A STUDY OF THE TOXIC PROPERTIES OF TUBERCULO-
       PROTEINS AND POLYSACCHARIDES

By FLORENCE R. SABJN, M.D., FRANKLIN R. MILLER, M.D., CHARLES A.
       DOAN, M.D., AND BRUCE K. WISEMAN, M.D.

(From the Laboratories of The Rockefeller Irtstitute fw Medical Research)

(Received for publication, Jury 25, 1930)

It was reported by White in 1928 (1) and confirmed by Sabin, Doan,
and Forkner (2, 3)' that a polysaccharide isolated from the tubercle
bacillus by Anderson (4) was toxic for tuberculous guinea pigs.

With doses of 10 mg. the guinea pig either died within a few hours, in which
&se there was a precipitous fall in temperature; or it survived and showed only a
temporary fall in temperature with subsequent rise above the normal level.
There was also a change in the blood cells characterized by a rise in the neutrophilic
leucocytes and a fall in lymphocytes.

  Similar phenomena follow the administration of tuberculo-protein.
Since the polysaccharide, as prepared, has a certain nitrogen content,
and since all the protein preparations contain carbohydrate, one must

resort to a biological titration in order to determine responsibility for
the intoxication. By giving the polysaccharide and the protein to
tuberculous animals, in decreasing doses, we have ascertained that the
temperature reaction is due to the protein, since amounts of poly-
saccharide which do not contain as much nitrogen as is present in a
temperature-altering dose of the protein fail to elicit the change.

In the course of our testing of fractions from tubercle bacilli, it has
become clear that we must consider not only fractions from merent
types of bacilli, according to the plan of the Research Committee of
the National Tuberculosis Association (S), but also the.varying mani-
festations of the disease in different species of animals.  To show the
relations of these Werent factors we have drawn up an outline
(Table I).  The attempt at any quantitative comparative estimation
of the factors of the disease must be considered as only an approxima-
tion, subject to marked variations in individual animals. For the

51


TUBERCULO-PROTEINS AND POLYSACCHARIDES

F. R. SABIN, F. It. MILLER, C. A. DOAN, B. K. WISE&MN   53

,

Tp)LE I

Phenomenu of Tuberculosis as Rektd to Chemical Fractions from the Bacilli

=
I

Production of lesions

Phenomena of the disease

CaseatiOtt

Probably protein

Hemorrhage

Protein

Tuber&a

Non-specific
connative tissue

Wax and protein

-

1

. -

Phosphatide

Relation of
phos hatide to
nti-p osphatidt
`I,

?

?

Protein

Relation of chemical fractions
from the bacilli to these
    phammena

Protein and
polysaccharide

rypc of bacillus

Fowls

Guinea
Piss

Rabbits

Monkey

Rat

Avian S
(Petroff)
Avian R
(Petroff)

BI andfrac-
tions of
&r

+

+++

+++

++++

++++

++++

Reversed
++++
slight
age

Reversed
++

++++

++

Stlld

Average

Average

Average

Reversed
++++

Average

                                             I

++++ ++               Large      /

     1-y. .l
       .,J                                    1

++++           ++++   h3e

Reversed
++++

Extreme

ACUte

++++

++

Minimal

+++

  f

+

  f

++++

++++

-I

  f

++

+++

  ++
Slight with
phosphatide in
normal

++++

Epithelioids
f3led with fat

                                                 1
                -
iuong the upper transverse line of the table are listed the phenomena of the disease itself and !
normal and tuberculous animals as far as they are known. The lower part of the chart records th
kactions from the bacilli.

subacute
and
dUOUiC

Acute or
dUOtiC

ACUte

Chronic

Protein 10-2.5
mg. and poly-
sac&ride

Protein up to
.OOOl mg.

Sensitive to
0. T.

++++
Lymph
nodes

++

++++

++

he second line are recorded the effects of chemical fractions isolated from tuberde bacilli on
,arying manifestations of the disease in animals, viewed in the light of studies with chemical


54       TTJBERCULO-PROTEINS AND POLYSACCHARIDES

data on the temperature reactions in the monkey we are indebted to
Dr. G. P. Berry whose work will soon be published.

The phase of the subject upon which we have done the most intensive work (3)
is that of the chemical factors causing the production of new tissue.  Both lipoids
and proteins in repeated doses have given rise in rabbits to the production of new
connective tissue cells, but a phosphatide designated A-3 by Anderson (6) has
proved to be so specific in the production of epithelioid cells and epithelioid giant
cells, the essential elements of the tubercle, that we may speak of this element in
the disease as the phosphatide reaction. In the tuberculous animal the phos-
phatide is probably liberated only from the bodies of dead bacilli.  The amount
of the reaction to the phosphatide is governed by two different factors, first, by
the number of dead bacilli, which can only be estimated; and second, by the
amount of antiphosphatide produced in the disease (7,8,9).

The most striking new material shown in the table is represented by the pre-
hinary analysis of the effects of the dissociation of the avian strain of bacilli by
Petroff (10, 11, 12). These two strains were given to us by Dr. Petroff for path-
ological studies. As shown in Table I, the two dissociated strains of the bacilli
produce different types of tuberculosis, that is, two different combinations of the
manifestations of the disease.  Roosters with the avian S strain had no tubercles
large enough to be seen with the unaided eye, but under the microscope there were
numerous tiny clumps of pure epithelioid cells, containing very large numbers of
tubercle bacilli. Smears stained for tubercle bacilli had too many organisms per
oil immersion field to be counted. From the small phosphatide reaction, we
estimate that there had been a relatively slight death rate of bacilli.  The animals
had negative skin tests but became extremely emaciated.

With the avian R strain, on the other hand, the skin test was marked and the
loss in weight only nominal, while tuber&s were comparable in sixe to those seen
in rabbits. Such striking diEerences in reaction to bacterial dissociation demon-
strate the significance of this method in the further study of tuberculosis.  A
comparison of the reaction with the avian S in roosters with the disease in rats is
also interesting. Both show a large number of bacilli, but in the case of the rat,
we estimate an increased death rate of bacilli from the extent of the phosphatide
reaction, the rat finally dying from the amount of epithelioid cells in the lungs
rather than from any toxic effect. The detailed account of the pathological
di.iIerences with dissociated strains will be published subsequently.

The data in Table I suggest, for the further testing of chemical
fractions, the selection of animals showing certain extreme reactions,
such as the loss of weight after the Petroff avian S in roosters, or the
caseation in monkeys. The hypersensitivity of the guinea pig to
tuberculo-protein has been the reason that this animal has been used
so extensively for the study of allergy, Through the studies reported


F. R. SABIN, F. I(. MILLER, C. A. DOAN, B. K. WISEMAN   55

here, it seems probable that the temperature in tuberculosis as indi-
cated in Table I, is related to proteins liberated from the bacilli.  The
tuberculo-proteins give a rise in temperature in the normal animal
but the reaction is increased in the sensitized animal.

The two types of temperature curves, one the fall which precedes
death, and the other the reaction from which the animal recovers,
are shown in Charts 1 to 7 for both proteins and polysaccharides.
The results of .the titrations of these two substances in tuberculous
guinea pigs are shown in Tables II and III.

Most of the experiments with the protein have been made with a preparation
from the H. K. Mulford Company, designated MA-100, which gfves an active
skin test in tuberculoua guinea pigs hi doses of 0.0001 mg. One guinea pig received
the Protein 304 and another an alkali-soluble protein, both prepared from the
bacilli by Johnson, Coghill and Renfrew (13,14).

Four preparations of tuberculo-polysaccharides have been used. First, a
sugar isolated by Anderson (15) from the water separated from the ether-alcohol
extraction of lipoids from H-37, and designated A-8; second, an analogous sugar
from the bovine strain; third, preparations from the Bacflli H-37, prepared by
Heidelberger, Hbg 511 and 514; and fourth, similar preparations made by the
H. K. Mulford Company, MB-200 (16).  The guinea pigs used in these experi-
ments were all tested with tuberculin (0.02 cc. 0. T. Saranac) and were negative
before the inoculation with tubercle bacilli.  They ah received l,OOO,OOO counted
organisms (l/50 mg.) of a 10 day culture of human Strain H-37 obtained from
Dr. S. A. Petroff. They were inoculated either intraperftoneahy or subcu-
taneously in the groin, as shown in Tables II and III.  The shin test was positive
to 0. T. in each instance before the injections of protein and polysaccharide were
begun.

The rabbits were given 0.5 mg. of the Strain B-l undissociated, intravenously
from a 13 day culture. The experiments with the proteins and the polysaccharides
were begun about 6 weeks after inoculation and were continued through 4 months.
One (R 1344) of the 20 gufnea pigs of the first series (inoculated Feb. 14, 1930),
died 18 weeks after fnocufation without having received any injection, and showed
caaeous fnguinal lymph nodes and extensive tuberculosis of liver, spleen, omentum
and lungs.  Other anfmals that died following the injection of both protein and
polysaccharlde have shown marked lesions of the liver and spleen. .In every
case, with one exception, the routine was to take a rectal temperature first, then
the specimens for the blood count and then give the injection.  The exception
was the iirst experiment on Chart 3, Guinea pig R 1323, in which the injection was
given first.  The amount of fluid injected was in each instance 1 cc.  The poly-
saccharide and the water-soluble Protein 304 were dissolved in freshly distilled
water. The alkali-soluble protein was dissolved in dlstllled water and then


DO!%

  ml.
10

5

2.5

1

17 2/14/3(  Mulford
R 1329 (i.p.)   MA-100

9 2/14/3(
R 1321 (i.p.)

40 3/22/3(
R 1398 (i.p.)

41 3/22/d
R 1399 (i.p.)

48 3/22/3
R 1406 (i.p.)

3 2/i4/3
R 1315 (i.p.)

"

`I

"

`L

304

TABLE II

Effects of Tubem&-Proteins in Tubercuhu Guinea Pigs

Type of
protein
mjected

Date .of
n jpm  pc$e+ of
protein   mpctmoa

--

:/11/30

l/10/30

i/ 5/30

i/ 5/30

i/ 5/30

i.v.

i.p.

i.p.

i.p.

i.p.

t/25/30 In tra-
     testic-
      ular

-10"

-7"

-3O

fl"

-1.3"

+l" -3"

  Blood count
I I
before infection. if
White blood cells
  per c. mm.

iii

97c

`651

03(

-

-

17ti

-

ii
-

63:

-
LY
-

13!

28!

la!

-

-

221

-

321

27(

-

-

34

-

llood count before
ejection of protein.
White blood cells
per c. mm.

-
`MN

-
LY

i-

6985

-

-

l651

-

-

-

153'

-

-

10%

-

-

-

104!

-

-

274

-

-

-

590.

-

Result

Death
in 2f
hours

Death
in 16
hours
Death
in 51
hours
Death
withii
24
hours

Death
within
24
hours

Survival


0.1

0.02

0.01

0.001

30  2/14/30
R 1342  (s.c.)

16  2/14/30
R 1328 (i.p.)

Alkali
soluble
protein
(John-
son)
Mulford
MA-100

3  2/14/30
R 1315 (i.p.)

30  2/14/30
R 1342 (s.c.)

31 _ 2/14/30
R 1343  (s.c.)

31  2/14/30
R 1343 (s.c.)

36  3/22/30
R 1394 (s.c.)
30  2/14/30
R 1342  (s.c.)
7  2/14/30
R 1319 (i.p.)

u

`8

"

`C

3/25/30 Intra-
     testic-
    ulaF

4/16/30  i.p.

4/21/30 i.p.

         .
4/22/30  i.p.

4/22/30  i.p.

3/25/30 Intra-
    tt?&c-
      uiar
4/23/3Q i.p.

4/25/30  i.p.

5/ l/30 Intra-
     denaai

f4.5"

-1.8' +2.2"
   -3.9"

- .8O +3o -5O

-1.5" +3.40

+2.9"

+3.2"

+2.5O

-.7" j-3.7"

Negative
-1.3" +1.40

588333301554 5200
588333301554 5200 91525616~197121888 507 Survival

41465934 1380 2782 1575 735 8532 756 1404
41465934 1380 2782                      I`

1768 1224 340 2366 1820 364 3960 176 110
1768 1224 340 2366                      0

5883 3330 1554 12198 147
5883 3330 1554 12198 14766 2568 19276 2254 483    ,,

67322856 408 5016 5700 684118081065 1152
67322856 408 5016 5700 684118081065 1152    "

6732 2856 4Of3 12243 6237 4389 14700 2952 980
6732 2856 4Of3 12243 6237 4389 14700 2952 980    ,`

---
---  1527 1560 162 4972 526 396
        1527 1560 162 4972 526 396    `4

58833330 1554 5475 9417 3285 16942 1576 985
58833330 1554 5475 9417 3285 16942 1576 985    "

1260 714 126 4380 1440 180 7296 498 1462
1260 714 126 4380 1440 180 7296 498 1462    `I

* These are serial numbers of the work of the department covering a term of years.


TABLE II--CofKh&d

WK.
0.0001

O.OOOC

31
R 134
3
R 131
30
R 134

    I
     IVliifOKl
I/14/30 ~-100 4/25/30  i.p.
(S.C.)
t/14/30 "   6/ 4/30 i.p.

!%J " /6 n/j01 ip.

16  z/14/30    ((

R 132  0. p.)
11  z/14/30 u
R 132  0.p.)
20  2114130    "

R133  (i.p.)
7  2/14/30    "

R 131  0.p.)

51 2130  i.p.

6/U/30  i.p.

6/10/30  i.p.

6/10/30  i.p.

PMN Ly M PYN Ly

                                                *
Negative    98914867 785 9398 76204318 2


F. a. SABIN, F. R. MILLER, C. A. DOAN, B. IL WISEMAN   59

enough 1 per cent NaOH added to make the solution neutral to litmus.  The
Mulford Protein MA-100 was received in sterile salt solution, IO mg. per cubic
centimeter, and it was diluted with freshly made, sterile salt solution.

Kjllilzg Power of Tuberculo-Protins and Polysaccharides in Tubercdous
            Guinea Pigs

As is shown in Table II, all five tuberculous guinea pigs which
received from 10 to 2.5 mg. of the protein died within 24 hours;
and all receiving less than 2.5 mg., seventeen injections in all, survived.
Thus the minimal lethal dose for tbis protein lies between 2.5 and 1 mg.

iu tuberculous guinea pigs. A report of the killing power of protein
prepared from the filtrate has been given by Seibert (17).  In Chart 1
is shown the type of temperature curve following a lethal dose of the
protein, for Guinea pig R 1329.

The injection was intravenous; the animal showed irregular and rapid breathing
within 2 minutes of the injection.  Subsequently there was involuntary defecation.
The temperature fell precipitously 10'.  The irregular breathing continued until
death 41 hours later.  The postmortem e xamination showed the omentum, spleen,
and liver massively involved with tuberculosis; the lymph nodes were markedly
caseous; the lungs had a moderate number of tube&s. There were numerous
fresh hemorrhages especially marked around the tuberculous lesions in the omen-
turn and in the lung. The lungs were markedly congested.


60        TWERCULO-PROTEINS AND POLYSAC-ES

A more gradual fali in temperature, 7" in 7 hours, is shown in Chart
2, of Guinea pig R 1321, which received 5 mg. of the Protein MA-100
intraperitoneally. .

The animal died in 16 hours after the injection. The postmortem examination
showed extensive involvement of omentum, spleen, liver, and inguinal lymph
nodes; there were a few tuber&s in the lung. Fresh hemorrhages were not
apparent at autopsy, but in sections there was marked congestion of the vessels
of the lung and some hemorrhage into the tubercles.  It will be noted in the chart
that there was a fall in neutrophiic leucocytes, lymphocytes, and monocytes
during the 7 hours that counts were made.  All but one of the animals, R 1399,
that died within 24 hours after the injection, had the marked fall in temperature.

6WO -                                             *
VXQ-
4cm-
3ooo,-
2owL
loM)-
  I

I

Th-

1

Feh 5 13 14 Ibc4llpr8             9             10             11
1930
                  bA.RT 2

In this instance the changes in temperature were but slight; there was a drop of a
degree and then a rise to the original level; the temperature had begun to fall
when last taken and the animal died in the night.  All of the animals that showed
the fall in temperature were obviously quite sick, showing a marked toxic effect of
the protein. The other animal that died after protein showed the same extensive
involvement of liver, omentum, spleen, and lymph nodes.  The lungs had a few
tuber&s and were markedly congested and in one case (R 1406) hemorrhagic.

The polysaccharides had also a certain kiiling power in sensitized
guinea pigs, though it was by no means as consistently related to
dosage as with the protein. In the original studies of Sabin, Doan,
and Forkner (2, 3) six tuberculous guinea pigs received 10 mg. of the
Polysaccharide A-8 intraperitoneally; two died within a few hours,
two during the night after the experiment, while the others survived.


F. R. SABIN, F. R. =-R,` c. A. DOAN, B. K. WISER   61

The plysaccharides in the present series did not show as marked a
l;iKng power for guinea pigs.

*is shown in Table III, one guinea pig, R 1324, died in 4f hours after an intra-
Pritoneal injection of 20 mg. of the A-8. The liver, spleen, omentmn, and
lymph nodes were on& moderately involved; the lungs were markedly congested;
but no hemorrhages were found in sections.  This animal had survived two
in+&- on the 2 preceding &YS of 10 WC. each of this polysaccharide,  None
of the animals receiving 10 mg. died; but R 1316 which had an intraperitoneal
dose of 5 mg. of the Heidelberger polysaccharide died in 19 hours, the fall in tem-
perature and the changes in the blood cells being shown in Chart 1.  At post-
mortem there was extensive involvement of liver, omentum, and spleen.  The
lungs showed many tuberdee, but were only moderately congested and had no
hemorrhages. It is important to consider whether there was sufficient protein
in this dose of the polysaccharide to account for the killing power and for the fall
in temperature. The problem with the polysaccharine is complicated through
not knowing the actual state of the nitrogenous contamination or its potency.
Dr. Heidelberger found the nitrogen content of this sugar to he 0.34 per cent, 90
that if ah of the nitrogen were calculated as protein, 1 mg. of polyssccharide would
contain 0.021 mg. of protein; and 5 mg. would have only 0.105 mg. protein, wh&$
is below the minimal lethal dose of the protein whose potency we have been
testing. On this basis it would take 100 mg. of the polysac&&de to cone 2.1
mg. of protein, 2.5 mg. having killed a guinea pig (Table II).

The question of a possible killing power of the polysaccbaride here
employed needs further study, with a larger series of experiments;
but these experiments suggest tbat it either has a certain killing power
in sensitized animals or it may enhance the killing power of a dose of
protein too small to kill by itself. It should be stated tbat the
manner of death is exactly like that of the lethal action of the protein.

In relation to the fall in temperature, on the other hand, it is
interesting to note that the animal (Guinea pig R 1315, Table II)
which received 0.1 mg. of the protein, t&e computed amount in the
5 mg. of polysaccharide, showed a fkl drop of 5" in temperature, so
that if the fall in temperature in Guinea pig R 1316 (Table III) which
succumbed to the polysaccharide was a direct eflect of the substance
injected rather than an indirect toxic effect on the animal, it might
have been due to the protein introduced with the sugar.

It will be noted that one other animal which received 5 mg. of
the sugar, R 1317, showed a fall in temperature and died 4 days later.


D@Sl

5

I-I-

12   2/14/30  A-8 original 3/13/30 i.p.
R 1324 (i.p.)    (Anderson)
31   2/14/30  Bovine (Au- 4/17/30 i.p.
R 1343 (SC.)   de&on)

15   2/14/30
R 1327 (i.p.)
12  2/14/30
R 1324 (i.p.)
12  2/14/30
R 1324 (i.p.)
15  2/14/30
R 1327 (i.p.)
15  2/14/30
R 1327 (i.p.)

18   2/14/30
R 1330 (i.p.)

A-8 purified 3/12/30 i.p.
(Anderson)
A-8 purified 3/11/30 i.p.
(Anderson)
A-8 original 3/12/30 i.p.
(Anderson)
Heidelberger 3/21/30 i.p.
mg 514
Bovine crude 3/13/30 i.p.
II (Ander-
-=I
Bovine crude 3/13/30 i.p.
I (Ander-
=n)

11   2/14/30
R 1323 (i.p.)

Mulford MB- 4/ 8130 i.v.   -0.9" +4.2"  98914876
                         98914876 785 No couuta
                               785  No couuta    No
                                              No counts
                                                      counts

200   I I

TABLE IU

Effects of Tubercdo-Po~yzccharid~ in Tuberculous Guinea Pigs

Effect on
t.empemtLue

I          I       I          I

  Blood count   Blood count before
         injection of   Maximum change
before infection.         in cells folloyi~
White blood cells &e$& &zi$$&&
  per e. mm.     pcrC.lIUU.      perc.mm.
B 3 4 ii     a 2
               a 2
       3% E
       3% E      a a
              3 %
              3 %
_----
_---- -----
               -----

26182796 476 5400 7501125 5525 378 252
26182796 476 5400 7501125 5525 378 252

6732 2856 408 8008.5434 572 20976 456
6732 2856 408 8008.5434 572 20976 456   0 0

-3O

    +4.10

    +2.7"

-0.8' +3.2"

-2.0" +2.g0

-1.2O +1.4"

Negative +0 .6"

+2.7"

5376 7056 2352 12954 4000 2095 5760 976 525
5376 7056 2352 12954 4000 2095 5760 976 525

26182796 476 522628211202 6732 624 288
26182796 476 522628211202 6732 624 288

26182796 476 4416 1449 966 2NHO 412 8800
26182796 476 4416 1449 966 2NHO 412 8800

5376 7056 2352 2300 5635 3220 6240 8.55 380
5376 7056 2352 2300 5635 3220 6240 8.55 380

5376 7056 2352 14418 1424 1246 2860 553 220
5376 7056 2352 14418 1424 1246 2860 553 220

21601480
21601480 240 586s 1870 595 8733 817 30
      240 586s 1870 595  8733  817  30

Radt

Death in 4f

~ houn
Survival

I,

u

"

`I

I`

u


5

2.5

1.0

0.1

0.01

4  2/14/30
R 1316  (ip.)
7  2/14/30
R 1319  (i.p.)
5  2/14/30
R 1317  (i.p.)

6  2/14/3O
R 1318  (i.p.)

IO  2/14/30
R 1322  (i.p.)
7  2 /I4130
R 1319 (i.p.)

19  2/14/30
R 1331  (i.p.)

11  2/14/30
R 1323  (i.p.)
20  2/14/30
R 1332  (i.p.)
7  2/14/30
R 1319  (i.p.)

Heidelberger  4/ 2/30  i.p.
Hbg 514
Mulford MB- 4/ 7130  ip.
2cul
Mulford MB- 3/31/30 i.p.

200   I I

Heide&erger  41 4/30 i.;.
Hbg 514

Mulford MB- 4/14/30 i.p.
200
Heidebrger  4128130 i.p.
Hbg514

Heideberger S/22/30 ip.
wg 514

Heide.lberger  5/29/30  i.p.
Hbg 514
Heidelberger S/23/30 ip.
Hbg 514
Heidelberger  6/ 3/20  i.p.
Hhg 514

                                 zhfrt blood
-4.5"      4160 1728 448  10803888 216 15164 6244 669

    +4.5'  1260 714 126 2982 1008 126 5172 232 1170

-3.7O      2K'S2709 516 5775 1750 1225 9625 5161625

    +2.1" 3168 1485 198 3136 1617  98 7830 130 720

-4.8O +4.7'  58564309 773 8532 5372 173826080 1810 530

    +1.8'  1260 714 126 33211968 738 4009 594 346
With tiesondary
rise 4129

    +2.3'  1971 1022 584 46745535 615 9130 2300 1660

Negative   98914867 785 13296 9418 2770 9747 2565 3591

    +2.3"  1026 3564 810 5960 7003 1937 7802 1034 470

-1" +l"   1260 714 126 3752 728 1064 8034 8241960

Death in 13
houra
Survival

Died 4 day
latex

sunrival

I`

`I

`I


64        TUBERCULO-PROTEINS AND POLYSACCHARIDES

At the postmortem examina tion, extreme involvement of liver, spleen, omentum,
and peripheral lymph nodes wss found together with many tuber&s in the lungs.
There were extensive hemorrhages.  They involved many of the lymph nodes
and some of the organs, as well as the vessels in the skin and in the subserosal
layers of the peritoneal lining.  It may be that a sustained fall in temperature
after the injection of either protein or polysaccharide indicates, even if the animal
survives 24 hours, that it is not likely to survive the disease for many days.

In general the animals which died after both protein and poly-
saccharide had extensive involvement of the liver, spleen, omentum,
and lymph nodes, and a moderate number of tubercles in the lungs.
They all showed congestion of the vessels of the lung but no edema;
most of them had hemorrhages around the tuber&s in the lung and
around the caseous areas in the omentum.  The lymph nodes were
congested.

Reactiom to Sublethal Doses of Protein and Polysaccharide ilz Tuberculous

G&ma Pigs

The animals which survived the injection of either of these sub-
stances did not show any marked effects except on the temperature
and on the blood cells.  They were not clinically ill.

The results were the same for both substances regardless of the route of injection,

whether intravenous, intraperitoneal, or intratesticular.  The type of the changes
in the blood cells and the temperature are shown in Tables II and III; but the type
of reaction is better indicated by curves ss shown in Charts 3 to 5.  A typical
temperature reaction involved a preliminary fall lasting from + to 2 hours, followed
by a gradual rise of from 2 to 4.5' with a return to the original level in approxi-
mately 5 hours.  The positive reaction was not only a change in temperature but a
specific type of curve. The fall occurred at once after the injection; it was,
however, sometimes much shortened or even suppressed, but the subsequent rise
and return to the original level were constant.  The sublethal reaction for the
polysaccharide is shown by the first curve in Chart 3, R 1323, the animal having
received 5 mg. of the Mulford polysaccharide intravenously.  The injection was
made before a prehminary temperature had been taken and no blood counts were
taken.  The animal had a slight chill but no other symptoms.  Similar curves for
the different proteins are shown in Chart 4, R 1342.  The animal received an
injection of 1 mg. of the alkali-soluble protein (Johnson) intratesticularly 5 weeks
after inoculation.  It was highly sensitized; the temperature fell slightly for an
hour and then rose 4.5' by five o'clock.  It was still above 103" the next morning,
the original level having been 102'.  It will be noted that the leucocytes rose in
this instance while both lymphocytes and monocytes fell.


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66        TUBERCULO-PROTEINS AND POLYSACCHARIDES

Bidogitxd Titration of the Tubercdo-Protein and Polysacchade in
Tubercdous Guinea Pigs. -Since the reaction of the temperature and
of the blood cells to these t&o substances was the same and since each
was contaminated with the other, it was necessary to see if one
substance could give the reaction in such small doses as to exclude the
other. The experiment with titrations of these two substances is
shown in Tables II and III.

All but one of the guinea pigs &eiving injections of the protein of from 1 to
0.9001 mg. gave the &amcte&tic temperature curve and fall in lymphocytes.
The one negative reaction was in Guinea pig R 1319, Chart 5, following the
injection of 0.001 rug. of the Mulford protein given by the intradermal route.
With the next dilution, containing 0.00001 mg. of protein, the ,=&ion became
varied; two were negative, one moderately and one markedly positive. This
dosage thus approaches the limits of the reaction to the protein.
On the other hand, fn Table III it is clear that the inconstant temperature
reaction was obtained with the dose of 0.01 mg. of the polysaccharide; one animal
was negative, a second showed a slight reaction and the third had a rise of 2.3".
On the basis that 1 mg. of the Heidelberger polysaccharide contains 0.021 mg. of
protein or protein degradation products, 0.01 mg. would contain 0.00021 mg. of
protein, which is twice the dose of protein which gave a consistently positive
reaction.                                    I

There was probably enough protein, or its degradation products, to
account for the temperature reaction in all of the experiments with
the polysaccharides, provided the protein was in an active form.

Variations in Temperature Induced in Tuber&o&s Guinea Pigs by
         Protein and Polysacchade

Certain interesting points can be made out in this connection.

In Chart 3 it will be noted that the guinea pig was highly sensitive to polysac-
charide (protein) as shown by the first temperature reaction after inoculation with
tubercle bacilli.  During the months of May and June the control temperature
curves were constant. These control periods are an important part of the experi-
ment for they show that there is for the most part relatively little fluctuation in
temperature from the disease itself.  In only one snimal of the entire series was
there an afternoon temperature during one of these control periods; in this animal,
R 1332, on one occasion the temperature was steady for the morning hours but
rose suddenly 3.2' between one-thirty and two-thirty and then fell gradually to
the original level.  The cause of this rise was not determined.  In Chart 3 it will
be seen that the temperature remained normal both after 0.01 mg. of the Heidel-


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70        TKJBERCULO-PROTEINS AND POLYSACCEARIDES

berger polysaccharide and after 0.00801 mg. of the Mulford protein; but onboth
of these days, the lymphocytes fell markedly and the neutrophilic leucocytes were
irregular.  Thus the reactions on the blood cells are not to be correlated with the
change in temperature.

Chart 4 represents a long experiment with Guinea pig R 1342. The first
experiment shows that there was no rise in temperature, but rather a fall after an
injection of 1 cc. distilled water intraperitoneally. The water was freshly distilled
from glass, boiled and used as soon as sufiiciently cooled.  A second control with
distilled water in another guinea pig, R 1332, showed a steady reaction.  The
intratesticular route of injection was employed m R 1342 to study the tissue
reaction as reported by Long (18). 21 days later the testis was removed and
showed the marked degeneration desaii by Long.

By the twenty-first of April the control temperature was moderately steady,
and the following day the animal gave a positive reaction to 0.02 mg. of the
Mulford protein. The rise in leucocytes and the fall in lymphocytes were extreme;
it is interesting that the lymphocytes had returned to 8000 cells per cubic milli-
meter by the next morning, though not to the original level  On the twenty-fourth
of April a test was made with salt solution which had been sterilixed and sealed
several days previously; there was a slight rise in temperature in the afternoon
which did not occur with freshly made salt solution. The rest of the experiment
had to do with the minute doses of the protein, 0.001 and 0.0001 mg., with less
reaction in temperature to the smaller dose.

In the intervening days, it will be noted that the level of the temperature of the
guineapigwasadegreehigheronthethirdof Junethanontheprecedingday.
This phenomenon has occurmd both with normal and tuberculous guinea pigs.
The mean temperatures for New York City, together with-three readings for
humidity, reported at 8 am., at 12 noon, and at 8 p.m., by the Weather Bureau,
United States Department of Agriculture, are included in the charts.  In Chart 4
it will be noted that with a rise of 6oF. between June 2 and 3, tbe temperature of
the guinea pig was a degree higher on the second day. Comparing the humidities
on June 2 and 3 it will be noted that on June 2 there was a fall of 13'points between
8 a.m. and noon, and on the next day a rise of about the same amount, namely 15
points, so that the change in temperature of the animal in this instance has run
parallel with the change in atmospheric temperature rather than with the variation
in humidity.

Chart 5 represents another long series of experiments. This guinea`pig, R 1319,
was markedly sensitive to an injection of 5 mg. of the Mulford polysaccharide on
April 7, about 7 weeks after infection.  The original control temperature was
somewhat irregular, as frequently happens the first thne temperatures are taken.
The change in humidity was not great.  It will be noted that the temperature of
the guinea pig was entirely steady on the day after the injection, but on the 14th
and 15th of April was irregular and ran nearly 2" lower on the 15th.  There was a
drop in atmospheric temperature of 11oF. The temperature of the animal became


F. R. SABIN, F. R. MILLER, C. A. DOAN, B. K. WISEI&AN   71

quite level again and on the 28th of April, it showed an average temperature
reaction to 1 mg. of the Heide-lherger polysaccharide.  The next day there was a
~condary rise, pra~tid~ a duplicate of the curve of the preceding day with no
change in the weather, and for 3 days the temperature of the animal was at a

TABLE IV

~ffec& of Tubercdo-Protean and Pd~sadaride on Nomwd Guinea Pigs

  m.
20
15

10

5

1

0.1

0.01

0.001

0.0001

O.ooool

Numbu Dsteot
& llljecti
--

R 1433 6/ 6/3
R 1430

R 1432 6/ 6/3
R 1425

+1.3o

f1.90

R 1425

R14.30

R1430

R 1430 4/30/3
R 1432
R1433
R 1436
R 1425

+1 so

R 1435 5/ l/3
R 1432 5/ 213

             +1.6"
        0      +2.4.'

R 1433 5/ Z/30   Negative
R 14365/ l/30 + .5" +l"

66
63

48
32

R 14345/ Z/30   NegatiE    76

70

61
6.5
77
69

higher level than before the injection. We have seen no other secondary rise of
this magnitude, but in three instances it has taken 3 days for the temperature to
come down to the previous level Q 1323, Chart 3, after 5 mg. of Mulford poly-
sac&ride; R 1331, (19) after 0.1 mg. of the Heidelberger polysaccharide; and
Rabbit R 1365, Chart 8, after the Heidelberger polysaccharide). On May 1,
R 1319, Chart 5, there was a negative reaction to an injection of 0.001 mg. of
protein MA-100 given intradermally. On the third of June there was a slight


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F. R. SABIN, F. R. MILLER, C. A. DOAN, B. K. WISE116;AN   73

reaction to 0.01 mg. of the Heidelberger polysaccharide, but 7 days later the animal
proved to be highly sensitive to the Mulford protein in a dilution of 10".  It will
be noted also that the temperature of the guinea pig during the control period of
June 9, was lower by 2oC. than the initial temperature of June 3.  Between these.2
days the environment diEered by lOoF.  This guinea pig showed low lymphocytes
thoughout the experiment but they fell with every injection except after the 0.01
mg. of the polysaccharide.

Ejects of Tuberczclo-Protein and Polysaccharides om the Temperature of

Nowd Gkzea Pigs

The results of experiments with two types of fractions are shown in
Table IV and Chat 6.

For ail of the work the Mulford Protein MA-100 and the Heidelberger Poly-
sac&wide 514 were used.  It wiii be seen that with the normai animal the rise of
temperature was .more moderate throughout than with the sensitised animai; the
type of curve was, however, the same. It is likewise ciear that the biological
titres of polysaccharide and protein exhibit the same relationship in the normal
guinea pig as in the sensitized; however the necessary amounts for normai animals
are 10 to 100 times greater.  For the polysac&aride, the reaction was either
negative or siight with doses of 0.1 and 0.01 mg.; while with the protein the iimit
of reaction wss 0.001 mg. and the dose of 0.0001 mg. was negative.  In Chart 6
it will be noted that during the control periods the temperatures were more irregular
but were not at a lower level than with the tubercuious animais.  There were more
of these irreguiar control temperatures in normai than in tubercuious guinea pigs.

EJects of Tuberdo-Proteins &d Polysacchardes an Tubercdous and
               Non& Rabbits

Fewer experiments were made with rabbits than with guinea pigs.  With two
tubercuious rabbits, it wss determined that the protein from the Strain H-37
gave a rise in temperature in small doses. Rabbit R 1366 was given 1 mg. of the
Protein MA-100 10 weeks after inocuiation with 0.5 mg. bovine Strain B-l; the
temperature feii 0.5" and then rose 2"; 2 days later the reaction to 0.001 mg. of
the same protein was similar but with a rise of only 1.2".  Rabbit R 1367, in-
ocuiated with tuberculosis at the same time as R 1366, was given 0.01 mg. of the
same protein in 10 weeks and showed a fail of 1.2O and a subsequent rise of 2.1";
while 2 days later the rise in temperature after the injection of O.ooOl mg. was of
the same magnitude, 2". After all four injections in these two animals, the
leucocytea rose and the lymphocytes aud monocytes fell. Considering the
difference in size of rabbit and guinea pig, the tubercuIous rabbit inoculated with
the bovine strain of organisms proved to be quite sensitive to the protein from the
human strain of tuberde bacillus.


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F. R. SABIN, F. It. MILLER, C. A. DOAN, B. K. WISEM.AN   75

In two other tuberculous rabbits, R 1364 and R 1362, a comparison was made
between the reaction of a polysacchan'de isolated by Anderson from the bovine
organism, analogous to the A-8 from the human strain and the polysaccharide
isolated by Heidelberger from the human shin.  In both instances the reaction
was pmct,ically identical and is shown in Chart 7 for R 1364.  A moremarked rise
in temperature following the Heidelberger polysaccharide was shown by another
rabbit, R.1365, Chart 8.  The chart illustrates VW wd the delayed return of the
temperature to the oti@d already noted.

co?ztrozs

The polysaccharides, the Protein 304, and the alkali-soluble protein were all
injected into the guht%t pig in 1 CC. water freshly distilled from glass.  In the
rabbits 2 cc. of fluid were used.  The Protein MA-100 was received from the H. K.
i\Iulford Company in sealed ampules in sterile salt solution.  It was thus necessary
to study the effects of these dihmnts.  The freshly distilled water or salt was boiled
and used as soon as sticiently cooled. Neither the water nor the freshly made
salt solution gave any rise in temperature. The curves of temperature for distilled
water and for a stock salt scduti~n are shown in Chart 4, and the records are given
in Table V. In general both the distilled water and the salt solution did not give a
positive curve in temperature. The distilled water was either negative or showed a
slight fall as on Chart 4. The stock salt solution, which had been kept in sterile
flasks gave a slightly more irregular curve, Chart 4, than the freshly prepared
solution.

In these experiments it has been found that control temperatures at hourly
intervals should be taken for 2 or 3 days pmceding each experiment and after each
experiment until the temperature reaches the or&al level. This may take 4 or
5 days.

Concerning these control temperature reactions, two factors must
be considered; fkst, the level at which the temperature runs, and
second, the steadiness of the reaction during the day. Our charts
show diEerences in level of as much as 2". This diBerence in the
animal is probably related to differences in atmospheric temperature,
which, of course, could only be tested when the animals were not in
artificially heated rooms. The irregular temperature reactions are
more difhlt to evaluate because more factors may be invokd.  It is
probable that variations in humidity a affect the temperature of the
animal, but abnormal conditions, such as accidental infections must
also be taken into consideration.


Animal
oumbcr

30
R 1342

14
R 1326

20
R 1332

30
R 1342

31
R 1343

57
R 1425

Date of
ofection with
ubercle bacilli
Ifar 1,060,006
orgaoisma

2/13/30

2/13/30

2/13/30

2/13/30

2/13/30

Normal

TABLE V

Effects of Distilled Water and Salt Solution in Tuberculmu Guinea Pigs

TypL$id Date>' Route
        in au00
(1 cc.) 1     of *II-
      o fluid j&ion

       --
Distilled 3/24/30 i.p.

i.p.

DiStilled
HSO

stock salt
solution

Fresh salt
0OlUtiOIl

Distilled
w

i.p.

i.p.

i.p.

i.p.

Effect on
temperature

Irregular
-1.9" +1.70

Negative
   +o.v

Negative
-0.7' +0.40

Irregular
Al.00

Negative

Negative

Bhodco~co~nefom   Blood count before   Maximom change
                       in cells foUowi
white blood &Is    in'ection of fluid.
      White blood cells   injstion of flu1 %
  per e. mm.       per e. mm.     White bhd cells    Result
                          per c. mm.

PMN Ly Mono PMN Ly Mono PbfN Ly Mono
---------
5883 3330 1554 10089 5564 1770 - -   - Survival

9638 13806 208411712 4224 2880 14980 2041 1365     "

1026 3564 810 5746 1774 929 7169 1422 1148     "

5883 3330 1554 4260 6390 1988 15870 2760 1525     "

6732 2856 408 6840 3720 1320 11160 1878 552     "

3504~ 16791 19711 - 1 - 1 - 1 - 1 - 1 - 1    "


F. R. SABIN, F. R. MILLER, C. A. DOAN, B. K. WISEMAN   77

Ejects of the Injection of the Tubetculo-Prohens and PoZysacchades
              on the Blood Cells

In this series of experiments only immediate effects of the injection
have been considered. The blood counts for tuberculous guika pigs
after protein and polysaccharide are shown in Tables II and III.

The total numbers of neutrophilic leucocytes, lymphocytes, and monocytes
are giveb before the infection with tuber& bacilli, in the tirst cohnnn; in the second
cohrmn are the corresponding 8gures for the count taken just before the injection
of either protein or sugar; while in the third are recorded the greatest change in
these three strains. This third column therefore does not represent a single count
a~ was true for the first two cohmms. After the injection of the protein there
was a rise in leucocytes in every guinea pig except the second snimal in Table II,
R 1321, which died in 16 hours; this was the only one of the fatal cases in which
~~tmts were made. In every animal there was a fall in lymphocytes, which for
the most part showed little or no tendency to recover; the monocytes have also
fallen in every instance. but have shown a tendency toward a subsequent rise
before the end of the 7 hours of the experhnent. The average fall in lymphocytes
in tuberculous guinea pigs after the protein, computing from the percentages rather
thanfromthetotalnumbers,was63percent.

After the injection of the polysac&Gdes the total numbers of lymphocytes fell
in every instance but one; this case was the last experiment in Table III, R 1319,
shown in Chart 5, after the injection of 0.01 mg. of the Heidelberger polysaccharide,
in which the lymphocytes were very low before the injection.  In this animal, the
total numbers of lymphocytes rose slightly but the percentages fell. In two
instances, the percentage of lymphocytes rose while the total numbers fell; (R 1324
after A-8 on 3/13/30, and R 1327 after the bovine polysacckide on 3/13/30).
Including these percentages in the average, the lymphocytes fell 45 per cent after
the polysaccharides, as is shown in Table III. Six normal guinea pigs showed a
fall in lymphocytes of 55 per cent in 7 hours after tuberculo-protein, and eight
showed a fall of 65 per cent after tuberculo-polysac&&de.

Non.-.!@eci$c Protein Fever

It has long been known that the injection of proteins causes fever
and a leucocytosis.

In 1890 Buchner (19) showed that bacterial proteins injected subcutaneously
gave a local reaction of aseptic pus. The mechanism of this phenomenon was
then studied by Roemer (20, 21) who found that an intravenous injection of
bacterial protein gave a leuaxytosk which was maximum in 8 hours. Gold-
&eider and Jacobs (22) then found that leucopenla preceded the lencocytosis
using an extensive series of stimuli, organ extracts, bacterial proteins as well as


78        TUBERCULO-PROTEINS AND POLYSACCEAIUDES

proteins from other sources.  Ameth (23) then demonstrated that 8 hours after
the injection of peptone intravenously in rabbits, at the height of the leucocytosk,
there were 3 per cent myelocytes in the blood stream, showing a replacement of
leucocytes from the bone marrow.  In 1923 Hussey (24) discovered that the same
phenomenon occurred after the injection of a considerable series of salts, sodium
chloride, sodium carbonate, potassium phosphate, lithium nitrate, and sodium
sulfate, and stressed the fall in mononuclear cells, finding that it was about 70
per cent in 3 hours. More recently Beard and Beard (25) have followed the blood
cells in eight rabbits every 10 minutes for 5 hours after the intravenous injection
offrom1Oto15cc.ofsaltsolu~nvslyingfrom1to2.5percent. Bymaking
the counts at such frequent intervals they demonstrated that all three groups of
the white cells, leucocytes, lymphocytes, and monocytes, fall immediately after
the injection and that the maximum leucopenia is reached in about an hour.
At this time the neutrophilic leucocytes start to rise, while the lymphocytes and
monocytes continue to fall.  The monocytes then begin to rise but in their experf-
ments did not reach their original level in 5 hours, while the lymphocytes were
lower by approximately 2000 cells at the end of the experiment.

This type of reaction is well shown for the tuberculous guinea pig, Chart 4,
R 1342.  By taking the count 2 hours after injection, the fall in the leucocytes
was missed but the subsequent rise is clear.  It will be noted that before each
injection shown in this chart, the lymphocytes were high and showed a marked
fall with the lowest level, with one exception, at the end of 7 hours.  They had
recovered in varying degrees by the next morning, but only in one instance did
they exceed the level of the time before the injection.  The monocytes varied
somewhat in reaction in these tuberculous guinea pigs, but as shown in Chart 4,
after a fall during the first 2 hours, they showed a tendency toward a temporary
recovery in 5 hours with subsequent fall at the seventh hour.

As is shown in Table V the eiIect of the injection of distilled water
on the blood cells may be slight as in Guinea pig R 1326, or negative
as in R 1332; while the injection of salt solution is followed by the
characteristic changes already described.

It is clear that the injection of proteins, salts and sugars has constant
ekcts on the blood cells at quite specifk time intervals.  There is an
immediate leucopenia followed by a leucocytosis, in which the three
strains of circulating white cells fall at difIerent rates and return at
different times.  The leucocytes start to return first; then the mono-
cytes, and finally the lymphocytes.  This reaction is elicited in the
tuberculous animal by ail the proteins tested; it also follows the
injection of polysaccharide in concentrations containing an amount
of nitrogen too smali to cause a temperature reaction. The rise


F. R. SAFIIN, F. B. WEB, C. A. DOAN, B. K. WISEMAN   79

h Ieucocytes in tuberculous animals is not consistently greater than
is recorded in the literature, though several counts of over 20,000
pseudo-eosinophik 1eucoCytes are shown in Tables II and III.  The
f& in lymphocytes of 65 per cent corresponds with the 70 per cent for
all mononuclear cells recorded by Hussey. The question suggests
itself of whether the tuberculo-protein and the polysaccharides may
not affect the proportion of monocytes to lymphocytes and so be
concerned with the cellular factors of resistance to infection.

S-Y

The temperature reaction in tuberculous and normal guinea pigs
and rabbits is elicited by the tuberculo-protein and probably not at all
by the polysaccharides. The polysaccharides may have some killing
power under certain conditions, but this is not as consistently related
to dosage as in the case of the proteins.  Both proteins and polysac-
&rides cause a change in the white blood cells when introduced
by any route.

BIBLIOGRAPHY

1. White, W. C., Tram. Asm. Am. Phys., 1928,43,311.
2. Sabin, F. R., Doan, C. A., and Forkner, C. E., Tram. of the 24th Am. iKeeL
  N. T. A., 1928,253.

3. Sabin, F. R., Doan, C. A., and Forkner, C. E., J. Erp. Me& 1930,52, suppL
  3,1-152.

4. Anderson, R. J., Ptm. Sot. I&*. Bid. and Me&, 1930,27,387,
5. White, D. C., N. T. A. Technical Series No. 9.
6. Anderson, R. J., J. Bid. Ch., 1927,74,537; 1929,85,351.
7. Doan, C. A., Trans. 2% Am. Meet. N. T. A., 1929,182.
8. Doan, C. A., Pm. Sot. I&$+. Bid. and Med., 1929,26,672.
9. Doan, C. A., and Moore, D., Trans. 26th Am d&et. N. T. A., 1930, 188.
IO. Pet&f, S. A., Proc:Soc. Exp. Bid. ad Med., 1927,24,633,956,958.
Il. Petroff, S. A., Branch, A., and Steenken, Wm., Jr., Ibid., 1927,25,14, and
  Am. Rev. T&m., 1929,19,9.

12. Petroff, S. A., and Steenken, Wm., Jr., J. l&p. itfed., 1930,51,831.
13. Coghill, R. D., J. Biol. Chess., 1926,70,449.
14. Johnson, T. B., and Renfrew, A. G., Am. Rev. T&em., 1928, b, 505.
15. Anderson, R. J., Trans. 26th Am. Meet. N. T. A., 1930, 181.
16. Masucci, P., McAlpine, K. L., and Glenn, J. T., I&i., 1930, 182.
17. Seiirt, F. B., Ibid., 1930,234.
18. Long, E. R., and Seyfarth, Ma&I.., Am. Rev. Tubem., 1924,9, 254.


80        TUBERCULO-PROTEINS AND POLYSACCHARIDES

19. Buclmer, H., Bd. Klin. Wochenschr., 1890,2?, 1084.
20. Roemer, F., Ibid., 1891,28,886,1189.
21. Roemer, F., Virch. Arch., 1892, 128,98.     .
22. Golds&eider, A., and Jacobs, P., Zeitschr. f. Klin. Med., 1894,25, 373.
23. Ameth, J., Die Qualitative Blutlehre, Leipzig, 1920.
24. Hussey, R. G., J. Gen. Physiol., 1923,5,359.
25. Beard, L. A., and Beard, J. W., Am. J. PhysioZ., 1928,85,169.