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Studies on the Locus of
Antibody Formation

1913

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IV

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STUDIES ON THE LOCUS OF ANTIBODY
FORMATION

BY

FREDERICK P. GAY AND G. Y. RUSK

From the Hearst Laboratory of Pathology and Bacteriology
University of California

Reprinted from the Transactions of the Fifteenth International

Congress on Hygiene and Demography, held at

Washington, D. €., September 23-28, 1912

(10) WASHINGTON : GOVERNMENT PRINTING OFFICE : 1913

•

STUDIES ON THE LOCUS OF ANTIBODY FORMATION.

FREDERICK P. GAY and G. Y. RUSK, from the Hearst Laboratory of Pathology
and Bacteriology, University of California.

Since the critical work of Knorr 1 on toxins it has been generally
accepted that antibodies are formed, not by a simple inversion of
antigens, but by a reaction on the part of the cells of the animal that
has received the antigen. Correlatively, it has been assumed that cer-
tain cells have a particular affinity for a given antigenic substance
and are presumably specifically fitted to produce the corresponding
antibody. Ehrlich's receptor hypothesis, while stating this assump-
tion more concretely, has in no instance given direct proof that any
particular type of cell gives rise to any given antibody. The experi-
ment of Wassermann and Takaki 2 that demonstrated the apparent
neutralization of tetanus toxin by brain substance is no longer re-
garded as a proof of the nerve-cell origin of tetanus antitoxin. In-
deed the work of Loewi and Meyer 3- would show that injection of
the toxin into nervous tissue produces an increased susceptibility of
the animal to tetanus toxin rather than an increased resistance.
The fact that tetanus toxin disappears rapidly from the circulating
blood of susceptible animals and may soon be demonstrated in the
central nervous system would not, it would seem, prove conclusively
that the toxin may not also have been fixed and neutralized by other
body cells. At least it would seem necessary to assume that the
cells responsible for the antitoxin formation must first fix the an-
tigen. This proof of antigen fixation, indeed, constitutes one of the
methods that have been employed in searching for the locus of an-
tibody formation. The only other apparent method of determining
antibody origin would seem to lie in the early demonstration of
antibodies in given cell groups before they are demonstrable in the
circulating blood.

Very little information, therefore, on the site of antibody forma-
tion has been gained from studies on toxin and antitoxin. Our
information, inconclusive as it is, has been obtained from the work
with other antibodies, and we would do well to consider first what
data have been accumulated in respect to each of the antibody

272713

2 SECTION I. HYGIENIC MICROBIOLOGY AND PARASITOLOGY.

RACTERIOLYSINS.

Pfeiffer and Marx4 inaugurated the first systematic attempts to
discover the origin of lytic antibodies. Their work would seem to
indicate very clearly that the protective antibodies directed against
the cholera spirillum are elaborated in the leucopoietic organs, par-
ticularly in the spleen, but to a less extent in the bone marrow, inas-
much as extracts of these organs protect guinea pigs from infection
before the blood serum does. Deutsch5 essentially corroborated
these findings with B. typhosus and Castellani 6 with B. dysenteriae.
Levaditi's7 conclusions from his work with the spirillum of hen
septicemia were likewise corroborative, but his conclusions would
not seem justified by the experimental evidence. These authors all
agree that the spleen is not essential, as its removal at best but
slightly inhibits antibody formation; the bone marrow and lymph
nodes are secondarily concerned.

A careful inspection of Levaditi's experiments would seem to point
to the blood stream as a possible source of antibodies, although his
conclusions are different. Several authors, however, have attempted
to disprove experimentally the local or leucocytic origin of the bac-
teriolysins. Thus Stenstrom8 found that the injection of bacteria
plus leucocytes led to less antibody production than the injection of
bacteria alone. Pfeiffer and Marx found less antibodies in the
ground leucocytes of immunized animals than in the plasma.
Deutsch found the lysins were not present in peritoneal exudate and
Paetsch8 extended this finding so as to include both pleural and
peritoneal exudates and the lining endothelia of these cavities.

Violle's 10 injections into the gall bladder for the rapid production
of antibodies indicate the possible function of the liver in this con-
nection, which is emphasized more distinctly in work with other
antibodies.

HEMOLYSINS.

Metchnikoff11 and Cantacuzene 12 originally suggested, on what
would seem to be largely philosophical grounds, that the hemolysins
may be formed by the leucocytes owing to the recognized phagocy-
tosis of blood cells by the mononuclears. The output of " fixatives,"
according to Metchnikoff, varies directly with the degree of phago-
cytosis. There is direct evidence that goes to prove, however, that
these bodies are not formed in the blood stream. McGowan 1S
showed that no leucocytosis follows the injection of alien blood and
Hektoen and Carlson 14 have shown by transfusion experiments that
the antigenic properties of foreign blood cells disappear from the
circulation within seven hours.

Gay and Rusk.] THE LOCUS OF ANTIBODY FORMATION. 3

Among the fixed tissues, the liver and spleen seem to have shared
the honors as the possible sites of hemolysin formation. Leuck-
hardt and Becht,13 following the work of Hektoen and Carlson,
found that the spleen alone of the organs in a dog that has received
goat or rat corpuscles 24 hours previously has the property of im-
munizing new animals. As a proof of the temporary location of the
red blood cells that have been injected, this evidence is undoubted,
although well recognized from other work; as a proof of the spleen
as the site of antibody formation it would seem to be negative. The
statement by London16 that splenectomy decreases the formation of
hemolysins is categorically denied by Jakusche witch.17 Brezina 18
found that a specific serum against the leucopoietic organs had no
effect in disturbing hemolysin formation. Carrel and Ingebristen 19
have produced hemolysins in the growing embryonic spleen.

The evidence in favor of the liver as the site of hemolysin for-
mation is more positive. Both Cantacuzene 12 and McGowan 13 have
shown the function of Kiipfers cells in the destruction of red blood
corpuscles. Muller,20 in an interesting paper, has apparently traced
normal hemolysin formation to the liver and has even been able to
stimulate its excretion in the liver suspended in Ringer's solution
outside the»body, by transfusing it with solutions containing iodine
(iodipin). A further contribution to the stimulating effect of iodin
compounds is given by Hektoen 21 who was able to increase the output
of hemolytic sensitizers in dogs by injecting sodium iodoxybenzoate.
Violles 10 method of producing antibodies by gall-bladder injection
has already been referred to under bacteriolysins.

AGGLUTININS.

Whereas the evidence for bacteriolysin formation seemed to favor
formation in the spleen or liver, the evidence for the locus of origin
of the bacterial agglutinins points distinctly to the blood stream.
Thus the work of Deutsch,5 Castellani,6 Rath,22 Weil and Braun,23
and Kraus and Schiffrnann 24 all shows that the agglutinins appear
in the blood serum before they are present in the extract of any
organ. Although Gruber25 originally suggested that the polymor-
phonuclears form the agglutinins, no experimental evidence goes to
prove this. The experiments of Achard and Bensaud,26 Widal and
Sicard,27 of Paetsch,9 and of Kraus and Schiffmann24 all seem to
disprove leucocj^tic or local origin.

There is some evidence of agglutinin formation in the spleen
offered by v. Emden,28 Jatta,29 and Girgoleff.30

PRECIPITINS (ANTIBODIES OF SOLUBLE ANTIGENS).

In the case of precipitin formation, again the evidence seems
divided. It is shown by two sets of observers (Petit and Carlson,81

4 SECTION I. HYGIENIC MICROBIOLOGY AND PARASITOLOGY.

Vaughan, Gumming, and McGlumphy32) that soluble antigenic sub-
stances like egg white or serum apparently disappear within a few
hours from the circulating blood. This is shown by the impossi-
bility of producing, in the one set of experiments, antibodies in
another animal that is liberally transfused with such blood, and,
on the other hand, by the failure to produce anaphylaxis in guinea
pigs to the substance originally injected (egg white, Vaughan and
collaborators). In apparent contradiction are the observations of
several observers on the relation of leucocytosis to precipitin forma-
tion. Thus both Cantacuzene 83 and Swerew 34 have noted a marked
hyperleucocytosis preceded by an absolute decrease in polymor-
phonuclears, which may reasonably be related to the liberation of
'precipitins. This observation fits in neatly with that of Hiss and
Zinsser,35 who obtained nonspecific bacterial precipitins from leuco-
cytic extracts, and that of Stenstrom.8 who found that hemologous
leucocytes injected with the precipitinogen increases precipitin out-
put. Kraus and Schiffmann 24 emphatically regard the blood as the
source of precipitins, whereas Cantacuzene. in spite of his evidence
in favor of the leucocytes, is inclined to trace precipitin formation
to the spleen.

The liver is by no means to be overlooked in discussing precipitin
formation. The work of Manwaring,36 of Nolf,37 and of Balizot,88
on anaph}rlactic shock, would seem to point to the liver as the seat
of action, and so, indirectly, owing to the relation that exists between
anaphylaxis and precipitins, as a possible location of the latter
substances.

It is evident from this brief survey of the literature that no general
statement can be made on the locus of antibody formation in gen-
eral. It may well be that each of the antibody types is produced
in a different place or places. But even when we consider the pos-
sible seat of origin of any particular class of antibodies, we are
struck by the apparent confusion in the acquired data. In the case
of any of them we may still say that the antibody may be formed
either in the blood stream or in the fixed tissues. There seems
greatest agreement on the point that antibodies are formed either
by the leucocytes or the leucocyte- forming organs. And yet a good
deal of recent work points with increasing emphasis to the liver, an
organ which, in view of its other functions, might logically likewise
serve to produce antibodies.

Our own studies on antibody formation have been actively in
progress for over a year. We regard them hitherto as largely pre-
liminary and they have led rather toward establishing certain meth-
ods of attack and the evolution of working hypotheses of possible
heuristic value than to any conclusion on the main subject at issue.

Gay and Rusk.] THE LOCUS OF ANTIBODY FORMATION. 5

Certain by-products of the investigation are in themselves of distinct
interest, although their results are negative in so far as explaining
antibody formation is concerned. Two incidental investigations
may first be summarized before dealing with the work that bears
more directly on the site of antibody formation :

I. THE EFFECT OF IODIPIN ON THE OUTPUT OF ARTIFICIAL HEMOLYSINS.38

Muller's20 experiments led him to the conclusion that both nor-
mal sensitizer (amboceptor) and alexin are formed in the liver,
but that their output depends on the stimulating action of the
iodin of the thyroid gland. This author finds that the injection of
thyroid preparations, or of various iodine preparations, notably of
iodipin (Merck, 25 or 10 per cent of iodin), produces a distinct
increase in from 24 to 36 hours of the normal hemolysins in rab-
bits and other animals. As already stated, this increase comprises
not only an increase in alexin, as tested on blood cells sensitized by
an artificial hemolysin, but an increase of the normal hemolytic
sensitizer. Hektoen 21 has further found an increase in hemolytic
sensitizers in dogs that received a single dose of goat's blood over
the amount produced in control dogs on giving injections of sodium
iodoxybenzoate. This alleged increase of normal hemolytic sensi-
tizers immediately suggested the importance of determining the
effect of iodin on artificial hemolysins, not only as a matter in itself
of theoretical and perhaps of practical importance, but as bearing
on the origin of antibody formation. Differences that might appear
in the hemolytic potency of the sera of immunized animals would
presumably be more striking than corresponding differences in nor-
mal hemolysins, owing to the much greater strength of immune sera.

Our experiments deal, first, with the effect of a single injection of
iodipin (Merck, 10 per cent) in rabbits that had been immunized by
several injections of washed guinea-pig corpuscles. In several ex-
periments a control animal that had been immunized in a similar
manner, but that received no iodipin, was carried through. The
animals with and without iodipin were bled before injection from the
ear and at intervals subsequently; the separate sera were heated at
once to 56° for one-half hour, and at the end of the experiment all
were tested for hemolytic units at the same time with the same cor-
puscles and alexin. In other experiments a critical intravenous re-
injection of the antigenic blood was given in two highly immunized
rabbits, and on the following day one of them was given iodipin.
Both sera were then tested at intervals for hemolytic potency. There
was no evidence from any of these experiments that the injection of
iodipin will increase the output of artificial hemolysins in the im-
munized rabbits.

6 SECTION I. HYGIENIC MICKOBIOLOGY AND PARASITOLOGY.

II. HISTOLOGICAL STUDIES OF THE TISSUES IN IMMUNIZED ANIMALS WITH
A COMPARISON OF CHEMICAL AND MICROCHEMICAL TESTS FOR GLYCO-
GEN.40

The histopathological studies of Gay and Southard41 in serum
anaphylaxis in the guinea pig have seemed an interesting contribu-
tion to the relation of structure to function. These authors found
that anaphylactic intoxication is accompanied by definite lesions in
the nature of hemorrhages and more particularly by fatty changes
in parenchyma and in the endothelium. These endothelial fat
changes could be produced in a few minutes following intravenous
injection and obviously bear direct relation to the cyclonic symptoms
of the syndrome. It occurred to us that similar evidence of func-
tional cellular activity might be histologically demonstrable in the
cells that are engaged in antibody formation. For this purpose
rabbits were highly immunized by repeated intravenous injection of
washed guinea-pig corpuscles and following a rest of two or more
weeks were given a critical reinjection of 1 cubic centimeter of
washed guinea-pig blood and at subsequent intervals bled and the
tissues fixed in various ways and stained by many methods. The
critical reinjection was aimed, obviously, to obtain the antibody-
producing cells in a condition of highest activity.

In the first experiment a series of immunized rabbits received
each the critical reinjection of antigen and were then bled at 1, 4.
and 24 hours and 4 and 6 days subsequently. A careful histological
study of tissues from this series showed in the 24-hour animal a very
marked increase of glycogen in the liver (Best's carmine stain, alco-
hol fixation). The animals bled before and after this period gave
a regular increase and decrease of glycogen to the 24-hour maximum.
A more careful control of this experiment has involved us in techni-
cal difficulties that seemed too time consuming and led, moreover, to
far less encouraging results than the first experiment. The question
of the effect of starvation on the amount of glycogen alone seemed
unsurmountable ; it was found, for example, that rabbits that have
not been fed for two days have stomachs stuffed with food. We are
still in doubt as to the significance of our first find of increased
glycogen. An attempt to correlate the microchemical reaction of
glycogen with a careful chemical analysis of total glycogen in the
liver has, however, led to results of importance. So far as we are
aware, there has been no systematic comparison between the chemical
analysis of an organ and microchemical staining reactions in a sam-
ple of it. Dr. Rusk has studied the amount and distribution of
glycogen in sections of 22 rabbit livers stained by the Ehrlich iodin
method and the Best carmine method, with a chemical analysis of a
greater (weighed) portion of the same livers, following Pfluger's

Gay and Rusk.] THE LOCUS OF ANTIBODY FORMATION. 7

method to the conversion of glycogen to glucose, and at this point
introducing Betrand's modification of Fehling's method as more
accurate in measuring the amount of copper reduced. It is found
that the microchemical method serves to give somewhat definite in-
formation as to the amount of glycogen present, but within a limited
range only, for when the chemical analysis showed very much or
very little glycogen the staining method was at times wholly
inadequate.

III. THE FATE OF HORSE SERUM INJECTED INTRAVENOUSLY IN NORMAL
AND IMMUNIZED RABBITS.

The two general methods that have been and may be employed in
seeking antibody origin are either to trace the course of the injected
antigen to some group of cells or to seek the precocious appearance
of antibodies in extracts of a given group of cells. The latter method
is the one that has been used most frequently, but it is the former
that we have employed. Our observations, some of which have
already been published,42 began with a study of the fate of horse
serum injected into the blood stream of rabbits that had been im-
munized against horse serum. They have since led to further studies
on the result of an initial injection of horse serum in normal rabbits.
In all instances our results deal with an injection of one cubic centi-
meter of serum intravenously.

In beginning the experiments with immunized animals it was neces-
sary first to determine the best method of detecting the antigen that
was reinjected. It was found that when horse serum is injected intra-
venously in rabbits that have a high precipitin content for horse serum
it nevertheless remains demonstrable by the fixation reaction or the
precipitin reaction for 24 hours. The reactions are carried out by
adding the antigen-containing antiserum to a pure antiserum. After
48 hours the antigen is no longer demonstrable. The persistence of
the antigen in the immune animal is accompanied by a fall in the
precipitin value of serum (negative phase). It is of interest to note
that although this antigen-containing antiserum will not precipitate
or fix alexin spontaneously, it will react with another antigenic anti-
serum as well as with a pure antiserum. It was rather surprising to
us to fail in any conclusive demonstration of the antigen by the
fixation reaction in extracts of the organs of these same immunized
animals (spleen, lymph nodes, liver, kidney, and muscles) either
at the same time the antigen is present in the blood or even 24 hours
later.

Of undoubtedly greater significance is the fact that neither the
antigen-containing antiserum, nor the organ extracts of the same
animal will sensitize guinea pigs to subsequent intoxication by horse

8 SECTION I. HYGIENIC MICROBIOLOGY AND PAEASITOLOGY.

serum. We compared the fixing values of pure horse serum and
antigenic antiserum, and although one fixing dose of the horse serum
will sensitize guinea pigs, many fixing doses of the antigenic anti-
serum fail to do so (at least 100). This would explain the results
of Vaughan, Gumming, and McGlumphy32 who found that egg
white apparently disappears from the circulation in a few hours
when tested for by the anaphylaxis reaction. It is perhaps also in
harmony with the work of Hektoen and Carlson14 and of Petit
and Carlson 81 who proved by transfusion that the antibody-incit-
ing factor in blood cells or in serum leaves the circulation in a few
hours. We might possibly assume that the factor in the antigen that
produces the antibody differs from the one that unites with it. (Cfr.
Bang and Forsmann.43

The results of injecting horse serum into normal animals are also
of interest. The horse serum is detectable by the precipitin and
fixation reactions for several days. It apparently does not sensitize
in large doses even after 24 hours. The fixation and precipittnogen
antigen is present not only in the blood, but also in the various organ
extracts (in this case carefully freed of blood) in uniform amounts
on the seventh, eighth, ninth, and tenth day. Of greatest importance
is the fact that the corresponding antibodies have begun to appear
in the serum two or three days before the antigen disappears. Simi-
lar facts have been noted by Hintze.44 It is evident then that not
all the antigen is used up in producing the antibody. We have to
imagine either that the antigen continues to unite with the cell or
stimulate the cell for some time after antibodies appear, or that the
antibody stimulant differs from the antigen fraction that unites with
the antibody. This latter hypothesis seems to us for the present
the most valuable for a working basis. It explains, moreover, our
own failure to produce anaphylaxis (cfr. also Vaughan and his
collaborators) , and also the results of Hektoen and Carlson. We are
continuing our work with this as a working hypothesis and also
with a possible further elucidation of the obscure phenomenon of
anaphylaxis in mind.

Fortschr. der Mediz., etc., vol. 15 (1897), p. 657.
'Wassermann and Takaki : Berlin. Klin. Wochen., vol. 35 (1898). p. 5.
'Loewi and Meyer: Archiv fur experiment. Path., vol. 59 (1908), p. 355.
4Pfelffer and Marx: Zeit fiir Hygiene, vol. 27 (1898), p. 272.
5Deutsch: Annales de 1'Inst. Pasteur, vol. 13 (1899), p. 689.

9 Ca Stella ni : Zeit. fur Hygiene, vol. 37 (1901), p. 381.
TLevaditi: Annales de 1'Iust. Pasteur, vol. 18 (1904), p. 511.
'Stenstrom: Zeit fiir Immunitatsforsch., vol. 8 (1911), p. 483.
* Paetsch: Centralblatt fiir Bakt, Grig. I, vol. 60 (1911), p. 255

10 Violie : Annales de 1'Inst. Pasteur, vol. 26 (1912), pp. 381, 467.

u Metchnikoff : L'immunite dans les maladies infectieuses, p. 103; Annales
de 1'Inst. Pasteur, vol. 13 (1899), p. 737.
" Cantacuz£ne : Annales de 1'Inst. Pasteur, vol. 16 (1902), p. 522.

Gay and Rusk.] THE LOCUS OF ANTIBODY

"McGowan: Journal of Pathology and Bacteriology, vol. 14 (1909), 379;
vol. 15 (1911), 262.

"Hektoen and Carlson: Transact. Chicago Path. Soc., vol. S (1909), p. 4;
Joura. of Infect. Diseases, vol. 7 (1910), p. 319.

15 Leuckhardt and Becht: Transact. Chicago Path. Soc., vol. 8 (1911), p. 202.

"London: Archiv fur biologische Wissenschaften (1901), p. 328.

17 Jakuschewitsch : Zeit. fur Hygiene, vol. 47 (1904), p. 407.

18Brezina: Wein. Klin Wochenschr. (1905), p. 905.

19 Carrel and Ingebristen: Journ A. M. A., vol. 58 (1911), p. 477.

"Muller: Central, fur Bakt, I. Abt, Orig., vol. 57 (1911), p. 577.

21 Hektoen: Transact. Chicago Path. Soc., vol. 8 (1911), p. 138.

22 Rath: Central, fiir Bakt, vol. 25 (1899), 529.

23 Weil and Braun: Biochem. Zeitschr., vol. 17 (1910), p. 337.

24 Kraus and Schiffman: Aunales de 1'Inst. Pasteur, vol. 20 (1906), 225.
^Gruber: Miinch. mediz. Wochenschr. (1897), Nos. 17 and 18.

26 Ac-hard and Bensaud : Archiv fiir med. Experiment (1896), p. 748.

27Wk!al arid Sicard: Annales de 1'Inst. Pasteur, vol 11 (1897), p. 353.

28 v. Emden: Zeit. far Hygiene, vol. 30 (1899), p. 19.

"•Jatta: Zeit. fur Hygiene, vol. 33 (1900), p. 185.

30Girgoleff: Zeit. fiir Imniunitatsforsch, vol. 12 (1912), p. 401.

81 Petit and Carlson: Jour. Infect. Diseases, vol. 10 (1912), p. 43.

82Vaughan, Cumming & McGlumphy: Zeit. fiir Immunitatsforsch., vol. 9
(1911), p. 16.

33 Cantacuzene : Annales de 1'Inst. Pasteur, vol. 22 (1908), p. 54.

34Swerew: Rusaky Wratsch. (1910), p. 367; Ref. Jahresbericht der Iminuni-
tatsforschung, VI2, p. 527.

36 Hiss and Zinsser: Jour. Med. Research, vol. 19 (1908), p. 399.

38Manwaring: Zeitschr. fiir Immunitatsforsch., vol. 8 (1910), p. 1.

3TNolf : Archives internat. de Physiolog., Vol. X (1910), p. 37.

38Blaizot: Compt. rend. heb. Soc. de Biol., vol. 70 (1911), p. 383.

38 Gay & Rusk: Univ. Calif. Publ. Path., vol. 2, No. 7 (1912).

40 Rusk: Univ. Calif. Publ. Path., vol. 2, No. 9 (1912).

43 Gay and Southard: Jour. Med. Research, vol. 16 (1907), p. 143.

42 Gay and Rusk : Univ. Calif. Publ. Path., vol. 2, No. 6 (1912).

48 Bang and Forssmann: Centralblatt fiir Bakt, I. Orig., XL (1905), p. 151.

"Hintze: Zeit fiir Immunitatsforsch., vol. 6 (1910), p. 113.

DISCUSSION.

Dr. W. H. PARK: Observations made on the injection of toxins and
antigens into the blood or into the subcutaneous or other tissues in-
dicate that for many antibodies several varieties of cells take part.
The blood in an animal highly immunized to diphtheria toxin contains
about 100 times the quantity of antitoxin as the tissue fluids. If toxin
is added to antitoxin, union slowly takes place and the combined toxin
is no longer able to excite the production of antitoxin. When a horse
having a strongly antitoxic blood is injected intravenously with a
definite amount of toxin very little production of antitoxin takes
place, because most of it is neutralized by the antitoxin in the blood.
The same amount injected in scattered spots subcutaneous!}^ will
produce a large amount of antitoxin. The part absorbed into the
blood would meet the same fate as that injected intravenously. It

10 SECTION 1. HYGIENIC MICROBIOLOGY AND PARASITOLOGY.

seems, therefore, certain that some or all of the cells in the area in the
subcutaneous tissues that the toxin reaches must take part in pro-
ducing antitoxin. If toxin is injected through the trachea into the
lungs it acts in the same way as when injected subcutaneously. I
have seen similar, though not as definite, results with the injection
of various bacterial antigens. These facts seem to suggest that
more varied cells than Dr. Gay indicates may take part in antibody
formation.

Dr. VAUGHN thinks that the ferment is formed by different cells
according to the sensitizer used. He referred to the work of J. W.
Vaughan on sensitization to cancer proteins in which it appears
that the ferment is formed in the large mononuclear leucocytes and
that the sensitization is transitory. The fact that the nonpoisonous
part sensitizes may be due to the presence of a minute trace of un-
broken proteins. However, this seems highly improbable because
* this part does not sensitize to itself.

O

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