THE ACTION OF THE LIVING CELL

cause death within fifteen to twenty minutes was al-
ways greater than in the case of guinea pig tissue ash.

Such differences in the toxicity of the tissue ash of
various species was also observed when they were in-
jected intravenously, the route which causes the most
rapid onset of shock. For comparative purposes 10%
solutions, in normal saline, of the ashes of various ani-
mal tissues were employed and were injected into the
jugular veins of the experimental animals.

When injected in this fashion into rats weighing
150-200 gms., a 10% solution of the ash of rat tissues
was found to cause the onset of shock and death in from
5 seconds to 2 minutes. When rats of the same size
received }i cc. of a 10% solution of cat tissue ash,
they developed a slight shock, but usually recovered
in about 10 minutes. This result shows definitely the
existence of a species specificity in the ash.

A large number of similar experiments were carried
out with cats, using 10% solutions of the ash obtained
from rat, horse, beef, and lion tissues. When 1 cc. of
such a solution of homologous tissue ash was injected
into cats weighing from 2500 to 3000 gms., it was
found that the animals always passed into severe shock
and frequently died immediately. When such was not
the case, the cats always died within two or three days
after the intravenous injection of this quantity of cat
tissue ash. When like quantities of the tissue ash of
horse, dog, beef, or lion muscle were substituted for
the cat ash solution, the injected cats of similar weight
often showed no ill effects whatsoever. At other times
they developed a mild shock from which they made
uneventful recovery within less than an hour.

For some curious reason the ash obtained from rat

NATURE OF CYTOST 237

tissues was remarkably toxic to cats. Three cats
weighing respectively 2790, 2115, and 2470 grams
died within less than a minute after the intravenous
injection of 1 cc. of a 10% solution of rat muscle ash,
while a fourth cat, weighing 2850 gms., developed a
mild shock from which it recovered in 10 minutes,
and remained in good health for two weeks, when it
was used in another type of experiment.

The species specificity which is evidenced in these
experiments is also brought out by the reaction of cats
to intraspinal injections of solutions of homologous
and heterologous tissue ash. Ten per cent solutions
of the various ashes were injected intraspinally into
cats. The solutions of homologous tissue ash when
administered in this fashion caused shock, paralysis of
the hind limbs, and in some instances death, whereas
equal quantities of the ash of heterologous tissues
caused only slight transitory symptoms from which
the animals promptly recovered. The results of repre-
sentative experiments of this type are recorded in
Table XIV. (Turck, 1921.)

These experiments again show the same type of
species specificity observed in the other experiments.
While 0.25 cc. of the solution of cat ash equivalent to
0.025 gm. of ash evokes distinct reaction, four times
this amount of the ash from human, horse, and lion
tissues causes the recipients but little inconvenience.
It is especially interesting to note that of the three
heterologous ashes employed in equal quantities, the
ash from lion tissue caused the most distinct reaction,
although this was far less severe than that caused by
an equivalent quantity of cat ash. Phylogenetically
the lion is closely related to the cat; hence this find-

238 THE ACTION OF THE LIVING CELL

TABLE XIV
Effects of Intraspinal Injections of Ash Solutions

Cat No.

Ash

Amount
Injected

Resot-t

149

Cat

I.O cc.

Deep shock for 30 minutes. Complete
paralysis of hindquarters. Died in
8 days.

iSS

Cat

I.OCC.

Deep shock, prolonged coma. Died in
12 hours.

158

Cat

0.25 cc.

Slight shock, paralysis of hindquarters.
Died in 7 days.

192

Cat

0.25 cc.

Deep shock, delirium, marked incoor-
dination. Recovered.

150

Human

1.0 cc.

Slight delirium only. Recovered in
30 minutes.

157

Horse

1.0 cc.

No evidence of any reaction.

i6r

Lion

1.0 cc.

Very slight shock. Recovered com-
pletely in I hour.

180

Lion

0.25 cc.

Marked delirium. Recovered in 30
minutes.

All the injections were made in the last lumbar space.

ing suggests the existence of a possible similarity be-
tween the composition of the ash of closely related
species. (See Figure 15, facing page 262.)

One must not, however, confound the apparent spe-
cies specificity of the various ashes with the remark-
able protein specificity of different species which has
been so carefully investigated by Nuttal (1904) in
his studies on the correspondence between immuno-
logical relationships and accepted zoological classi-
fications. Thus we have shown that the ash obtained
from rat tissues, when injected intravenously, is about
as effective as cat tissue ash in causing shock and death.
Further, in the experiments on guinea pigs it was
shown that following intraperitoneal injection all

NATURE OF CYTOST 239

the heterologous ashes could cause death, provided
the dosage was sufficiently high. The relative species
specificity became strikingly apparent only when we
compared the minimal lethal doses of the heterolo-
gous ashes with that of the homologous ash.

It follows from this that the apparent species spe-
cificity which we have observed in our experiments
is due not to a particular component of the ash unique
to a given species, but rather to the quantitative dis-
tribution of a component common to the ash of several
species. Thus if it be assumed that a certain concen-
tration of this component per unit weight of ash is
necessary to elicit in the cat the various symptoms
which follow injection, it may be imagined that the
concentration of this component in the ash of rat tis-
sues is comparable to that which exists in the ash of
cat tissue, in consequence of which solutions of the
same strength of these two ashes will be about equally
toxic when administered to cats by any of the routes
we have mentioned above. Conversely, we may as-
sume the ash of other heterologous tissues which we
have used in our experiments to contain less of the
active component than cat or rat tissues; hence when
injected into cats a larger dosage of such ashes will
be necessary to evoke reactions comparable to those
obtained with cat and rat tissues.

This concept is purely speculative, but is sugges-
tive of further experiments. Thus, if it is true, one
should be able to ascertain a quantitative relation-
ship between the toxicity of a series of heterologous
ashes for animals of different species.

As yet we have no inkling as to the nature of the
toxic component present in tissue ash. One is strongly

240 THE ACTION OF THE LIVING CELL

tempted to postulate that it is inorganic in nature,
since it resists the temperatures incident to ashing.
Our experiments have shown this to be unlikely, how-
ever, for, as mentioned earlier in this chapter, the
ash may be inactivated if heated to too high a tem-
perature during the ashing procedure. While very
high temperatures might lead to the slow volatiliza-
tion of some inorganic compounds, it is highly im-
probable that this would be great enough to cause
the complete loss of an inorganic compound which
determined the toxicity of the ash. Furthermore, in-
jection of various salts of the metallic elements com-
monly found in tissue ash does not elicit shock in ani-
mals; hence it seems that such substances cannot be
responsible for the effects observed in the experiments
we have discussed above.

Since the ordinary methods of chemical analysis
might fail to detect the presence of minute quantities
of various elements present in the ash, we have sub-
jected various samples of the active ash to spectro-
graphic examination. This was carried out with a
Hilger quartz spectrograph and by the arc method.
For the most part high purity copper was used as
electrodes, although in some instances aluminum and
carbon were substituted. Typical spectrograms of the
ash obtained in this manner are shown in the accom-
panying spectrographs, wherein the lines corre-
sponding to particular elements have been marked
with their respective symbols.

Ash obtained from the tissues of the cow, cat, hen,
fox, guinea pig, horse, lion, rabbit, rat, dog, and human
were examined. Examination of the spectrograms
discloses the presence of sodium, potassium, calcium,

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NATURE OF CYTOST 241

magnesium, and phosphorus in all samples examined.
Rubidium was present in all the tissues except that
of the dog. Traces of boron were found present in
the ash of rabbit, rat, dog, and human tissues, traces
of lead in the lion tissue, and traces of zinc in the
fox tissue, and of aluminum in the cow and horse
tissues.

While these findings were of no especial interest in
connection with the cytost problem, the presence of
rubidium in all the tissues examined excepting those
of the dog is interesting. So far as the writer is aware,
no biochemical significance has been attached to this
element, although various investigators have from
time to time noted that rubidium salts may be substi-
tuted for those of potassium in so-called physiologi-
cal balanced salt solutions. The traces of heavy metals
found in some of the tissues examined are probably
attributable to the retention of metals derived from
the utensils used in feeding and watering the animals.

For the present we must conclude that when tissues
are ashed at low temperatures in the neighborhood
of 300° C, they retain some markedly thermostable
organic constituent which is responsible for the physi-
ological reactions which follow the injection of solu-
tions of the tissue ash into animals. As we have men-
tioned before, heating of the tissue ash of all species
to 700° in an electric furnace causes the ash to lose
its toxic properties. This is exactly what one would
expect if the toxic moiety present in the ash were really
an organic compound.

While, as was pointed out at the beginning of the
text, we are in complete ignorance as to the exact
chemical nature of cytost, the facts stated above testify

242 THE ACTION OF THE LIVING CELL

to its unique nature and will perhaps serve as an ori-
entation for any chemically minded investigator who
wishes to extend our experiments. At any rate, the
study of the effects of tissue ash upon the activities
of cells and tissues appears to offer an interesting field
of experimentation.

In this connection it is of interest to note the recent
investigations of Alexander, Weaver, and McConnell
(1930), who found that the size of wheals caused by
the subcutaneous injection of histamine was consid-
erably enlarged if the latter were mixed with an aque-
ous extract of skin prior to the injection. In a later
paper the same authors report that the substance in
the skin which augments histamine wheals resists ash-
ing temperatures, and on the basis of their experi-
ments they suggest that it is calcium sulphate.
(Weaver, McConnell, and Alexander, 1931.)
Further, they found that the wheal forming powers
of atropine and codeine are likewise augmented by
skin extracts, and they state that it is likely that con-
stituents of the ash other than calcium are capable
of augmenting the localized action of histamine.

At first glance these conclusions may not appear
relevant to our investigations. But if the reader will
recall that it has been pointed out previously in con-
nection with our discussion of shock that extracts of
autolyzed tissue possess a histamine-like action, this
discussion may strike a responsive chord. Various
considerations discussed in earlier chapters have led
to the conclusion that cytost is always present to a
greater or lesser extent in the normal animal. Such
being the case, it might be imagined that the introduc-
tion of tissue ash into the circulation of an animal

NATURE OF CYTOST 243

brings about an intensification of the action of cytost
already present in much the same fashion as that in
which Weaver and his associates have found the ash
to intensify the localized action of histamine.

While this is an hypothesis vs^hich merits serious
consideration, the author feels that it is incorrect.
This objection is based upon the observation previ-
ously mentioned that ignition of a tissue at 700° C.
leads to the formation of a virtually non-toxic ash.
Such an ash must contain all the inorganic substances
which were present in the original tissue. Conse-
quently the toxicity of the ash obtained at lower tem-
peratures cannot be due solely to the augmenting
effects of such inorganic substances upon the cytost al-
ready present in the tissues of the experimental animal.
It is obvious that further experimentation will be
necessary to settle this point definitely.

Chapter XII

CYTOST IN THE PLANT WORLD

In the preface the writer has drawn attention to his
earliest experiments with plants, conducted over forty
years ago. At that time nothing was known of the
role of cytost in animal physiology. In view of the
experiments with animals it is interesting to recall
these early experiments, for, curiously enough, they
are capable of interpretation from the standpoint of
the cytost hypothesis.

For many years it has been generally recognized
that plants cannot be grown in the same soil for an
indefinite succession of years, a fact commonly ex-
pressed in the agriculturists' "law of the minimum,"
which states that as soon as the concentration of cer-
tain elements in the soil has reached a minimum level,
plants are unable to withdraw those elements in a
quantity sufficient to permit growth. If, however,
such elements are added to the soil, normal growth of
the plants ensues. This is the essence of the well-
known effect of commercial fertilizers on plant
growth.

In the author's experiments referred to above it was
observed that upon the addition of rich virgin soil
to exhausted earth, healthy growth of transplanted

244

CYTOST IN THE PLANT WORLD 245

fronds did not ensue until the ratio of virgin soil to
exhausted soil exceeded one half or two thirds. Of
itself this observation is of no moment; but inci-
dent to the comparison of growth in various mixtures
of exhausted and virgin soil it was observed that trans-
planted fronds grew more rapidly in a mixture of vir-
gin soil with 5 to 10 per cent of the exhausted soil
than in the virgin soil alone. This curious observa-
tion, which went unexplained for some years, may be
readily understood if viewed in the light of our ex-
periments with animal cells.

It will be recalled that in the latter case it was
found that small quantities of homologous cytost ap-
pear to stimulate the anabolic metabolism and growth
of cells, whereas higher concentrations of the same
substance exert a distinctly toxic action. Transfer-
ring this concept to the plant world, we may reason-
ably assume that the accelerated growth of plants
in the soil mixtures containing small quantities of the
exhausted soil is due to the fact that the latter con-
tains homologous cytost, which stimulates the growth
of the plant cells.

Again, as noted above, considerable virgin soil must
be added to exhausted soil in order to render it capa-
ble of supporting a rich growth. Much smaller quan-
tities of the virgin soil should suffice to replenish the
exhausted essential elements ; hence we must conclude
that the high ratios of virgin soil found necessary to
stimulate plant growth play a dual role. Aside from
supplying essential elements, it must have acted as a
diluent for toxic products present in the exhausted
earth. That is, by the addition of the virgin soil, the
concentration of cytost present in the exhausted soil

246 THE ACTION OF THE LIVING CELL

must have been lowered to a level compatible with the
growth of the plant.

Recently H. E. Dolk and K. V. Thimann (1932)
have been able to isolate a substance capable of accel-
erating plant growth by culturing Rhizopus suinus in
such manner that a constant stream of culture fluid
passes through the vessels in which growth takes place.
The continual passage of the culture fluid through
the apparatus washes away the "growth hormone,"
as it is formed. From their experiments the growth-
accelerating substance which they obtained appears
to be an organic acid easily susceptible to oxidation,
whereby it loses its growth-accelerating activity. This
easy destructibility by oxidation indicates that this
substance differs markedly from the active principle
present in the various cytost preparations used in the
author's experiments. But, as has been pointed out
previously, in the case of animal cytost a marked
species specificity appears to exist. Fundamentally
such species specificity must depend upon differences
in the chemical constitution of the various cytosts. In
consequence, there is no reason why the chemical con-
stitution of plant cytost or "growth hormones" should
not vary markedly from that of the corresponding
animal compounds.

Unfortunately, since the development of the cytost
theory the author has not had available the facilities of
a greenhouse wherein further experiments with plant
cytost could be made. Nevertheless a considerable
literature has accumulated in recent years which indi-
cates that endocellular products such as cytost are
of importance in plant physiology.

In the first chapter attention was drawn to the fact

CYTOST IN THE PLANT WORLD 247

that phototropic, stereotropic and chemotropic re-
sponses in plants depended upon the release of some
endocellular agent by light, contact, pressure, or cau-
terization. This view was championed by the late
Jacques Loeb and the earlier literature on this sub-
ject is reviewed in his book Forced Movements,
Tropisms and Animal Conduct. (1918.) More re-
cently, Cholodny (1927) has reviewed this problem,
with particular reference to plants. He points out
that all the various tropistic responses of plants, which
are essentially growth changes, can best be explained
upon the assumption that, due to pressure, injury, or
light, the plant cells give rise to a "Wachshormone"
which, by diffusion to various parts of the plant,
brings about alterations in growth, the asymmetrical
growth characteristic of tropic curvatures being due
to an uneven distribution of the growth-promoting
substance.

A few years ago Paal (1918) presented evidence
for the presence of such a substance in the tips of
coleoptiles of grasses. Later Went (1928a) succeeded
in extracting such substances from the coleoptile tips
and devised a quantitative method of estimating the
activity of such extracts in accelerating the growth
of plants. The latter author (Went, 1928b) has also
demonstrated that the quantitative differences in
growth on the two sides of a seedling which follow
unilateral illumination must be due to changes in the
concentration of the growth-promoting substance on
the illuminated side.

When plants are subjected to continual friction,
fungus infection, or chemical irritants, they frequently
give rise to pathological outgrowths or intumescences.

248 THE ACTION OF THE LIVING CELL

Wallace has recently made a careful histological ex-
amination of such outgrowths, following the action of
ethylene on the buds and stems of the apple, Pyrus
Malus, Var. Transparent. Under the action of 1%
ethylene in air, it was observed that the walls of the
cells were corroded away by a sort of solution process
(autolysis) induced by the ethylene. Individual cells
and groups of cells then separated from the tissues.
This process involves all the living elements between
the cambium and phellogen, and the cells may be
stimulated to division or become hypertrophied. In
some instances cell counts in the intumescences pro-
duced at the ends of cutting showed a thirty-three
per cent increment in numbers.

It is interesting to compare these findings of Wal-
lace with the writer's observations on the prolonged
irritation of the gastric mucosa by mustard. In the
latter case, it may be remembered that a somewhat
analogous situation was found, the mustard oils first
causing a degeneration and loosening of the mucosal
cells, whereas later even though the mustard was re-
moved, the underlying tissues were found to show
evidences of active proliferation. It was deduced
(page 33) from the experimental findings that the
proliferation and other changes were secondary
changes which arose not from the direct action of the
mustard but because of the presence of cytost lib-
erated from the mucosal cells injured by the mus-
tard.

If we transfer this concept to the problem of plant
intumescences, we may imagine the phenomena ob-
served by Wallace to take place as follows : The plant
cells in contact with the ethylene suffer injury in much

CYTOST IN THE PLANT WORLD 249

the same fashion as the mucosal cells in contact with
mustard. As in the latter instance, the injury leads to
autolysis and the consequent liberation of cytost. This
substance in turn attacks adjacent cells, thereby giv-
ing rise to the "solution efifect" described by Wallace,
and those cells not exposed to too high a concentration
of the plant cytost are stimulated to division.

Conceivably any other injury capable of liberating
cytost from the plant tissues should bring about a
similar situation. This is testified to by the well known
fact mentioned above, that the stimulus to intumes-
cence formation may take the form of abrasion, fun-
gus infection or chemical substances; although so far
as the writer has been able to ascertain the effects of
such stimuli have not been examined histologically
in a manner such as Wallace employed in his study
of the action of ethylene. This would appear to open
an interesting field of study and it is hoped that such
investigations will be carried out.

The localized stimulation of growth which takes
place in the formation of intumescences is presum-
ably identical in nature with the various growth proc-
esses which accompany the tropistic bendings of
plants under various physical influences, and it may
be worth while to consider a few of these. The coil-
ing of the tendrils of phanerogams, the most sensitive
of all plant structures, has long been of interest to
botanists. The investigations of this curious phe-
nomenon by Darwin (1882) andPfeffer (1885) have
thrown considerable light upon the conditions pre-
requisite for such coiling. At the outset it was deter-
mined that tendrils are stimulated to depart from a
linear path of growth by contact with a solid body

250 THE ACTION OF THE LIVING CELL

such as a stick or plant stock. This of course sug-
gested frictional irritation as the factor responsible
for the onset of a more or less circuitous growth path.
Experiments, notably those of Pfeffer, disclosed that
contact with any rigid body suffices to induce ten-
dril-coiling, but that contact with moist gelatin, water,
oil, or mercury did not lead to a similar thigmotropic
response. Further experiments of Peirce (1894)
showed that, while contact with glass rods led to thig-
motropy, such was not the case if the rods were cov-
ered with a layer of moist gelatin. On the other hand
if sand is incorporated into the gelatin, then a normal
coiling response ensues. Peirce found further that
dry gelatin stimulated the coiling of tendrils in much
the same fashion as did any other solid body; there-
fore the failure of the gelatin coated rods to excite
such coiling cannot be attributed to any specific chem-
ical character of the gelatin.

In consequence it must be concluded that the sur-
face of the gel covered rods was not possessed of suffi-
cient rigidity to permit the development of the degree
of friction necessary to excite the usual thigmotropic
response.

It seems reasonable to assume that such frictional
force must attain a certain minimum value in order
that the surface cells may become sufficiently irritated
to discharge their cytost. In this connection it is of
interest to recall the experiments of Sir Thomas Lewis
and his collaborators on the production of a skin tache
and subsequent wheals by stroking the forearm with
a blunt instrument. A considerable amount of such
stroking is necessary in order to elicit capillary dila-
tation and subsequent wheal formation. Nor is this

CYTOST IN THE PLANT WORLD 251

surprising, since the human skin is constantly sub-
jected to mild friction from clothing, etc. In other
words, from Lewis's experiments it appears that the
degree of friction is important in determining the ex-
tent of the cellular irritation which leads to the dis-
charge of the endocellular substance responsible for
wheal formation. There seems to be no valid reason
why a similar state of affairs should not exist in the
plant, although of course the cells of the tendrils must
be considerably more sensitive to friction than are
those of the human skin. Indeed, it is recorded by
MacDougal (1896) that the tendrils of Echinocystis
will respond to the frictional forces incident to con-
tact with the delicate thread of a spider's web.

Similar stereotropic responses in the roots of seed-
lings have been observed by Sachs (1873) and many
later investigators. This author found that when a
pin was placed in contact with the rapidly growing
-roots of various seedlings the growing region became
concave within about ten hours. More recent investi-
gations have disclosed that in such stereotropic re-
sponses the actual growth processes leading to the
tropic curvature takes place, not at the site of contact
between the stimulus and the growing root tip, but
at a short distance from it. In this respect the be-
havior is comparable to that which obtains in photo-
tropic bending.

Curiously enough, however, it is only the delicate
growing root tip which is able upon contact to give
rise to the growth-promoting substance — a phenome-
non comparable to the observation that decapitated
coleoptile tips do not respond to light. In this con-
nection the experiments of Cholodny (1926) are il-

252 THE ACTION OF THE LIVING CELL

luminating. This author found that when the root
tips of Zea were decapitated stereotropic responses
could not be elicited. When, however, coleoptile tips
from the same plant were transplanted to the decapi-
tated root tips, restoration of stereotropism followed.
Conversely, when the root tips were placed upon the
decapitated coloeptiles, it was found that restoration
of growth ensued.

These ingenious experiments show conclusively that
the sensitive tissues of both coleoptile and root tips
are capable of elaborating a substance which controls
the growth of plant tissues, and the inference follows
that in both instances the growth-stimulating sub-
stance is the same. If this be accepted, then it is clear
that both phototropic and stereotropic behavior de-
pend upon a common factor, a substance liberated
from the sensitive tissues by the external physical
stimulus.

Traumotropism, or responses to wounding, is closely
akin to thigmatropism, and this we should expect, on
the basis of the cytost hypothesis; for, as has been
shown, in the case of animals the same cell substance
appears to be liberated regardless of the agency em-
ployed to bring about the injury of the cell. Long
ago Darwin ( 1 88 1 ) injured bean radicles by the local-
ized application of silver nitrate. Within a day the
radicles exhibited a marked curvature away from the
wounded side. A variety of other caustics, such as
copper sulphate and potassium hydroxide, have been
found to lead to the same type of response; and
Spaulding (1894) achieved the same end by the cut-
ting of a thin slice from the tip of the radicle, and by
allowing steam to strike on one side only.

CYTOST IN THE PLANT WORLD 253

As a result of these experiments Spaulding con-
cluded that traumotropism follows the application
of any agent which irritates strongly without killing
the plant. This conclusion coincides nicely with the
deductions made by the author as a result of his ex-
periments with animals.

Numerous investigations by various botanists have
shown conclusively that the actual growth process
which accompanies the phototropic, thigmotropic,
and traumotropic responses of plants take place, not
at the site of injury, but at some distance from it, due
presumably to the diffusion of cytost through the
plant. In this respect, then, we find a rather crude
analogy to the behavior of cytost in the animal body,
where, it will be remembered, the distinctive action
of cytost becomes manifest in the splanchnic area at a
considerable distance from the original site of in-
jury.

Loeb (1924) in his exhaustive experiments on re-
generation in Bryophyllum calycinum has shown that
a quantitative relationship exists between the amount
of regenerated shoots and roots and the mass of leaves
and stems exposed to illumination. This was to be ex-
pected, for the mass of material employed in the
regenerative process must arise from the photosyn-
thetic activities of the leaves. Loeb accounted for the
fact that mutilation leads to growth in portions of the
organism where no growth would have occurred
otherwise by assuming that, following mutilation, an
increased concentration of sap took place at the site
of the injury. These conclusions of Loeb's are satis-
factory in so far as they account for the accretion of
the material necessary for the new growth, but they

254 THE ACTION OF THE LIVING CELL

do not explain why the new growth follows. In other
words, given an adequate supply of the basic sub-
stances necessary for the formation of new plant tis-
sue, one must also account for their utilization in
the formation of new cells — that is, for the excitant
that leads to cellular multiplication at the site of
injury.

In our discussion of the tropistic responses of plants
we have seen that injury apparently leads to the liber-
ation of a growth-promoting substance, presumably
derived from the injured cells. Hence one is tempted
to postulate that it is such a substance which is liber-
ated from mutilated tissue that leads to regeneration
at the site of injury. The accretion of sap at this point
would tend to hinder the diffusion of the growth ex-
citant from the site of injury, and thus permit the
neighboring cells to receive the full brunt of its ac-
tion. This is, of course, only speculation ; but it should
be capable of experimental test in some fashion.

In view of these conclusions it is of interest to recall
the early observations of Jost ( 1 893 ) , who found that
if potatoes were injured in close proximity to the
eyes, germination was accelerated. A similar end was
achieved by Schneider (1925) by the simple expedi-
ent of inserting near the eyes sticks coated with various
chemicals. Schlumberger (1926), in a reinvestiga-
tion of this curious phenomenon, came to the conclu-
sion that the chemicals employed by Schneider were of
little or no consequence, the accelerated germination
being due to the trauma incident to the insertion of
the sticks into the potatoes. In conformance with this
conclusion, Schlumberger found that a simple me-
chanical jolting, or the application of pressure to seed

CYTOST IN THE PLANT WORLD 255

potatoes, leads to a noticeable increase in the rate of
germination.

If it be conceded that such injuries lead to the liber-
ation of cytost, we may draw a somewhat crude anal-
ogy between the acceleration of germination and the
beneficial effects of homologous cytost upon the breed-
ing of laboratory animals. It would be exceedingly
interesting to study the effects of cytost extracts pre-
pared from potatoes upon the growth of the latter.
In this connection it may not be amiss to suggest that
the current practice of plowing under those portions
of plants which are of no economic importance may
possess benefits aside from the return of nitrogen and
mineral salts to the soil. Conceivably the autolysis of
such plant material within the soil may lead to the
accumulation of a certain amount of cytost which is
actually beneficial to the growth of succeeding gener-
ations of plants.

Before leaving the topic of trauma in plants, it may
be worth while to note that Buenning (1926), in an
investigation of the effects of mechanical irritation on
Aelium ascolonicum, has observed that such injury
and excessive heat both lead to the liberation from
the injured tissues of some substance which is capa-
ble of affecting neighboring cells, leading to coagula-
tion and increased permeability. This is in harmony
with the author's observation that similar effects fol-
low the exposure of animal cells to the abnormal con-
centrations of homologous cytost.

As a result of his experiments, Buenning suggests
that the substance responsible for the histological
changes which he observed may be inorganic salts
freshly liberated from the plant tissues as a result of

256 THE ACTION OF THE LIVING CELL

the injury. This may be true, but as yet the experi-
mental data available are insufficient to warrant any
definite statement on this point.

Taken together, the experimental facts enumerated
above appear to substantiate the hypothesis that in
the plant w^orld some endocellular product plays a
role similar to that of cytost in the animals.

Chapter XIII

CYTOST IN MEDICINE

One can easily imagine that the introduction of cy-
tost into the human body would result in the onset of
pathological changes similar to those induced in the
lower animals which we have investigated in the lab-
oratory. Direct experiments concerning this conclu-
sion are impossible for obvious reasons. There are,
however, a number of pathological conditions com-
monly found in man that closely parallel those pro-
duced in animals as a result of cytost intoxication, and
one can hardly escape the conclusion that many of
these are due to just this cause.

For instance, the shock which may be easily pro-
duced in animals by the methods we have described
earlier is of common occurrence in men who have
been subjected to trauma, particularly crushing in-
juries, severe burns, and unduly prolonged anesthesia.
Such shock is unquestionably due to the cytost re-
leased from damaged tissue, as is the case in lower
animals. Early in the text it has been pointed out that
the excision of injured tissues, if performed before
extensive autolysis has progressed, is a fairly effec-
tive prophylactic against the onset of traumatic shock.
This fact is well recognized by surgeons, who fre-

257

258 THE ACTION OF THE LIVING CELL

quently employ debridement, cauterization, and elec-
trocoagulation as practical means of preventing the
onset of shock during the repair of traumatic injuries.

Our experiments have shown that the continued
introduction of sub-shock producing quantities of
cytost into animals may result in degenerative changes
in the organs of entodermal origin. Clinical experi-
ence suggests that an exactly parallel situation may
exist in man. For example, individuals who have suf-
fered severe burns frequently develop a nephritis
which very often proves fatal. It seems clear in such
instances that the nephritis is caused solely by toxic
substances released from the damaged tissues. While
this has long been surmised by pathologists, we wish
to make it clear that such kidney damage closely paral-
lels that which may be induced in animals by a prop-
erly regulated series of cytost injections.

This is of interest because it indicates that a dis-
eased condition of non-bacterial origin may be caused
by an individual's own tissue products, or cytost.
Since clinical experience with burns justifies this con-
clusion in the case of kidney degeneration, it seems
entirely logical to extend this concept to other patho-
logical conditions involving the organs of entodermal
origin.

There are a number of diseases, sometimes classi-
fied as metabolic, whose obscure origin strongly sug-
gests that cytost is their causative factor. Among
these we may include such chronic ailments as arthri-
tis deformans, neuritis, certain types of paralysis,
arterial sclerosis, and nephritis. At the outset we wish
to make it clear that such diseases may be caused by
factors other than cytost, but that, as will be shown

CYTOST IN MEDICINE 259

presently, they may be caused by cytost also. For
an example we may consider nephritis, which may
be caused experimentally by various means. Various
metallic poisons, such as mercury and uranium, may
cause the onset of this condition. Likewise there is
considerable evidence that kidney damage may re-
sult from infections of one sort or another. In the case
of nephritis following burns, however, it is clear that
such factors are not the causative agent, and one is
forced to assign this role to cytost. One may reason
similarly concerning the other pathological conditions
enumerated above.

In the case of arthritis deformans, this has been
recognized by Nathan (1917), for he states:

"The principal gross and microscopical findings
in both acute and chronic joint disease, whether of
known or unknown origin, are fundamentally simply
phenomena of inflammatory reaction.

"They are all of osseous origin. There are certain
peculiarities characteristic of so-called Arthritis De-
formans which are apparently very difficult to bring
into correlation with an infectious origin. Indeed the
peculiar deformities of hands and fingers, the acro-
paresthesia and premonitory weakness and spasm,
cannot be ascribed to an infectious focus within the
joint at all. It may show involvement of cord and
spinal roots, definite signs of peripheral nerve ab-
normalities, — at most Perineuritis, Ulnar neuritis,
etc.

"It must be assumed that all polyarthritis (neu-
ritis) , is due to infections, or, that all deleterious sub-
stances cause fundamentally the same general changes
in articular structures. It cannot be denied that there

260 THE ACTION OF THE LIVING CELL

is some foundation for the interpretation of these
phenomena, for it is well known that traumatic con-
ditions near, or in the epiphysis, induce the phenomena
of inflammation, congestion, vascularization, bone de-
calcification, and absorption, and, at times, tissue
proliferation, which may lead to fibrous or bony anky-
losis."

Similarly Harding (1921) reports that in a study
of 300 cases of arthritis he found blood cultures to be
negative, and states that he is "not at all convinced
that germs in tooth or tonsil are causative factors in
joint conditions."

Failure to obtain positive blood cultures does not
necessarily indicate that focal infections are of no
importance in connection with arthritis. Indeed,
various statistical studies have shown that there ex-
ists a close correlation between foci of infection and
various pathological conditions. On the other hand
competent men have frequently failed to find any evi-
dence of focal infection in patients apparently suf-
fering from the same maladies as those in whom foci
are found. Further, it frequently happens that the
removal of foci of infection does not result in allevia-
tion of the patient's condition. Such findings as these
are disconcerting. If, however, we assume that the
cellular degeneration is caused by cytost these appar-
ent discrepancies may be obviated. In our discus-
sion of the relationship of bacterial infection to cytost,
it was pointed out that proteolytic microorganisms
may cause the liberation of the tissue toxin. Obviously
then, a focus of infection may cause a more or less
continual release of cytost into the circulation, and
this may lead to the onset of various pathological con-

CYTOST IN MEDICINE 261

ditions. Now the same end should follow the release
of cytost by any means whatsoever.

In consequence one may regard a focal infection
simply as one means of causing the accumulation of
excessive quantities of cytost in the individual. If this
view be accepted, we may readily account for the ab-
sence of a bacteriemia in individuals supposedly suf-
fering from the effects of foci of infection. Further,
this concept enables one to understand the fact that
the removal of such foci does not necessarily guar-
antee the patient relief, for he may suffer from cytost
arising from another cause, or from the cytost released
by the infection, which is not removed from his body
when the focus is cleaned up.

In order to ascertain the possibility of causing in
animals a condition similar to that of arthritis in man,
cytost preparations were injected into the bone mar-
row of cats. This was accomplished by trephining
small openings in the proximal end of the tibia and
the distal end of the femur. One eighth of a cubic
centimeter of a 10% solution of an active cat tissue
ash was then aseptically injected directly into the
bone marrow through each of the two openings. As
a control, other cats were injected similarly with cor-
responding quantities of horse and lion cytost. The
cats receiving the cat cytost in this way developed a
marked lameness, with stiff and sensitive joints, which
lasted for several days. The animals injected with
horse cytost showed no ill effects, while those which
had been injected with lion cytost developed a slight
lameness which lasted for two days only. (Turck,
1921.)

Further experiments showed that the injection of

262 THE ACTION OF THE LIVING CELL

homologous cytost intramuscularly directly adjacent
to the sciatic or ulnar nerve resulted in the onset of
a condition resembling neuritis and causing marked
sensitivity and lameness, the duration of v^hich in any
individual case was found to depend upon the amount
of cytost injected. The accompanying photograph
(Fig. 16) shows a cat suffering from neuritis of the
hind limbs induced by this means.

Microscopic examination of the affected nerves
showed that the injections of cytost had caused con-
gestion and extension of the vessels in the tissues sur-
rounding the nerves and in the nerve sheath, although
the nerve itself did not appear to be affected. This is
shown in figure 17, wherein one may clearly discern
three areas distinctly showing this fact. The onset of
neuritis in the experimental animals is probably to be
attributed to the stagnation caused by the cytost, which
then causes extension of the vessels and consequent
pressure upon the nerve, rather than to any direct ef-
fect upon the nervous tissue.

The results of these simple experiments indicate
that cytost intoxication may actually be an important
causative factor in the occurrence of analogous condi-
tions in man. If this be the case, how is one to diagnose
and treat patients suffering from this cause?

The diagnostic procedure is of necessity somewhat
unsatisfactory. First, of course, one must examine the
patient in order to ascertain the absence of any gross
pathologies which may give rise to the cytost, and these
must be cleared up by the usual medical and surgical
procedures. The writer has found that intramuscular
injections of homologous cytost at the site where pa-
tients complain of pain frequently leads to a marked

Figure 1 5.

Photograph of a cat with complete paralysis of the hind
quarters caused bv the intraspinal injection of a solution of
homologous tissue ash as described in the text.

Figure 16.

Photograph of a cat with "neutritis" of the hind limbs
resulting from intramuscular injections of cytosa directly
over the sciatic nerve.

Figure 17.

Section showing sciatic nerve. Taken from the limb of
a cat which had received an intramuscular injection of
homologous cytost adjacent to the nerve a few days previ-
ously. Note the congestion of the tissues about the nerve,
and in the sheath, which gives rise to pressure on the nerve.

CYTOST IN MEDICINE 263

increase in pain and sensitiveness. For this purpose
we have employed a saline extract of the gray ash of
human muscle. When heterologous cytost is injected
similarly the only additional pain experienced is that
which normally follows the intramuscular injection
of almost any non-irritating solution.

The fact that injections of homologous cytost mark-
edly accentuate the pain and soreness for which pa-
tients seek treatment is of diagnostic significance, for
it indicates that the patient's tissues are hypersensitive
to cytost. This may be due to either of two causes : his
tissues may be naturally hypersensitive — i.e., his re-
sistance to cytost is low; or because of an unduly large
amount of cytost already present, the additional quan-
tity introduced in the injection may raise its concen-
tration in the tissue fluids to an abnormally high de-
gree. In either case the end result is the same. The
writer believes that when an individual is found to
be hypersensitive to cytost the conclusion is war-
ranted that such an individual is suffering from cytost
intoxication.

Now the question arises as to how one is to over-
come this condition, — that is, can we ofifer the patient
any relief from the effects of cytost upon his tissues?

The answer to this question is to be found in the
results of our laboratory experiments on animals. It
may be recalled that we were able to obtain evidence
that an animal's resistance to cytost may be materially
raised by a properly regulated series of injections of
homologous cytost. Because of this it appears logical
that one should be able to do the same with humans.
Elsewhere the writer has described the treatment of
various diseases by this means.

264 THE ACTION OF THE LIVING CELL

As cytost obtained from human tissues is not readily
obtainable, we have taken advantage of the fact that
an animal may be caused to liberate cytost at v^ill by
the simple expedient of causing a small localized tis-
sue damage by the subcutaneous injection of chloro-
form or ether at selected sites. In the author's experi-
ments upon patients it has been customary to inject
0.25 cc. of chloroform at one or tw^o points, usually in
the lumbar region. This causes the patient no incon-
venience, and the small quantity injected causes but
little tissue damage, and consequently the release of
but a small quantity of cytost. Several such injections
spaced several days apart apparently have the same
effect as similar injections of human cytost.

Following such treatment a number of striking
"cures" have been obtained both in acute and chronic
cases of migraine, arthritis, rheumatism, etc. Some of
these have been described elsewhere (Turck, 1919,
1921, 1922), and we shall not discuss them here, for
the writer well recognizes that general conclusions
cannot be drawn from the results obtained with indi-
vidual patients.

Strictly speaking, one can determine the utility of
a given method of treatment only when it is applied
under more or less constant supervision to a sufficiently
large number of patients to allow the deduction of
statistically valid conclusions.

Through the courtesy of L. E. Feldman, director
of the Russian Red Cross (old organization) in Bul-
garia, such an opportunity was offered at the Hos-
pital and Dispensary in Sofia and the Asylum for old
people and chronic invalids in Shipka. All diagnoses,
treatment, and observations were carried out by the

CYTOST IN MEDICINE 265

physicians associated with these institutions for a
period of several years. The writer's only connection
with this practical experiment consisted in outlining
the method of treatment described in the preceding
pages, and supplying human cytost at the beginning of
the venture. From time to time reports were received
from the Russian Red Cross physicians, which pre-
sented evidence that the cytost treatment was actually
found to be of benefit.

Typical of these reports is one from Dr. R. Berzin
of Sofia, under the date of July 20, 1929, from which
we quote: "During the last three years I have had
more than 100 patients treated with cytost and anti-
cytost. The first group (79 patients) were suffering
from arthritis, especially from arthritis chronica de-
formans and polyarthritis chronica, 48 of them were
men, 31 women, age from 35-68 years. The majority
of them received the treatment in the Hospital of the
Russian Red Cross, only a few were treated in their
homes. Nearly all of the patients had been bedridden
for weeks or months. Several of them had been inva-
lids from two to four years. Everyone had been treated
with mineral baths, etc., for years without any lasting
results.

"Our treatment was very simple: We gave our pa-
tients only cytost and anticytost alternatively with an
interval of 10 days, and nothing more. The dose va-
ried from 1-5 cc. of anticytost and 0.4—0.5 cc. of cy-
tost each time. The total dose was from 10-50 cc. of
anticytost and 2-3 cc. of cytost.

"The results were excellent. We had no complica-
tions at all, neither local nor general disturbances.
On the contrary the patients affirmed that they felt

266 THE ACTION OF THE LIVING CELL

much better, that not only afifected joints were cured,
but their very essence of life, their vitality, was in-
creased in a very considerable degree."

The author has received similar reports from other
physicians who state that they have consistently found
that patients suffering from arthritis are benefited by
injections of cytost or chloroform. In some instances
similar treatment has been found of distinct benefit in
cases of arteriosclerosis, asthma bronchialis, neuras-
thenia, and various ailments which may be classed as
senile changes. Further discussion of such cases
would be out of place here, but will be reported else-
where in the future.

Quite recently others have recognized the useful-
ness of tissue extracts in the treatment of disease. Thus
Ludwig (1931) states that extracts of both heart and
skeletal muscle are of distinct utility in the treatment
of various cardiac troubles, particularly angina pec-
toris. Similarly Weiss (1931) found that in four cases
of angina pectoris an immense improvement followed
treatment with an extract of ox hearts. Weiss states
that the cases which he treated seemed to experience
a renewal of physical capacity and eventually lost all
indications of pressure symptoms.

This is of particular interest since this observation
apparently substantiates the writer's conclusions that,
when properly administered, cytost actually brings
about a general increase of physical well-being.

In our discussion of the stimulating effects of cy-
tost upon animals it was pointed out that a properly
regulated course of cytost injections gives rise to the
observed beneficial effects, solely because it raises the
animal's natural resistance towards its own cellular

CYTOST IN MEDICINE 267

toxins. There appears to be no reason why this concept
should not be carried over to humans. Indeed the fact
that patients suffering from various diseases of ob-
scure origin are distinctlybenefitedbycytost or chloro-
form injections appears to substantiate this conclu-
sion.

With this in mind we may briefly extend the present
discussion of the possible application of our experi-
mental researches to medicine. For example, let us
consider infections of the respiratory tract. It may
be recalled that respiratory disturbances of non-bac-
terial origin may be easily induced in animals by
exposure to cytost-laden air. Once such a disturbance
is produced, bacterial infection readily follows. On
the other hand, it is by no means easy to induce res-
piratory infections in healthy animals not previously
exposed to cytost. (Turck, 1919b.) It follows from
this that exposure to cytost-laden air may be a predis-
posing cause of the onset of various respiratory dis-
eases in humans.

It has frequently been stated that the incidence of
respiratory infections is higher in urban communi-
ties than elsewhere. This in part is due to greater
chances of contact with infected individuals and may
in part be due to the fact that in congested areas the
air actually contains human cytost sufficient to pro-
voke a reaction in the sensitive respiratory endothe-
lium of the less resistant members of the community.
In favor of this concept is the fact, frequently
remarked by Arctic and Antarctic explorers,
that respiratory disturbances are exceedingly rare
amongst the members of their expeditions. This is
especially interesting since during the winter months

268 THE ACTION OF THE LIVING CELL

the members of such expeditions are closely crowded
together in their small huts. It would seem that the
absence of respiratory disease among the members
of such expeditions is in part due to the fact that the
air of the polar regions must be free of human cytost;
hence the men are not exposed to this predisposing
cause.

In this connection it is of interest to note that Dr.
S. E. Jones, a physician who accompanied the Aus-
tralasian Antarctic Expedition led by Sir Douglas
Mawson, records in his medical report that an epi-
demic of influenza broke out on the ship and that the
recovery of the convalescents was much more rapid
after arrival in the Antarctic than on the ship. (See
Mawson, 1916.) This is in agreement with the well
known fact that patients suffering from various res-
piratory disturbances recover more rapidly in spa-
cious country sanitoria than in congested city hos-
pitals, a fact which suggests that removal from
cytost laden air maybe distinctly beneficial in the treat-
ment of such conditions. Indeed, if possible, this
would seem to be of value in the treatment of any type
of disease in which cytost intoxication may be an im-
portant factor.

By analogy with our experiments with animals, it
should be apparent that any measures which may in-
crease an individual's resistance to cytost should be
of value both in the prevention and the treatment of
disease. In our discussion of so-called natural re-
sistance it was pointed out that such factors as proper
diet and adequate exercise seem to raise one's natural
resistance by raising his resistance to cytost. Con-
versely, improper diet and lack of exercise tend to

CYTOST IN MEDICINE 269

decrease resistance to cytost and consequently are pre-
disposing causes of disease.

During and following the Great War, large num-
bers of various European populations were forced to
subsist upon rations little short of actual starvation.
In consequence, it is not surprising that many Euro-
peans, particularly children, developed many obscure
and rather ill-defined ailments indicative of a general
lack of body tone and diminished resistance com-
parable to that found in animals as a result of partial
inanition. In animals, as we have shown, this leads
to cytost intoxication and the degeneration of various
tissues. It seems highly probable therefore that in
many instances the poorly defined ailments of the war
refugees may have been due to a similar cause.

In this same class we may place those who suffered
shock from the severe wounds of war but did not fully
regain their health upon recovery from the primary
injuries. Such individuals were frequently found to
be suffering from various ill defined chronic com-
plaints whose very nature suggested that they were
basically due to a more or less chronic cytost intoxica-
tion.

With this in mind the physicians of the Russian
Red Cross have at the writer's suggestion treated such
individuals with a view to increasing their resistance
to cytost. In order to obtain comparative data such
patients were divided into two groups. Both groups
were well fed and housed and given whatever general
medical treatment was indicated in particular cases.
The members of one group were given injections of
chloroform, such as have been described earlier in
the chapter, with a view towards raising their resis-

270 THE ACTION OF THE LIVING CELL

tance to cytost, while the members of the other group
did not receive such injections. According to the
medical men who observed these individuals, those
receiving the chloroform injections were, on the aver-
age, restored to health more rapidly than were the
members of the control group. This experience again
testifies to the fact that immunization to cytost is a
very useful adjunct to common medical treatment.
The few facts relative to medicine which have been
considered in this chapter have been presented in the
hope that they may be of use to others who desire to
make use of them. It must be remembered, however,
that such treatment is not a panacea for all human ills,
but is to be considered only as a supplement to the more
common methods of medical treatment of proven
value.

Chapter XIV

CONCLUSIONS

Since the major aspects of the writer's experiments
have been discussed in the preceding pages in a more
or less topical form, it seems advisable to review and
integrate these investigations in such a way as to form
a harmonious picture of the experimental findings
and the theoretical conclusions deduced therefrom.

Strictly speaking, the various topics which have
been considered are the logical outgrowth of the
writer's early investigations concerning the nature of
so-called shock in animals. Thirty odd years ago this
was considered to be of psychoneurogenic origin. The
earliest investigations of the writer indicated that
such was not the case, but rather that the train of physi-
ological events termed shock are primarily due to the
liberation from injured tissue of a toxic entity, first
termed shock toxin, and later cytost.

Initially this concept arose from the following ob-
servations. In all cases of shock, regardless of the ap-
parent cause, the onset of typical symptoms was ac-
companied by a marked stagnation of blood in the
splanchnic area of the experimental animals. This
was easily observed in animals whose abdomen was
open during the course of an experiment, and in other

271

272 THE ACTION OF THE LIVING CELL

animals was indicated by the drop in surface tempera-
ture and blood pressure. Such reactions as these,
which always accompany shock, were never found to
follow immediately after the infliction of an injury, as
would be expected if the shock were due to a nervous
mechanism of any sort. In general, regardless of the
nature and magnitude of the inflicted injury, shock
does not supervene until approximately two hours fol-
lowing the injury. This fact of course suggested the
intervention of some relatively slow chemical process,
rather than a nervous one, and since the onset of shock
is the direct outcome of the injury, such a chemical
process must take place in the injured tissues.

This conclusion was substantiated by three lines of
experimental evidence which have been discussed in
detail in the early chapters of this monograph. First,
ligation of an injured limb above the site of an injury
was found to prevent the onset of shock because it
prevented the absorption of materials from the wound.
If after several hours the ligature was loosened, then
the various symptoms of shock rapidly appeared, thus
indicating that the shock was due to the passage of
substances from the site of injury to the splanchnic
area. Secondly, histological examination of the in-
jured tissues disclosed the fact that, following injury
of any sort, the affected tissue cells undergo autolysis.
Such a process requires a definite time, and it is this
interval which must elapse between the infliction of
the injury and the onset of shock. From this observa-
tion, it was deduced that the primary cause of shock
was an endocellular toxin liberated by the tissues
incident to their autolysis.

The validity of this conclusion was subsequently

CONCLUSIONS 273

confirmed by the observation that severe shock and
death could be induced in intact animals by the injec-
tion of aqueous extracts of homologous tissues w^hich
had been permitted to undergo sterile autolysis in
vitro, while aqueous extracts of freshly excised tis-
sue, when injected similarly, did not cause the animals
any apparent inconvenience.

These results clearly substantiated the author's
conclusion that shock was of toxic origin, and, since
the World War, others, to whom reference has al-
ready been made, have reached essentially similar
conclusions.

Since the extract of autolyzed tissue proved to be
such a highly toxic substance when introduced into
the circulation of an animal, it became of interest to
investigate the effects of sublethal quantities of cytost.
So far as could be determined, the injection in a single
dose of a minute quantity of cytost does not as a rule
lead to any distinct or definite reaction. However,
when such injections were repeated every few days
for a protracted period of time, it was found that tem-
porarily the injected animals gained in weight more
rapidly than control animals of the same age under
the same conditions. Furthermore, as far as could
be judged from external observation, during the
period of rapid gain in weight the injected animals
appeared to be more active and in better health than
the controls. As the injections were continued, the
animals lost this advantage, and after a period during
which they lost considerable weight, they rapidly be-
came ill, and died.

At autopsy, such animals were found to have suf-
fered marked degenerative changes in the organs of

274 THE ACTION OF THE LIVING CELL

endodermal origin, the lungs, liver, kidneys, etc.
Thus, by the long continued action of sublethal quan-
tities of cytost, it was found that one may induce
chronic pathological changes in the same tissues, and
that they respond most rapidly to the action of cytost
during shock. The rate at which such degenerations
may be induced was found to depend upon both the
dosage of cytost employed in the experiments, and
the frequency of the injections.

It is of especial interest to note that the degenera-
tive conditions which can be induced by the often re-
peated injection of cytost are similar to those which
are of the utmost importance in pathology, and whose
cause is frequently the most obscure. The writer is of
the opinion that such conditions arise most frequently
from the action of cytost derived from injured tissues.
As is well known, the kidney involvements which
sometimes occur in individuals who have been badly
burnt are commonly attributed to "tissue toxins," and
this is in harmony with the writer's contention.

From the investigations which we have discussed
earlier, it should be clear that since any injury, trau-
matic, chemical or bacterial, may result in the libera-
tion of cytost which will affect the visceral organs in
greater or lesser degree, if long continued, this may
eventually lead to pathological conditions of appar-
ently obscure cause. It follows, therefore, that a mild
more or less chronic injury may finally result in such
pathological conditions at a distance from the actual
site of injury.

With this in mind, one may easily trace the possible
connection between foci of infection and disease of
obscure origin, such as nephritis, or arthritis. That

CONCLUSIONS 275

such a connection exists has been established with a
high degree of probability by clinical observers, but
the actual details of the relationship have not been
satisfactorily established. In our discussion of the
relation of bacterial infection to cytost intoxication,
it was pointed out that bacterial infection, although
localized in a particular area, may cause the liber-
ation of considerable quantities of cytost in much
the same manner as would any other localized tissue
damage.

If such a condition persists for some time, the ani-
mal is continually subjected to a concentration of
cytost analogous to that which occurs when sublethal
quantities of this substance are injected at frequent
intervals during a protracted period of time. In con-
sequence, the cytost liberated by the activities of the
bacteria in foci of infection may be expected to bring
about a degeneration of the tissues of endodermal
origin in much the same manner as was found to be
the case in our experiments. The writer feels confi-
dent that an investigation of this mechanism would
yield results of considerable interest in the interpre-
tation of some disease processes.

When a large number of animals is injected with
equal quantities of the same extract of autolyzed tis-
sue, it is found that they react to the injection in vary-
ing degrees. If the quantity is sufficiently great, the
larger proportion of the animals develops shock and
dies. Certain other members of the group develop a
severe shock, and recover, apparently none the worse
for their experience, while a lesser number reacts but
slightly to the injection. This, of course, is a common
finding in all types of biological experimentation, and

276 THE ACTION OF THE LIVING CELL

is commonly ascribed to individual variations in re-
sistance or susceptibility.

These variations in resistance to cytost injections,
however, are especially interesting since they repre-
sent a resistance to a product of the animal's own cells,
and suggest that, other things being equal, an animal's
health and well-being may depend upon his resistance
to the toxic products resulting from the activities of
his body cells.

This line of thought led to two series of investiga-
tions concerned respectively with the toxic effects of
sublethal quantities of cytost, and the stimulation of
natural resistance to that substance. These have been
discussed at length in previous chapters, where it was
shown that, depending upon both the quantity of cy-
tost injected, and the number of injections, an animal
may be caused to suffer an actual decline in weight and
health, or stimulated so that it gains in weight and
general appearance.

The latter result appears to depend upon a general
stimulation of the metabolism of the body cells of the
animal, as well as the production of an increased tol-
erance for cytost. The apparent stimulation of metab-
olism may really be due to the latter, for it is con-
ceivable that in the case of an animal having a low
resistance to cytost, the accumulation of the latter in
its body fluids may impair the functioning of the body
cells so that their activities are below normal. If, on
the other hand, the animal's resistance to cytost is
raised by a course of immunizing injections, such im-
pairment of function is largely removed and the
growth of the body cells assumes a more normal
course. This aspect of the problem is one worthy of

CONCLUSIONS 277

further investigation, since it should lead to a better
understanding of bodily function, both in health and
disease. This seems all the more certain, since, as we
have shown, a number of pathological conditions of
admittedly obscure origin can be easily induced by
a series of sublethal injections of homologous cytost.

These effects of cytost on the intact animal are but
the expression of the integrated effects upon the indi-
vidual tissue cells. This follows from our experi-
ments on tissue cultures and upon the growth of para-
mecia, where, it may be recalled, small quantities of
homologous cytost were found to exert a distinctly
stimulating effect upon the cells, while the character-
istic toxic action of cytost became manifest if its con-
centration in the media was too high.

These two actions of cytost may seem paradoxical,
but since they are attested to by the results of literally
hundreds of experiments, they cannot be questioned.

Indeed, nature seems to have endowed all biological
systems with apparently paradoxical reactions to
baffle the investigator. Thus, the life of the majority
of animal cells is adapted to a definite partial pres-
sure of oygen ; oxygen tensions less than or greater than
the optimum lead to anomalous activity frequently
resulting in death. The same is true of the ionic bal-
ance of salts in the fluid media surrounding such cells.

It is not illogical, therefore, to postulate that a cer-
tain optimal concentration of cytost is compatible
with the normal functioning of cells, whereas when
this optimum is exceeded in one direction or the other
anomalous behavior results. When such anomalous
behavior becomes excessive, a pathological condition
ensues.

278 THE ACTION OF THE LIVING CELL

Care must be taken not to press such analogies too
far, for while isolated cells or tissues are unable to
control such factors as oxygen tension, they are, in a
measure, able to control the cytost content of their
immediate environment, since, as we have seen, cytost
is an endocellular autosynthetic product. On the other
hand, our experimental results have shown that the
liberation of cytost in quantities sufficient to affect
the cells in one way or another is dependent upon an
injury of some sort; that is, although the living cell
contains cytost or its immediate precursor, mobiliza-
tion of this material in quantity sufficiently large to
cause an observable alteration in the activity of cells
does not take place until an injury of some sort occurs.
It should be noted in this connection that we use the
term injury in a very general sense, including any
response to various physical and chemical agencies
which we have discussed previously. It follows there-
fore that so-called "mild stimulation" as well as physi-
cal destruction falls in this category.

Since such injury provokes the liberation of cytost
to a greater or lesser degree, and since, as our experi-
ments have shown, an optimal quantity of cytost in
the surrounding media appears to lead to maximal
cellular activity, we are enabled to see a definite rela-
tionship between cellular activity and alterations in
the physical environment. Assuming that such an
environment is well adapted as regards oxygen, sources
of nutriment, and so forth, the activity of a cell, tissue,
organ, or organism is to a considerable degree condi-
tioned by alterations in any environmental factor
which may lead to a liberation of cytost.

Let us first consider the beneficial action of such

CONCLUSIONS 279

environmental factors as temperature and sunlight.
In all likelihood such environmental agencies affect
an organism in manifold ways and it is difficult to
subject their effects upon an organism to complete
analysis. Nevertheless the writer's experience has
convinced him that in part the action of such agencies
depends upon their ability to cause the liberation of
small quantities of cytost from the exposed cells and
thus effect a general stimulation of the body.

Because of this conviction the writer (Turck, 1899)
long ago recommended the introduction of hot water
into the colon and stomach as a means of increasing
the activity of the body cells in general. This method
was introduced before the writer's cytost theory was
formulated as clearly as it now is, but the results ob-
tained by this method of treatment were in complete
harmony with what we should expect on the basis of
this theory. Sluggish individuals subjected to such
treatment consistently manifest an increased activity
following the brief exposure of their gastro-intestinal
tube to temperatures considerably higher than that
normal to the body.

Biologists at large are inclined to attribute the re-
sponse of an organism to changes in temperature solely
to the effects of temperature upon the rate of the
chemical reactions taking place within the cells.
Within certain limits this view is undoubtedly cor-
rect, but when temperatures are attained which cause
cellular injury we know from our experiments with
burns that considerable quantities of cytost are liber-
ated. Such being the case, it does not seem unreason-
able to assume that even at temperatures considerably
below that necessary to cause rapid cellular disinte-

280 THE ACTION OF THE LIVING CELL

gration, cytost may be liberated from the exposed cells
in sufficient quantity to evoke a response in neighbor-
ing cells.

Much the same argument obtains in the case of sun-
light, whose therapeutic value has been definitely
recognized since the time of Hippocrates. Today it
is common knowledge that the ultra-violet portion of
the spectrum of sunlight is of importance in connec-
tion with the synthesis of the antirachitic vitamin, but
the beneficial efifects of exposure to sunlight cannot
be attributed to this fact alone.

Experiments have shown that the penetration of
sunlight into the skin is limited; hence the systemic
effects which follow prolonged exposure to such radi-
ation must be due to the absorption of cellular prod-
ucts liberated from the radiated area. That such prod-
ucts, including cytost, are liberated is evident, since
prolonged exposure to sunlight frequently leads to a
severe sunburn and the eventual onset of symptoms
typical of cytost intoxication. It seems highly prob-
able that even brief exposures to sunlight must like-
wise result in the release of cytost, for the character-
istic erythema resulting from such appears to be
identical with that which follows the release of cytost
by any other mild localized injury, such as has been
discussed in the previous text.

Upon extending this line of thought, it is readily
conceived that the magnitude of the cytost released
under the influence of such environmental agencies
must vary in some quantitative manner with the in-
tensity of such injury. Thus when one unaccustomed
to intense sunlight is foolish enough to expose a con-
siderable area to the sun's rays for a protracted period.

CONCLUSIONS 281

the quantity of cytost liberated may greatly exceed
the quantity sufficient for healthy stimulation of the
tissues, and a mild toxic action may become apparent
some hours afterwards. In some degree the severity
of such a toxemia is found roughly to parallel the ex-
tent of the tissue injury caused by the sunlight. Of
course individuals vary in this respect. Some active
persons can contract a markedly destructive and pain-
ful sunburn v^ithout any apparent general ill effects.
Presumably such individuals, through previous activi-
ties, have developed a resistance to cytost comparable
to that v^hich follov^s a series of immunizing injec-
tions.

The essential point to be gleaned from the fore-
going is that cytost occupies a unique role in the
activity of cells, for through its action such activity
is closely determined by alterations in the environ-
ment. Of course the response of cells to all conceiv-
able environmental modifications cannot be dependent
solely upon the action of cytost, but the evidence v^hich
the w^riter has accumulated points strongly to the fact
that many changes in cellular activity are uniquely
controlled by the liberation of cytost under the influ-
ence of environmental factors. This is a point not
generally appreciated by v^^orkers in the biological
sciences at the present time, and the w^riter is con-
vinced that the adoption of this viev^ may do much
to effect a rational explanation of many apparently
uncorrected biological facts.

It is the v^riter's earnest hope that other investiga-
tors w^ill attempt to repeat and extend his observations.
Certain of our researches, although prosecuted to the
fullest extent w^hich available facilities permitted, still

282 THE ACTION OF THE LIVING CELL

require re-investigation and extension. Thus, the ex-
periments upon immunity towards cytost and the
relationship of such immunity to natural resistance
and breeding might well be extended to larger num-
bers of animals and particularly to species other than
those used in our laboratory.

The writer has not carried out any experiments with
invertebrate forms other than protozoans, but there
is every reason to suppose that in such animals cytost
must play a role similar to that in higher animals. In-
deed, Child (1915) has presented some evidence that
this is so in the case of planaria, for he states (page
66) : "There is no doubt that a relation exists between
the general metabolic condition of organisms and
their susceptibility to a large number of substances
which act as poisons, i.e., which in one way or another
make metabolism impossible." He further points out
that the action of such poisons resembles that of "cer-
tain soluble products of metabolism," and in so doing
comes close to the writer's concept that the toxic action
of many cellular poisons is largely due to their ability
to cause the liberation of cytost. Throughout his book
Child stresses the possible relationship between the
"soluble products of metabolism and the activity of
the animal." Thus (page 188) : "Fatigue, the de-
crease in rate of metabolism which follows continued
stimulation, is generally believed to be due to the
accumulation of toxic products of metabolism." And,
again: "Various metabolic intoxications are prob-
ably very similar in character . . ."

In the chapter on the possible role of cytost in the
activities of the plant, we have presented evidence
that endocellular products which are liberated as a

CONCLUSIONS 283

result of injury are of importance in determining the
response of plants to their environment. Most of this
evidence gleaned from the vs^ritings of others should
be reinvestigated, using extracts of plant tissues in a
manner similar to that vs^hich we have employed in
our animal experiments.

If by such methods it can be established definitely
that cytost is of equal importance in the plant and
animal worlds, we shall have at our disposal a deeper
appreciation of an important factor in the physiologi-
cal activity of living systems.

Our experiments have shown cytost to be a substance
of remarkable thermostability, but we have failed to
identify it as a distinct chemical substance. This is a
biochemical problem which still awaits solution.

So far we have been unable to find any chemical test
for the presence of cytost in a tissue extract. The only
means of detecting this substance remains the physio-
logical reactions described in the text. For laboratory
purposes the induction of shock and death in animals,
and the localized reaction in the lungs remain the
most satisfactory methods for the assay of various cy-
tost preparations. While these methods yield positive
and unquestionable evidence for the presence of cy-
tost, it may be desirable to expand them by the study
of the action of cytost on isolated organs. Such a study
may give rise to a quantitative method of assay better
suited for routine laboratory experiments.

The development of such methods may be of ex-
treme value in future investigations of the action of
cytost and its relation to the physiology of the cell,
both in health and disease. Certain of our experi-
ments have shown that it is possible in young animals

284 THE ACTION OF THE LIVING CELL

to produce retrograde senescent changes by the fre-
quent injection of cytost. This opens the way to the
possibility of obtaining important information con-
cerning the nature of senility, a problem of immense
importance in biological science.

It has been shown that by an appropriate series of
immunizing injections, one may raise an animal's re-
sistance to cytost, and we have deduced that this is
of importance in determining an animal's so-called
natural resistance. This is of importance from both
the theoretical and practical standpoints, and conse-
quently is deserving of further investigation. In this
connection we have postulated the existence of an
anticytost whose formation within the animal pro-
tects the latter from the action of cytost. Since anti-
cytost is an antibody which protects an animal from
a toxin developed by his own cells, a new concept
has been added to the science of immunology.

The writer is confident that the various types of
experiments described in the text are capable of sub-
stantiation in the hands of other experimenters. It
must be remembered, however, that animals vary
markedly in their susceptibility to injury of any sort;
but as we have seen, the gross effects which result
from various types of injury have in common the
liberation of the tissue substance which we have
termed cytost. In consequence, the individual dif-
ference between animals may be largely explained
as due to their difference in susceptibility to cytost;
or, in other words, to put it crudely, to their available
anticytost, with which they are able to combat the
action of cytost itself. Bearing this in mind it is
obvious that the experimenter working with a limited

CONCLUSIONS 285

number of animals may fail to obtain results com-
parable with those which we have described. How-
ever, if a sufficiently large number of animals is sub-
jected to a given experimental procedure, it will be
found that results similar to ours will be obtained in
a sufficient majority of instances to warrant the con-
clusions which we have stated. This of course is a
common experience in biological investigations of
all kinds.

As an aid to any investigator who wishes to repeat
or extend such experiments, we have appended a short
selected list of procedures taken from our protocol
books in the hope that these may prove of service to
anyone who wishes to venture into this interesting field
of investigation. These experiments have not been
chosen because of the particular value of the results
obtained, but rather as an illustration of the technique
developed in our laboratory.

The writer fully appreciates that many of his de-
ductions and concepts will meet the opposition usu-
ally accorded new theories. This is desirable, since
such opposition frequently constitutes the impetus
necessary to provoke experimental researches which
will finally prove or disprove the validity of the con-
cept.

APPENDIX

SOME REPRESENTATIVE
EXPERIMENTS

The experiments described below in the form of
the actual protocols are representative of many which
show the toxic nature of shock induced in various
ways. These have been presented as a further il-
lustration of some of the concepts discussed in the
text. Experiments concerning the influence of cytost
upon the growth, development, and breeding of ani-
mals have not been included because the descrip-
tions of these in the text are believed to be sufficiently
detailed to permit of a proper understanding of their
technical details.

TRAUMATIC SHOCK

Cat, Male, Weight 4,300 grams. Temperature
39.5°. Room Temperature 21°. Ether anesthesia be-
gun at 9.15 A.M.

9.30 A.M. Temperature 38.5°. Esmarch bandage ap-
plied so as to exert pressure on internal iliacs and ab-
dominal aorta, thus keeping blood from lower ex-
tremities. Animal placed on radiator to keep warm.

287

288 APPENDIX

Thigh and gluteal muscles severely bruised by means
of bone forceps. Toe cut, no bleeding indicated block-
ing of circulation effective.

10.15 A.M. Temperature (esophageal and gastric)
36°. Temperature taken in this fashion because of
interference v^ith circulation.
11.00 A.M. Intragastric temperature 35.5°.
11.20 A.M. Intragastric temperature 35°. Bandage
removed, and the bruised tissue v^as occasionally mas-
saged to reestablish circulation.
11.40 A.M. Intragastric temperature 34°. Anesthetic
discontinued.

1.55 P.M. Intragastric temperature 35°. Limbs mas-
saged. Onset of shock.

3.00 P.M. Temperature 32.5. Mucous membranes
of mouth and nose were pale and cold.
4.00 P.M. Intragastric temperature, 32°, rectal tem-
perature 32°.

6.00 P.M. Rectal temperature 30°. Cat died shortly
afterv^^ards. Immediate autopsy. Hepatic lobules well
marked. Congestion of pyloric region. Lower small
intestines and colon pale. Histological examination of
liver showed stagnation in portal veins with marked
distension of the portal capillaries and well marked
periportal changes in the parenchyma of the liver.

Cat, male, weight 2,523 grams, rectal temperature
40° C.

9.00 P.M. Under anesthesia, the upper and inner
sides of the left leg were shaved. Muscle tissue taken
from the mangled leg of another cat which had been
in profound shock was aseptically inserted between
the muscles through incisions made through the

APPENDIX 289

shaved area of the subject. This was accomplished
by separating the leg muscles with the blunt end of
a knife and forceps in order to prevent laceration and
hemorrhage. After insertion of the mangled tissue
(40 grams) from the other cat, the wounds were
closed by suture. Anesthetic discontinued at 9.30
PM.

930 P.M. Rectal temperature 38°.
10.15 P.M. Rectal temperature 37°.
11.15P.M. Rectal temperature 37°. Beginning symp-
toms of shock indicated by gradual onset of inactivity
and weakness.

Following day. S A.M. Rectal temperature 36.5°.
12.00 M. 35°. Animal very weak, apathetic, reflexes
nearly abolished, heart weak, respiration shallow, gen-
eral pallor and coldness.

Third day. 6.30 P.M. Rectal temperature 34°. Pro-
found shock, died at 8. P.M. Immediate autopsy dis-
closed the usual shock picture. Congestion and edema
of the liver, stomach, upper part of small intestines
and pallor of lower splanchnic area. Microscopic
examination of sections showed marked venous en-
gorgement with extravasated cells in the submucous
tissue (Zona Transformans) of the pyloric end of
stomach; active venous stasis and periportal extrava-
sation in the portal zone of the liver; peripulmonary
arterial congestion in the lungs; and straight tubular
venous stasis, hemorrhagic areas and agglutination in
the glomerular region of the kidney.

Rabbit, male, weight, 1,400 grams, temperature 40°.
Anesthesia commenced at 9.15 A.M. Left leg tied ofif
above the great trochanter. Leg mangled and badly
bruised with bone forceps.

290 APPENDIX

10.15 AM. Anesthetic stopped. Ligature released.
Animal depressed so that no anesthesia needed.
11.00 A.M. Difficult shallow breathing, temperature
35°. Rapid heart.

12.45. Exit. Immediate autopsy showed liver, py-
loric end of stomach and small intestines markedly
congested.

SHOCK PRODUCED BY BURNS

A rabbit (No. 1 ) under ether anesthesia was burned
over the back by means of a hot iron. The burned area
was immediately removed and grafted onto the back
of another rabbit (No. 2) and the normal skin of the
latter was transferred to No. 1 , from which the burned
tissue had been removed. Rabbit No. 2 showed signs
of shock about two hours after the insertion of the
burned tissue graft, and died in shock after 68 hours,
while rabbit No. 1 showed but slight signs of shock
and made an uneventful recovery from its wound.

A cat was slightly burned along the spinal region,
the burned skin dissected out and transplanted to a
second cat. The latter developed shock after four
days, probably due to delayed absorption. At autopsy
the post-mortem findings were typical of shock. This
experiment illustrates the necessity of keeping ex-
perimental animals under observation for several days
before passing judgment on the outcome of an ex-
periment.

"SHELL SHOCK"

8.15 A.M. A guinea pig was wrapped in a small cloth
bag and the latter bound to a stand placed within a
few centimeters of the revolving arm of a centrifuge

APPENDIX 291

making 10.000 r.p.m., for Yi minute. Animal ap-
peared depressed with slight symptoms of shock. Ob-
served for 10 minutes and then placed in proximity
to centrifuge arm for another half minute. Well de-
veloped shock, eyes dull, fur staring.
845 AM. Temperature 35°. Marked trembling.
1.05 PM. Temperature 36°.

3.45 P.M. Animal in crazed condition, began to eat
feces and chewed frantically at steel forceps under
jar.

6.00 P.M. Autopsied. Upper part of intestines and
stomach markedly congested and hemorrhagic. Liver
showed extensive small hemorrhagic areas. Kidney
and spleen hemorrhagic. Brain and spinal cord find-
ings were practically negative although the mem-
branes were congested.

A guinea pig, weight 775 gms., placed in proximity
to centrifuge arm as above described for two ^ min-
ute periods spaced 5 minutes apart. Slight symptoms
of shock from which the animal recovered. Although
kept upon a normal diet this animal lost weight. 10
days after the experiment it weighed 649 grams (loss
126 grams). Was dull and listless with dull, rough
coat.

This experiment offers evidence of cytost intoxica-
tion following an injury of a sort which leaves no
definite external indication of its nature.

EXPERIMENTS WITH AUTOLYZED TISSUE

For experiments such as those described below,
sterile autolyzed tissue extracts are easily prepared
as follows : The desired tissue is removed aseptically

292 APPENDIX

and transferred to a sterile vessel which in turn is
placed in a covered desiccator containing a small ves-
sel of chloroform. The vapor of the latter serves to
inhibit any bacterial growth which might otherwise
take place. The dessicator and its contents are then
placed in an incubator at 37.5° C. for twenty-four
hours or longer in order that autolysis may proceed
at a reasonable rate.

At the expiration of this time the autolysate may be
diluted with a convenient volume of isotonic saline
solution and centrifuged to remove suspended matter.
This process need not be carried out aseptically, for,
as stated in the text, the cytost extracts prepared in
this fashion may be boiled and filtered through a
Berkefeld filter without any loss of potency.

Before using such an extract for any particular
experiment it is well to test its toxicity. This may be
conveniently accomplished by determining the mini-
mum lethal dose when injected intraperitoneally into
guinea pigs, as described in Chapter XI, or by direct
injection into the mesenteric vein of animals with
opened abdomen. From the standpoint of general
utility the former method is perhaps the most satis-
factory.

Rabbit, weight 1,500 gms. Abdomen opened under
ether anesthesia. One drop of homologus muscle
autolysate (previously diluted with 10 volumes of
saline) was injected into the liver. This caused a
slight turgescent reaction. 1 cc. of the extract was
then injected into the gastric branch of the right epi-
ploic vein. This caused the onset of shock — the symp-
toms developing slowly in the same sequence as is
found in traumatic shock. Upon removal of the anes-

APPENDIX 293

thetic, the animal regained consciousness promptly,
but severe shock soon supervened. The injection was
made at 4.30 P.M. when the rectal temperature was
39.4°. Following the injection the temperature
dropped regularly, reaching 34.1 ° at 8.45 P.M., when
the animal died in shock.

Rabbit, weight 1,200 gms. 1:30 P.M. Rectal tem-
perature 39°. Injected 2.5 cc. of the autolysate into the
marginal vein of the ear. Symptoms of shock began
at 2.00 P.M., the animal appearing apathetic. 2.30.
Eyes dull, gums pale. 3.30. Reflexes diminished,
marked pallor of mucous membranes of mouth, nose,
and conjunctivae, pulse, rapid and weak, labored
breathing, rectal temperature 37°. Temperature 34°
2.1 8. 45P.M. T)'itd9.00. Immediate autopsy disclosed
marked congestion of stomach, duodenum, upper
small intestines, and liver.

That extracts of charred tissues obtained from
burns behave similarly to tissue autolysates is shown
by the following experiments:

One half gram of charred tissue was dissected from
the burned area on the back of a cat which had passed
into shock as a result of a burn. This was ground in
a mortar with 5 cc. of saline and filtered. 2 cc. of this
extract were then injected into the jugular vein of a
cat weighing 2,200 gms. Shock developed immedi-
ately following the injection, but the animal recovered
after several hours.

One cc. of this extract was injected into the mesen-
teric vein of a cat weighing 2,500 grams. Death fol-
lowed within 3 minutes.

Ten grams of rabbit muscle were charred in a
crucible over a Bunsen flame. The black mass was

294

APPENDIX

extracted with 10 cc. of sterile water, boiled for a
few minutes and filtered. 5 cc. of this extract were
injected into the ear vein of each of two rabbits weigh-
ing 1500 grams. Both developed immediate shock
with diminished reflexes, nystagmus, respiratory and
circulatory failure. One animal died after 2 hours
in profound shock and the other after a lapse of six
hours.

The experiments presented above are typical of
literally hundreds of similar ones recorded in our
laboratory notebooks.

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35. Woodruff. L. L. ( 191 1 ) : Jour. Expt. ZooL, 10, 551-581.

36. Woodruff, L. L. (1914) : Jour. Expt. ZooL, 14, 575-582.

37. Wright, G. P. (1925) : Jour. Expt. Med., 39, 577.

Chapter VII

1. Bainbridge, F. a. (1919): "The Physiology of Muscular

Exercise," Longmans, Green and Co., New York.

2. Bayliss, W. (1918) : Proc. Roy. Soc. Med., 12, 1-34.

3. Cannon, W. B. ( 1902) : Jour, of Physiol., 8, No. 5.

4. GiLMAN, A. (1903) : Jour, of Anat., 2, No. 2.

5. Lewis, T. (1924) : Heart, 11, 119.

6. Lewis, T., and Grant (1924) : Heart, 11, 209.

7. Manwaring, W. H. (1929) : Science, N. S., 70, 1-7.

8. Petersen, W. F. (1928): In Jordan and Falk's "Newer

Knowledge of Bacteriology and Immunology,'" Univ. of
Chicago Press, Chicago.

9. Turck, F. B. (1903): "A Study of Fatigue in Gastric

Muscle," The Med. Standard, 26, 623-627.

10. Turck, F. B. (1905) : "Atony and Catarrh of the Gastro-

intestinal Tract," Trans. Tenn. State Med. Assn., 72d
Session, 146—163.

11. VON Uexkull, J. (1895) : Zeit. f. Biol., 31 and 32.

12. Vincent, S., and Sheen, W. (1903): Jour. Physiol., 29,

242-265.

13. Wells, H. G. (1925) : "The Chemistry of Immunity," The

Chemical Catalogue Co., New York.

302 BIBLIOGRAPHY

14. Zinsser, H. (1928) : Bull. New York Acad. Med., 4, 351-
358.

Chapter VIII

1. Ehrlich, p. (1891) : Deut. med. Woehnsehr., 17, 976, 1218.

2. Ehrlich, P. (1892) : Zeit. f. Hyg. u. Infectionskrankh, 12,

183-203.

3. Hartman, C. G. (1931) : Sci. Monthly, 33, 17-27.

4. Holland (1920) : Am. Jour. Dis. Child., July, 1920.

5. Muller, H. J. (1925) : Genetics, 10, 470.

6. TuRCK, F. B. (1918) : Reference 28, Chapter II.

7. TuRCK, F. B. (1919) : "Tissue Necrosis and Autolysis in Re-

lation to Toxaemia: Experimental and Clinical Observa-
tions," Int. Clinics, 4, series 29.

8. TuRCK, F. B. (1921a) : Reference 9, Chapter V.

9. TuRCK, F. B. (1921b) : "Metabolic Diseases Caused by Tis-

sue Extract," Wisconsin Med. Jour., 20, 271-277.

10. TuRCK, F. B. (1922) : Reference 26, Chapter III.

11. TuRCK, F. B. (1923) : "The Pathological Reaction of Tissue

Extract Liberated in Pregnancy," Am. Jour. Obs. and Gyn.,
5, 139-155.

12. Weichardt, F. (1912): Ueber Emiidungsstoffe, Stuttgart.

Chapter IX

1. Allee, W. S. (1931): "Animal Aggregations," Univ. of

Chicago Press, Chicago, 1931.

2. Greenman, M. J., and Duhring, F. L. (1923) : "Breeding

and Care of the Albino Rat for Research Purposes," The
Wistar Institute, Philadelphia, 1923,

3. King, Helen (1919) : "Studies on Inbreeding," The Wistar

Institute, Philadelphia, 1919.

4. TuRCK, F. B. (1906) : "Ulcer of the Stomach: Pathogenesis

and Pathology," J. A. M. A, 56, 1753-1763.

5. TuRCK, F. B. (1908): "Experimental Studies on Round

Ulcer of the Stomach and Duodenum," Jour. Med. Res.,
17, 365-377.

6. TuRCK, F. B. ( 1919) : "Etiology of Pneumonia and its Serum

Treatment," Med. Rec, 95, 719-721.

BIBLIOGRAPHY 303

Chapter X

1. Besredka, a. (1931) : Ann. Inst. Pasteur, 46, 542-557.

2. Irons, E. E. (1931) : J. A. M. A, 96, 1289-1293.

3. Manwaring, W. H. (1930) : Jour. Immunol., 19, 155-163.

4. SiMMONDS, J. P. ( 1928) : Chapter LVII in Jordan and Falk's

"The Newer Knowledge of Bacteriology and Immunology,"
Univ. of Chicago Press, Chicago, 1928.

5. TuRCK, F. B. (1903): "A Study of Fatigue of Gastric

Muscle," Medical Standard, 26, 623-627.

6. TuRCK, F. B. (1904) : Reference 22, Chapter II.

7. TuRCK, F. B. (1908) : Reference 5, Chapter IX.

8. TuRCK, F. B. (1909) : "Zur Aetiologie und Pathologic des

Runden Magen und Duodenal Geschwures," Proc. Int.
Med. Congress, Budapest, Sec. 3, 196.

9. TuRCK, F. B. (1910) : "Zur Aetiologie und Pathologic des

Runden Magen und Duodenal Geschwures, Neue Experi-
mente," Zeit. f. Expt. Path. u. Therap. (4) 7.

10. TuRCK, F. B. (1914) : "The Diffusion of Bacteria into the

Intestinal Wall," Trans. Am. Gastro-Enterol. Assn., 198-
219.

11. TuRCK, F. B. (1917): "Intestinal Venous Stasis," Boston

Med. and Surg. Jour., 176, 663-669.

12. Zinsser, H. (1931): "Resistance to Infectious Diseases,"

4th Ed., Macmillan Co., New York.

Chapter XI

1. Alexander, H. L., Weaver, W. K., and McConnell, F. H.

(1931) : Proc. Soc. Expt. Biol, and Med., 27, 486.

2. NuTTALL, G. (1904): "Blood Immunity and Blood Rela-

tionship," Cambridge University Press, Cambridge, England.

3. Petrie, Flinders (1932) : "Seventy Years in Archeology,"

Henry Holt, New York.

4. TuRCK, F. B. ( 1921 ) : "Metabolic Diseases Caused by Tissue

Extract (Cytost)," Wisconsin Med. Jour., 20, 271-277.

5. Weaver, W. K., McConnell, F. H., and Alexander, H. L.

(1931) : Proc. Soc. Expt. Biol, and Med., 28, 468.

304 BIBLIOGRAPHY

Chapter XII

1. BuENNiNG, E. (1926) : Bot. Archiv., 15, 4-60.

2. Cholodny, N. (1926) : Jahr. Wiss. Bot., 65, 447-459.

3. Cholodny, N. (1927) : Biol. Zentralbl., 47, 604-626.

4. Darwin, C. (1881) : "The Power of Movement in Plants,"

New York.

5. Darwin, C. ( 1882) : "The Movements and Habits of Climb-

ing Plants," London.

6. DoLK, H. C, and Thimann, K. V. (1932): Proc. Nat.

Acad. Sci., 18, 20.

7. JosT (1893) : Cited by Schlumburger.

8. LoEB, J. (1918) : "Forced Movements, Tropisms and Animal

Conduct," J. B. Lippincott Co., Philadelphia.

9. LoEB, L (1924): "Regeneration," McGraw-Hill Co., New

York.

10. MacDougal, D. T. (1896) : Ann. of Bot., 10, 373-402.

11. Paal, a. (1918) : Jahr. wiss. Bot., 58, 406.

12. Pfeffer, W. (1885) : Unters. a. d. bot. Inst. Tiibingen, 1,

483-535.

13. Peirce, G. J. (1894) : Ann. of Bot., 8, 53-117.

14. Sachs, J. (1873) : Arb. bot. Inst. Wurzburg, 1, 385-474.

15. Schlumburger, O. (1926): Angewandte Bot., 8, 62-274.

16. Schneider (1925) : Cited by Schlumburger.

17. Spaulding, V. M. (1894) : Ann. of Bot., 8, 423-452.

18. Went, F. W. (1928a) : Rec. trav. bot. neerl., 25, 1.

19. Went, F. W. (1928b) : Rec. trav. bot. neerl., 25, 483-489.

20. Wallace, R. H. (1928) : Am. Jour. Bot., 15, 509-523.

Chapter XIII

1. Harding, M. C. (1921): Calif. State Journ. Med., 19,

26-28.

2. LuDWiG, W. (1931) : Klin. Wochenschr., 10, 1531.

3. Mawson, Douglas (1914) : "The Home of the Blizzard,"

J. B. Lippincott Co., Philadelphia.

4. Nathan, P. W. (1917) : Jour. Med. Res., 36, 197-223.

5. TuRCK, F. B. (1919a): "Tissue Necrosis and Autolysis in

BIBLIOGRAPHY 305

Relation to Toxemia: Experimental and Clinical Observa-
tions," Int. Clinics, 4, series 29.

6. TuRCK, F. B. (1919b) : Reference 6, Chapter IX.

7. TuRCK, F. B. (1920) : "Treatment of Borderline and Ob-

scure Cases," N. Y. State Jour. Med., 20, 82-87.

8. TuRCK, F. B. (1921) : Reference 4, Chapter XI.

9. TuRCK, F. B. (1922) : Reference 25, Chapter III.
10. Weiss, R. F. (1931) : Med. Welt., 5, 1100.

Chapter XIV

1. Child, C. M. (1915): "Senescence and Rejuvenescence,"

Univ. of Chicago Press, Chicago.

2. TuRCK, F. B. (1899) : "Treatment of the Abdominal Viscera

through the Colon," J. A. M. A., 33, 880-886.

INDEX

Activity and growth, lS9ff., 163
Anesthetics, 69-72
Animals, breeding of, 174, 193ff.
Animals, captive, 173
Animals, crowding of, 197
Anticytost, 169

in pregnancy, 183

and fecundity, 208
Antienzymes, 69-71
Archusia, 130
Arthritis, 259ff.
Ash cytost, 232ff.
Ash, of tissues, 240ff.
Atrophy, 164

Autointoxication, 21, 55, 62
Autolysis, 22, 49

by injury, 51, 248

by irritants, 31-36, 64-67

by ligation, 58-62

by starvation, 49, 103ff.

in plants, 248

mechanism of, 50-52, 57

uterine, 180

B

Bacillus colt, feeding of, 104, 196

pathology from, 105
Bacteria, growth of, 136-138

and cytost, 214ff.
Bacterial infection, 212
Bios, 5

Cytost, effect on,
kidney, 89ff.
respiratory system, 84
splanchnic circulation, 29, 76
tissue culture, 122ff.
Cytost, in air, 198
in tissue ash, 232
in pregnancy, 115, 181
intoxication, 262

treatment of, 264
immunity to, 98, 223
liberation of,

by bacteria, 213, 230

by irritants, 98

by trauma, 51, 248
plant, 245ff.

specificity of, 89, 111, 237
stimulation by, 112ff.

D

Debridement, 24
Desmones, 130
Diet, 194ff.

high fat, 106
Dust, cytost in, 198ff.

mummy, 231

E

Egg production, 173
Electrosurgery, 46
Endoderm and cytost, 83
Exercise, 158, 194, 205

Capillary permeability, 81

stasis, 80ff.
Cells, death of, 14, 15

interdependence of, 15
Cell toxins, 5
Crowding, 197
Cytost, extract of, 94

toxicity of, 95

Fatigue, 15 Iff.
antitoxin, 170
resistance to, 151
toxin, 154

Glomerulitis, 92
Growth hormone, 246
Gyromele, 27

307

308

INDEX

H

Histamine, 44, 242

tache, 150
Hormones, division, 130

growth, 246

Imhotep, 10

Immobilization, 165

Immunity, to cytost, 151, 168
to mustard, 147
transmission of, 177, 191, 207ff.

Immunization:

to cytost, active, 168, 176
to cytost, passive, 170, 176
to cytost and fertility, 208
to shock, 168

Inaction, 107

Inanition, partial, 104ff., 209

Intumescences, 247ff.

Lung, experiments on, 84ff.
Lung paste, 87

M.

Mitosis, stimulation of, 122, 248
Monkeys, diet of, 106ff.
Mutilation, 9

N

Natural resistance, 139ff.
Neuritis, experimental, 261
Nutrition and cytost, 107

Photochemical products, 7, 8, 247
Plants, growth of, 11, 245
Pneumonia, 87
Potentiation of cytost,

by bacteria, 218ff.

by salts, 242
Pregnancy, toxemia of, 180ff.
Protozoans, growth of, 131-136

Q

Quiescence, enforced, 107fl.

Resistance, natural, 139, 159, 167ff.

Senescent changes, 108ff.
Shock, 17

anaphylactic, 219

from anesthesia, 37^3

from burns, 23ff.

postural, 18

primary cause of, 26

toxic nature of, 17, 23, 43ff.

traumatic, 17, 20ff., 269

wound, 17, 20ff., 269
Species specificity, 239
Splanchnic circulation, 18, 19, 29, 30,

76
Starvation, 103ff.
Stasis, capillary, 80fl.
Stereotropism, 251
Stomach mucosa,

reaction to irritants, 28-36, 53
Sunburn, 141, 280

Tissue ash, 232

effects on tissue cultures, 126-128

specificity of, 235

toxicity of, 233
Tissue culture, 117ff.

growth stimulants in, 119-121

action of cytost on, 122ff.
Toxemia of pregnancy, 180ff.
Traumatropism, 252
Trephones, 130
Tropisms, 247ff.

U
Ulcers, gastrointestinal, 102, 196

W

Wound healing, 10, 142, 171

Zona transformans, 227
effect of shock on, 228

1