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Патент USA US3097151

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United States Patent 015 ice
1
3,097,141
INHVIUNOLOGICAL IWETHOD
Elizabeth N. Wilcox Kidwell, 16 E. 9th St,
New York, N.Y.
No Drawing. Filed Feb. 26, 1959, Ser. No. 795,601
Patented July 9, 1963
2
but is easily determined for each species.
Thus 56° C.
has been found to be the optimum for Staphylococcus,
Streptococcus, E. coli, tubercle bacillus; 60° C. for Bru
cella; 64° C. for the poliomyelitis virus; 100° C. for the
6 tobacco mosaic virus. For biochcmicals I employ 58‘ C.
for animal use.
16 Claims. (Cl. 167-78)
The temperature and time of the heating step in reduc
ing the toxicity of antigens while retaining their anti—
genicity according to my invention may vary respectively
This invention relates to a method of modi?cation of
anaphylactogens which reduces toxicity while retaining
antigenicity.
3,097,141
10 from about 50° to 100° C. and about 15 to 28 hours.
It is an object of this invention to provide antigenic
materials of reduced toxicity.
The optimum heat modi?cation temperature and time
may be determined by a simple leukocyte response test
It is a further object to provide a means for desensitiza
which I have discovered. A suspension of cells in serum
tion to anaphylactic shock.
is drawn from a fresh sample of whole clotted blood of
An additional object is to provide novel means for 15 the species to which the modi?ed substance is to be
studying and preventing hypersensitivity.
administered. A small drop of serum is placed on a slide
Other objects will in part be obvious and will in part
and a small drop of “Viability Stain” is added and
appear hereinafter.
mingled with the blood. (The Viability Stain is prepared
My modi?cation of biological substances involves essen
from 0.1 g. Toluidin blue powder, 0.02 g. Janus green
tially four steps in the following order: pepsin digest, 20 powder and 1 ml. of absolute alcohol. The mixture is
oxidation, heat, and ion exchange. This modi?cation
ground and washed through ?lter paper with 200 ml. of
procedure provides useful biochemicals which retain sub
distilled water.) A drop of the modi?ed agent is then
stantial antigenicity with reduced toxicity and minimum
introduced, a coverslip is placed over the mixture, and
anaphylactic or sensitizing properties and which will re
the slide examined under oil immersion. The immediate
main stable at room temperature for a prolonged period, 25 and complete disappearance of all leukocytes with no
e.g. 11 years.
debris remaining indicates c?'ective response to a well
When modifying bacteria, a pure strain of bacteria is
modi?ed agent.
?rst grown on agar slants and harvested in saline at the
An even more effective method for determining opti
point of optimum growth. It is then centrifuged at about
snlntzim modi?cation is through the use of hemobiological
1000 rpm. for one minute to remove agar fragments, 30
and is then killed with heat or phenol prior to beginning
the modi?cation. As discussed below, an endotoxin may
be prepared by including the additional step of sonic
1 es.
This technique involves the use of a specimen of whole
clotted blood, tested with antigenic substances prepared
in known concentrations. A chamber 22x40 mm. is
marked with liquid plastic on a slide. The slide and
In carrying out my modi?cation, a solution is prepared 35 everything which comes in contact with the blood is
disruption.
by dissolving pure crystalline pepsin, sterilized by repeat
?amed.
A piece of clot from the blood sample is placed
edly freezing and thawing, in distilled water. For a
on the slide, and three drops of blood and one drop of test
suspension of about 5 billion organisms per cc., 1 mg. of
substances are added. The preparation is covered, with
pepsin per cc. is adequate. Proteolytic enzymes other
air excluded, with a coverslip, which is sealed in place with
than pepsin, such as papain, may also be used. The pH 40 impervious plastic. The slides are preserved under re
is promptly adjusted with 10% HCl to a pH of approxi
duced oxygen until resistant response appears sometimes
mately 4-5, suitably 4.6. The mixture is then placed in
as early as one week, or pathologic changes develop about
an incubator at about 37‘l C. for about 8-24, preferably
6 or more weeks later.
16 hours, after which it adjusted with 10% NaOH to a
This test is not so quickly performed as those described
45 above, but is particularly useful in determining when the
pH of about 7.
In the second step of modi?cation according to my
patient is suffering from disease of an organ or tissue, and
invention the incubated, pepsin-digested cells are oxidized,
the blood, especially the clot, is damaged in the manner
for example by addition of equal quantities of 30%
prescribed by substances either bacterial or biochemical,
sodium nitrite and 30% acetic acid. 5 cc. of each for
speci?c to the diseased area. Biochemicals found capable
every 3 cc. of bacterial suspension is adequate. After 50 of damaging blood as described are believed to act as an
about four hours at room temperature the vaccine is
allergen to which the body establishes a pathologic re
centrifuged and washed twice with sterilized distilled
spouse by acquiring sensitivity under circumstances of
water and twice with sterilized N saline. When working
stress. The immunologic treatment of chronic disease
with virus vaccines it is preferred to continue the oxida
would
therefore involve modi?cation of each substance in
65
tion process for twenty-four hours.
accordance with this invention and the use of such a modi
The oxidizing agents noted above tend to destroy some
?ed agent for desensitization to the corresponding normal
biochemicals. When working with such materials it is
substance.
preferable to use commercial H303 as the oxidant in an
A well modi?ed substance will produce a uniformly
amount approximating one-third the volume of the sus
pension to be modi?ed. The preparation is placed in an 60 more resistant response in hemobiological slides than dow
the corresponding normal one. The response to the op
incubator overnight, the exact time being unimportant.
timumly modi?ed substance will be more super?cial and
The third step is heat modi?cation. The oxidized prcp~
produce more hemoglobin crystals and less clot damage
aration, suitably contained in covered glass jars which
than does the unmodi?ed substance. It is preferred to
are sealed, is placed in a water bath at a temperature of
from about 50 to 100° C. I have found that the optimum 65 test a group of blood samples from several patients.
temperature for each bacterium may vary to some degree
A more antigenic toxoid is produced if heat modi?ca
3,097,141
4
where R is a substitnent selected from the class consisting
tion is carried out for only 6 hours. Such toxoid may
satisfactorily be used with all
species.
of hydrogen, chlorine, bromine, monovalent alkyl radi
cals, monovalent aralkyl radicals, monovalent aryl radi
Bacterial substances are next subjected to ultrasonic
disruption to provide a toxoid. ‘Biochemicals and viruses
are not subjected to this treatment, however. The ultra
cals and monovalent alkaryl radicals, and is described in
Patent 2,340,111, granted January 25, 1944.
A
IRA-400 is the reaction product of (l) a halomethyl
ated copolymer of a major proportion of a monovinyl
sonic disruption of the cells is suitably effected by subject
ing the heat-modi?ed vaccine to the in?uence of ultrasonic
aromatic hydrocarbon with from 0.5 to 20.0% of a divinyl
aromatic hydrocarbon, and ( 2) a tertiary monoamine and
waves for a period from about 20 minutes to about 90
minutes and, desirably, at a temperature not above 30° C.
More particularly, a rectangular vat, 12" x 8" x 4"
deep, having a 4" diameter focused piezo electric element
is described in Patent 2,591,573, granted April 1, 1952.
IRA-410 is the reaction product of (1) a benzene
built into the bottom, may be used as the ultrasonic trans
insoluble copolymer containing in chemically combined
ducer. The vat is partially ?lled with transformer oil, in
such a way that the ultrasonic power generated by the
piezo electric element is propagated through the 01]
form from 0.5 to 40 percent by weight of a polyvinyl
aromatic hydrocarbon and from 99.5 to 60 percent of a
monovinyl-aromatic compound of the class consisting of
monovinyl-aromatic hydrocarbons having the vinyl radi
toward a focal region within the oil. A cooling coil may
be immersed in the oil, thereby providing a means of
cal directly attached to a carbon atom of the aromatic
maintaining the oil temperature at approximately 4° C.
nucleus and nuclear halogenated derivatives thereof,
which copolymer contains an average of from 0.2 to 1.2
The coil should be positioned so that it does not inter
fere with the propagation of the sound energy from the 20 halomethyl ‘groups per aromatic nucleus in the copoly
concave surface of the piezo electric element toward the
mer, with (2) a tertiary amine selected from the group
consisting of the tertiary mono- and di-alkyl N-substituted
_
Small quantities (15 cc.) were sealed in 35 cc. th-m
alkanol and alkanediol amines, and is described in Patent
walled glass ?asks. The bottoms of these ?asks were
2,614,099, granted October 14, 1952.
spherical in shape, and were placed in the oil transmission 25
IR-4B is the product obtained by reacting by con
medium in such a way that the focal region of the piezo
densing together in an aqueous medium a polyphenylol
electric transducer fell inside the flask within the material.
alkane, one-half to two mols of an alkylene polyamine
In this position, sound energy from the concave surface of
per phenylol group present in said alkane, and formalde
the piezo electric element approaches the ?ask normal to
hyde in an amount at least equivalent both to the phenylol
its surface, and maximum transmission of energy through 30 groups and to the mols of polyamine, and is described
focal region.
the glass with minimum re?ection and de?ection is thereby
in Patent 2,356,151, granted August 22, 1944.
possible.
lR-45 is a haloalkylated copolymer of a mixture of an
A frequency of 800 k-ilocycles per second was employed,
aromatic monovinyl hydrocarbon and an aromatic divinyl
hydrocarbon, said mixture containing a predominant
and the material was radiated for a period of about 20
minutes. Sound intensity in the focal region within the 35 amount of said monovinyl hydrocarbon and said copoly
material, measured by means of a radiation balance (com
mer containing on the aromatic nuclei substituent groups
pensated for liquid ?ow), was in excess of 1,000 watts per
square centimeter. The focal region of high intensity
of the general formula:
1
sound energy encompassed a volume of approximately
10 x 10 x 10 mm.
It may be mentioned that the passage 40 in which n is an integer of value one to four and X is a
of the material through the focal region is a random phe
member of the group consisting of chlorine and bromine,
nomenon.
After about 20 minutes‘ radiation, approxi
is reacted with an amine containing a hydrogen atom on
mately ninety percent of the material was found to have
been subjected to the high intensity ultrasonic energy.
Satisfactory results have been obtained at high frequen
the nitrogen atom whereby a polymeric amine salt is
formed, and is described in Patent 2,591,574, granted
45 April 1, 1952.
Further IR-1l2 and lR-120 are strongly acidic sul
phonic acid group cationic resins; IRC-SO is a weakly
heat-modi?ed substance with an ion exchange resm.
acidic carboxylic cationic resin; IRA-400 and IRA-410
Non-bacterial substances are so treated directly following
are strongly basic quaternary amine anionic resins, and
heat modi?cation. Bacterial substances from which a 50 lR-4B and lR—45 are weakly basic polyamine anionic
toxoid has been prepared by sonic disruption are ?rst
resins. It is to be understood that the term “Amberlites"
combined with a portion of the undisrupted, heat-modi
as used herein and particularly in the claims refers to
?ed bacteria from which it was prepared, and the combi
resins of the above identi?ed kind.
' nation is contacted with an ion exchange resin. It is pre
Equal quantities of cationic and anionic resins which
cm.
The ?nal modi?cation step comprises contacting the
ferred to use a mixture of cationic and anionic resins. 55 have been sterilized, suitably by autoclaving, may be
Resins produced by Rohm & Haas and designated “Am
4
berlites". Ill-112; IR-l20; IR-SO; IRA-400; IRA-410;
used.
The time of contacting with the ion exchange resin is
111-48 and “IR-45" preferably “IR—120” and "IRA-410”
critical as will be shown below. For this reason the
have been found suitable.
exact time at which contacting is initiated should there
The "Amberlites" just above mentioned may be more 60 fore be carefully noted. In order to determine the
particularly identi?ed as follows:
optimum period it is convenient to employ the ?brinag
IR-112 and IR-120 are water-insoluble sulphonated
glutination technique which I have discovered. This
polymerizates of a mixture of a polyvinyl aryl compound
technique demonstrates a mechanism of immunological
and a monovinyl aryl compound, and are described in
response for which hitherto there has been no laboratory
Patent No. 2,366,007, granted December 26, 1944.
65 test. This method may be used to determine the point
IRC-SO contains a substantial amount of an insoluble,
of optimum modi?cation and is a particularly useful
infusible copolymer of a polymerizable mixture compris
method in practicing my invention. By use of this tech
ing (1) at least one compound having a polymerizable
nique it is possible to remove the modi?ed substance
from the resins at the point of optimum clumping, at
CH,=C<
grouping and at least one other polymerizable grouping, 70 which time they are of the greatest e?icacy. Two stains
are prepared as follows:
the unsaturated bonds of said polymerizable groupings
being non-conjugated with respect to each other, and (2)
at least one acrylic acid having the structural formula
Viability Stain
orn-G-ooou
75
Toluidin blue powder _______________ __gm-_ .1
Janus green powder ________________ __gm__ .0
Absolute alcohol ___________________ .._ml__ .1
3,097,141
5
6
.
Grind and wash through ?lter paper with 200 ml. of
?brinagglutination point, e.g.: Streptococcus, Staphylo
distilled water.
coccus and Bruoella on the 24th day and Mycobacterium
tuberculosis on the 25th day. The staining technique
Antigen Stain
for ?brinagglutination is thus useful as an aid in bac
Bluish eosin ....................... ..mg_.. 15
Absolute alcohol _____________________..ml__ 1
terial classi?cation.
7
Allergy or hypersensitivity is an altered reactivity of
Grind and wash through ?lter paper with 50 ml. of dis
the tissues toward particular substances, not inherent,
but acquired by the introduction into the body by any
tilled water.
The Janus green is not essential for the counter-stain
route of a foreign substance, usually protein in nature.
ing reaction, but its presence makes the Viability stain 10 The mechanism of hypersensitivity, while subject to many
also a useful method for the study of leukocyte activity.
variations, generally involves the ?xation of antigen tot
The ?brina-gglutination method is then practiced as fol
tissue cells or free cells in the blood, followed after an
lows:
interval by the local production of speci?c antibody,
'
A solution of whole blood is prepared by placing a
which remains attached to the sensitized tissue. A sub
drop of ?ngertip blood in 10 ml. saline and diluting to 15 sequent dose or doses of the specific antigen reacts with
approximatedly 1:10,000. Heparinized blood or plasma
the latent speci?c antibody, producing results which may
may be used, but not clotted blood or serum. A small
not be clinically obvious or may cause marked pathologic
drop of Viability stain is placed on a clean slide and
changes.
covered with a drop of the dilute blood. A drop of the
Certain laboratory animals, notably the guinea pig, are
red stain is placed separately on the slide. The modi?ed 20 susceptible to acute, vfatal systemic shock, termed anaphyl~
substance to be tested is removed from the resins with a
loop, added to the blue drop and mingled for a moment
before the red stain is mixed in. The blue dye binds the
?brin particles. The modi?ed agent, if not at a point
axis. The phenomenon has received intensive study, and
the suitable antigens, dosage, routes and sensitizing inter
vals are well known.
Repeated small subcutaneous doses,
especially in rabbits, produce the Arthus phenomenon
of optimum modi?cation, will, when added to the dilute 25 with tissue damage similar to that found in many dis
blood and mingled with the red stain, result in a clear,
ease states.
pinkish suspension. If the test is made on the day and
do the present state of the art, there is no previously
hour which is optimum for the particular substance or
developed method of modi?cation which ful?lls the con
group of substances being modi?ed, and at 9, 17, 39 or
ditions of the classical experiment in anaphylaxis, which
47 minutes past the hour at which the substance was ?rst 30 requires that, with suitable dosage and sensitizing inter
placed on the resins, and the substance removed immedi
ately or at the next clumping point, the modi?ed agent so
val, the following results shall be obtained:
TABLEA
obtained will be one which exerts a strong attraction for
?brin particles. On the slide, the test substance, clotting
elements and stain form profuse purple clumps which, 35 Group..__.‘_
if removed at the optimum time of contact, are grossly
visible. At times of contact other than the optimum,
Segsitlzing
A
B
C
D
NormaL-
Normal _____ -_
Modi?ed ____ __
Shorkdose-. .__do ____ _.
Modi?ed ____ __
Normal _____ _-
Result ____ _.
Reaction sur-
Reaction sur-
Modi?ed.
ose.
the clumps, if any, will be small and innumerable. The
object accordingly is to determine the point at which the
Death.___
vlval.
clumps are few, deep purple and compact. The modi 40
?ed substance exposed to the resins may exhibit this ag
viral.
do.
Reactionsur
vival.
A failure of reaction in groups B or C would‘ indicate
glutinating force for a matter of a few seconds to a few
that antigenicity has been altered. Death in B, C and D
indicates that toxicity has not been reduced. A failure
the resins is lowered a point or two.
The ?uctuation of clumping may continue for about 45 of reaction in these 3 groups indicates that antigenicity
has been destroyed.
two hours to several hours. Prior to this period, clump
I have found that by employing horse serum modi?ed
ing appears to be uniform at all times. After an inter
in accordance with my method anaphylactic shock in
val of ?uctuation, clumping may disappear entirely or
minutes, during which time the pH of the suspension on
guinea pigs can be mitigated or avoided. Table B shows
may again become uniform. There may be more than
one day at which a zone of ?uctuation appears, as for 50 that the animals to which normal horse serum was ad
ministered with adequate timing and dosage suffered
example, the 8th, 19th and 30th. The majority of pro
teins and lipids clump on the 19th day, carbohydrates
severe anaphylactic shock and even death, while those
who received one dose of normal serum and the other dose
on the 14th, albumins on the 24th, histamine on the
of my modi?ed serum generally su?ered only mild ef
fects. Those who received my modi?ed serum in both
doses exhibited little or no anaphylactic response.
10th, 19th and 30th, acetylcholine on the 7th, minerals
on the 8th and 19th, urea on the 14th.
It seems pos
sible that each bacterial species may have a distinctive
TABLE B
Pattern of Anaphylactic Response in 18 Guinea Pig
Experiments With Norma! and Modi?ed Horse Serum
Group _________________ ,,
Number of animals-.-" ..
_
A
8
Normal serurn._..
Normal serum. . . .
Shock syndrome;
death it dosage
was adequate.
B
4
Normal serum. - - .
Modi?ed serum...
Shock syndrome;
recovery.
O
4
Modi?ed serun1_ __
Normal serum. _ _ _
Shock syndrome;
recovery.
D
4
Modi?ed serum.
Modi?ed scrum.
No shock syndrome;
ttalarm
reaction in
we.
Degree of shock!
.............. -
l Symbols are as tollows: 0=none; I x-dyspnea; 2 x=convulsions or eyanosls; 3 :- coma; 4 := death.
3,097,141
8
symptoms. In vivo changes which have been identi?ed
Further evidence of the desensitizing eifect of my modi
?cation method is shown by the results of controlled
anaphylaxis experiments on guinea pigs, using horse
in studies of classical shock are: fall in blood pressure,
decreased coagulability of blood, leukopenia and decrease
in platelets, diminution of complement, release of hist
amine and acetylcholine from tissues, heparin from the
liver and S-hydroxytryptamine from platelets. Relaxa
tion of smooth muscle is followed'by contraction which
serum. Sensitizing and shock dosages, both with un
modi?ed serum, at an interval c; one week resulted in
the typical shock syndrome and death in ?ve minutes.
In a second animal, a sensitizing dose of normal serum,
followed by a shock dose of horse serum modi?ed by the
method of my invention resulted in a mild reaction of
causes the hair to bristle.
It also produces venus and
arterial spasm, which in presently accepted theory is the
sneezing and dyspnea, and the animal recovered. 24 10 cause of the reduction of hemoglobin, evidenced by
cyanosis.
hours later the latter animal was given a further shock
dose of normal serum; there was slight snuf?ing, and the
animal survived. 24 hours thereafter the latter animal
In acute shock, antigen accumulates around the bron
chii, causing a broncotetany, and the guinea pig dies
with lungs distended. The chemical changes in pro
was given an additional dose of normal serum, followed
in one week by a shock dose of normal serum; there was 15 tracted shock cause damage to many tissues.
3% weeks later
The lungs
are often collapsed, edematous and hemorrhagic.
the same animal was given a shock dose of normal serum,
' My own studies in hemobiological slides made on the
slight snu?iing and the animal survived.
blood of guinea pigs before and after shock have shown
and experienced no reaction.
that the speci?c antigen oxidizes and reduces hemoglobin.
Thus the modi?ed serum caused some initial reaction,
showing its antigenicity, but the reaction was mild. 20 At autopsy the blood and tissues of guinea pigs dead in
protracted shock are cyanotic. Controls, if killed by con
Moreover, the animal had been desensitized to a shock
cussion, have bright red blood and tissues of normal
dose of normal serum administered shortly thereafter.
color. I have made numerous in vitro studies of ‘blood
7 Even more surprisingly, subsequent sensitizing and shock
and tissues of ana-phylactic and normal animals and
doses, both of normal serum, failed to produce the nor
mal shock syndrome and death, but only a slight snui?ing. 25 have found that proteolysis with the release of amino
acids begins during shock. Similar changes in healthy
And a ?nal shock dosage gave no reaction.
tissues appear after several days.
I have modi?ed ovalbumin by my method as previously
In the present state of the art no method is known for
inducing prolonged desensitization in the manner I have
now provided. My modi?cation procedures accordingly
described, including contact with ion exchange resins for
24 days, 11 hours, and 52 minutes. My modi?ed speci?c
provide a novel and useful means of reducing or avoid
ing anaphylactic shock and constitute a useful means for
antigens do not damage hemoglobin or cause proteolysis,
even in the case of the death of a guinea pig caused by the
studying such reactions.
intracardial injection of a preparation of bovine albumin
The classical anaphylactogen is ovalbumin. Em
not adequately modi?ed. Hemobiological slides of the
ployed by the usual intravenous route, with a sensitizing 35 animal‘s blood showed none of the destructive changes
period as short as a week, it has produced acute, fatal
produced by the unmodi?ed speci?c antigen in the
systemic shock in guinea pigs with doses as small as 0.1
blood of anaphylactic guinea pigs.
mg. When both injections are given by the intra
Experiments which I have made in anaphylaxis with
perintoneal route, much larger doses are tolerated.
normal and modi?ed ovalbumin have employed 3 strains
Shock is protracted, and there is more time to observe 40 of guinea pigs and are summarized in Tables C and H.
TABLE C
Shock Syndrome Produced By Normal or Modi?ed
a
Crystalline ovalbumin
TWO EXPERIMENTS IN ANAPHYLAXIS WITH MALES OF INBRED LH STRAIN
GP
sensitizing dose
Treatment
Interval
No.
Temp.,
Shock dose
I
Result
Active phase or Until death
° F.
Temp. ?uctu
stlon, degrees
375....
10 rn1z.N ...... ..
0 ............ ._
29
105. 8
10 mg. N__-_-__.
1054;. “3
369-...
10 mg. M _____ ..
0 ............ __
29
102. 7
10 mg. N ...... ..
101 9_1o0_ 5
GP!
No.
Ery-
Surface Digging Rectal
Dysp-
therna Eating irrilta- hiding bleeding Miscellaneous
375____ ______________ __
x
369". _ .............. _ _
x
Air
Oya—
Loss at Oonvul-
or: or hunger nosis tonicity sicns
t on
History aiter shock or autopsy
?ndings
es
................ ._
x
x
xxx!
................................ _-
x
xx
X
------ . -
1
xxx!
us
.............. -_
All
organs
and
tissues
eyanoscd;
lungs collapsed in?amed.
Recovered;
ealthy.
TABLE D
Shack Syndrome Produced By Normal or Modi?ed
Crystalline ovalbumin
EIGHT EXPERIMENTS WITH FEMALES 0F INBRED MF STRAIN
GP
sensitizing dose
Treatment
Interval
No.
'I‘emp.,
Shock dose
‘‘ F.
A and B .... ..
Result
|
Active phase or Until death
Temp. ?uctu
ation, degrees
20
103
10 mg.
H)
21
23
23
23
23
42
42
42
42
102. 5
103. 5
103
103
103. 7
102. 0
104. 7
104. 0
105. 7
102. 9
10 mg.
10 mg.
10 mg.
10 mg.
10 mg.
10 mg.
10 mg.
10 mg.
10 mg.
10 mg.
103
N
M
M
M
N
N
- 98.0
102. 0- 98. 2
102. 7-103. 6
103 -102
103 —102. 5
103. 7— 00. 3
102. 13-101. 6
104. 7-101 8
104. 0- 99. 6
105. 7-101. 0
102. 0-101
3,097,141
TABLE D--C0ntin,ued
GP
Ery-
Surface Di
3 Rectal ‘
Dysp-
No. them Eating irélntw 1115115; bleeding Miscellaneous
88)....
xx
xx
r
xx
______ --
...... ._
xx
x
Cya- Lose 0! Oonvul-
History alter shook or autopsy
Cryin x______ -_
xxx
........................ -_
iindinga
es
g
382.--.
Air
n3 or hunger nosie touioity alone
n
x
xxx:
xxxx
‘
'
xxx:
xxxx
xx
xx
All organs and
tissues cyanoaed'
lungs collapsed, etalectasls; Io‘:
x
nu
A1elmall spots 011111781‘.
Organs
an
tissues
eymoeed'
lungs distended; 2 small spots or;
ver.
385.---
Recovered".
386..-387.-..
Do.
Do.
._-.
ht lever.
' an;
All organs and tissues oyanoeed;
luggs in?ated and in?amed; 1
n1
3%..--
erate spot in liver.
Recovered; healthy.
-._
Kiigedhall
eooran
andttilllseues
cture‘nolrmnl
unp
grosss
collapsed.
388.---
____________ ._
x
........................................ __
xx
xxx:
xx
xx
All
organs
'
and
tissues
eyanoeed;
spleen smaller than normal; by
terior of liver dark chocolate brown;
lungs moderately distended, areas
or otaleotlmis.
381-..-
___________ -_
xxx
________________________________________ _.
xx
xxx:
xx
xxxx
All organs and tissues cyanosed, but
health ;
spleen
smaller
than
norms ; lungs ballooned.
391-...
....-
x
x
...... --
x
Alarm x_______________________________________________ ._
Kl11ed;a]l
and tissues normal
in color an gross structure; spleen
slightly enlarged; lungs eollapeed.
TABLE E
Shock Syndrome Produced By Normal or Modi?ed
Crystalline Ovalbumin
EXPERIMENTS IN ANAPHYLAXIS IN GUINEA PIGS 0F INBRED LH STRAIN
Group A-Sensitized and Shocked With Normal Ovalbumin
GP
No.
sensitizing dose
Interval Temp, °F.
in days
18
102. 9
20
Shock dose
Result
Active phase
10 mg. N ________ __
Severe __________ __
or
Until death
35 min ______________________________ __
.
Temp.
?uctuation,
degrees
10815-1021!
.
103. 3-100. ll
22
...... -.
101.1- 00.8
24
25
25
25
25
25
23
40 min
20 min
70 min
50 min
70 min
65 min
27 min
101. 9- 90.8
2. 9-101. 7
102. 4- 95. 2
102. 3- 98. 3
102. 7- 08. 0
103. 4- 98. 8
103. 4-101. 8
z;
________ _-
29
30
2% 1101118Many hours.._.__.
103. 8-10). 9
105. 8- 04. 3
104. 7- 04.6
Group B-Sensitiwd With Normal, Shocked With Modi?ed Ovalbumin
102
20 mg. M _______ _-
Moderate ....... __
102. 1-1“). 5
2a
25
102. 5
10 mg. M - _.
Slight- _
102 -l01. 4
25
25
102. 6
102.5
10 mg. M - __
.-.._do._
i0mg.M-._..___- ...._do ___________ __
102. 0-101
1927.102
Group C-Sensitizcd With Modi?ed, Shocked With N ormel Ovalbumln
25
102. 9
20 mg. N ________ -- M1nimal___-_.--_
25
102. 8
.
25
25
102. B
102. 4
.
.
29
102. 7
25
101. 9
102. 8-102. 1
.
.
10 mg. N ........ -_
.
Minimal ________ -.
75 m
_-
___
.
102
—101.!
103
403.9
Group D-Sensitized and Shocked With Modi?ed Ovalbumin
26
103.
20mg.M ....... ..
Moderate _______ _. 30 min.
26
103. 2
20 mg. M ....... _.
Minimal ________ ._
26
20
103. 2
103.
10 mg. M...
10 mg. 111.. _
Fatal ____ __
(10
do
__ __________________ __
5hr.10mln ..... .-
102.5- 90.4
55 min ______________________________ __
102. 5403.8
55 min. .
40 mln_.
103. 2-102. 2
103. 7-103. 0
3,097,141
12
11
TABLE E-Continued
Group A-Bensitized and Shocked With Normal Ovalbumin-Continued
Sur-
GP Er ho- Estlng
N0.
Fur
nyita
iaoo
Dysp-
Di
g Rectal Crying
nee
lrrita- n15 ng bleeding
tion
1
Air
or
Cya-
Loss 01 Convul-
Auto y ?ndin
hunger nosis touicity sions
p5
Ea
rales
x
x
xx
xxx
x
x
______ ._
XX
xx
XXX
xxx
XXX
xxx
I!
X
xx
xxxx
xxxx
xx
xx
xx
xxxx
xxx
...... __
xxxx
Lungs ballooned.
xxx
xxxx
xxxx
xxxx
xxxx
Lungs collapsed.
xx
xxxx
xxxx
xxx
xxx
Lungs collapsed; slight areas of
xx
xxxx
xxxx
xxxx
xxxx
Lungs slightly in?ated.
x
xxx
xxxx
xxxx
xxxx
Lung? sli ghtly in?ated; largo spot
x
xxxx
xx
xx
xxxx
Lung: ballooned; small spots on
Lungs slightly in?ated; slight
areas of atslectasls.
atalectasis.
on
vet.
ver.
xxxx
x
xx
xxx
...... -_
x
xxxx
x
x
xxx
xxxx
xxxx
xxx
xxx
.
Lungs collapsed; in?amed.
Lun
moderately distended;
0
II I :-|l ed,
Group B—8ensitized With Normal, Shocked With Modified Ovelbumin-Continued
355...
Slook.__-.-__
x
.__._do
_..
x
_
x
.
Chloroformed; organs normal.
x
Chloroiormed; 1 lung diseased;
iew small spots in liver.
Group C-Sensitlned With Modi?ed, Shocked W ith Normal Ovslbumln—-Gonti.nued
Diarrhea; killed; organs normal.
360--.
Lungs collapsed.
36L"
362-363.-
TABLE F
E?ect of Secondary Shock on LH Survivors
Previous Days after
GP No.
dosage
1° shock
351 L--. N/N
“8A.... MIN
349 A._.. N/N
3560..-IN
3570.--- MIN
359
MIN
366 0.--- MIN
14
19
24
18
18
18
18
18
361
362
-_-_ M/M
-__- M/M
18
18
363 D..__ M/M
18
355B.___
'I‘emFp.I
°
.
Shock dose
8111GP
E
-
Eating
No. theme
Fur
rough
lace
Result
Rectal
or
Until death
Temp.
?uéztuation,
agrees
Dysp
Digging bleed- Crying nea or
i|£r1lta~ hiding
Active phase
ing
Air
Cya-
Loss oi Convul-
Autopsy ?ndings
rules hunger nosis tonicity slons
on
351A
xxx
348A.
x
xx
On face.
xx
x
.............. ..
xx
xxx
x
xx
xxxx
Lungsoollapsodmtalectasisontipsoi
xxx
x
______________ ._
xx
xxxx
xxxx
xxxx
xxxx
Lurésmgxlodarately distended; arms
349A-
xxx
xxx
Brie?y__
xxxx
xx
355B.
x
x
xx
x
xxx
8500.
x
...-.-..
xx
x
3570.
x
...... ..
xxxx
lobes; spots on liver.
______ _. _._do_____
11
______ _.
x
.
xxxx
xx
x
...... _.
x
.............. _.
xx
xxx
xx
xx
xxxx
Lungs niodcrately distended; organs
Kiliedmrransnormai.
...................... --
x
xxxx
xxxx
xxx
xxxx
14111111.]? slightly distended; large spot
xxxx
______ __
xx
xx
...... __
Killedmrgsnsnormal.
x
xx
x
...... ._
Kiiied;smnll spot on liver.
Killed; very large spots on liver.
norms .
n
x
xx
xx
x
...... _.
x
xxx
xx
__...._.............. __ --..____
xxx
xxx
.............. .x
xx
x
...... __
xxx
x
x
...... ..
x
xx
xx
x
x
xx
x
xxxx
.............. ..
xx
...... ..
xxxx
xx
...... .-
x
xx
xx
xxxx
xxx
xx
ver.
Killed;tip oillung dlsoased;exhan
sive discoloration in liver.
, ______________ _.
xxx
Killed:slightdisoolorstioninliver.
Do.
3,097,141
13
14
TABLE 0
Shock Syndrome Produced By Normal 0:‘ Modi?ed
Crystalline Ovalbumin
OVALBUMIN SHOCK IN S STRAIN GUINEA PIGS BY ANAPHYLACTIC GBOUPINGS
Group A-Sensltined and Shocked with Normal Ovslbumin
GP No.
Interval
in days
Sensitixing dose
Tom .
°F.p '
Shock dose
Result
I
or
Active plume
Temp.
?ugtuation,
Until death
B81’ 665
. N-
26
102. 5
10 mg. N
Fatal
3! min
. N
26
102. 6
5 mg. N_________ __
Severe .......... --
. N.
37
102
10 mg. N
Fain!
52
31
37
Neverreeovered-.-
102 5-101
9 days _________ __
102. 6-100
32 min
102. 8-101
Moderate. __.'__._. Many hours.-.___. ________________ __
Minimal
50 min
14 dayq
‘
102
10 mg. M__._---_. Slight ........... ._ 40 min ............................ -_
102. 9-104. 5
10L 7-103
102 -103
Group (‘J-sensitized With Modi?ed, Shocked With Normal Ovalbmnin
26
g
103
10 mg. N
103
Fatal
10 mg. N
as
25 min ________ _-
do
26 min..
103. 1-100. 6
.
36
an
Group D
396 H ____ -_ 10 mg. M _______ -_
42
1
101113. M__
Blight
60min__
109 .1012
393 III_._._ 10 mg. M-.-
52
102 6
10 mg. M ............ -_do.--.----_---- Many hours.. _____________________ __
1oz. 5-103 8
399 III_-_._ 10 mg. M
41
1
10 mg. M
101_ 5.102‘ 5
3
_____do
60 min
Group A-Sensitimd and Shocked With Normal Ovalbumln-Coniinued
GPNo. gm
em» n ating
392 I ............ -.
g
393 I
tlon
mg.g llfleod- Crying pm}
n“
"a zomot
vul-—
nee
unger
n
odlng
lug
tales
m
lty
alone
n
u
n
------ --
In!
xx
xx
xx
xx
x
xx
______ __
xxx
x
x
xxx
xx
xxxx
xx
Histor after ahonx oranm
I
pay an
dim
Lass distended; alight ntaleotasls and
ems.
Lived 9 days without recovering normal
respiration or appetite.
403 IV____
x
______ _-
Lungs ballooned.
Group B-Bonsitined With Normal, Shocked With Modi?ed Ovalbumin-Oontinued
401 IV___-
39511_____ .Q ____ __
x
xxx
xxx
xx
x
402 IV. .._
x
x
x
x
x
xx
x
x
irecovereg;
' I0
eoovere ' i‘ilebliihyd
ow y eve
"
x
do
condition; died on 14th
_x_
man
Recovered but for chronic lever.
Group Hensltixed With Modi?ed, Shocked With Normal Ovalbumln-Oontinued
394 1
397 II_____
400 III
404 V-..
405 v ___________ __
__ ___
xxx
xxxx
xx
x
xxx
xxx
x
xxxx
xxx
xx
x
xxx
x
xxx
xxx
xxx
xxxx
xx
m
x
x
x
xx
x
xxxx
xx
xxx
xxxx
xxxx
Lungs distended; few spots on liver,
Lungs ballooned; many spots on liver.
Lungs ballooned; iew hemorrhages,
xx
x
xxxx
xx
Lungs partially distended.
Lungs ballooned; several spots on llver.
_x_
Recovered; degenerative condition ap
poured on 5th day; died on 13th day.
Group D-Contlnued
I!
I
I
Recovered.
Do.
Do.
8,097,141
TABLE H
Secondary Shock m Survivors of MF Stram
GP
No.
Previous
treatment
Shock
dosage
369w. None ____________ __
386__-_ Aand B ________ __
387.... And B ________ __
390.," A _______________ ._
GP
No.
369....
Ery-
MIN
N/M
N/M
N/M
16
19
19
23
Temp,
° F.
xx
i
tion
xxx
ding bleedlug
x
Shock dose
Result
Active phase
102.5
103.5
103.6
Not taken
Surface Digging Rectal Dysp-
theme Eating
x
Days after
1° shock
nea
tales
Misoal-
Air
Oya-
Loss of
laneous
hunger
nosis
tonicity
Convulsions
xx
xx
x
______ __
xx
Climbing___ ...... -_
...... _.
Xx
Alarm
Days till
sacri?ce
Temp.
eihange,
egrees
28
9
9
1025-1003
l03.5—l(]0
103. 6-l01.6
9
103 402.4
Autopsy ?ndings alter death by
concussion
Blood and tissues normal in color; all
organs normal in color and gross struc
ture; lungs normal, collapsed.
386...-
x
x
"
__ ...... ._
Blood and tissues normal in color; all
organs normal in color and gross struc
ture; lungs well aerated, slight local
engorgement on pleura.
387,--.
x
m
390...-
x
______ -_
______________________ _-
xx
Shivering.--
.. .............. ..
Blood and tissues normal in color; all
xx
Alarm ..................................... --
Blood and tissues normal in color; all
organs normal in color and gross struc
ture; few light yellowish areas on sur
face 0! liver; lungs normal, collapsed.
x
x
...... .-
organs normal in color and gross struc
ture; large, pale, super?cial areas on
liver; lungs aerated and moderately
engorged on pleura and in interior.
Sensitization to shock depends upon genetic factors, 30 ditions were maintained with modi?ed anugen, shock
was slight and the symptoms super?cial. Animals in
and strains of guinea pigs di?er in degree of sensitivity to
group
C sensitized with modi?ed antigen and given 20
particular anaphylactogens. The LH strain has been in
mg. of normal ovalbumin experienced minimal shock
bred for about 15 years from an original herd of 100.
syndrome. If the modi?cation process had merely de
The MP and S strains have each been inbred from a single
pair for 13 years. The S strain shows a marked resist 35 stroyed a large part of the protein, these animals would
have died.
ance to foreign globulins, but unusual sensitivity to al
Two animals which received a shock dose of 20 mg.
bumins. All animals used were albinos.
of modi?ed antigen experienced severe reactions. No.
The differing effects of modi?ed and normal antigens
355 which had been sensitized with the normal antigen
appear most obviously when the temperature readings are
recorded every 5 minutes and every symptom noted. Each 40 (Table E, group B), scratched until it fell on its side re
peatedly, tore constantly at its mouth with its hind claws
animal in shock is observed closely for one hour, and
and exhibited bright red erythema on its skin and ex
hemobiological slides are made before and after shock.
posed
parts. It had severe dyspnea throughout the re
By classifying and tabulating the symptoms as shown in
action, but did not once display air hunger.
the tables, I have found that the eifects of protracted
No. 360, sensitized with modi?ed ovalbumin (Table E,
shock from normal ovalbumin relate to anoxia of the 45
group D) reacted to the shock dose much as other guinea.
tissues. The guinea pigs of LH strain, all males, which
pigs did in the same dosage (group D), except that its
had no protective treatment, showed the most typical syn
ear veins turned slightly bluish and it kicked twice in 5
drome (Table E).
minutes. It apparently recovered, lay quietly for about
As soon as the shock dose is administered, the animal,
10 minutes, and then went into a second and different
50
which has been fasting for 24 hours, is placed in a cage
phase,
with alternating apearance of cyanosis and cry
with fresh lettuce. It does not eat, but sits quietly star
thema, bristling and smooth fur, loss and recovery of
ing. It rubs its nose and ears, or scratches with its hind
muscle tone. After 5 hours it displayed abdominal ten~
foot (surface irritation), begins to pant (dyspnea),
derness, vomiting, white extremities and extreme pros
scratches the ?oor of the cage or pokes its head under the
lettuce (digging and hiding), occasionally displays alarm 55 tration and weakness, but died without convulsions. No.
361 (Table B, group D) survived an equal dose with al
by circling rapidly. These symptoms are brief and tran
most no symptoms. It is probable that in No. 360 the
sitory. Its coat becomes rough and cannot be smoothed.
massive dose of antigen exhausted antibody and permitted
At about 20 minutes the fatal symptoms begin, sometimes
a gradual accumulation of biochemical imbalances. This
with a convulsive kick. The ear veins turn blue. The
breathing deepens, and the animal stretches its neck and 60 experiment demonstrated that modi?ed ovaibumin is
strongly antigenic.
throws back its head in a typical gesture (air hunger).
Choking and rales may occur, and the guinea pig moves
restlessly and uncomfortably, sometimes trying to hide
its eyes from light. In the last 10 minutes the entire
that the toxicity of the antigen has been greatly de
cyanosis, the muscles become increasingly limp, the ani
mal lies prostrate, taking slow, gasping breaths, develops
altered. A slight chemical change, even the addition of
a single side chain, during the modi?cation process, would
The 3 other animals in group D of Table E illustrate
creased. The fact that shock occurred in the animals in
groups C and D proves that antigenicity had not been
skin, particularly the ears, turns a bluish shade, due to 65
have altered the antigen so that a. di?erent antibody would
have been evoked. In that event, there would have
70 been shock in groups A and D, but not in groups B and C.
5° or 6'.
Following recovery from sublethal primary shock a
In guinea pigs which received one or both doses of
guinea pig for the next month is in a state of enhanced
modi?ed ovalbumin, only the primary symptoms devel
convulsions and dies. Sometimes strangulation cuts
short the terminal symptoms. The temperature may fall
oped. A sensitizing period of 25 days and sensitizing and
sensitivity. Between the second and third week, death
from the normal antigen is said to be almost invariable,
shock doses of 10 mg. were the conditions found to pro
duce fatal shock with normal antigen. When similar con 75 as shown in secondary shock experiments on No. 351 and
3,097,141
17
18
No. 348, Table F. The eleven 11H guinea pigs which
symptons, with the addition that its eyes exuded pus. Its
survived the experiments listed in Table B were sub
jected to secondary shock, as recorded in Table F, those
fever was 106° F.
which had received modi?ed ovalbumin all being in
jected on the 18th day. Two of this group died, 'one of
tected by modi?ed albumin, but during fatal anaphylaxis
S strain guinea pigs in secondary shock were not pro
developed a wheezing sound in their bronchials, similar
to the asthmatic syndrome. It is believed that ovalbumin
shock may convert the animals’ albumin to a pathologic
form, and that the use of modi?ed ovalbumin may induce
which was No. 355, which had severe primary shock.
The other, No. 356, had a large spot on the liver. These
pale areas may be fatty in?ltrations or abscesses and
were not identi?ed by culture. As shown in Table F, the
a chronic state. Asthma has never been produced in ex
7 remaining animals survived secondary shock. If the 10 perimental animals, and my ?ndings make available a
new means of laboratory work upon the problem.
original modi?ed dose had not exerted a protective eilect,
No. 357, No. 359 and No. 365 would certainly have died.
This group of 3 animals should also include No. 369,
The blood of allergic patients in hemobiological slides
invariably becomes liqui?ed, suggesting some change in
the plasma proteins. This change appears progressively
an LH male used as contrast in experiments on MF strain
(Table H) which had been protected with modi?ed blood 15 in the blood of guinea pigs during sensitization.
Experiments with ovalbumin shock in a group of MF
female guinea pigs were combined with the additional
factor of protection against shock with modi?ed blood
ment of the second stage or fatal symptons of classical
proteins. Eleven guinea pigs were treated for a month,
anaphylaxis, ?uctuating over a period of many hours
which extended into the night. They displayed intermit 20 as shown in Table I. A sensitizing dose of 10 mg. of
normal ovalbumin was given in the ?nal week (Table D).
tent cyanosis, loss of muscle tone, rapid, deep, hard
The modi?ed substances used were: bovine albumin,
breathing and abdominal tenderness. All were lively and
globulins and ?brinogen, also histamine and guinea pig
normal on the following day.
hemoglobin. All were used in the 50th dilution.‘ ‘This
No. 349 (Table F) received the N/N/M dosage and
responded with chills and fever, which suggested that in 25 dosage apparently had no eifect on primary shock, but
the difference appeared in secondary shock (Table H).
?ammation was being discharged from the tissues, result
constituents. The group of 4 LB guinea pigs which re
ceived the MIN/N dosage all showed a partial develop
TABLE I
Treatment of Eleven Female Guinea Pigs of
MF Strain
Dosage A.—Modi?ed histamine, bovine albumin, globulins, ?brinogen, modi?ed guinea pig hemoglobin.
Dosage B.—Modi?ed equine cytochrome C.
Dosage A administered orally in 50th dilution every 3 days from 8/19 to 9/16, 4 weeks. Dosage B administered orally, 3 doses
in 50th dilution, between 9/8 and 9/16. sensitizing dose of 10 mg. normal ovalbumin iniected lntraperitoneally 9/8. Shock dosage
10 mg. each, normal or modi?ed ovalbumin as indicated.
GP No.
Dosage
9/8
_
'
10/7
10/14
1° shock
Interval,
N
Interval,
(days)
an
Feta ...................... ..
__
42
.-.._do__-._.__._.__ ________ __
A and 13----
20
42
N
N
N
Result
(days)
N
______ __
___
N
0 ____ __
___
N
0_. . . _ . _ . _ _ . .
_ . . . . _ . _ _ _ _ .
N
Aand B.-..
A.andB.-__
N
0
0 .... ..
. . _ _ __
__
2° shock
21
16
23
19
10/20 N.
Z3
19
10/20 M.
42
23
,
________ ._
~---_
___-
________ _
N
23
Slight ........... ..
N
42
Minimal killedon ________ __
23
10/24 N.
Autopsies on the last 8 animals listed in Table I were
ing in a permanent impairment of the mechanism of tis
made and tissue sections prepared from the shock or
sue respiration. This phenomenon appeared also in ex
gans. In guinea pigs which died of classical shock, both
periments on S strain (Table G).
blood and tissues were cyanosed. No. 384 and No. 391,
Four animals, No. 358, No. 366, No. 364 and No. 367
(Table B, group B and A) received sensitizing doses of 55 which had received a shock dose of modi?ed ovalbumin
and survived, were killed by concussion. Their blood was
100 mg. of normal ovalbumin. When shock is adminis
cyanosed, but organs and tissues were normal in color
tered by the intravenous route, a massive sensitizing dose
will prevent fatal primary anaphylaxis. In the experi
ments with intraperitoncal shock, the large sensitizing
and gross structure.
changed antigenicity of the antigen.
did not receive it.
‘
Four survivors were given secondary shock, as shown
60 in Table I. All survived and were killed by concussion.
dose did not seem to exert any blocking eifect.
The blood was bright red and the tissues and organs of
Table G records the results of experiments made with
normal color. The 3 MP‘ guinea pigs which had re
the S strain of guinea pigs. Five litters of 4 males each
ceived modi?ed blood constituents had displayed almost
were used. No di?erence in resistance between litters
no shock syndrome, apparently not sui?cient to reduce
was detected. The unusual sensitivity of this strain to
albumin was demonstrated by the fatal results in group 65 their hemoglobin. The administration of cytochrome C
C. This experiment illustrates that modi?cation has not
apparently did not contribute to protection, since No. 391
Three animals in this group exhibited what appeared
Fever and inflammation of the pleura in this group
to be degenerative changes due to shock. No. 393,
were due to the fact that they were transported on an
given sublethal N/N shock, never recovered. I-t dis 70 hour’s trip for demonstration. Lesions on the liver were
played permanent dyspnea, anorexia and alarm and died
not identi?ed. Histologic studies showed normal tissues
after 9 days. No autopsy was performed. No. 401 died
in all animals which survived. One animal which died
of a slowly developing morbidity with respiratory dis
irli slsock showed demonstrable changes in the adrenal
tress and at autopsy, evidence of pathology of lungs,
g an .
liver, stomach and intestine. No. 404 developed identical 75 These experiments demonstrate that the modi?cation
8,097,141
20
19
EXAMPLE II
process which I have invented affords a new opportunity
for the study of hypersensitivity in the medium of ana
The preparation of Example I in a dilution of 4 billion
phylactic shock.
organisms per ml. was used in ?eld trials on 15 cows with
Even more striking are the results of experiments in
volving animals, all of which had received a sensitizing
dose of normal ovalbumin, followed by a shock dose of
active Bang's disease, proved by agglutination titre and
positive milk cultures. Five similarly diseased but un~
treated animals were subjected to concurrent agglutination
tests as controls. The experiments were planned by New
my modi?ed ovalbumin and a further shock dose of
normal ovalbumin. Two animals (#386 and #390, Table
H) received modi?ed blood proteins, as shown in Table I,
York State Veterinary College along conventional lines
Two other ani 10 to test the effect of a modi?ed agent directed at the speci?c
infection and use in massive dosage. The majority of
mals, #355 (Table F) and #374, had not received the
animals showed a reduction in titre 1 month after treat
modi?ed blood proteins.
ment, but in 2 animals other infections were present and
As shown in Table H, #386 and #390 exhibited only
advanced‘ to the point where the animals were destroyed
slight reaction to the secondary shock dose. #355 and
#374, ‘however, exhibited a violent reaction and died 15 as useless. All injections were given subcutaneously.
prior to administration of ovalbumin.
The experiment indicates that antibody could be ex
hausted by massive dosage and that no permanent results
in the chronic state could be obtained by the modi?ed
within 65 minutes and 40 minutes, respectively.
This comparison shows that the modi?ed blood pro
teins afforded e?ective protection against the speci?c an
speci?c agent alone.
tigen in the period of extreme sensitivity following pri
mary shock.
20
EXAMPLE III
Sensitization remains one of the principal problems
of modern medicine. There are few techniques to aid in
A 2-year heifer, vaccinated as a calf with Strain #19,
developed typical Bang's disease. She was treated with
the understanding of the subject. During only the past
15 years life expectancy has been greatly increased by
immunotransfusion, with the blood of a healthy cow vac
the development and use of new drugs and antibiotics, 25 cinalted in vitro, followed by a course of chloromycetin,
but the same measures have greatly increased the number
but the titre of 1:200 persisted without clinical improve
of allergic patients and of the allergens to which they
ment.
are sensitive. Diabetics become sensitized to insulin.
Penicillin sensitivity alone causes hundreds of deaths a
Brucella vaccine and toxoid, oxidized and heat modi
?cd by my method was given subcutaneously as follows:
1 ml. and 1.5 ml. in 2 successive doses, after which the
titre was 1:100. A small daily dose was given for the
year. Bacterial allergy, naturally acquired or from the
use of unmodi?ed vaccines, causes hypersensitive states
that are practically untreatable.
A recent study has shown that when rats are treated
for a period of weeks with one of a number of drugs in
common pharmacologic use, including some of the hor 35
mones, damage is done to the hypothalamus from which
following week.
remained.
The titre then dropped to 1:50 and
EXAMPLE IV
A polyvalent vaccine of several of the known etiologic
agents of mastitis, viz. E. coli, Straphylococcus aureus
the rat never recovers. The hypothalamus, or mid-brain,
is the location of many control centers, and damage to it
hemolyticus, and streptococci zooepid'emicus, agalactiae,
is manifested by cyclic manifestations in the animals’
moods and in their behavior patterns in eating, drinking, 40 and uberis, was oxidized and heat modi?ed. No toxoid
had been prepared. Five ml. containing 5 billion or
sleeping and mating. Damage is also demonstrable on
ganisms
per ml. were injected intramuscularly into an
autopsy.
animal suffering from acute mastitis of 48 hours‘ dura
The use of my hemobiological techniques and modi?ed
bacterial and biochemical agents opens a new opportunity
for observing the interrelationship of the complex factors
tion.
In 24 hours the udder was again soft and in 48
The animal remained
45 hours it ‘was practically normal.
well.
of disease and health, and for the management in vivo of
This experiment showed that in recent, acute infection,
immunologic response.
The following examples demonstrate the usefulness of
my modi?cation method in elucidating the mechanism of
a modi?ed vaccine against the invasive factor of the etio
logical agent was effective.
I have also found my modi?ed substances to be useful
immune response and in demonstrating new methods for 50
in the treatment of other animals such as dogs, as the
evoking it in such a way as to promote resistance and
following examples show.
healing in vivo, especially in chronic states. When refer~
ring to various dilutions I mean a concentration which
EXAMPLE V
may be expressed at 10“, where n is the number of the
dilution. Thus the ?rst dilution is» 1:10, the eighth is 55
1:l00,000,000, etc.
EXAMPLE I
A Shetland Sheep dog, 9 years old, had a streptococcal
infection at 4 years, nephritis at 7 years, and after 2 years
more, he could urinate with evident pain only a few drops
at a time, vomited 3 or 4 meals a week, had no appetite,
A modi?ed vaccine was prepared from a virulent strain 60 an enlarged prostate, and a belly row with dermatitis which
kept him continuously scratching. He had cataract in
of Brucella abortus. A suspension of whole cells was
both eyes and could hardly go downstairs unaided. An
heated for 22 hours at 60° C. A similar suspension was
acute illness with fever and prostration caused the veteri
narian to recommend that he be destroyed.
I made a heat-modi?ed vaccine from a strongly chromo
The combined preparation was diluted to approximately 05
genic Staphylococcus isolated from his vomit. After the
5 billion organisms per cc. This preparation was in
third injection, the itching stopped and the lesions on the
jected into a healthy cow. The dosage was as follows:
belly healed. An oxidized, beat-modi?ed vaccine was
then used, and digestion, urination and appetite returned
Ml.
Nov. 18, 1949 _______________________________ __ 2 70 to normal. He had what appeared to be a focal response
from the sinus. He panted a good deal, drank unusual
Nov. 22, 1949
5
quantities of water and slept most of the time but after
Jan. 16, l950
_
5
heated for 6 hours at 60° C. and disrupted by ultrasound.
The toxoid thereby obtained was added to the vaccine.
several weeks appeared fairly well. Gradually the cata
There was at all times no evidence of toxicity or ana
phylactic shock.
racts cleared, and his vision seemed to give no trouble
75 up to the time of his death, nearly 4 years later.
3,097, 141
21
22
EXAMPLE V1
?ed by my method. Before treatment she ran a tempera
ture above 103° R, an established symptom of disease
A beagle puppy, 6 weeks old, had been given raw meat.
in guinea pigs. The preparation was administered to the
animal every few days. The animal lived for six weeks
The membranes of mouth, eyes and ears were much in
?amed, lymph glands were swollen and had ruptured ex
ternally.
There was practically no pus.
and in the last week appeared to eat_ nothing _and was in a
Cultures 011- 5
tained *by scraping and probing the lesions grew only
Straphylococcus albus. Fungus cultures were negative.
State of extremeweakness and-respiratory difficulty. On
autopsy she exhibited *1 <f°ndltl°n of advanced patholcfgy
Brucella infection was diagnosed clinically and by hemo-
Suoh I had never Seen "1 cases of minimally ocoumng
biological slides. The dog had grown worse under penicil-
death The who‘? chest “MW “'3? necmuci the heart
lin treatment, but when mixed vaccines and toxoids pre- 10 enlarged to 3 01' 4 "mes 31¢ normal 511:» was 3 mm wane}!
pal-ed by my method from straphylowccus and strap“).
coccus canis, Brucella abortus, and E. coii in 7th dilution
sac easily punctured by slight pressure, the blood was thin
and dad}; the lungs almost completely atrophlsd with
were given orally, it cleared up completely in 2 weeks.
EXAMPLE VII
alatectasls; the liver ‘Ya-s dafk and mPmed_
,
In several other animals in extremis, by employing'this
15 mode of treatment I have been able to prolong morbidity
A terrier, 13 years old, was senile, practically blind
and develop advanced pathology. This may also be ac
from cataract, its kidneys almost non-functioning, and
covered with dermatitis.
it to live more than 3
modi?ed by my method
tion, including vaccines
complished by using the 8th dilution, which apparently
The veterinarian did not expect
exhausts antibody. ‘It is possible that the use of a modi
days. It was given substances
?ed vaccine retards the invasiveness of the infection and
in approximately the 30th dilu- 20 that pathology is due to the toxins, for which no toxoid
and toxoids of Straphylococcus
was used.
aureus canis, Streptococcus hemolyticus canis, E. coli,
Brucella aborrus, acetylcholine, eserine, choline esterase,
glucuronic acid, histamine, the amino acids, uric acid,
A group of 8 RF male guinea pigs developed respira
tory disease 5 weeks after receipt. ‘They were treated only
with a Streptococcus C vaccine modi?ed according to my
urea, vitamins, adrenal and sex hormones. It was treated 25 invention and appeared to recover.
orally for about 6 months and made what appeared to be
a complete recovery.
I have found that bacterial substances modi?ed in ac-
In the ?fth month
four survivors were again ill; two were destroyed and the
other two were again successfully treated. The results are
summarized in the following table.
TABLE I
Immunizing Treatment of RF Strain Guinea Pigs With
Streptococcus C Autovaecine 0n Eight Males
{Temperatures (°,F.) on dates of experiments]
gP
10/16 10/21 10/11
11/23
11/30 12/6
12/16 12/29
1/8
1/10
1/15
1/21
2/20
3/8
3/20
4/11
4/22
5/22
0.
297..-. 1103.3
298-... 1104.4
299..__1103.2
300.... I104
301.___ 1 102.9
Lymphadenitis' -
1103.4
(1“)
303.... 1103.6
304--.. 1102.6
1 Pulmonary symptoms
Acute l l ____ _.
1 Vaccine given.
ment with temperature over-102.0 F.
acid.
5 End of experiment, animal died in shock or was chloroiormed. Animal not used for erper 1
I Supplementary treatment with modi?ed ?brinogen, globulin, albumin, histamine, acetylcholtne, glucuronl c
I have also discovered that protein synthesis in vivo
may be induced by the administration of proteins modi?ed
by my method. In experiments with albumin, I have
study a little-known factor in human disease such as 55 found that younger guinea pigs experience a greater in
crease than do older animals. In the tables below, nor
pathology due to toxins absorbed from a distant focus
cordance with my invention exhibit a striking utility when
used to produce advanced pathology by inducing pro
tracted morbidity. This method otters an opportunity to
such as the sinus or intestinal tract.
mal and modi?ed albumin were given in a dosage of 2
I isolated a short-chain Streptococcus from the lungs
of an RF guinea pig, dead of pulmonary infection, and
made a vaccine therefrom by vmy four step method dis
drops (0.1 ml.) orally in the 8th dilution. An average
total serum protein (T.S.P.) of eight untreated, healthy
guinea pigs weighing between 300—500 gm. had been found
closed above. This vaccine was administered to a female
to be 4.71 gm. percent.
guinea pig of the RF strain, a breed which has proved
TABLE K
to be a carrier of Streptococcus C. This low grade infec
tion, non-pathogenic for humans, impairs the usefulness
of the animal for research. 'I‘he infection is manifested
as a pulmonary disease. often fatal, or in chronic form,
commonly a lymphadenitis. The animal treated was one
of 6 females received in a lot; 2 of the animals had died
of respiratory disease, one on arrival and one shortly
thereafter. The animal in question had developed acute 70
respiratory symptoms and was near death. Her supply of
antibody was apparently near exhaustion. She was treated
with the 20th dilution of the modi?ed vaccine prepared
as stated above, with which was combined albumen,
globulin, ?brinogen, histamine, and strontium, all modi 75
T.S.P. at
GP No.
Dose
Interval
(days)
Clinical state
10th day
(gram- r
cent
264i‘ ......... __
1normal___.
10
healthy ....... __
265i..-
-_._
o ...... __
10
___do __________ -_
267111..
____do ______ __
10
lymphudenitis"
262i____.
2631"...
274m ....... -_
Average.
1 modified"
eulthy ________ -_
7.6
___.do ________________ __
6.4
lymphadenltis... ____ ..
6.6
________________ ..
5.37
687
3,097,141
00TH DILUTION
GP No.
GP N 0. Weight during expertinent
Dose
Number
of doses
Interval
in days
alter last
dose
299 .... __
1,000+ gm ...... __
1 __________ __
10
315 ____ _.
From3wks.to534
22in6wks__
28
221n5wks.-
28
l8tn7wks__
D
Total protein
Mum‘
?ed Normal
5.5 ...... -.
______ _.
5.1
gm.
317 .... ..
From3wks.to520
323 ____ _.
263to505gm .... ..
5.7
______ ._
gm.
______ __
5.15
It will be seen that the modi?ed strontium had a very
signi?cant and marked effect upon protein synthesis of
the animals to which it was administered. A de?ciency
Average ’I‘.8.P. tor modi
?ed dose ........................ .. -__. ____ __
5.61
5.71
____ ._
5.2
5.2
.... -_
Average T.S.P. for nor
mal dose ________________________ _. ____ .... _.
in protein synthesis may thus be adjusted by the practice
of my invention.
A further study was made on strontium deposition in
the femurs of guinea pigs. Strontium in bone is known
The results with the smaller animals given in Table K 25 to be in a state of continuous replacement. In the rat
show that those treated with modi?ed albumin had a total
increased deposition appears within 20 days when excess
serum protein of 6.87 gm. percent, as contrasted with 5.37
strontium is fed. During my studies, it became evident
gm. percent for those dosed only with normal albumin, or
that the metabolic instability of the guinea pig a?ects its
28% greater.
30 capacity to deposit strontium. The normal for the two
The larger animals (Table L) demonstrated a some
strains RF and MF, appears to be between 0.06% and
what lesser, although striking, increase in total serum pro
0.07%. Fetal strontium is normally lower than adult
tein. Those tested 10 days following administration
strontium. ‘Since bone strontium in the guinea pig seems
showed totals of 5.61 gm. percent for modi?ed albumin as
to be more labile than that of other species, factors which
compared with 5.2 gm. percent for unmodi?ed, and on the
affect increase and decrease may be studied.
llth day, 5.71 gm. percent as against 5.2 gm. percent; 8%
A solution of unmodi?ed strontium hydroxide was
and 10% greater, respectively.
prepared in a concentration of 30 mg. per ml., and a
These results are believed to be the ?rst demonstration
in vivo of the principle of reversibility of enzyme action.
solution of comparable density was made of strontium
It is known that protein synthesis is essential to the life 40 modi?ed in accordance with my invenion. The 8th
process. My modi?ed substances accordingly provide a
dilution was prepared of each, and this strength was used
valuable means of laboratory study of the mechanism of
for all treatment of experimental animals, unless other
protein synthesis in blood.
I have further found that, surprisingly, minerals may
be modi?ed by the method of my invention above related.
They exhibit the same clumping activity during the ion
exchange step of my‘ method as shown by the ?brinag
glutination technique. The etfectiveness of these modi
?ed minerals is believed related to the catalytic e?ect
of minerals in the body.
wise indicated. Treatment was given by mouth, 2 drops
(0.1 ml.) constituting a dose. When “cone. Sr" is re
ferred to in Tables N and 0, it indicates a concentration
of 30 mg. per ml.
Strontium enters the body mainly from dietary sources.
Prepared feed used by breeders' and in my laboratory
contains only traces of strontium. Unwashed lettuce and
other vegatables are given daily at my laboratory, but at
the farm where RF guinea pigs are raised, they receive
lettuce only once a week. MF guinea pigs receive it
several times a week. The rate of strontium deposition in
days, 6 hours and 39 minutes. The total serum protein
of guinea pigs was determined before and after admin 55 young animals depends in part on dietary sources.
istration of the modi?ed strontium (in the 8th and 60th
Differences in increase in young animals are illustrated
dilutions). The results are summarized in Table M.
in Table N by two obtained directly from the breeder of
the RF strain, No. 318 and No. 319, compared with No.
TABLE M
308 and No. 309 of MP strain, bred and raised in my
Determinations of Total Protein (gm. percent) on Sera 60 laboratory from a dam which had been dosed with modi
?ed strontium in the 8th dilution approximately every 3
of Guinea Pigs Treated With Normal or Modi?ed
Strontium
days throughout pregnancy. The MP strain is in general
Strontium was modi?ed by my method by treating with
pepsin for 16 hours, H202, heating for 17% hours at
58° C., and contacting with ion exchange resins for 8
more resistant to disease than the RF strain.
8TH DILUTION
Seven RF female guinea pigs, received from the breed
Weight dur- Interval in
lng experl- days after
ment, gm.
dose
GP N0.
631 to 730..-.
_
Modi?ed Normal
10%
6
692 to 729..-.
11
6. 5
284 to 502...
Baseline
404 $0740....
er at approximately 6 weeks of age, were maintained on
Total protein
4.8
________ -_
4. 6
l0
10%
5. 7
5. 2
________ ..
........ -_
ll
5. 1
________ _ _
11
5.9
11% ........ ..
5.0
B. 3
my laboratory diet for 6, 10 or 12 weeks without treatment
of any kind. All exhibited fever during a part or all of
the interval under observation, and all exhibited some
evidence of chronic lesions during life or at autopsy.
Two died of pulmonary disease, 5 were chloroformed.
The femurs were retained and the percent strontium of the
bone ash was determined. In two cases separate deter
minahtions were made on each femur. In all cases the total
strontium was below normal levels.
3,097,141
25
26
TABLE N
Experiments in Strontium Depositton in Bones of Healthy
Guinea Pigs Treated With Normal or Modi?ed Stron
u'um
(1 P
N 0.
a13___
Sex
M
Treatment
Number
doses
Duration
None
Total
dosage
Incubation
period
_
_ _
Date
01
death
Age at
death
Percent
SR in
bone ash
Comments
4/2
4 wk ______ ._
0.0116
Received from term at 4 wk.,
4/2
0 wk _____ _.1.
0. 033
Received from farm at 9 wk_,
RF strain.
319__- M
-_-__do
-
________________________ __
RF strain.
297___ M
_____do____.__
303..-
_...- do
__-_
11/30
12 wk _____ __
0. 07
Received from farm at 6 wk.,
2/8
18 wk _____ -_
0. 06
Had been used in anaphy.
RF strain.
M
.... ..
lads experiment, RF.
2o
.06 pg.-- __ 19 days ____ ._
11/1
0. 07
g
2/16
0. 055
29
&
2/8 ‘
2/16
0. 06
0.07
20
.
MF Strain bred to #252, also
MF. Litter born 10/13,
weaned 11 1.
From 10/13 litter of I28].
.
Do.
D0.
Littermate 01 I281, bred to
#252, MF. Litter born
10/14, weaned 11/1.
35
.105 pg__._ 3 days _____ __
4/11
10% mu_._-_
0. 031
Litter born 4/8; septic par—
From 10/14 litter o! #232,
tnrltlon, fever 104.5° F;
chloroiormed.
30s___ F
_.___do _____________ __
10/18
1
.003 “5.-.. 20 days ____ __
11/7
3 wk. 3 da..-
0. 05
009..- M
-----do ............. ._
10/18
1
.003 93.... 64 days ____ __
12/11
8 wk. 2 da___
0.06
At. birth"--- ,
0. 013
320_-_ M
Nona
__
4/8
Do.
From 4/8 litter 01'282; chlo
rotormed 2 liwermates died
on ma day site: birth.
235___ F
Modl?ed Sr _______ __
6/24-8/18
12
‘
.036
____
RF strain, bred to RF male.
pg
Normal Br ________ __
size-ore
4
Modi?ed Sr (heat-
111MB
42
14327;) born 9/26, weaned
1.2 #8:---
.126 pg.._. ____________ ._
...... __
1/0
-_ ........ --
1 yr. 8 mo...
0.042
'
In late pregnancy, ev1denun
modi?ed beyond
opumum 101 GP
01 dlsease and lack of fetal
life; died with slight respi
blood ,
ratory
miecnon;
other
organs normal; 4 normal,
full-grown fetuses.
294___ F
Modi?ed Sr...
Normal Sr, con
“[4
1
296___ M
Normal Sr_.__
0128-1111/25
12
9 wk. 6 da._
11!!
1
____________ ..
____________ _.
Normal Sr, con
. 252--.
M
9/3-10/25
12
Modi?ed Sr, eone_ _ _
11/4
1
Modi?ed Sr _______ __
1/23-2/23
15
Normal Sr. _ _ _
Modi?ed Sr___
.-___do _____________ __
2ss___ M
Normal Sr ________ __
302___ M
Modi?ed Sr _______ __
9 wk. 6 da.-.
0.08
From 9/26 litter of #2315.
.
0055
Do.
2/23-3/0
6
4/44/24
10
4/24-5/18
8
.
2/28-4/22
27
.081 new- 0 .......... --
4/22
8 mo ...... _.
0. 088
B15011 8165M total protein,
2/28-4/22
27
.081 #3..-- 0 .......... .,
4/22
7 mo. 3 wk_-
0.063
Bgsd 2:611:11‘ total prot/eln,
______________________ __
lyr. 5 1110...
0.06
.
MF Strain, bred to #281 and
cc. e
00. ea
200___ M
..---do ............. _-
12/0-4/21
3
.009 pg____ ____________ __
5/22
8 mo. 3 wk__
0.060
237___
F
.
Bised Similar total proteln,
00. e
00th (111-..- 4 wk.
.
.
_--.-do_____________ __
4/21
1
____.do ____________ __
6/24-8/18
12
Normal Sr ________ __
8l23~0l2
4
Litter born 9/6, weaned 0/24.
Normal Sr, cone. __
12/2-12/5
1/15-3/15
2/26-3/8
0
30
6
Lit/$01’ born 12/20, weaned
Litter born 3/14I weaned 4/11.
1136113.--.
__
. . . . . _..
. . _ _ . . _ . __
RF; stirain, bred to I252, M!‘
s re 11.
289_._ M
Modi?ed Sr _______ __
0/24-11/2
15
.045 ug_ _._
291___
Modified 81', cone-.Normal Sr ________ __
Normal Sr, oonc.___
11/4
0/24~11/2
1
15
11/4
1
3 mg .... __
. 0-15 pg____
3mg ____ __
1/10-3/19
1/10-4/11
34
55
M
312-.. F
313-" F
314--. F
None--.
Modi?ed Sr _______ __
__-_.d0 ............ ._
--.__ .
.102 pg____ 1 day _____ __
-16510E__-_ 2 days _____ _.
315_,_ F
4/11-4/24
15
.015 pg_-__
4/24-5/18
12
60th dil- _
317-__ F
4/11-4/24
6
.015 pg__._
1124-5118
12
1212-12/15
6
239.__
F
Normal Sr, cone. _ _,
60111 d
From 9/0 litter of #237.
Do.
1/10
3/20
4/11
3 wk ______ __
12 Wk. 5 do“
15 wk. 5 (1a..
0.04
0. 058
0. 04
From 12/20 litter of #237.
Do.
Do.
From 3/14 litter of #237.
Do.
.._
18 mg. -_-_
. .. _. . .
. . . . . _ . _ __
Litter
born
12/16
weaned
us; RF, bred m 1252.
_.__.do ............ __
1/9-5/20
60
180 mg. _-_
5/20
1 yr. 8 1110...
0.025
Pr'eegnant about 1 mo. or 34
l/B
3 Wk. 2 dB...
009
From 12/16ll1ter 011230.
4/8
12 Wk. 6 da__
0. 034
rm.
310___
M
311_-- F
None . _ _ . _
Modi?ed S _
_ _ . . . _ . _ . . . --
1/8-4/8
Do.
s,o97,141
27
TABLE 0
Untreated Guinea Pigs of RF Strain, With Fever During
Period in Laboratory, Evidence of Chronic Lesions and
Irregularities in Strontium Deposition
GP
Sex
Age at Percent SR in bone ash
death,
wks.
Femurs Right Left
Date
Date
rec'd
death
324- ._ _ F
4/24
6/31
326- __ . F
327-.-- F
328--.- F
4/24
4/24
4/24
6/1
7/8
7/8
342-... F
6/4
8/13
16
0. 05
Iniec‘ged eye; slight lever in early weeks; organs normal at
343..-- F
_.-_ F
6/4
5/4
8/13
8/13
16
18
0. 02
0. 03
Fever of 103.4° at 12 weeks; etalectasis at lobe of 1 long.
Intermittent lever, pus in vagina; swelling of pubic area.
N 0.
12
0. 039
Comment
Caged with #326; died 0! respiratory infection; lungs extensively
i In!‘
s: ‘a I ,.
12
0. l0
18 ...... .18 ...... ._
0. 03
0. 03
0. 06
0. 03
Died or respiratory infection; lungs extensively
Fever through life; lungs extensively diseased.
Fever through life; lungs moderately d.
an
.
psy.
The results indicate that the animals may be prone to 20 a massive dosage totalling 105 mg. of normal strontium,
which without protective treatment would have resulted
infection because of dietary lack of strontium in utero and
in increased deposition. The strontium level at 18 weeks
in infancy, or that birth in an infected environment pre
was 0.055.
vents a normal deposition of strontium, and clearly show
In the litter of No. 235, 2 were fed 12 doses of modi
the involvement of strontium in the mechanism of disease.
The case of No. 297, a healthy RF guinea pig which at 25 ?ed strontium and l a similar dosage of normal strontium.
taincd a normal strontium level after 6 weeks on labora
tory dict illustrates that there is probably no genetic factor
which restricts strontium deposition.
Several experiments were conducted on the effects of
At the conclusion of this treatment, each was given a
single “shoc " dose of concentrated strontium, followed
by a latent period to allow time for deposition. Each
showed a di?crcnt balance, the only normal one being
feeding normal and/or modi?ed strontium. Experiments 30 0.07% for the animal which had received only modi?ed
strontium.
on adult female guinea pigs suggested that strontium may
be of importance in pregnancy. An MF female was fed
modi?ed strontium through pregnancy and sacri?ced 19
days after the litter was born. Her strontium level was
No. 253 was fed the 8th dilution of a preparation of
modi?ed strontium which had been heat-modi?ed for a
period greater than the optimum period (17% hours)
35 for guinea pigs. It had rcceivml pepsin digest, H303,
heat for 24 hours at 56° C. and exposure to ion exchange
Evidencethat strontium may be low in pregnancy was
0.07%.
shown by a healthy MF female, No. 239. During the
resins for 8 days, 1 hour and 17 minutes, a point at which
it produced a weak ?brinagglutination. Over-modi?ed
latter part of her ?rst pregnancy she was fed a total of
substances tail to evoke leukocyte response in hemo
18 mg. of normal strontium. One of her litter, sacri?ced
at 3 weeks, 2 days, showed a strontium level of 0.09%. 40 biological slides and are believed to sensitive and damage
leukocytes.
No. 239 was fed concentrated normal strontium for 4%
In the last stages of pregnancy the animal grew visibly
months, a total of 180 mg., while caged with successive
weaker without clinical evidence of disease. She ceased
eating, there was no sign of fetal life, and in the last days,
half term pregnant. In a normal, non-pregnant animal 45 she was prostrate and showed respiratory distress.
males. When sacri?cd, her strontium level was 0.025%,
and she was found to be approximately one month or one
this dosage would produce increased dcpoa'tion.
No. 282 received the same dosage of modi?ed strontium
as her littcrmate, No. 281, through her ?rst pregnancy,
and was put on the same dosage again after beginning
her second pregnancy. Near the end of term she showed 50
evidence of pelvic infection. The belly was almost
denuded, and she ran a high fever daily. Her litter of
3 was emaciated, undersized and feeble.
One on the
Autopsy showed 4 fully developed and apparently normal
fetuses. The dam showed slight congestion in the lobe
of one lung, but I could ?nd no other evidence of
pathology.
The conclusions to be drawn from these exploratory
experiments are that strontium is concerned in such
important life processes as immunity to disease and
pregnancy. It is also evident that modi?ed strontium
may be used in oral dosage to protect or restore the
day of birth had a strontium level of 0.031; the other 2
died 2 days later. The dam had a strontium level of 55 normal strontium level in bone tissue, even when massive
0.031. The most probable explanation of this experiment
appeared to be that a prolonged overdosagc with the 8th
dilution exhausted antibody and caused the animal to incur
infection.
doses of normal strontium are fed, as in No. 252 and
No. 305.
Presently accepted knowledge of strontium metabolism
shows that when massive doses of stable strontium are
Several experiments demonstrated that modi?ed stron; 60 fed, strontium-90 is diluted in the total strontium intake,
tium may be used to preserve normal strontium balance
in bone.
and the amount deposited in bone tissue is decreased.
But if a large dosage of stable strontium is continued,
serious metabolic disturbances ensue. The usefulness
No. 252, a healthy MF male guinea pig, subjected to
of‘modi?cd strontium in exploring a method for protec
treatment with modi?ed strontium for 4 months, was
given 18 mg. of normal strontium and showed a strontium 65 tion against strontium-90 is demonstrated by these ex
level in bone of 0.06%, evidently within normal limits.
periments.
I have studied the modi?cation of the majority of
Three litters bred in the laboratory were given com
minerals which occur in the body. All reach a zone of
parative treatment. No. 308 and No. 309 received modi
?uctuating ?brinagglutination between the 7th and 8th
?ed strontium in utero and showed a rapid increase
toward adult levels.
70 day, and within a period of 14 hours, each displays
optimum ?brinagglutination. There is another such zone
The litter of No. 281 was treated with modi?ed stron
on the 19th day. In general I have found that the longer
tium in utero and then from weaning until the time of
a modi?ed substance remains on the resins, the more
sacri?ce. No. 306 and No. 307, given only this treat
permanently it retains its capacity to evoke ?brinegglm
ment, showed at 17 and 18 weeks respectively strontium
levels within normal limits. No. 305 received in addition 75 tination after it has been removed.
3,097,141
29
Each mineral has a point of optmium modi?cation,
peculiar to itself, which can be obtained in successive
runs on the same mineral. Although I have found several
minerals appear to have the same point of clumping,
e.g.: cobalt, copper, potassium and silicon, each displays
individual differences in the schedule of its ?uctuations,
during which it reaches several points at which clumps of
1 mm. or more in size are produced.
30
and heat modi?cation of a strain of Brucella as described
in Example VIII is then contacted with an ion exchange
resin in the following manner:
The resins employed may be those designated as “IRA
410” an anionic resin and “IR-120“, a cationic resin. The
resins should, if necessary, be dried until they will pour
readily, following which they are separately sterilized.
The sterilization is brought about by placing 1 cc. quan
titles of each resin in small vials which are sterilized in an
As exemplary of the manner in which an organism
may be modi?ed in accordance with my invention to 10 autoclave at a pressure of about 15 pounds per sq. in. and
for a period of about 20 minutes. The vials in which the
provide a modi?ed vaccine and endotoxoid, the following
“IR-120” is sterilized are screw top and when cool and
examples are presented.
ready for use, the vials are opened under a sterile lamp.
EXAMPLE VIII
1 cc. of the "IRA-410" is poured into the screw top
A strain of Brucella aborrus, known as the Perry strain, 15 vial containing the “IR-120" whereupon the vaccine to be
isolated from a case of brucellosis, was implanted on agar
slants and grown for 48 hours at 37.5 ° C. It was then
further modi?ed is added in a quantity not greater than
about 10 cc. The mixture is then well shaken and placed
in an incubator at a temperature of about 37° C. for about
harvested in normal saline, transferred to sterile centri~
3% weeks. The optimum time of contacting with the
fuge tubes and centrifuged for about 1 minute at about
1,000 r.p.m. The agar particles were thus removed. It 20 ion exchange resin may conveniently be determined by
my ?brinagglutination technique above described. In the
was then transferred to test tubes which were sealed by
case of the Perry strain of Brucella abortus of Example
?ame and immersed in a water bath at 60° C. for about 30
VIII an optimum time was found to be 24 days, 1 hour,
minutes. The organism suspensions were transferred to
and 17 minutes, as shown by formation of large purple
sterile test tubes and tested for growth. Those which were
found to be sterile were combined and treated with the 25 clumps on the test slide.
When a biochemical is utilized, as hormones, amino
addition of pure crystalline pepsin, about 5 mg. or less
acids, vitamins, and glucuronic acid, it is desirable to
per cc. of vaccine. The pH was adjusted to 4.6 with 10%
oxidize with hydrogen peroxide rather than as described
HCl and the vaccine incubated for from 18 to 24 hours
in Example VIII, and in accordance therewith about one
at 37.5“ C. The pH was then adjusted with 10% NaOH
to 7.0 and the vaccine oxidized by adding 5 cc. of 30% 30 half volume of sterile commercial 3% hydrogen peroxide
sodium nitrite and 5 cc. of 30% acetic acid for each 3 cc.
may be added to a suspension or solution of the biochemi
of the suspension.
After maintaining at room temperature for about 4
cal and the mixture heated to a temperature of about
37° C. for about 24 hours.
hours, the suspensions were centrifuged, washed twice
Hence, the modi?cation procedure includes the use of
with sterile distilled water and twice with sterile isotonic 35 a proteolytic enzyme such as pepsin and papain, and it
may be added to a bacterial suspension prior to or after
sodium chloride solution. The oxidized suspensions were
the oxidation and heating. Thus 2 mg. of pepsin may be
standardized to 5 billion Bruoella per cc. by the McFar
‘added to about 10 cc.ofaheavy bacterial suspension. The
land barium chloride method, and then heated at about
pH thereof is adjusted to about 4.6 and the suspension
60° C. for a period of about 24 hours. There was thus
obtained a heat-modi?ed vaccine derived from the 40 placed in an incubator at about 37 ' C. for about 24 hours.
Following incubation, the pH value is adjusted to neu
Brucella abortus and a portion thereof was subjected to
trality (pH 7), whereupon it is contacted with an ion ex
the in?uence of ultrasonic waves as previously described.
change resin as hereinbefore described.
The usual quantity of vaccine so treated has been 10
Toxoids which are prepared by ultrasonic disruption of
cc. The endotoxic toxoid following ultrasonic disruption
was transferred to suitable ?asks and each ?ask was 45 vaccines which have been pepsin digested, oxidized and
checked for growth in broth. An equal quantity of the
oxidized and heat-modi?ed vaccine was then added.
EXAMPLE II
heat-modi?ed, may be treated further by contact with an
ion exchange resin. Thus, a toxoid may be prepared by
adding pepsin to a bacterial suspension followed by oxi
dation, heating to a temperature from about 56° C. to
A strain of Staphylococcus aureus was employed and 50 about 64° C., ultrasonically disrupting, and contacting
the procedure followed was that above described in Ex
with an ion exchange resin.
ample VIII for the Brucella, with the exception that the
Further, heat modi?cation gradually decreases the
heat modi?cation was carried out at a temperature of 56°
toxicity of the exo- and endotoxins, but the vaccines are
C. for a period of 24 hours. The strain utilized was apleo
increased in toxicity if heated for less than the time opti
morphic light cream-colored coccus of fairly uniform size, 55 mum for a species and sulfer a decrease in antigenic
mannitol and coagulase negative and non-hemolytic,
properties if the heating is prolonged beyond the time
which had been isolated from a chronic sinus infection.
This strain has a special property of broad speci?city since
optimum for particular species. Accordingly, in one
it has produced ?brinagglutination whenever tested in
pathogenic organisms through pepsin digestion, oxidation
blood.
‘
It may be added that a toxoid may be produced ‘only
embodiment, my invention entails a detoxication of
60 and ‘heat modi?cation which renders them well adapted
as vaccines, and for the ultrasonic disruption of the or
gansims so modified with the production of endotoxic
or when the disrupted suspension of a heat killed orga
toxoids. Additionally, the vaccines, toxoids and bio
nism is contacted with an ion exchange resin. Other
chemicals may be further modi?ed by contact with an
methods of modi?cation destroy the endotoxins.
65 ion exchange resin.
In accordance with the foregoing there was obtained a
Furthermore, modi?cation reduces the capacity of bio
toxoid preparation from pathogenic organisms through
logical substances to damage the blood from pathologic
pepsin digestion, oxidation and heat modi?cation thereof,
states by the in vitro oxidation and reduction of hemo
followed by ultrasonic disruption. It was found that heat
globin and inhibition of lysis of the clot as seen in hemo
ing to occasion coagulation of the protein material prior 70 biological slides. The modification of bacterial, viral
to disruption yielded a toxoid which was suitably modi
and biochemical substances entails a physical change,
?ed and remained stable for extended periods of time, as
such as alteration of color and viscosity, increased resist
at least three years.
ance
of vaccines to ultrasonic disruption and increased
EXAMPLE x
agglutinability of vaccine in serum.
A vaccine prepared through pepsin digestion, oxidation 75 This application is a continuation-in-part of my appli
from protein hardened by modification before disruption,
31
3,097,141
‘
32
cation S.N. 129,495, ?led November 25, 1949, now
abandoned, and my application S.N. 342,260, ?led March
3, 1953, now abandoned.
I claim:
1. A method of reducing the toxicity of an antigen
disrupted portions of the organism and contacting the
while retaining its antigenicity, which comprises treating
proteolytic enzyme, oxidizing the digested histamine,
said combined portions with an ion exchange resin.
8. A method of increasing ?brinogen content of ani
mal blood, which comprises administering to animals
histamine which has been modi?ed by digesting with a
the antigen by adding ‘thereto a proteolytic enzyme, add
heating the oxidized and digested histamine for about
ing to the treated antigen an oxidizing agent, heating said
20-28 hours at about 55°-65° C., and contacting the
oxidized and treated antigen for about 15-28 hours at a
resultant with an ion exchange resin.
temperature in the range of about 50°-100° C., and con 10
9. As a composition of matter, a microbiological or
tacting the heated resultant with an ion exchange resin.
gansim which causes an infection in animals, which or
2. A method of reducing the toxicity of an antigen
ganism has been modi?ed by proteolytic enzyme digest,
while retaining its antigenicity, which comprises digesting
oxidation, heating at about 55"-65° C. for about 20-28
the antigen with pepsin at a pH in the range of about
hours, disruption of the cells of a portion of the treated
3-5 at a temperature of about 37° C. for about 16-24 15 organism by ultrasonic waves, and contacting the dis
hours, adjusting the pH of the resultant mixture to about
rupted and undisrupted portions with an ion exchange.
7, oxidizing the digested antigen for about 4-24 hours,
resin.
heating the oxidized and digested antigen for about 15-28
10. The composition of matter of claim 9 wherein the
hours at a temperature in the range about 55°-65° C.
organism is a brucella.
and contacting the heated resultant with a mixture of 20
11. The composition of matter of claim 9 wherein the“
anionic and cationic ion exchange resins.
organism is a staphylococcus.
12. The composition of matter of claim 9 wherein the
3. In a method of reducing the toxicity of an antigen
while retaining its antigenicity, which includes the steps
organism is a streptococcus.
of proteolytic enzyme digest, oxidizing, heating, and con
13. The composition of matter of claim 9 wherein the
tacting with an ion exchange resin, the improvement 25 organism is Escherichia coli.
which comprises terminating contact with the ion ex
14. A composition of matter comprising an anaph
change resin at the point at which the substance in con
ylactogen which has been modi?ed by proteolytic enzyme
tact with the ion exchange resin causes maximum ?brinag:
digest, oxidation, heating at about 55°-65" C. for about
glutination in blood.
20-28 hours, and contacting with an ion exchange resin.
4. In a method of reducing the toxicity of an antigen 30
15. The composition of claim 14, wherein the anaph
while retaining its antigenicity, which includes the steps
ylactogen is ovalbumin.
‘
of proteolytic enzyme digest, oxidizing, heating and con
16. The composition of claim 14, wherein the anaph
tacting with an ion exchange resin, the improvement
ylactogen is an animal serum.
which comprises maintaining said contact with the ion
exchange resin until material in contact with the ion ex 35
change resin exhibits maximum ?brinagglutination when
admixed with blood and toluidin blue dye and bluish
eosin dye is added to said admixture.
5. In a method of reducing the toxicity of an antigen
while retaining its antigenicity, which includes the steps 40
of proteolytic enzyme digest, oxidizing, heating and con
tacting with an ion exchange resin, the improvement for
determining optimum heating which comprises admixing
material so heated and blood leukocytes, whereby opti
mum heating is determined by disappearance of all said 45
leukocytes from said admixture.
6. A method of reducing the toxicity of an anaphylac
togen while retaining its antigenicity, which comprises
treating the anaphylactogen by adding thereto a proteo 50
lytic enzyme at a ‘pH of about 4.6, oxidizing the treated
anaphylactogen, heating said oxidized and treated anaph
ylactogen at about 55°-65° for about 20-28 hours, and
contacting the heated resultant with an ion exchange
resin.
References Cited in the file of this patent
UNITED STATES PATENTS
2,123,198
Parfentjev ___________ __ July 12, 1938
2,230,997
Chambers ____________ .._ Feb. 11, 1941
2,288,738
2,368,464
Parfentjev ____________ _- July 7, 1942
Gerlough ____________ __ Jan. 30, 1945
2,461,505
2,461,506
2,578,491
Daniel ______________ __ Feb. 15, 1949
Daniel ______________ __ Feb. 15, 1949
Shropshire ___________ __ Dec. 11, 1951
468,676
493,101
682,768
Great Britain __________ __ Oct. 9, 1934
Great Britain ________ -_ Sept. 30, 1938
Great Britain _________ __ Nov. 19, 1952
FOREIGN PATENTS
OTHER REFERENCES
Bronfenbrenner: Pros. Soc. Expt. Biol. Med., XXVII,
pp. 734-735, April 1930.
Myers: Ind. and Eng. Chem, June 1941, pp. 697-706,
55 vol. 33, No. 6.
Foshay et al.: American Jour. of Public Health, vol.
32, October 1942, pp. 1131-1145.
Gershenfeld: Bacteriology and Allied Science, Mack
Publishing Co., Easton, Pa., 1945, pp. 294-295.
comprises modifying the organism by digesting said or
Green: J. Bacter., April 1946, pp. 487-493.
ganism with a proteolytic enzyme, oxidizing said digested 60
Taub: J. Am. Pharm. Assoc. Sc. Ed., vol. 37, 1948,
7. A method of reducing the toxicity of a microbio
logical organism which causes an infection in animals,
while retaining the antigenicity of said organism, which
organism, heating the oxidized, digested organism at
pp. 24e2so.
about 55°-65’ C. for about 20-28 hours, subjecting a
portion of the resultant to ultrasonic waves to disrupt
Drug and Cosmetic Industry, February 1948, p. 245.
Zinsser’s Textbook of Bacteriology, 1948, pp. 918 and
939.
cells of the organism, combining the disrupted and un
UNITED STATES PATENT OFFICE
CERTIFICATE OF CORRECTION
Patent No. 3,097, 141
July 9, 1963
Elizabeth N. Willcox Kidwell
It is hereby certified that error appears in the above numbered pat
ent requiring correction and that the said Letters Patent should read as
corrected below.
In the grant, lines 1 and 12, and in the heading to
the printed specification, line 3, for "Elizabeth N. Wilcox
Kidwell , " each occurrence,
read —— Elizabeth N. Willcox Kidwell,
——; column 1, line 64, for "sealed" read —— unsealed ——;
column 4, line 75, for ". 1" read —— l ——; column 6, line 11,
for "tot" read —— to ——; column 8, line 8, for "venus" read ——
venous ——; columns 11 and 12, TABLE E‘, under the column
heading "Convulsions" in the line relating to GP No. 359C,
for "X" read —— XX ——; columns 21 and 22, TABLE J, in the
heading to the third date box, for "10/11" read —— ll/ll ——;
column 211, line 50, for "vegatables" read -—— vegetables —-;
column 28, line 40, for "sensitive" read —- sensitize ——;
column 29, line 1 , for "op tmium" read —— optimum —-; column
30, lines 61 and 62, for "organsims" read —- organisms ——.
Signed and sealed this llth day of February 1964.
(SEAL)
Attest:
ERNEST W. SWIDER
A'ttesting Officer
EDWIN L. REYNOLDS
Acting Commissioner of Patents
UNITED STATES PATENT OFFICE
CERTIFICATE OF CORRECTION
Patent No. 3,097, 141
July 9, 1963
Elizabeth N. Wil lcox Kidwell
It is hereby certified that error appears in the above numbered pat
ent requiring correction and that the said Letters Patent should read as
corrected below.
In the grant, lines 1 and 12, and in the heading to
the printed specification, line 3, for "Elizabeth N. Wilcox
Kidwell , " each occurrence, read —— Elizabeth N. Willcox Kidwell,
——; column 1, line 64, for "sealed" read —— unsealed ——;
column 4 , line 75, for ". 1" read —— 1 —-; column 6, line 11,
for "tot" read —— to ——; column 8, line 8, for "Venus" read ——
venous -—; columns 11 and 12, TABLE F, under the column
heading "Convulsions" in the line relating to GP N0. 3596,
for "X" read -- XX -—; columns 21 and 22, TABLE .I,
in the
heading to the third date box, for "10/11‘I read -— ll/ll ——;
column 24, line 50, for "vega tables" read —— vegetables ——;
column 28, line 40, for "sensitive" read —— sensitize ——;
column 29, line 1, for "op tmium" read —— optimum ——; column
30, lines 61 and 62, for "organsims" read —— organisms —-.
Signed and sealed this llth day of February 1964.
(SEAL)
Attest:
ERNEST W. SWIDER
A'ttesting Officer
EDWIN L. REYNOLDS
Acting Commissioner of Patents
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