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Further results of attempts to desensitise tuberculous guinea-pigs.
398
TUBERCLE
[June, 1933
AMEI~ICAN SECTION. Under the Editorship of Dr. A. K. Krause.
FURTHER RESULTS OF ATTEMPTS TO DESENSITISE TUBERCULOUS GUINEA-PIGS. By JO~N WEINZIRr,, Ph.D., J. I)URWARD THAYER, M.S., and JOY HIRSCHMANN, B.S,, Alice McDermott Foundation, University of Washiq~gton, Seattle, Washington. INTRODUCTION.
Two reports [1, 21, have been made previously presenting results of attempts to desensitise tuberculous guinea-pigs; these reports covered trials made with bacilli killed by physical and chemical means, and with tuberculin and tuberculoproteins. It was found that the method used to kill the bacilli influenced the value of the antigen for purposes of desensitisation; for example, bacilli killed by iodine or by light were practically useless as desensitising antigens, while those killed with carbolfuchsin or with ether gave better results. Old tuberculin proved highly unsatisfactory, but tuberculo-proteins appeared more promising. Some degree of desensitisation was always obtained, but in all the trials made we were never able to secure 100 per cent. protection against a fatal dose of either old tuberculin (OT) or Seibert's synthetic medium tuberculin (SMT). Attempts at desensitisation were continued, however, in order to determine more fully the value of our most promising antigens. There can be no doubt any longer that sensitisation plays an important r61e in tuberculosis, for its importance has been demonstrated many times. If tuberculin, or tubercle bacilli, are injected into normal animals they produce no immediate effect ; on the other hand, if either are injected into tuberculous animals, serious symptoms soon develop and may lead to the death of the animals. In our experiments deaths have occurred many times from such injections. The failure of Koch's " lymph " in 1891 was due to the fact that the patients were sensitised to tuberculo-protein and consequently a dose of tuberculin caused fever, malaise and other undesirable conditions which actually hastened the death of the patients. If it were possible to overcome the sensitisation, the body might become immunised, and, as a result, might conquer the tubercle bacilli. From the results obtained by desensitising against serum sickness and hay fever, it would seem that desensitisation in tuberculosis should be possible if suitable antigens are found. In the present work we are presenting some protocols repeating those of earlier trials, but with certain modifications, in order to determine the true value of these antigens. We are also reporting on some new antigens, one of which has given complete protection against lethal doses of tuberculin. ~IETHODS EMPLOYED.
In our previous work the systemic test proved sufficiently satisfactory and we have continued to employ it. According to this test, one or more
June, 1933]
DESENSITISATION
OF T U B E R C U L O U S
399
GUINEA-PIGS
fatal doses of OT or SMT are administered at the close of the desensitisation period. If the tuberculous animals have been sufficiently desensitised they will withstand the fatal dose without exhibiting shock. We have continued to make Mantoux skin tests, mainly to show that the animals were sensitive, and incidentally to confirm the systemic tests as far as possible. In these experiments we have continued to use different sized doses of antigen in order to discover which gave the greatest desensitisation. We have administered the antigen at weekly intervals, but work now in progress seems to indicate that a shorter interval gives better results. The antigens have been given by the intraperitoneal route mainly as a matter of convenience. Tuberculous controls have been included in each protocol. ~:~E SULTS
OBTAINED.
In our last report [2] on desensitisation, we found three antigens which gave similar results, and which presented the greatest possibilities of twenty or more antigens previously tested. The three antigens were : (1) ether-killed H 37; (2) ether-killed 520; and (3) ether-killed Mycobacterium sraegmatis. We decided to repeat these trials in order to eliminate the less promising antigens. We also added some trials with new antigens. Desensitisatiou of tuberculous guinea-19igs by means of ether-killed tubercle bacilli, H 3 7 . - - I n the first protocol, 30 guinea-pigs of 200 grm. weight or over were infected with I-I 178, a strain of low virulence, then divided into five lots of six animals each. One lot was kept as tuberculous controls, while the others received varying doses of the antigen killed by ether and treated with chloroform. Two lots were given constant doses of 1"0 mg. and 0"1 rag. weekly, and the other two varying doses starting with 1"0 nag., one lot decreasing by 0'1 rag. weekly, and the other increasing by 0"1 mg. weekly. In this and all subsequent protocols, both PROTOCOL I.--DESENSITISATION OF TUBERCULOUS GUINEA-PIGS WITH ETHER. KILLED TUBERCLE BACILLI~ H 37.
Unit
Animals Average w e i g h t L i v i n g bacilli H 178" Average sk in t e s t .. D e s e n s i t i s i n g dose .. Doses of a n t i g e n .. Average s k i n test .. Average w e i g h t .. Systemic test .. Tested .. Survived ,. .. S u r v i v a l ratio Second ~y~temie t e s t Tested .... Survived .. .. Survival r a t i o ..
Day
NO.
Gm. Mg. Deg. Mg. No. Deg.
G'm. Mg. No. No. Mg. No. No.
1 1 21 28 42 63 91 96
103
Tuberculou: eonLrols
Test animals
6 312 0"l 2.5+ 1"0 10 2"3 + 2"4 + 412 0"4 5 4 0"800 0.8 4 0"500
6 358 0"1 2"5 + 0"1 10
2"5 + 9-8 +
531 0"4 5 2 0"400 0'8 2 0 0'000
6 320 0"1 2"3 + 1"0--0"1 10 2"6 + 2"6 + 545 0'4, 3 3 1"000 0'8 3 fi 0'666
6 324 0'1
6 352 01
IO 2'2+ 2'0 + 423 0% 3 2 0'666 0'8 2 1 0"500
0 0 2"6+ 2'3 + 573 0"4 6 5 0"888 0.8 5 2 0"400
2.8 + 1"o+o'1
2-5 +
400
[June,
TUBERCLE
1933
the infecting dose and the antigen were given by the intraperitoneal route primarily as a matter of convenience. The results for the individual guinea-pigs have been combined for each lot and are given in Protocol I. The results of this trial showed very little desensitisation of the test animals. The controls showed a survival ratio (number surviving divided by number tested) which indicated that the infecting dose had been too small. The infecting organism used was a culture obtained indirectly from the New York State Laboratory, and it was less virulent than the strain previously employed. If we compare the test groups, the 0"1 rag. dose gave the poorest result, even indicating increased sensitisation; the 1"0 mg. gave greater desensitisation than the 0'1 mg. and the decreasing dose gave a slightly better result than the increasing dose. However, probably none of the figures is significant. Protocol I was repeated with certain changes; the infecting dose of tubercle bacilli was doubled, and only the varying doses of antigen were employed. It was hoped that the trial might decide between the relative value of the increasing and the decreasing doses of antigen. Results are given in Protocol II. PROTOCOL II.--DESEI~SITISATIOI~ OF TUBERCULOUS GUINEA-PIGS WITH ETHER-KILLED TUBERCLE BACILLI, H37. Unit
knimals . . . . average weight hiving H 178 kverage skin test ) e s e n s i t i s i n g dose Doses of a n t i g e n kverage s k i n test
.. .. .. .. .. ..
kverage w e i g h t .. ~ystemic test .. [ested . . . . ~urvived . . . . ~urvival ratio ..
:No. Gm. Mg. Deg. Mg. :No. Deg. Gin. Mg. :No. No.
Day
1 1 21 27 46 67 88 88 92
Tuberculous controls
Test animals
6 344 0'2
2.1 +
1'0 + 0'1 9 2'1+ 20+ 2'0 + 397 0"8 1 0 0'000
6 317 0'2 2"1 + 1 " 0 - 0"1 9 2"5 + 3"2 + 3"0 + 313 0"8 4: 1 0'250
6 310 0"2 2 3 -40 0 9'5 + 2'5 + 2"6 + 434 0"8 5 0 0"000
Eight of the animals were lost by intereurrent infections during the experiment, thus leaving a small number for the systemic test. The test showed practically no difference between the treated and the control animals. Between the increasing and decreasing doses, the advantage, though slight, was with the decreasing dose, the same as before. The two Protocols I and II led us to abandon the ether-killed bacilli as an antigen. Evidently the treatment of the bacilli with the ether denatured the protein sufficiently to render it practically valueless for desensitising purposes.
Desensitisation of Tuberculous Guinea-pigs by Means o] Avirulent Tubercle Bacilli, 520.--In previous trials this organism had given similar results to those obtained with ether-killed Mycobacterium tuberculosis, strain H 37; hence it was desirable to continue the trials in order to
June, 1933]
DESENSITISATION
OF
TUBERCULOUS
GUINEA-PIGS
401
determine its ultimate value. P r o t o c o l l I I was set up paralleling Protocol I in all respects except that the antigen was different. W e had previously determined that this organism was not virulent. By some accident a saprophytic organism was designated as a tubercle bacillus, human strain. The trial, therefore, merely tests the value of a non-specific antigen, presumably containing proteins common to the tubercle bacillus, for desensitising purposes, as compared with a specific antigen (Protocol I). The results are given in Protocol III. ]~)ROTOCOL I I I .
DESENSITISATION OF TUBERCULOUS GULNEA-PIGS WITH :ETHER-KILLED AVIRULENT TUBERCLE BACILLI, 5c20.
UnIB
Animals Average weight L i v i n g H 178 A v e r a g e s k i n tes~ Desensitising dose D o s e s of a n t i g e n Average skin test
.. .. .. .. .. ..
Average weight .. Systemic test .. Tested . . . . Survived Survival ratio ..
~O. Gm. Mg. Deg. Mg. No. Deg.
G;L Mg. No. No.
1 1 97
3t 55 84 9l 97
Ttl/}eretll()lls eontru|s
Test animals
Day
'
6 514 0'I
6 545 01
6 518 0'1
3'0 +
2"3 +
2"6 +
1'0 9 3"5 + 3'33 539 0"4 2 1 0 "500
0'1 9 ~t.=t + 3"4 + 580 0"4 5 0
1 ' 0 + 0.1 9 4"0 + '2"9 + 486 0'4 5 5
0-000
1'009
i
6 503 0"1 2"6 1'09 4"3 3'3 484 0'4: 3 3
6 633 0'1 + 0"1 + +
i'000
2.8 + 0 0 2'8 § 3'0 + 577 0'4 6 5 0'833
The systemic test showed, as in Protocol I, that the infecting dose was too small, since five of the six controls survived. That all the animals receiving the constant dose of 0"1 mg. of antigen weekly should be killed bv the systemic test, while all but one of the other treated animals survived, would seem to indicate that the larger doses had some desensitising powers ; that this is the case cannot be stated definitely because nearly all of the controls also survived the test. J)ROTOCOL IV.--DEsE-NSITISATION OF TUBERCULOUS GULNEA-PIG~ 1VITH ETHER.KILLED AVIRULENT TUBERCLE BACILLI, 520.
Unit
Animals .. Average weight L i v i n g H 178 Average skin test Desensitising dose D o s e s of a n t i g e n Average skin test
Average weight Systemic test Tested . . . . Survived . . . . gurvival ratio
26
.. .. .. .. .. .. ..
.. ..
..
No. Gin. Mg. Dog. Mg. No. l)eg.
GB1. Mg. No. No.
Day
1 1 21 27
41 67 9O 90 98
Tuberculous colltrols
Test animals
6 591 0"2 2'8 + 1.0 + 0 ' 1 9 3'3 + 2"5 + 3"0 + 825 08 2 1 0-500
6 739 0"2 30 + 1 ' 0 -- 0"1 9 3"5 + 9.9. + 2"8 + 800 0"8 5 1 0"200
6 567 0"2 2'3 + 0 0 1'6+ 2'5 + 1'8 + 659 0"8 6 4 0.666
402
TUBERCLE
[June, 1933
The test of culture 520 as a desensitising antigen was repeated in Protocol IV, but the details were made to parallel those of Protocol II. The results of this trial indicated no desensitising value for the nonspecific antigen, although they are not clear cut since two-thirds of the controls survived the test. The evidence appeared ample for the rejection of this antigen.
Desensitisation of Tuberculous Guinea-pigs by Mea~s of Ether-killed Mycobacterium smegmatis.--Previous trials had not definitely determined the value of Myco. smegmatis as a desensitising antigen. Some work by Sewell [3~ had indicated that this organism exerted an antagonistic effect upon Myco. tuberculosis. Protocol u was designed to parallel Protocol I. PROTOCOL V.---DESENSITISATION OF TUBERCULOUS GUINEA-PIGS WITH ETHER-KILLED SI~IEGMA BACILLI.
Unit
Animals
. . . . Average weight L i v i n g H 178 A v e r a g e skin test
.. .. .. Desensitising dose . . Doses of antigen . . Average skin test . . ~
~
~
No. Gm. Mg. Deg. Mg. :No. Deg.
9o
Average weight .. Systemic test .. Tested . . . . Survived . . . . Survival ratio ..
Gm. Mg. :No. :No.
1 1 21 23 49 75 91 95 95
Tuberculous
Test animals
Day
6 306 0"1 3"1 1"0 10 4"0 4"3 3"5 441 0"4 6
6 343 0'1
~ 6 -~-
-i-.
0'1 10 3"142'5 + `2.4 + 476 0.4 5 3 0"600
-;+ +
2 0"333
controls
6 '284 0q 3"8 + 1"0 - - 0-1 10 3.5 q.. 2"5 § 3"1 -I450 0q 6 4 0"666
6 346 0'1 3"0 + 1-0 -{-- 0"1 10 3"3 + `2'8 + '2-8 -b 503 0"4 6 4 0"666
6 325 0'1 2'8 + 0 0 3"8 -42 ' 5 -b 2"8 -+515 04 6 2 0 333
As in Protocols I and III the amount of infection was inadequate for a satisfactory test. The results again seemed to show considerable desensitisation of the treated animals, however, the lot receiving the 1"0 nag. dose weekly were apparently not desensitised. Obviously the results are inconclusive. Protocol V was repeated but in a form paralleling Protocol I I . PROTOCOL VI.--DESE~-SITISATION OF TUBERCULOUS GUINEA-PIGS WITH ETHER-KILLED SI~IEGMA BACILLI.
Unit
Animals .. Average weight
.. .. L i v i n g H 178 .. Average skin test . . Desensitising dose . . Doses of antigen . . Average skin test . .
Average weight
..
Systemic test .. Tested . . . . Survived .. .. Survival ratio ..
l~o. Gin. Mg. Deg. Mg. No. Deg.
Gin. Mg. No. No.
Day
1 1 '21 23 45 68 90 90 92
Ttlberculous controls
Test animals
6 344 0'2
3'0
4-
l'0--O'l 9 3'4 + 2 "6 + 2-8 + 385 0'8
6 331 0"2 3'0 + 1.0+0"1 9
4"0
+
5
2 ' 3 ~2"3+ 48~: 08 3
2 0 "400
0'666
6 305 0"2
2.4
+
0 0 4"'2 -~ 4"0 + 4"0 -q453 0"8 5 1 0"200
June, 1933~
DESENSITISATION
OF T U B E R C U L O U S
GUINEA-PIGS
403
The results seemed to indicate a slight degree of desensitisation. Since Myco. smegmatis is a non-pathogenic organism, and since ether apparently denatures proteins, rendering them unsatisfactory for desensitisation purposes, it seemed desirable to carry out a protocol using living bacilli as antigen. The new protocol paralleled Protocol V, but for the two varying doses there was substituted a single varying dose of antigen of 0"1 rag. increased by 0"1 mg. each week. This dose fell between the other two doses and might shed light upon the problem of dosage9 The results are given in Protocol VII. PROTOCOL V I I . - - D E S E S S I T I S A T I O N OF T U B E R C U L O U S G U I N E A - P I G S WITH L I V I N G SMEGBIA B A C I L L I .
Unit
Animals . . . . Average w e i g h t L i v i n g H 178 Average s k i n t e s t D e s e n s i t i s i n g dose Doses of a n t i g e n Average skin test
INTO.
.. .. .. .. .. ..
Gin. Mg. Deg. Mg. ~o, Deg.
Average weigh~ .. S y s t e m i c ~est .. Tested . . . . Survived . . . . Sur v ival ratio ..
G~I. Mg.
1 1
19 22 40 64 89 118 118
Tuberculous controls
T e s t animals
Day
4 415 02
4.0.+
1"0 14 3"5 + 3'0 + 3"6 + 517 0 "74
:NO.
2
Iq'o.
0 0'000
4 437 0"2 3"7+ 0'1 14 2'5 9 + 3"3+ 4"0 + 561 0'74 3 0 0"000
4 40~ 0"2 2'7 + 0'1 + 0-1 14 3'5 + 3'7 + 3'0 + 445 0"74 2 0 0"000
4 425 0"2
2'5 + 0 0 4'0 + 3"2 + 3"7 + 505 0'74 4 1 0"250
The results of this trial appear clear cut, and they indicate that a living, non-specific antigen failed to desensitise in any degree. Consequently the use of Myco. smegmatis as a desensitising antigen for tuberculosis was abandoned. Desensitisation of Tuberculous Guinea-pigs by Means of Mycobacterium tuberculosis, H 178, Killed by Salt.- A study was made of the killing PROTOCOL V I I I . - - D E S E N S I T I S A T I O N
OF T U B E R C U L O U S G U I N E A - P I G S WITH S A L T - K I L L E D
TUBERCLE BACILLI, I[178.
Unit
A n i m a l s .. Average weig ht L i v i n g H 178 Average sk in test D e s e n s i t i s i n g dose Doses of a n t i g e n Average skin tes~ )J
))
NO.
o.
Gin,
9. 9 ..
Mg. Deg. Mg. .No.
))
avdago w;igh~" Systemic test T6sted .. Survived .. Sur vival ratio
..
.. .. o, oo
Day
i
;7
Gin. Mg. No. INTO.
1 i 21 25 44 66 87 117 1'20
Tuberculous controls
Test animals
6 327 0"2 3"3 + 1"0 13 3"0 + 30+ 3'2 + 445 0'74 4 1
0"250
6 328 0"2 3'S + 0"1 13 3'3 43'5 + 3"3 + 396 0'74 4 1 0"250
6 297 () '2 3O + 0"1 + 0'1 13 3'5 + 3"1 + 2"5 + 335 0"74 2 0 O'CO0
6 3"20 0"2
'26+ 0 0 3'8+. 3'1+ 3"5 + 431 0'74 4 1
0 "'250
404
TUBnRCLE
[June, 1933
power of sodium chloride upon the tubercle bacillus, and it was found that a solution containing 35 grm. NaC1 per 100 c.c. of water killed in six to ten days, 25 grin. per 100 c.c. killed in six to twelve days, 15 grin. per 100 c.c. killed in ten to twenty days, and 10 grin. per 100 c.c. killed in ten to twenty-five days. It was decided to test the desensitising power of bacilli killed by a salt solution of 35 grin. per 100 c.c. water. Protocol V I I I was planned to parallel Protocol VII. In this trial the results failed to indicate any desensitisation. Apparently the salt denatures the protein of the bacilli sufficiently to render them useless as desensitising agents.
Desensitisation of Tuberculous Guinea-pigs by Means o/ Seibert's Synthetic Medium Tuberculin.--In our earlier trials with OT desensitisation was unsatisfactory; small doses failed to desensitise and large doses killed the animals. The new tuberculin of Seibert [-4], SI~IT, is an entirely different product which might give different results. Our preliminary trials with tuberculo-proteins were promising. It was decided to set up a protocol paralleling Protocol I, using a desensitising dose of SMT smaller than the lethal dose for guinea-pigs. The results are given in Protocol IX. PROTOCOL IX.--DESENSITISATION OF TUBERCULOUS GUINEA-PIGS WITH ~EIBERT'S S M T .
Unit
nimals . . . . verage weight lying bacilli H 178 veragc skin test . . ~esensitising dose . . ~oses
.
verage skin test
..
"
~
verage w i g h t ~stemic test ested . . . . urvived . . . . urvival ratio
.
.. ..
..
NO. Gm. Mg. Deg. Mg. No. Deg. ~Ig. lqo. No.
!
Day
1 ] 21 50
74 111 117 117
Tuberculous controls
Test animMs
6 '/90 0"1 3"6 + 0"2 10 2"5 + 17+ 466 0.4: 5 5 1 '000
6 309 0"i
3'6 + 0"02 10 9'5 + 3'0 + 434 0"4 6 6 1 "000
6 9~96 0"1
3"0 +
0"2- 0"02 10 2"0 + 2"2 + 508 0"4 6 r 1 "000
6 3g0 0'i 3'8+ 02 + 0.02 10 9"0 + ]'2 + 465 0"4 4 1 "00'~
6 336 01
2.6 + 0 o 3"0 + 3.2+ 481 0"4 5 2 0-,t00
As in the other paralleling protocols the infecting dose of bacilli was too small so that all of the controls were not killed by the lethal dose of SMT. However, all the treated animals survived the systemic test. Not 0nly did they survive but they showed no inconvenience or unfavourable symptoms, while all of the controls were sick and three of them died. The results indicated complete desensitisation of the treated animals to the test dose. The results of Protocol IX were so favourable that it was repeated, but with double the infecting dose of bacilli. The results are given in Protocol X. Again complete desensitisation was obtained in the treated animals. Unfortunately all but one of the controls also recovered, so that the figures do not give a satisfactory impression of the trial. The animals used in this protocol were about twice the size of those previously employed ; this fact may account for the recovery of the controls when given two lethal
June, 1933]
DESENSITISATION
OF
TUBERCULOUS
405
GUINEA-PIGS
doses of S]V[T. The treated animals showed no distress or other un-favour~ble symptoms during the systemic test, while the controls were decidedly ill. PROTOCOL X - - D E S E N S I T I S A T I O N OF TUBERCULOUS GUINEA-PIGS WITH SEIBERT'S S ~ I T .
Unit
Animals Average w e i g h t L i v i n g bacilli H 178 Average sk in t e s t .. D e s e n s i t i s i n g dose .. Doses.. Average s k i n ' / e s t :2 Average weig ht Systemic test Tested .... Survived Sur vival ratio"
.. .. .. ..
No, Gm. Mg. Deg. Ms. No. Deg.
Gm. Mg. No. No.
1 I 24 .29 48 71 91 9.2 92
Tuberculous controls
Test animals
Day
6 583 0'2 .2"3 + 0' 2 9 1"6 + 2'0 + 2"2 + 626 0"8 6 6 1 "000
6 603 0'`2 .2"0 + 0 '0`2 9 1"6 + 2'2 + `2-0 + 713 0'8 6 6 1 "000
6 618 0"2 2"3+ 0 ' 2 - 0'02 9
6 668 0"2
.2.0+
1-o +
0"2+0"02 9 1-0 +
1"8 + 2'0 + 67O 0'8 5 5 1 "000
1"6 + 708 0'8 5 5 1 000
1"3 +
3 595 0'2 2" 0+ 0 0 '2"5 + 2"4 + 26+ 689 0"8 5 4 0'800
A third protocol was carried out with SMT, but paralleling Protocol I I as closely as possible. The increasing dose was started with only 0"02 mg. of SMT, but it was increased each week by 0"02 mg., thus making the final dose 0'26 mg. The results are given in Protocol XI. PROTOCOL XI.--DESENSITISATION OF TUBERCULOUS GUINEA-PIGS WITH SEIBERT'S SI~{T.
Unit
~nimals ~-verage weight Living H 178 ~_verage skin test Desensitising dose Dose of a n t i g e n Average skin t e s t ~verage w e i g h t ~ystemic test rested .... 3urvived .. Survival ratio
.. .. .. .. .. .. .. ..
~h~O.
Grn. Mg. Deq. Mg. No. Deg.
dm Mg. 1'{o.
.. ..
No.
1 1 19 22 7l 95 1'20 15l 151
Tuberculous controls
Test animals
Day
6 3~8 0"2 2"6 + 0'.2 14
2"1 +
2'8 + 1"0 + 373 0"74 3 3 1"000
6 328 0"2 3"3 + 0"02 14 2"5 + 2"8 + 1'0 + 374 0"74 3 3 1 000
6 33l 0'2 3'9 + 0"02 + 0"02 14 2'3 + 1"8 + 1"2 + 446 0"74 3 3 1"000
6 352 0'2 2"8 + 0 0 3"6 +
3"5 +
3"4 + 419 0"74 5 1 0 "200
This protocol was marred by intercurrent infection, but the results showed complete desensitisation in all the groups of treated animals, while all but one of the controls were killed. It should be noted that the Mantoux skin test also showed a marked degree of desensitisation, although it was never reduced to zero. DISCUSSION. The present report covers trials with three antigens previously employed and showing some promise for desensi~isation purposes, and also trials with
406
~rUBERCLE
[June, 1933
four new antigens. Tubercle bacilli and organisms of the genus Myco. have been killed in many ways, both by physical and by chemical means, in our attempts to prepare a satisfactory ant~igen for desensitisation purposes. Thus far all these attempts have failed. W h e n non-specific living bacilli were employed as antigens they also failed to desensitise sufficiently to save the animals when the systemic test was given. We have found, however, that tuberculo-protein of the type of SMT as prepared by Seibert is capable of desensitising tuberculous guinea-pigs against several fatal doses of S~vIT. Since the same protein was employed in both the treatment and the test, it may be contended that the animals were immune to the test substance. Doubtless such immunity was developed, but this does not invalidate the fact that the tuberculous animals were freed from the tuberculin reaction exhibited by the controls. In freeing tuberculous animals from the tuberculin reaction we have accomplished an important step toward relieving them from the killing effect of tuberculo-protein. This leaves the animal free to combat the growth of the tubercle bacillus. W h e t h e r we can capitali.~e on this success as a means of treatment for h u m a n tuberculosis cases remains to be determined. SUMMARY AND CONCLUSIONS.
(1) The paper records eleven protocols carried out in attempts to desensitise tuberculous guinea-pigs by means of various antigens. (2) The antigens tried were : (a) ether-killed Myco. tuberculosis, H 37 ; (b) ether-killed saprophytic Mycobacterium 520; (c) ether-killed Myco. smegmatis ; (d) living Myco. sr ; (e) salt-killed Myco. tuberculosis, 178 ; and (f) Seibert's Synthetic Medium Tuberculin. (3) Bacillary antigens killed by chemical agents proved unsatisfactory ~s desensitising antigens when employed on tuberculous guinea-pigs. (4) Non-specific bacillary antigens, either killed or living, proved unsatisfactory as desensitising antigens. (5) Only one of the antigens tested SMT gave complete desensitisation when two to four lethal doses of SMT were administered after ten weeks of treatment. (6) Tuberculous guinea-pigs were desensitised sufficiently to protect them against two to four fatal doses of SMT. ACKNOWLEDGMENT.
The authors desire gratefully to acknowledge the assistance of 5{rs. Lourene Olson, Miss Alice Borden, Miss Genevieve Gamble, and Mr. Edwin Anderson in carrying out the protocols. REFERENCES. [1] WEINZIRL, JOHN, and BOHAR%RUBY,M. Amer. Bey. Tub., 1931, 23, 393. [2] WEINZIRL,JOHN, and THA~R, J. DURWARD. Tubercle, 1931, i2, 488. [31 SEWELL, HENRY, and D~ SAVITSCH, EUGENE. Trans. Nat. Tub. Assoc., 199,8, 24, 234. [4] SEIBERT, FLORENCEB. Jozg7"n.Biol. Chem., 1928, 78, 345.
TUBERCLE
[June, 1933
AMEI~ICAN SECTION. Under the Editorship of Dr. A. K. Krause.
FURTHER RESULTS OF ATTEMPTS TO DESENSITISE TUBERCULOUS GUINEA-PIGS. By JO~N WEINZIRr,, Ph.D., J. I)URWARD THAYER, M.S., and JOY HIRSCHMANN, B.S,, Alice McDermott Foundation, University of Washiq~gton, Seattle, Washington. INTRODUCTION.
Two reports [1, 21, have been made previously presenting results of attempts to desensitise tuberculous guinea-pigs; these reports covered trials made with bacilli killed by physical and chemical means, and with tuberculin and tuberculoproteins. It was found that the method used to kill the bacilli influenced the value of the antigen for purposes of desensitisation; for example, bacilli killed by iodine or by light were practically useless as desensitising antigens, while those killed with carbolfuchsin or with ether gave better results. Old tuberculin proved highly unsatisfactory, but tuberculo-proteins appeared more promising. Some degree of desensitisation was always obtained, but in all the trials made we were never able to secure 100 per cent. protection against a fatal dose of either old tuberculin (OT) or Seibert's synthetic medium tuberculin (SMT). Attempts at desensitisation were continued, however, in order to determine more fully the value of our most promising antigens. There can be no doubt any longer that sensitisation plays an important r61e in tuberculosis, for its importance has been demonstrated many times. If tuberculin, or tubercle bacilli, are injected into normal animals they produce no immediate effect ; on the other hand, if either are injected into tuberculous animals, serious symptoms soon develop and may lead to the death of the animals. In our experiments deaths have occurred many times from such injections. The failure of Koch's " lymph " in 1891 was due to the fact that the patients were sensitised to tuberculo-protein and consequently a dose of tuberculin caused fever, malaise and other undesirable conditions which actually hastened the death of the patients. If it were possible to overcome the sensitisation, the body might become immunised, and, as a result, might conquer the tubercle bacilli. From the results obtained by desensitising against serum sickness and hay fever, it would seem that desensitisation in tuberculosis should be possible if suitable antigens are found. In the present work we are presenting some protocols repeating those of earlier trials, but with certain modifications, in order to determine the true value of these antigens. We are also reporting on some new antigens, one of which has given complete protection against lethal doses of tuberculin. ~IETHODS EMPLOYED.
In our previous work the systemic test proved sufficiently satisfactory and we have continued to employ it. According to this test, one or more
June, 1933]
DESENSITISATION
OF T U B E R C U L O U S
399
GUINEA-PIGS
fatal doses of OT or SMT are administered at the close of the desensitisation period. If the tuberculous animals have been sufficiently desensitised they will withstand the fatal dose without exhibiting shock. We have continued to make Mantoux skin tests, mainly to show that the animals were sensitive, and incidentally to confirm the systemic tests as far as possible. In these experiments we have continued to use different sized doses of antigen in order to discover which gave the greatest desensitisation. We have administered the antigen at weekly intervals, but work now in progress seems to indicate that a shorter interval gives better results. The antigens have been given by the intraperitoneal route mainly as a matter of convenience. Tuberculous controls have been included in each protocol. ~:~E SULTS
OBTAINED.
In our last report [2] on desensitisation, we found three antigens which gave similar results, and which presented the greatest possibilities of twenty or more antigens previously tested. The three antigens were : (1) ether-killed H 37; (2) ether-killed 520; and (3) ether-killed Mycobacterium sraegmatis. We decided to repeat these trials in order to eliminate the less promising antigens. We also added some trials with new antigens. Desensitisatiou of tuberculous guinea-19igs by means of ether-killed tubercle bacilli, H 3 7 . - - I n the first protocol, 30 guinea-pigs of 200 grm. weight or over were infected with I-I 178, a strain of low virulence, then divided into five lots of six animals each. One lot was kept as tuberculous controls, while the others received varying doses of the antigen killed by ether and treated with chloroform. Two lots were given constant doses of 1"0 mg. and 0"1 rag. weekly, and the other two varying doses starting with 1"0 nag., one lot decreasing by 0'1 rag. weekly, and the other increasing by 0"1 mg. weekly. In this and all subsequent protocols, both PROTOCOL I.--DESENSITISATION OF TUBERCULOUS GUINEA-PIGS WITH ETHER. KILLED TUBERCLE BACILLI~ H 37.
Unit
Animals Average w e i g h t L i v i n g bacilli H 178" Average sk in t e s t .. D e s e n s i t i s i n g dose .. Doses of a n t i g e n .. Average s k i n test .. Average w e i g h t .. Systemic test .. Tested .. Survived ,. .. S u r v i v a l ratio Second ~y~temie t e s t Tested .... Survived .. .. Survival r a t i o ..
Day
NO.
Gm. Mg. Deg. Mg. No. Deg.
G'm. Mg. No. No. Mg. No. No.
1 1 21 28 42 63 91 96
103
Tuberculou: eonLrols
Test animals
6 312 0"l 2.5+ 1"0 10 2"3 + 2"4 + 412 0"4 5 4 0"800 0.8 4 0"500
6 358 0"1 2"5 + 0"1 10
2"5 + 9-8 +
531 0"4 5 2 0"400 0'8 2 0 0'000
6 320 0"1 2"3 + 1"0--0"1 10 2"6 + 2"6 + 545 0'4, 3 3 1"000 0'8 3 fi 0'666
6 324 0'1
6 352 01
IO 2'2+ 2'0 + 423 0% 3 2 0'666 0'8 2 1 0"500
0 0 2"6+ 2'3 + 573 0"4 6 5 0"888 0.8 5 2 0"400
2.8 + 1"o+o'1
2-5 +
400
[June,
TUBERCLE
1933
the infecting dose and the antigen were given by the intraperitoneal route primarily as a matter of convenience. The results for the individual guinea-pigs have been combined for each lot and are given in Protocol I. The results of this trial showed very little desensitisation of the test animals. The controls showed a survival ratio (number surviving divided by number tested) which indicated that the infecting dose had been too small. The infecting organism used was a culture obtained indirectly from the New York State Laboratory, and it was less virulent than the strain previously employed. If we compare the test groups, the 0"1 rag. dose gave the poorest result, even indicating increased sensitisation; the 1"0 mg. gave greater desensitisation than the 0'1 mg. and the decreasing dose gave a slightly better result than the increasing dose. However, probably none of the figures is significant. Protocol I was repeated with certain changes; the infecting dose of tubercle bacilli was doubled, and only the varying doses of antigen were employed. It was hoped that the trial might decide between the relative value of the increasing and the decreasing doses of antigen. Results are given in Protocol II. PROTOCOL II.--DESEI~SITISATIOI~ OF TUBERCULOUS GUINEA-PIGS WITH ETHER-KILLED TUBERCLE BACILLI, H37. Unit
knimals . . . . average weight hiving H 178 kverage skin test ) e s e n s i t i s i n g dose Doses of a n t i g e n kverage s k i n test
.. .. .. .. .. ..
kverage w e i g h t .. ~ystemic test .. [ested . . . . ~urvived . . . . ~urvival ratio ..
:No. Gm. Mg. Deg. Mg. :No. Deg. Gin. Mg. :No. No.
Day
1 1 21 27 46 67 88 88 92
Tuberculous controls
Test animals
6 344 0'2
2.1 +
1'0 + 0'1 9 2'1+ 20+ 2'0 + 397 0"8 1 0 0'000
6 317 0'2 2"1 + 1 " 0 - 0"1 9 2"5 + 3"2 + 3"0 + 313 0"8 4: 1 0'250
6 310 0"2 2 3 -40 0 9'5 + 2'5 + 2"6 + 434 0"8 5 0 0"000
Eight of the animals were lost by intereurrent infections during the experiment, thus leaving a small number for the systemic test. The test showed practically no difference between the treated and the control animals. Between the increasing and decreasing doses, the advantage, though slight, was with the decreasing dose, the same as before. The two Protocols I and II led us to abandon the ether-killed bacilli as an antigen. Evidently the treatment of the bacilli with the ether denatured the protein sufficiently to render it practically valueless for desensitising purposes.
Desensitisation of Tuberculous Guinea-pigs by Means o] Avirulent Tubercle Bacilli, 520.--In previous trials this organism had given similar results to those obtained with ether-killed Mycobacterium tuberculosis, strain H 37; hence it was desirable to continue the trials in order to
June, 1933]
DESENSITISATION
OF
TUBERCULOUS
GUINEA-PIGS
401
determine its ultimate value. P r o t o c o l l I I was set up paralleling Protocol I in all respects except that the antigen was different. W e had previously determined that this organism was not virulent. By some accident a saprophytic organism was designated as a tubercle bacillus, human strain. The trial, therefore, merely tests the value of a non-specific antigen, presumably containing proteins common to the tubercle bacillus, for desensitising purposes, as compared with a specific antigen (Protocol I). The results are given in Protocol III. ]~)ROTOCOL I I I .
DESENSITISATION OF TUBERCULOUS GULNEA-PIGS WITH :ETHER-KILLED AVIRULENT TUBERCLE BACILLI, 5c20.
UnIB
Animals Average weight L i v i n g H 178 A v e r a g e s k i n tes~ Desensitising dose D o s e s of a n t i g e n Average skin test
.. .. .. .. .. ..
Average weight .. Systemic test .. Tested . . . . Survived Survival ratio ..
~O. Gm. Mg. Deg. Mg. No. Deg.
G;L Mg. No. No.
1 1 97
3t 55 84 9l 97
Ttl/}eretll()lls eontru|s
Test animals
Day
'
6 514 0'I
6 545 01
6 518 0'1
3'0 +
2"3 +
2"6 +
1'0 9 3"5 + 3'33 539 0"4 2 1 0 "500
0'1 9 ~t.=t + 3"4 + 580 0"4 5 0
1 ' 0 + 0.1 9 4"0 + '2"9 + 486 0'4 5 5
0-000
1'009
i
6 503 0"1 2"6 1'09 4"3 3'3 484 0'4: 3 3
6 633 0'1 + 0"1 + +
i'000
2.8 + 0 0 2'8 § 3'0 + 577 0'4 6 5 0'833
The systemic test showed, as in Protocol I, that the infecting dose was too small, since five of the six controls survived. That all the animals receiving the constant dose of 0"1 mg. of antigen weekly should be killed bv the systemic test, while all but one of the other treated animals survived, would seem to indicate that the larger doses had some desensitising powers ; that this is the case cannot be stated definitely because nearly all of the controls also survived the test. J)ROTOCOL IV.--DEsE-NSITISATION OF TUBERCULOUS GULNEA-PIG~ 1VITH ETHER.KILLED AVIRULENT TUBERCLE BACILLI, 520.
Unit
Animals .. Average weight L i v i n g H 178 Average skin test Desensitising dose D o s e s of a n t i g e n Average skin test
Average weight Systemic test Tested . . . . Survived . . . . gurvival ratio
26
.. .. .. .. .. .. ..
.. ..
..
No. Gin. Mg. Dog. Mg. No. l)eg.
GB1. Mg. No. No.
Day
1 1 21 27
41 67 9O 90 98
Tuberculous colltrols
Test animals
6 591 0"2 2'8 + 1.0 + 0 ' 1 9 3'3 + 2"5 + 3"0 + 825 08 2 1 0-500
6 739 0"2 30 + 1 ' 0 -- 0"1 9 3"5 + 9.9. + 2"8 + 800 0"8 5 1 0"200
6 567 0"2 2'3 + 0 0 1'6+ 2'5 + 1'8 + 659 0"8 6 4 0.666
402
TUBERCLE
[June, 1933
The test of culture 520 as a desensitising antigen was repeated in Protocol IV, but the details were made to parallel those of Protocol II. The results of this trial indicated no desensitising value for the nonspecific antigen, although they are not clear cut since two-thirds of the controls survived the test. The evidence appeared ample for the rejection of this antigen.
Desensitisation of Tuberculous Guinea-pigs by Mea~s of Ether-killed Mycobacterium smegmatis.--Previous trials had not definitely determined the value of Myco. smegmatis as a desensitising antigen. Some work by Sewell [3~ had indicated that this organism exerted an antagonistic effect upon Myco. tuberculosis. Protocol u was designed to parallel Protocol I. PROTOCOL V.---DESENSITISATION OF TUBERCULOUS GUINEA-PIGS WITH ETHER-KILLED SI~IEGMA BACILLI.
Unit
Animals
. . . . Average weight L i v i n g H 178 A v e r a g e skin test
.. .. .. Desensitising dose . . Doses of antigen . . Average skin test . . ~
~
~
No. Gm. Mg. Deg. Mg. :No. Deg.
9o
Average weight .. Systemic test .. Tested . . . . Survived . . . . Survival ratio ..
Gm. Mg. :No. :No.
1 1 21 23 49 75 91 95 95
Tuberculous
Test animals
Day
6 306 0"1 3"1 1"0 10 4"0 4"3 3"5 441 0"4 6
6 343 0'1
~ 6 -~-
-i-.
0'1 10 3"142'5 + `2.4 + 476 0.4 5 3 0"600
-;+ +
2 0"333
controls
6 '284 0q 3"8 + 1"0 - - 0-1 10 3.5 q.. 2"5 § 3"1 -I450 0q 6 4 0"666
6 346 0'1 3"0 + 1-0 -{-- 0"1 10 3"3 + `2'8 + '2-8 -b 503 0"4 6 4 0"666
6 325 0'1 2'8 + 0 0 3"8 -42 ' 5 -b 2"8 -+515 04 6 2 0 333
As in Protocols I and III the amount of infection was inadequate for a satisfactory test. The results again seemed to show considerable desensitisation of the treated animals, however, the lot receiving the 1"0 nag. dose weekly were apparently not desensitised. Obviously the results are inconclusive. Protocol V was repeated but in a form paralleling Protocol I I . PROTOCOL VI.--DESE~-SITISATION OF TUBERCULOUS GUINEA-PIGS WITH ETHER-KILLED SI~IEGMA BACILLI.
Unit
Animals .. Average weight
.. .. L i v i n g H 178 .. Average skin test . . Desensitising dose . . Doses of antigen . . Average skin test . .
Average weight
..
Systemic test .. Tested . . . . Survived .. .. Survival ratio ..
l~o. Gin. Mg. Deg. Mg. No. Deg.
Gin. Mg. No. No.
Day
1 1 '21 23 45 68 90 90 92
Ttlberculous controls
Test animals
6 344 0'2
3'0
4-
l'0--O'l 9 3'4 + 2 "6 + 2-8 + 385 0'8
6 331 0"2 3'0 + 1.0+0"1 9
4"0
+
5
2 ' 3 ~2"3+ 48~: 08 3
2 0 "400
0'666
6 305 0"2
2.4
+
0 0 4"'2 -~ 4"0 + 4"0 -q453 0"8 5 1 0"200
June, 1933~
DESENSITISATION
OF T U B E R C U L O U S
GUINEA-PIGS
403
The results seemed to indicate a slight degree of desensitisation. Since Myco. smegmatis is a non-pathogenic organism, and since ether apparently denatures proteins, rendering them unsatisfactory for desensitisation purposes, it seemed desirable to carry out a protocol using living bacilli as antigen. The new protocol paralleled Protocol V, but for the two varying doses there was substituted a single varying dose of antigen of 0"1 rag. increased by 0"1 mg. each week. This dose fell between the other two doses and might shed light upon the problem of dosage9 The results are given in Protocol VII. PROTOCOL V I I . - - D E S E S S I T I S A T I O N OF T U B E R C U L O U S G U I N E A - P I G S WITH L I V I N G SMEGBIA B A C I L L I .
Unit
Animals . . . . Average w e i g h t L i v i n g H 178 Average s k i n t e s t D e s e n s i t i s i n g dose Doses of a n t i g e n Average skin test
INTO.
.. .. .. .. .. ..
Gin. Mg. Deg. Mg. ~o, Deg.
Average weigh~ .. S y s t e m i c ~est .. Tested . . . . Survived . . . . Sur v ival ratio ..
G~I. Mg.
1 1
19 22 40 64 89 118 118
Tuberculous controls
T e s t animals
Day
4 415 02
4.0.+
1"0 14 3"5 + 3'0 + 3"6 + 517 0 "74
:NO.
2
Iq'o.
0 0'000
4 437 0"2 3"7+ 0'1 14 2'5 9 + 3"3+ 4"0 + 561 0'74 3 0 0"000
4 40~ 0"2 2'7 + 0'1 + 0-1 14 3'5 + 3'7 + 3'0 + 445 0"74 2 0 0"000
4 425 0"2
2'5 + 0 0 4'0 + 3"2 + 3"7 + 505 0'74 4 1 0"250
The results of this trial appear clear cut, and they indicate that a living, non-specific antigen failed to desensitise in any degree. Consequently the use of Myco. smegmatis as a desensitising antigen for tuberculosis was abandoned. Desensitisation of Tuberculous Guinea-pigs by Means of Mycobacterium tuberculosis, H 178, Killed by Salt.- A study was made of the killing PROTOCOL V I I I . - - D E S E N S I T I S A T I O N
OF T U B E R C U L O U S G U I N E A - P I G S WITH S A L T - K I L L E D
TUBERCLE BACILLI, I[178.
Unit
A n i m a l s .. Average weig ht L i v i n g H 178 Average sk in test D e s e n s i t i s i n g dose Doses of a n t i g e n Average skin tes~ )J
))
NO.
o.
Gin,
9. 9 ..
Mg. Deg. Mg. .No.
))
avdago w;igh~" Systemic test T6sted .. Survived .. Sur vival ratio
..
.. .. o, oo
Day
i
;7
Gin. Mg. No. INTO.
1 i 21 25 44 66 87 117 1'20
Tuberculous controls
Test animals
6 327 0"2 3"3 + 1"0 13 3"0 + 30+ 3'2 + 445 0'74 4 1
0"250
6 328 0"2 3'S + 0"1 13 3'3 43'5 + 3"3 + 396 0'74 4 1 0"250
6 297 () '2 3O + 0"1 + 0'1 13 3'5 + 3"1 + 2"5 + 335 0"74 2 0 O'CO0
6 3"20 0"2
'26+ 0 0 3'8+. 3'1+ 3"5 + 431 0'74 4 1
0 "'250
404
TUBnRCLE
[June, 1933
power of sodium chloride upon the tubercle bacillus, and it was found that a solution containing 35 grm. NaC1 per 100 c.c. of water killed in six to ten days, 25 grin. per 100 c.c. killed in six to twelve days, 15 grin. per 100 c.c. killed in ten to twenty days, and 10 grin. per 100 c.c. killed in ten to twenty-five days. It was decided to test the desensitising power of bacilli killed by a salt solution of 35 grin. per 100 c.c. water. Protocol V I I I was planned to parallel Protocol VII. In this trial the results failed to indicate any desensitisation. Apparently the salt denatures the protein of the bacilli sufficiently to render them useless as desensitising agents.
Desensitisation of Tuberculous Guinea-pigs by Means o/ Seibert's Synthetic Medium Tuberculin.--In our earlier trials with OT desensitisation was unsatisfactory; small doses failed to desensitise and large doses killed the animals. The new tuberculin of Seibert [-4], SI~IT, is an entirely different product which might give different results. Our preliminary trials with tuberculo-proteins were promising. It was decided to set up a protocol paralleling Protocol I, using a desensitising dose of SMT smaller than the lethal dose for guinea-pigs. The results are given in Protocol IX. PROTOCOL IX.--DESENSITISATION OF TUBERCULOUS GUINEA-PIGS WITH ~EIBERT'S S M T .
Unit
nimals . . . . verage weight lying bacilli H 178 veragc skin test . . ~esensitising dose . . ~oses
.
verage skin test
..
"
~
verage w i g h t ~stemic test ested . . . . urvived . . . . urvival ratio
.
.. ..
..
NO. Gm. Mg. Deg. Mg. No. Deg. ~Ig. lqo. No.
!
Day
1 ] 21 50
74 111 117 117
Tuberculous controls
Test animMs
6 '/90 0"1 3"6 + 0"2 10 2"5 + 17+ 466 0.4: 5 5 1 '000
6 309 0"i
3'6 + 0"02 10 9'5 + 3'0 + 434 0"4 6 6 1 "000
6 9~96 0"1
3"0 +
0"2- 0"02 10 2"0 + 2"2 + 508 0"4 6 r 1 "000
6 3g0 0'i 3'8+ 02 + 0.02 10 9"0 + ]'2 + 465 0"4 4 1 "00'~
6 336 01
2.6 + 0 o 3"0 + 3.2+ 481 0"4 5 2 0-,t00
As in the other paralleling protocols the infecting dose of bacilli was too small so that all of the controls were not killed by the lethal dose of SMT. However, all the treated animals survived the systemic test. Not 0nly did they survive but they showed no inconvenience or unfavourable symptoms, while all of the controls were sick and three of them died. The results indicated complete desensitisation of the treated animals to the test dose. The results of Protocol IX were so favourable that it was repeated, but with double the infecting dose of bacilli. The results are given in Protocol X. Again complete desensitisation was obtained in the treated animals. Unfortunately all but one of the controls also recovered, so that the figures do not give a satisfactory impression of the trial. The animals used in this protocol were about twice the size of those previously employed ; this fact may account for the recovery of the controls when given two lethal
June, 1933]
DESENSITISATION
OF
TUBERCULOUS
405
GUINEA-PIGS
doses of S]V[T. The treated animals showed no distress or other un-favour~ble symptoms during the systemic test, while the controls were decidedly ill. PROTOCOL X - - D E S E N S I T I S A T I O N OF TUBERCULOUS GUINEA-PIGS WITH SEIBERT'S S ~ I T .
Unit
Animals Average w e i g h t L i v i n g bacilli H 178 Average sk in t e s t .. D e s e n s i t i s i n g dose .. Doses.. Average s k i n ' / e s t :2 Average weig ht Systemic test Tested .... Survived Sur vival ratio"
.. .. .. ..
No, Gm. Mg. Deg. Ms. No. Deg.
Gm. Mg. No. No.
1 I 24 .29 48 71 91 9.2 92
Tuberculous controls
Test animals
Day
6 583 0'2 .2"3 + 0' 2 9 1"6 + 2'0 + 2"2 + 626 0"8 6 6 1 "000
6 603 0'`2 .2"0 + 0 '0`2 9 1"6 + 2'2 + `2-0 + 713 0'8 6 6 1 "000
6 618 0"2 2"3+ 0 ' 2 - 0'02 9
6 668 0"2
.2.0+
1-o +
0"2+0"02 9 1-0 +
1"8 + 2'0 + 67O 0'8 5 5 1 "000
1"6 + 708 0'8 5 5 1 000
1"3 +
3 595 0'2 2" 0+ 0 0 '2"5 + 2"4 + 26+ 689 0"8 5 4 0'800
A third protocol was carried out with SMT, but paralleling Protocol I I as closely as possible. The increasing dose was started with only 0"02 mg. of SMT, but it was increased each week by 0"02 mg., thus making the final dose 0'26 mg. The results are given in Protocol XI. PROTOCOL XI.--DESENSITISATION OF TUBERCULOUS GUINEA-PIGS WITH SEIBERT'S SI~{T.
Unit
~nimals ~-verage weight Living H 178 ~_verage skin test Desensitising dose Dose of a n t i g e n Average skin t e s t ~verage w e i g h t ~ystemic test rested .... 3urvived .. Survival ratio
.. .. .. .. .. .. .. ..
~h~O.
Grn. Mg. Deq. Mg. No. Deg.
dm Mg. 1'{o.
.. ..
No.
1 1 19 22 7l 95 1'20 15l 151
Tuberculous controls
Test animals
Day
6 3~8 0"2 2"6 + 0'.2 14
2"1 +
2'8 + 1"0 + 373 0"74 3 3 1"000
6 328 0"2 3"3 + 0"02 14 2"5 + 2"8 + 1'0 + 374 0"74 3 3 1 000
6 33l 0'2 3'9 + 0"02 + 0"02 14 2'3 + 1"8 + 1"2 + 446 0"74 3 3 1"000
6 352 0'2 2"8 + 0 0 3"6 +
3"5 +
3"4 + 419 0"74 5 1 0 "200
This protocol was marred by intercurrent infection, but the results showed complete desensitisation in all the groups of treated animals, while all but one of the controls were killed. It should be noted that the Mantoux skin test also showed a marked degree of desensitisation, although it was never reduced to zero. DISCUSSION. The present report covers trials with three antigens previously employed and showing some promise for desensi~isation purposes, and also trials with
406
~rUBERCLE
[June, 1933
four new antigens. Tubercle bacilli and organisms of the genus Myco. have been killed in many ways, both by physical and by chemical means, in our attempts to prepare a satisfactory ant~igen for desensitisation purposes. Thus far all these attempts have failed. W h e n non-specific living bacilli were employed as antigens they also failed to desensitise sufficiently to save the animals when the systemic test was given. We have found, however, that tuberculo-protein of the type of SMT as prepared by Seibert is capable of desensitising tuberculous guinea-pigs against several fatal doses of S~vIT. Since the same protein was employed in both the treatment and the test, it may be contended that the animals were immune to the test substance. Doubtless such immunity was developed, but this does not invalidate the fact that the tuberculous animals were freed from the tuberculin reaction exhibited by the controls. In freeing tuberculous animals from the tuberculin reaction we have accomplished an important step toward relieving them from the killing effect of tuberculo-protein. This leaves the animal free to combat the growth of the tubercle bacillus. W h e t h e r we can capitali.~e on this success as a means of treatment for h u m a n tuberculosis cases remains to be determined. SUMMARY AND CONCLUSIONS.
(1) The paper records eleven protocols carried out in attempts to desensitise tuberculous guinea-pigs by means of various antigens. (2) The antigens tried were : (a) ether-killed Myco. tuberculosis, H 37 ; (b) ether-killed saprophytic Mycobacterium 520; (c) ether-killed Myco. smegmatis ; (d) living Myco. sr ; (e) salt-killed Myco. tuberculosis, 178 ; and (f) Seibert's Synthetic Medium Tuberculin. (3) Bacillary antigens killed by chemical agents proved unsatisfactory ~s desensitising antigens when employed on tuberculous guinea-pigs. (4) Non-specific bacillary antigens, either killed or living, proved unsatisfactory as desensitising antigens. (5) Only one of the antigens tested SMT gave complete desensitisation when two to four lethal doses of SMT were administered after ten weeks of treatment. (6) Tuberculous guinea-pigs were desensitised sufficiently to protect them against two to four fatal doses of SMT. ACKNOWLEDGMENT.
The authors desire gratefully to acknowledge the assistance of 5{rs. Lourene Olson, Miss Alice Borden, Miss Genevieve Gamble, and Mr. Edwin Anderson in carrying out the protocols. REFERENCES. [1] WEINZIRL, JOHN, and BOHAR%RUBY,M. Amer. Bey. Tub., 1931, 23, 393. [2] WEINZIRL,JOHN, and THA~R, J. DURWARD. Tubercle, 1931, i2, 488. [31 SEWELL, HENRY, and D~ SAVITSCH, EUGENE. Trans. Nat. Tub. Assoc., 199,8, 24, 234. [4] SEIBERT, FLORENCEB. Jozg7"n.Biol. Chem., 1928, 78, 345.