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Antigenic studies by the dale test
The Journal. bf Allergy VOL. 11
JANUARY, 1940
No. 2
original Articles ANTIQENC
STUDIES
BY THE DALE
TEST0
11. THE ANTIQENICITY OF House DUST STANLKY F. HAMPTON, M.D., APD A~~wc NEW YORK, N. Y.
STULL, PH.D.
T
HE purpose of this investigation’ was to study the antigenicity of house dust extract with particular reference to two factors: (1) the part played by common household inhalants other than dust and (2) the “unknown factor” as an antigen, first pointed out by Cooke? when he introduced house dust as a causative allergen in bronchial asthma. The study was carried out by the Dale test. In order to prevent confusion iu the text of this presentatiou, the term “inhalant” is used to denote air-borne allergens other than house dust. Earnsdell aud Walzef were unable to sensitize guinea pigs to house dust extract. Coulson and Stevens,’ however, recently reported the production of gross anaphylaxia in five guinea pigs sensitized by alumprecipitated house dust extract. Five of t.he seven normal control animals showed symptoms simulating anaphylaxis, which the authors called “anaphylactoid” and attributed to a “nonspecitlc toxic factor.” YETHODS
Four extracts were prepared from dusts of different sources. A-dust was a sample collected from 40 different sour(apartments, houses, and ofices). B-dust was a sample collected from a single patient’s house, in which no animals were allowed because of the patient’s sensitivity. C-dust was collected from a physician’s oflee, in which no animals were admitted, but in which there were a few feather pillows. D-dust was a sample of dust from several different house sources (approximately one-fifth the number of A-dust). The dusts were treated and extracted as follows: Equal quantities of B-, C-, and D-dusts were washed with carbon tetrachloride, and after its removal by filtration and evaporation, extracting fluid (0.5 per cent sodium chloride and 0.3 per cent sodium bicarbonate) was -*Prom the Department ot Allergy. The Roosevelt Preeented In part before the medIng of the Society AUW Condltlonr. April 19. ION. Atlantic City. N. J. 109
Ho~pltal. for the
New
Study
York. ol Aathmn
and
to ewlr irk 500 c’.c. auwuuts to give n thick Jrn~tl conoisteucy. Ext.ract.ioa was cwrierl out. uuder tolueue for 24 hours at To C. The c~xtrwts were sepirated froilr tlw solid niatericil iu 21 JJleat press aid dialyzed apaiust rwniu g water uacler toliietbe for 24 1i011rs. Firetenths per ceent sotliuul c4~loricle aml 0.3 per cent. sodium bicarbonate were added, ~JMI the extra& wwe passed through Seitz lilters airtl pl*oucd sterile by the iisud c~ulturd methods. A-dust VW treated u~rcl extracted in the swue wag. except that a hwger quaut.ity of dust WHS used alid phe110l to O.-l per wit wrwi~tratiou ws utltlecl. So lhcriol was usctl in B-, (‘-. HIP II-dusts. All extrwts were staudurtlixed” 011 H lwoteiw uitrogeu basis” for IISC iu the Dale tests (Table I). ;I portion of euch of thcl four extrwts OHS lnwipitntetl hy the mltlitiou of H~IJU (Al& (S04)~.2411JO) to 0.3 per wut roweutrcdiou, awl the precipitates were wutrifuqalizetl. WHSIIC~ with 0.~5 per cent stwile saline, &led
nutl suspe~~clatl iu O.H.5 lw
sterile saliw. A II these procedures ioiis. Tlw suspeikms were analyzed for protein uitrapeu by l~lloy,llotuiI~sti~ a&l pre+tcltiw (‘TabIt: I) iwl were theu iisetl for swsitixntiw of puiw4 pips.
were done with sterile.
.-~-~--.
_-
---
-y_
--A
AN.uSW ;. ..-..--;
SERIES
POST EXTRA(T
i
.\
A-dust
-
IS
H-dU8L ( !4lllrt
L
-.... -_ 1hluRt . -.
wit
]WWi~llt
T.lR1.E I OF DUST c=-:
IUTE Ob’ I’ElWAI:.\TIOP
I'/
.I/:{),
r/1
z/39
I
- .. --.-.4/1”/?9 --__
n
IWITEIS UC.
I=
SITI:WEN I’Kl; (‘A’. em c. c
-I-
ILlSi
:‘I-
:E’T.:: AJ . KU-PRtXWJT.\TED . Sl’SPENSION-PROTEIK :SIT’):WiEX MC. PER CC.
I
0 "5
0.11
ii0
Il.115
d/JL!/W . . 1
EXTRACIS
cz:.--’
I -.--
0.20
0.0-l . ..-
0.15
-
Viryirl I’eniiilc~ guiliw pigs (250 to 3Oi) pIi. ill wiylit 1 WV11 wwiwcl II single illtl’il-Nh(l(~tllitls.1 illjwtiorl of WC of the puritie~l iIlutlt-precipitated dust. vxtrwts. 4\~~ muouut roiitiiiiiiii y 50.000 protc*iri uitropeu I)iilc tests ww ~wrfoi~iirecl. folJlJlitS WilS iii,jcctctl into cvrc4raiiiiual. hwiug t.he twhnique tleswihrtl iu the rrport of aatiplir st.uclit!s uf polleu.” 111 the cxpt~ritwiits with IL. t ‘-. nutI I)-dust vstrwts the two uteriiie 1~0r11swew tcstetl siilliiltHiieoiisl?. The wtioii of iiilwltiiits II~OII the cliisl-selihitiw utwiw ~t~iisrles ~IIS stiiclietl ii1 several puine4I pigs iii w~11 seriw by t.estiug the uterine harus with the v~~lt~t~~oii iiihaltiiit eSttw4s iI1 the order illlCl streii~th
listed iu the prot0ds.t The iuhaliiut extracts usetl for testing wew lhe routiue extrwto prepared ad stautlartlizecl on 8 protein uitrogell wit..‘. i III additioJl. hurnau clr~~clcr was cdlectetl fmn hair cowbiugs nucl U-W sitnilarly extracted and stmtlartlizetl. It cmtaiiwtl 13,000 iids per whit veiitimetw. --w *I unit = 0.00001 mg. wwtein nitrown. tlhte to lack of space for publlcntlnn. toml ?. Swier B. are given in this nrtirlc.
only
Protncnl
1. Swier
.\.
and
part
nf
IV-o-
HAMPTON
AND
STUIJ,:
ANTIGENIC
STUDIES
BY
DALE
111
TEST
In most instances half the group of inhalants were tested upon one horn and half upon the ot.her, in varying strengths as indicated in Protocol 1, Series A, and the part of Protocol 2, Series B. When a reaction was obtained with an inhalant., however, the ot.her uterine horn was usually tested with that particular inhalant as well.
f
nap.*
r.,o.o
,-a,*
#0.“,r...r, SL
::
cgrr.
““.‘a t W&I ,qou.J
Cer*hrrs
‘$4”.
Fig.
In the tested in from pigs ments the
1.
remaining guinea pigs, B-, C-, and D-dust extracts were equal strengths for their capacity t.o cross-neutralize uteri experisensitized to these dusts. Iu the cross..neutralization muscle was finally test.ed with a total of 30,000 units of the
sensitizing extra&, after it had beeu show to be neutralized to 10,000 uuits of a diflereut dust extract. A-dust was cousidered to cotlt.aiu all antipew co111rw1~ to house tlust since it was prepared Proiu dust. f~*oiu so inaiiy tliffereilt sources. The guinea pigs ii1 series il (sel&tiziiq atltigeil ? A-tlust.) wew testctl first, aiid siiwe the). failed to show sensitivity to wttouseed, p~~rethruia. tobacco, orris, silk, wool, 11o1*sc tlaluler, , tlf cmuriu, ragweed, kapok, and cottoii linters, these inhalant est.racts wore tested upon only a few of the uterine innscles ~II series IS, C? a11t1 I). ‘I’hose iiiliala~~ts, irainel,~-. dog and cat. dander and feathers,* which did produce posit ivc rcavtion in series 1\? however. were t.estetl in series l% (sensitizing antigen, IL dust )) series (1 (sensitiziii g antigens C-dust.), and series D (sensitizing antigen, D-dust.), exc+ept v-hell the observations were limited to cross relat.ionship of the dust extrac+ts. Furthermore, in series l.3, C, and I), huinan dander rstrwt8 ant1 seven molt1 ext.racts (illfornutitr., Moriiliu ctllkws, Nuc~or. Asl~etyill~6s, Ilo~~~~codc~~dr~~~~~, I’c~~icilliz~~,~,and DC~rmtitotr) were tested. (ilne and jute extracts were testetl in a few experiments. 3.IS-J% Dear- Date lPq-ar;‘-cnr
Series
A.
r)tvisif
izbag
AJi~gcn
, il-D/d.
(Protocol
1 md [I’ubla II.)-
All of the ten guinea pigs in this group were fouud sensitive by the Dale test. Six of the eight uteri tested with feather extract gave positive reactions. Three of the ten uteri test.ed with dog dander extract gave posit.ire react.ions ; four of the ten uteri tested with cat dander gave positive reactions. These findings are illustratetl in the reproductions of the Dale curves (Figs. 1, 2. atnl :i).
HAMPTON
AND SCULL:
ANTIGENIC
STUDIES BY DALE TEST
113
In seven of nine experiments in which reactions were obtained to one or more inhalants, the muscle reacted to dust extract after becoming neutral to the reacting inhalants (Figs. 1 and 2). The muscle failed to react in two experiments, suggesting that the inhalants
Fig. 4.
neutralized the uterine antibodirn to dust (k’ip. 4). III one instance, t.hc horns of the uterus failtd to react to all inhc3ant.s t.cst.ed, but one reacted to the test with the sensitizing dust (guinea pig 7). In the teats of guinea pigs 8 MINI 12, a fifth dnst (No. 2163’3) obtained from a single source was used. This dust extract produced a
HAMPTON
AND
BTULJ.
:
PWTOCOL QUINEA NO.
PI4
DATE
1
TESTRE
1
3/ l/39
STUDJEB
TEWlNO ANTIGEY
Low I&lgwec Rltrrllaricr
Silk Fcuthem Horse dander Dog dnndet J)ust nv. 2liX Aduai Cnt dander Histamine Silk Kapok Orris Dog dmder Dog dander A.dnet A-dust
A-dust A-dust
a/ 3/30 (See
zg.
1.
BY
DALE
SERIES A-CONT’U
HORN
3/ 2/30
9
1.
UTERlNE
2
8
ANTIOkXIC
SJTR( OEN UWlTS
'ROTEIN
10,000 10.000 2;wl 10,000 10,onn 10,000 1 cd?. 10,000 10,000 2,500 10.000 1o;oclo 10,000 lfi,OM IO,00 10,000 IQ,000
i n,onn
Feuthers Feat hers Dog dander Cat dander Cat dander A-dust A-dust Iiiatamioe - Cvttooseell .~IIfP%Wifl Horse dantlrr Dog tlnnder Dug clantlrr Dug tlnnder l+‘entherc Cat daotlet Pyrethrum Tobncw Otris A.dust A-aust Hibtumine
1 n,nno
Wool Featkers Feathers Silk Cat dander Dog dander Dug dander Kapok A-dust A-dust Hwtamine
10,000 10,000 1i),OllO 10,000 10,000 10,nnn 10,000 10,000 10,000 10,000
1 n,ooo 10,000 10,000 1:qo0 10,000
in,000 10,000
in,ooo 1n,noo I~),000 10,000
w,ooo 10,000 10,ono
10,000 10,000 10,000 10,000 10,000
‘l’Ji2j’J’
115
THE JOURNAL
116
PROTOCOL GUINEA PIG NO. 11
UTERI.VE HORS 1
DATE TESTED 3/ C/3!1
(See Fig. 4.
OF AI,LERGP
1. SERIES A-CONT'D TESTING ANTIGEN
\YRDTElN SITKO , OEN ukars
Cottonseed \Vool Kapok Horse tlandcv Feathers Fe&hers
lU,ooU 10.000 10;000 10,000 10,000 10,000 10,000 10,000 10,000 1 n,ooo 1n,ooo 10,000 10,000 10,000 10,000 10,000
.i1tsrnn#ic1
Tohncco Pyrethrum Flaxreetl Silk Cat &i.nder Dog danilei Ragweed A-dust Adust IIist.amine Cat dander Dog dnndel Cottonseed .4Itenmrin Tobacw Orris Pyrethrum Silk Ragweed E’rathers A~dust .\-dust .4ltmlclrin Hist.amine Cottonseed
I 1
3/ o/39
ROOI
.4lteraaria Horse tlantlel Feathers Feathers Cat dander Cat dander Dog tlan~ler Silk Kapok Tohrcco Orris Pyrethrum Ragweed Dust No. 21WI Dust No. 2163: X-dust Histamine 2
I (
A-dust A-tlust Feathers Dog dander Cat dawler Dust No. 21(i3! Histamine
I
10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 liJ,OOO 10,000 10,000 10,000 li),OOU 10,000 10.000 10;000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 1 c.(‘. 1 r.c. l(l,OOO 10,000 10,000 10,000 10,000 10,000 1 C.C.
= _- ,
HAMPTON
AND
STULL:
PROTOCOL GUINEA Pit NO.
13 (See Fig. 2
14
(See Eg. 3.
UTERINE HORN
1
DATE TESTED
3/15/39
3/17/39
3/M/39
ANTIGENIC
STUDIES
1. .%RIES
A--CONT
TESTING ANTIGEN
BY
DALE
'D GEINKITR( QEN UNITS
Cottoneeed Dog dander Feathers Feathers Cat dander A-dust A-dust Histamine
10,000 10.000 10;000 10,000 10,990 10,000 10,000
Cott.onseed Dog dander Feathers Feathers Cat dander A-dust A-dust Histamine Cotton linters Kapok Dog dander Dog dander Cat dander Feathers A-dust A-dust Histamine
10,000 10,000 10,000 10,990 10,000 10,000 10,000
Cat dander Feathers Dog dander Dog dander A-dust A-dust - 1Histamine Cotton linters Pyrethrum Cat dander Cat dander Cat dander Dog dander Cottonseed Feathers Feathers A-dust A-dust Histamine
10,990 10,990 10,990 10,000 10,ouo 10,990
Dog dander Dog dander Cat dander Cat dander Feathers A-dust A-dust Histamine
117
TEST
3,000 10,990 10,000 10,000 10,000 10,000 10,000 10,000
10,000 10,000 10,000 10;000 10,990 10,990 10,000 10,000 10,990 10,000 10,000 10,000 10,000 10,000 10,990 10,990 10,990 10,990
I!TERINE CONTRACTION 0 0 ttt 0 0 t tt .I. 0 t+t I. 0 0 t I) 0 ttt 0 ttt 0 0 t 0 z tttt 0 tttt 0 0 0 0 + t t t 0 t-l-t+ 0 0 tttt tt 0 0 0 tit 0 ;: t+tt 1: tttt 0 0 0
0 +++t
=5_... I
Orrip Silk.
%j,UO
:1n11 1 O.OOI)
SO.
OF VTEt:l ‘IXSTEI)
--_--_
IO JO h s i ci i 7 .i 4 -I 4 s .,
Cnits
1.
-io.
-~ ‘-.-P-P
OP POSIrlVE REhC’l’IONS
I
1 Jo
Fig.
---_
--
:: -I (i 0 0 0 0 0 0 (I 0 0 0 0 0 i -__-..
3.
(Pvolocol 2 und Tahlc Six of the SCV~I~ guinea pigs ill this series were found sensitive Dale test. Oile of tlw four uteri tested wit.li featlwr extract positive reaction (Fig. 5). lhg and cat. dander extracts failed tliicc! wt~tiwtioiis. All of the four uteri t,ested with hnan retract gwe positive reactions (Fig. 6). Scrics
L’. Ncmifiziwg
.l,ltigcn,,
h’-D&t.
III.)by the gave a to prodander
HAMPTON
-4ND
ANTIQENIC
STULL:
STUDIES
DY
Dhl.E
TFST
110
In three of t.he four experiments in which reactions were obtained to use or more inbalauts, the muscle reacted to a dust extract after it was neutral to the reacting iuhalailts, although the dust rciic4ious wrre stnall a.ud obtaiued upou only one Iioru iu two iustames (Pigs. 5 iintl 6). (J-dust .aml D-dust extracts produc+etl a contr’actiou ant1 neutralizetl the uterine muscle to H-dust exQract (seusitisinp antigen) in two crossneutralization esperiments (Figs. 7 am1 8).
,NO+NQ t Xirrn
,a,..0
-rPp-s*Ar.r
I.,...
“.ol
,.,eoo
Fig.
Hr..‘.
C,rr,.rw9.
rgeee
6.
of the Series C. Sensitizing Antigen, C-D~tst. (Table IV.)-Five seven guinea pigs in this series were found sensitive by the Dale test. One of the three uteri tested with dog dander extract gave a poskive
++ -T+=======+
.-._
_-.
+ 2.
-
+ f-L
. 7’
1 8
, -f
I
=5=-i
+ +
t-.,===.t
4 -I
HAMPTON
AND STULL:
ANTIGENIC
STUDIES BY DALE TEST
121
T~BLF, III GUINEA
Pros TESTING 10,000
INJECTED
SUMMARY OF REACTIONS OF SERIES B WITH 50,000 UNITS ALUM-PRSCIPITATED
ANTIGEN UNITS
NO. OF UTERI TESTED
Feathers Cat dander Dog dander Human dander Cottonseed Kapok Orris Horse dander Pyrethrum Tobacco Silk Wool BItemaria MmiZia M-lbcm
NO. OF POSITIVE REACTIONS
f !J 4
2 2
2 2 2 2 2 2 2 2 2 2 2
dbicans
Aspergillzss Eormodendn4m
Penicillium Denuatium Ragweed Cotton linters Glue Jute* B-dust C-dust D-dust *Jute contained too small a quantity
B-DUST EXTRACT
i 2 2”
1 ; 2 of protein
for
analyses.
Testing
strength
1 cc.
B-dust produced a contraction and neutralized the uterine muscle to C-dust (sensitizing antigen) in three experiments (Fig. 11).
122
THE
JOURNAL
OF
ALLERGT
fkrics Il. Sc7rsitiziny .hfige?l, D-Dust. (l’uble IT).---All seven pigs in this series were found sensitive by the Dale test. Three of the four uteri tested with feather extract gave positive reactiotls (Fig. 12). So reactions were obtained with cat and dog tlalldcr extracts. Three of t.he four uteri testetl with human tlan(lel gave positive wavtioiis (Fig. 13). .J-- 1 J-- 39. &Sq-. ode lQfp--cqo.0 urih oh- P’*=‘p-+*” O&w&P P’) IhI0.l cT(Ow.* o,aI- *‘?r;e;;r-~ J=Y
Fig.
9.
In two of the threv experiments in which reilctions were obtained with inhalants, the muwle reacted to dust extract after it was neutral to the respective inhalants (Figs. 12 and 13). One uterine muscle rcnctd to the scllsitizing dust but to none of tlw inhalants tctstcd.
HAMPTO.\’
.2x1)
STt-I . I Ad: f
ANTIGENIC
SUNNAKY
--
Pros
INJECIWI
TESTING (10,000
WIT~I
OF REACIWK~ 50,000
ANTIGEN KNITS)
UNITS SO.
BY
DA1.E:
OF
SEIUES
C:
AWN-PHECIPIT.\TED
OF I:TEKI TESTED
Feathers Cat dander Dog dander Human dander Cottonseed Orris Horse dander Pyrethrum Tobacco Silk Wool
C-DUST NO.
0 0
1
0
:
0 0 0 0 0 0 8 0 0 0
1 2 1
i 0 0 0 0 3 3 1
Ragweed Cotton linters Glue JUte” C-dust B-dust D-dust containe~l
EXTKACT
OF POSITIVE REACl-IONS
4 3 3 4
1 0
dlternaricc illonilia albicans Nwor Aspergillus IIormodendrum Pentillipvm Dcmatium
*Jute
123
TL.T
IV
TABLE
GUINEA
STUDIES
too
small
a quantity
of protein
Fig.
for
analyses.
Testing
strength
1 C.C.
10.
C-dust extract prQduced a contraction and neutralized the uterine muscle to D-dust extract (sensitizing antigen) in two experiments (Fig. 14). B-Dust extract produced a contraction and neutralized to D-dust extract in one experiment (Fig. 15).
124
THE
JOURNAL
OF
ALI,ERGT
DISCUSSION
Humau dander, although we of the antigens for guinea pigs in house dust, was not the only autigeuic factor in dust extract other than the common inhalants, since, in four of the fire experiments in
Fig.
12.
which human dander was the only inhalant eausiug muscular contraction, dust extract produced a contraction after the muscle was shown to be neutralized to human dander. There was no evidence of a histamine-like substance in the human dander extract or the dust extracts.
HAMPTON
AND
STULL:
ANTIGENIC
STUDIES
BY
DALE
TEST
125
In order to determine that human dander was antigenic for guinea suspension of human dander Pigs, 50,000 units of alum-precipitated were injected into each of four guinea pigs. The uteri of two guinea pigs were reactive to the dander extract. B-dust produced a positive reaction in the only instance in which it was tested on a uterine muscle, sensitive to human dander, but it did not completely neutralize to this dander extract in 10,000 unit strength (Fig. 16).
Fig.
14.
In the experiments in which a different dust extract produced a reaction prior to testing with the sensitizing dust extract, the uterine
‘l’lllt
JOI’RNAI,
Fig.
012
.II,I,ER(;T
15.
muscle was c+ompletcly ncwtralizerl ill all of twelre hstalices to the sensitizing dust. #Six of this proup were st,rili.cht c~~oss-,lerltralization c*xperiments.
HAMPTON
AND
STULL:
ANTIGENIC
STUDIES
BY
DALE
TEST
127
In the experiments in which the sensitizing dust extract produced a reaction prior to testing with another dust. extract., t.he uterine muscle was completely neutralized in twelve of thirteen instances to another dust. Six were straight cross-neut.ralization experiments. In one experiment (guinea pig 23) in which a uterine horn was sensitized by D-dust .extract, C-dust. extract produced a small contraction after the muscle was shown to be neutralized to D-dust.
Fig.
16.
In carrying out Dale tests in the past, using antigens other than dust, it has been an unusual occurrence for one uterine horn to be sensitive and the ot.her insensitive. It must be pointed out, however, that in this study wit.h house dust, the two uterine horns frequently failed to respond alike. For examples, in series A, horn No. 1 of guinea pig 15 (Fig. 3) reacted to feathers while horn No. 2 did not, and horn No. 2 of guinea pig 7 reacted to dust extract while horn No. 1 failed to react? although in each instance the horns gave the normal response to histamine. In fact, in series A, in six of the ten experiments, the two ut.erine horns of the respective guinea pigs failed to react similarly. This was the case in series B, C, and D (guinea pig 33) in a small number of inst.ances. However, the respective horns were not previously tested with exactly the same inhalant antigens. It is interesting to note that the two horns reacted similarly in the cross-neutralization experiments in which only dust extracts were used. This suggested that the dust extracts might contain minute quantities of various inhalant antigens, and, although the inhalants themselves did not protluce a muscular contraction, they may have neutralized the smooth
128
THE
JOURNAL
OF
ALLEROY
muscle antibodies. Consequently, the failure of dust extract to produce a contraction may have been due t.o the accumulative neutralizing activity of the several inhalants. This might suggest that the only antigens in dust. extract were the inhalants which were tested. That this was not true, however, was shown by the many experiments itr which dust extract produced positive Dale tests after t.he uterine muscle was shown to be neutralized to the reacting inha1ant.s. On the other hand, it is possible that inhalant ext.racts cont.ain traces of house dust, and it may thus be the accumulative effect. of the dust factor in the various inhalants t.hat. brings about neutralization without producing contractions. The presence of 0.4 per cent phenol in the inhalant extracts was another possible cause of inhibiting reactions after multiple tests. This was very unlikely in view of the many instances in which the muscles were tested with many inhalants and subsequently reacted to dust extract as well as to histamine. One might assume that multiple sensitization occurred with such a composite antigen as house dust, but why, in some instances, one horn was found sensitized to one antigen, while the ot.her horn wa.s apparently sensitized to a different one, is not explained in the present study. The “nonspecific t.oxic factor” producing anaphylactoicl symptoms in gross anaphylaxis, described by Coulson and Stevens, was not observed in these experimends in which the Dale test was used. However, their extracts were not said to be dialyzed, wherea.s the ext.racts used in this study mere dialyzed. Precipitin tests were performed with the dust extract used for sensitization in sera from guinea pigs 25 (D-dust), 2i (D-dust)? 22 (Cdust), 26 (C-dust), 21 (B-dust)? and 28 (B-dust), and the sera failed to show precipibins. SUMMARY
Twenty-eight of 31 guinea pigs , given a single intraperitoneal injection of purified alum-precipitated house dust extract from four different sources, were found sensitive by the Dale t.est. Positive Dale tests were obta.ined with feabher extracat in 10 of %I experiments, with dog dander in 4 of 20 experiments, and with cat dander in 4 of 20 experiments. Cottonseed, cotton linters, kapok, raw silk, orris, glue, l)yrethrum, wool, tobacco, ragweed, jute, horse dander, and the molds, Alterw~k. Aspergillus, Yztcor, Non,ilia. albicans,, Denmtiwn~., Penicillium, and Hornzodendrunc, failed to produce positive Dale tests in a total of 147 tests on dust-sensitized guinea pigs. Uterine muscles, sensitized by dust. extract, gave positive Dale reactions to dust extracts after the muscles were nantralised to certain rea.ct.ing inhalants in 13 of 18 instances.
HAMPTON
AND STULL:
ANTIGENIC
STUDIES BY DALE TEST
129
With the use of 3 dust extracts, 2 prepared with dusts from individual sources (B-dust and C-dust) and one from several sources (D-dust), neutralization to the sensitizing dust extract was effected by one or by the other two dusts in all of the 12 experiments. The sensit.izing dust extract neutralized to one of the other dust extracts in 12 of 13 experiments. Human dander extract gave a positive Dale test in 8 of 12 uteri sensitized by dust extract, but completely neutralized to dust extract in only one instance. Alum-precipitated human dander extract sensitized 2 of 4 guinea pigs injected, and one of t.hese two was sensitive to dust extract. House dust extract contained an antigen other than that found in the known inhalants tested, and this antigen was common to three different dust extracts as determined by the Dale test.
1. Hampton, S. F., Stull, A., and Cooke, R. A.: Antigenie Studies by the Dale Test (Abstract), J. ALLERC+Y 10: 465, 1939. 2. Cooke, R. A.: Studies in Hypersensitiveness : IV. New Etiologic Factors in Bronchial Asthma, J. Immunol. 7: 147, 1922. 3. Ramsdell, 8. G., and Walzer, hf.: On Antigens; Antigenie Properties of Glue, Wheat and House Dust, J. Immunol. 14: 207, 1927. Sensitization of Guinea Pigs to Cotton Liters 4. Coulson, E. J., and Stevens, H.: and House Dust Extracts, Proc. Sot, Exper. Biol. & Med. 40: 457, 1939. 5. Cooke, R. A., and Stull, A.: The Preparation and Standardization of Pollen Extract for the Treatment of Hay Fever, J. ALLERQY 4: 87, 1933. 6. Hampton, 5. F., Stull, A., and Cooke, R. A.: Antigenie Studies by the Dale Test : I. The Antigenic Relationship of Certain Pollens, J. ALLERGY 10: 417, 1939. The Protein Nitrogen Unit for 7. Stull, A., Cooke, R. A., and Glidden, M.: Standardization of Inhalant Extracts for Diagnosis and Treatment of Allergic Diseases, J. ALLEK~Y 8: 454, 1937.
JANUARY, 1940
No. 2
original Articles ANTIQENC
STUDIES
BY THE DALE
TEST0
11. THE ANTIQENICITY OF House DUST STANLKY F. HAMPTON, M.D., APD A~~wc NEW YORK, N. Y.
STULL, PH.D.
T
HE purpose of this investigation’ was to study the antigenicity of house dust extract with particular reference to two factors: (1) the part played by common household inhalants other than dust and (2) the “unknown factor” as an antigen, first pointed out by Cooke? when he introduced house dust as a causative allergen in bronchial asthma. The study was carried out by the Dale test. In order to prevent confusion iu the text of this presentatiou, the term “inhalant” is used to denote air-borne allergens other than house dust. Earnsdell aud Walzef were unable to sensitize guinea pigs to house dust extract. Coulson and Stevens,’ however, recently reported the production of gross anaphylaxia in five guinea pigs sensitized by alumprecipitated house dust extract. Five of t.he seven normal control animals showed symptoms simulating anaphylaxis, which the authors called “anaphylactoid” and attributed to a “nonspecitlc toxic factor.” YETHODS
Four extracts were prepared from dusts of different sources. A-dust was a sample collected from 40 different sour(apartments, houses, and ofices). B-dust was a sample collected from a single patient’s house, in which no animals were allowed because of the patient’s sensitivity. C-dust was collected from a physician’s oflee, in which no animals were admitted, but in which there were a few feather pillows. D-dust was a sample of dust from several different house sources (approximately one-fifth the number of A-dust). The dusts were treated and extracted as follows: Equal quantities of B-, C-, and D-dusts were washed with carbon tetrachloride, and after its removal by filtration and evaporation, extracting fluid (0.5 per cent sodium chloride and 0.3 per cent sodium bicarbonate) was -*Prom the Department ot Allergy. The Roosevelt Preeented In part before the medIng of the Society AUW Condltlonr. April 19. ION. Atlantic City. N. J. 109
Ho~pltal. for the
New
Study
York. ol Aathmn
and
to ewlr irk 500 c’.c. auwuuts to give n thick Jrn~tl conoisteucy. Ext.ract.ioa was cwrierl out. uuder tolueue for 24 hours at To C. The c~xtrwts were sepirated froilr tlw solid niatericil iu 21 JJleat press aid dialyzed apaiust rwniu g water uacler toliietbe for 24 1i011rs. Firetenths per ceent sotliuul c4~loricle aml 0.3 per cent. sodium bicarbonate were added, ~JMI the extra& wwe passed through Seitz lilters airtl pl*oucd sterile by the iisud c~ulturd methods. A-dust VW treated u~rcl extracted in the swue wag. except that a hwger quaut.ity of dust WHS used alid phe110l to O.-l per wit wrwi~tratiou ws utltlecl. So lhcriol was usctl in B-, (‘-. HIP II-dusts. All extrwts were staudurtlixed” 011 H lwoteiw uitrogeu basis” for IISC iu the Dale tests (Table I). ;I portion of euch of thcl four extrwts OHS lnwipitntetl hy the mltlitiou of H~IJU (Al& (S04)~.2411JO) to 0.3 per wut roweutrcdiou, awl the precipitates were wutrifuqalizetl. WHSIIC~ with 0.~5 per cent stwile saline, &led
nutl suspe~~clatl iu O.H.5 lw
sterile saliw. A II these procedures ioiis. Tlw suspeikms were analyzed for protein uitrapeu by l~lloy,llotuiI~sti~ a&l pre+tcltiw (‘TabIt: I) iwl were theu iisetl for swsitixntiw of puiw4 pips.
were done with sterile.
.-~-~--.
_-
---
-y_
--A
AN.uSW ;. ..-..--;
SERIES
POST EXTRA(T
i
.\
A-dust
-
IS
H-dU8L ( !4lllrt
L
-.... -_ 1hluRt . -.
wit
]WWi~llt
T.lR1.E I OF DUST c=-:
IUTE Ob’ I’ElWAI:.\TIOP
I'/
.I/:{),
r/1
z/39
I
- .. --.-.4/1”/?9 --__
n
IWITEIS UC.
I=
SITI:WEN I’Kl; (‘A’. em c. c
-I-
ILlSi
:‘I-
:E’T.:: AJ . KU-PRtXWJT.\TED . Sl’SPENSION-PROTEIK :SIT’):WiEX MC. PER CC.
I
0 "5
0.11
ii0
Il.115
d/JL!/W . . 1
EXTRACIS
cz:.--’
I -.--
0.20
0.0-l . ..-
0.15
-
Viryirl I’eniiilc~ guiliw pigs (250 to 3Oi) pIi. ill wiylit 1 WV11 wwiwcl II single illtl’il-Nh(l(~tllitls.1 illjwtiorl of WC of the puritie~l iIlutlt-precipitated dust. vxtrwts. 4\~~ muouut roiitiiiiiiii y 50.000 protc*iri uitropeu I)iilc tests ww ~wrfoi~iirecl. folJlJlitS WilS iii,jcctctl into cvrc4raiiiiual. hwiug t.he twhnique tleswihrtl iu the rrport of aatiplir st.uclit!s uf polleu.” 111 the cxpt~ritwiits with IL. t ‘-. nutI I)-dust vstrwts the two uteriiie 1~0r11swew tcstetl siilliiltHiieoiisl?. The wtioii of iiilwltiiits II~OII the cliisl-selihitiw utwiw ~t~iisrles ~IIS stiiclietl ii1 several puine4I pigs iii w~11 seriw by t.estiug the uterine harus with the v~~lt~t~~oii iiihaltiiit eSttw4s iI1 the order illlCl streii~th
listed iu the prot0ds.t The iuhaliiut extracts usetl for testing wew lhe routiue extrwto prepared ad stautlartlizecl on 8 protein uitrogell wit..‘. i III additioJl. hurnau clr~~clcr was cdlectetl fmn hair cowbiugs nucl U-W sitnilarly extracted and stmtlartlizetl. It cmtaiiwtl 13,000 iids per whit veiitimetw. --w *I unit = 0.00001 mg. wwtein nitrown. tlhte to lack of space for publlcntlnn. toml ?. Swier B. are given in this nrtirlc.
only
Protncnl
1. Swier
.\.
and
part
nf
IV-o-
HAMPTON
AND
STUIJ,:
ANTIGENIC
STUDIES
BY
DALE
111
TEST
In most instances half the group of inhalants were tested upon one horn and half upon the ot.her, in varying strengths as indicated in Protocol 1, Series A, and the part of Protocol 2, Series B. When a reaction was obtained with an inhalant., however, the ot.her uterine horn was usually tested with that particular inhalant as well.
f
nap.*
r.,o.o
,-a,*
#0.“,r...r, SL
::
cgrr.
““.‘a t W&I ,qou.J
Cer*hrrs
‘$4”.
Fig.
In the tested in from pigs ments the
1.
remaining guinea pigs, B-, C-, and D-dust extracts were equal strengths for their capacity t.o cross-neutralize uteri experisensitized to these dusts. Iu the cross..neutralization muscle was finally test.ed with a total of 30,000 units of the
sensitizing extra&, after it had beeu show to be neutralized to 10,000 uuits of a diflereut dust extract. A-dust was cousidered to cotlt.aiu all antipew co111rw1~ to house tlust since it was prepared Proiu dust. f~*oiu so inaiiy tliffereilt sources. The guinea pigs ii1 series il (sel&tiziiq atltigeil ? A-tlust.) wew testctl first, aiid siiwe the). failed to show sensitivity to wttouseed, p~~rethruia. tobacco, orris, silk, wool, 11o1*sc tlaluler, , tlf cmuriu, ragweed, kapok, and cottoii linters, these inhalant est.racts wore tested upon only a few of the uterine innscles ~II series IS, C? a11t1 I). ‘I’hose iiiliala~~ts, irainel,~-. dog and cat. dander and feathers,* which did produce posit ivc rcavtion in series 1\? however. were t.estetl in series l% (sensitizing antigen, IL dust )) series (1 (sensitiziii g antigens C-dust.), and series D (sensitizing antigen, D-dust.), exc+ept v-hell the observations were limited to cross relat.ionship of the dust extrac+ts. Furthermore, in series l.3, C, and I), huinan dander rstrwt8 ant1 seven molt1 ext.racts (illfornutitr., Moriiliu ctllkws, Nuc~or. Asl~etyill~6s, Ilo~~~~codc~~dr~~~~~, I’c~~icilliz~~,~,and DC~rmtitotr) were tested. (ilne and jute extracts were testetl in a few experiments. 3.IS-J% Dear- Date lPq-ar;‘-cnr
Series
A.
r)tvisif
izbag
AJi~gcn
, il-D/d.
(Protocol
1 md [I’ubla II.)-
All of the ten guinea pigs in this group were fouud sensitive by the Dale test. Six of the eight uteri tested with feather extract gave positive reactions. Three of the ten uteri test.ed with dog dander extract gave posit.ire react.ions ; four of the ten uteri tested with cat dander gave positive reactions. These findings are illustratetl in the reproductions of the Dale curves (Figs. 1, 2. atnl :i).
HAMPTON
AND SCULL:
ANTIGENIC
STUDIES BY DALE TEST
113
In seven of nine experiments in which reactions were obtained to one or more inhalants, the muscle reacted to dust extract after becoming neutral to the reacting inhalants (Figs. 1 and 2). The muscle failed to react in two experiments, suggesting that the inhalants
Fig. 4.
neutralized the uterine antibodirn to dust (k’ip. 4). III one instance, t.hc horns of the uterus failtd to react to all inhc3ant.s t.cst.ed, but one reacted to the test with the sensitizing dust (guinea pig 7). In the teats of guinea pigs 8 MINI 12, a fifth dnst (No. 2163’3) obtained from a single source was used. This dust extract produced a
HAMPTON
AND
BTULJ.
:
PWTOCOL QUINEA NO.
PI4
DATE
1
TESTRE
1
3/ l/39
STUDJEB
TEWlNO ANTIGEY
Low I&lgwec Rltrrllaricr
Silk Fcuthem Horse dander Dog dnndet J)ust nv. 2liX Aduai Cnt dander Histamine Silk Kapok Orris Dog dmder Dog dander A.dnet A-dust
A-dust A-dust
a/ 3/30 (See
zg.
1.
BY
DALE
SERIES A-CONT’U
HORN
3/ 2/30
9
1.
UTERlNE
2
8
ANTIOkXIC
SJTR( OEN UWlTS
'ROTEIN
10,000 10.000 2;wl 10,000 10,onn 10,000 1 cd?. 10,000 10,000 2,500 10.000 1o;oclo 10,000 lfi,OM IO,00 10,000 IQ,000
i n,onn
Feuthers Feat hers Dog dander Cat dander Cat dander A-dust A-dust Iiiatamioe - Cvttooseell .~IIfP%Wifl Horse dantlrr Dog tlnnder Dug clantlrr Dug tlnnder l+‘entherc Cat daotlet Pyrethrum Tobncw Otris A.dust A-aust Hibtumine
1 n,nno
Wool Featkers Feathers Silk Cat dander Dog dander Dug dander Kapok A-dust A-dust Hwtamine
10,000 10,000 1i),OllO 10,000 10,000 10,nnn 10,000 10,000 10,000 10,000
1 n,ooo 10,000 10,000 1:qo0 10,000
in,000 10,000
in,ooo 1n,noo I~),000 10,000
w,ooo 10,000 10,ono
10,000 10,000 10,000 10,000 10,000
‘l’Ji2j’J’
115
THE JOURNAL
116
PROTOCOL GUINEA PIG NO. 11
UTERI.VE HORS 1
DATE TESTED 3/ C/3!1
(See Fig. 4.
OF AI,LERGP
1. SERIES A-CONT'D TESTING ANTIGEN
\YRDTElN SITKO , OEN ukars
Cottonseed \Vool Kapok Horse tlandcv Feathers Fe&hers
lU,ooU 10.000 10;000 10,000 10,000 10,000 10,000 10,000 10,000 1 n,ooo 1n,ooo 10,000 10,000 10,000 10,000 10,000
.i1tsrnn#ic1
Tohncco Pyrethrum Flaxreetl Silk Cat &i.nder Dog danilei Ragweed A-dust Adust IIist.amine Cat dander Dog dnndel Cottonseed .4Itenmrin Tobacw Orris Pyrethrum Silk Ragweed E’rathers A~dust .\-dust .4ltmlclrin Hist.amine Cottonseed
I 1
3/ o/39
ROOI
.4lteraaria Horse tlantlel Feathers Feathers Cat dander Cat dander Dog tlan~ler Silk Kapok Tohrcco Orris Pyrethrum Ragweed Dust No. 21WI Dust No. 2163: X-dust Histamine 2
I (
A-dust A-tlust Feathers Dog dander Cat dawler Dust No. 21(i3! Histamine
I
10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 liJ,OOO 10,000 10,000 10,000 li),OOU 10,000 10.000 10;000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 1 c.(‘. 1 r.c. l(l,OOO 10,000 10,000 10,000 10,000 10,000 1 C.C.
= _- ,
HAMPTON
AND
STULL:
PROTOCOL GUINEA Pit NO.
13 (See Fig. 2
14
(See Eg. 3.
UTERINE HORN
1
DATE TESTED
3/15/39
3/17/39
3/M/39
ANTIGENIC
STUDIES
1. .%RIES
A--CONT
TESTING ANTIGEN
BY
DALE
'D GEINKITR( QEN UNITS
Cottoneeed Dog dander Feathers Feathers Cat dander A-dust A-dust Histamine
10,000 10.000 10;000 10,000 10,990 10,000 10,000
Cott.onseed Dog dander Feathers Feathers Cat dander A-dust A-dust Histamine Cotton linters Kapok Dog dander Dog dander Cat dander Feathers A-dust A-dust Histamine
10,000 10,000 10,000 10,990 10,000 10,000 10,000
Cat dander Feathers Dog dander Dog dander A-dust A-dust - 1Histamine Cotton linters Pyrethrum Cat dander Cat dander Cat dander Dog dander Cottonseed Feathers Feathers A-dust A-dust Histamine
10,990 10,990 10,990 10,000 10,ouo 10,990
Dog dander Dog dander Cat dander Cat dander Feathers A-dust A-dust Histamine
117
TEST
3,000 10,990 10,000 10,000 10,000 10,000 10,000 10,000
10,000 10,000 10,000 10;000 10,990 10,990 10,000 10,000 10,990 10,000 10,000 10,000 10,000 10,000 10,990 10,990 10,990 10,990
I!TERINE CONTRACTION 0 0 ttt 0 0 t tt .I. 0 t+t I. 0 0 t I) 0 ttt 0 ttt 0 0 t 0 z tttt 0 tttt 0 0 0 0 + t t t 0 t-l-t+ 0 0 tttt tt 0 0 0 tit 0 ;: t+tt 1: tttt 0 0 0
0 +++t
=5_... I
Orrip Silk.
%j,UO
:1n11 1 O.OOI)
SO.
OF VTEt:l ‘IXSTEI)
--_--_
IO JO h s i ci i 7 .i 4 -I 4 s .,
Cnits
1.
-io.
-~ ‘-.-P-P
OP POSIrlVE REhC’l’IONS
I
1 Jo
Fig.
---_
--
:: -I (i 0 0 0 0 0 0 (I 0 0 0 0 0 i -__-..
3.
(Pvolocol 2 und Tahlc Six of the SCV~I~ guinea pigs ill this series were found sensitive Dale test. Oile of tlw four uteri tested wit.li featlwr extract positive reaction (Fig. 5). lhg and cat. dander extracts failed tliicc! wt~tiwtioiis. All of the four uteri t,ested with hnan retract gwe positive reactions (Fig. 6). Scrics
L’. Ncmifiziwg
.l,ltigcn,,
h’-D&t.
III.)by the gave a to prodander
HAMPTON
-4ND
ANTIQENIC
STULL:
STUDIES
DY
Dhl.E
TFST
110
In three of t.he four experiments in which reactions were obtained to use or more inbalauts, the muscle reacted to a dust extract after it was neutral to the reacting iuhalailts, although the dust rciic4ious wrre stnall a.ud obtaiued upou only one Iioru iu two iustames (Pigs. 5 iintl 6). (J-dust .aml D-dust extracts produc+etl a contr’actiou ant1 neutralizetl the uterine muscle to H-dust exQract (seusitisinp antigen) in two crossneutralization esperiments (Figs. 7 am1 8).
,NO+NQ t Xirrn
,a,..0
-rPp-s*Ar.r
I.,...
“.ol
,.,eoo
Fig.
Hr..‘.
C,rr,.rw9.
rgeee
6.
of the Series C. Sensitizing Antigen, C-D~tst. (Table IV.)-Five seven guinea pigs in this series were found sensitive by the Dale test. One of the three uteri tested with dog dander extract gave a poskive
++ -T+=======+
.-._
_-.
+ 2.
-
+ f-L
. 7’
1 8
, -f
I
=5=-i
+ +
t-.,===.t
4 -I
HAMPTON
AND STULL:
ANTIGENIC
STUDIES BY DALE TEST
121
T~BLF, III GUINEA
Pros TESTING 10,000
INJECTED
SUMMARY OF REACTIONS OF SERIES B WITH 50,000 UNITS ALUM-PRSCIPITATED
ANTIGEN UNITS
NO. OF UTERI TESTED
Feathers Cat dander Dog dander Human dander Cottonseed Kapok Orris Horse dander Pyrethrum Tobacco Silk Wool BItemaria MmiZia M-lbcm
NO. OF POSITIVE REACTIONS
f !J 4
2 2
2 2 2 2 2 2 2 2 2 2 2
dbicans
Aspergillzss Eormodendn4m
Penicillium Denuatium Ragweed Cotton linters Glue Jute* B-dust C-dust D-dust *Jute contained too small a quantity
B-DUST EXTRACT
i 2 2”
1 ; 2 of protein
for
analyses.
Testing
strength
1 cc.
B-dust produced a contraction and neutralized the uterine muscle to C-dust (sensitizing antigen) in three experiments (Fig. 11).
122
THE
JOURNAL
OF
ALLERGT
fkrics Il. Sc7rsitiziny .hfige?l, D-Dust. (l’uble IT).---All seven pigs in this series were found sensitive by the Dale test. Three of the four uteri tested with feather extract gave positive reactiotls (Fig. 12). So reactions were obtained with cat and dog tlalldcr extracts. Three of t.he four uteri testetl with human tlan(lel gave positive wavtioiis (Fig. 13). .J-- 1 J-- 39. &Sq-. ode lQfp--cqo.0 urih oh- P’*=‘p-+*” O&w&P P’) IhI0.l cT(Ow.* o,aI- *‘?r;e;;r-~ J=Y
Fig.
9.
In two of the threv experiments in which reilctions were obtained with inhalants, the muwle reacted to dust extract after it was neutral to the respective inhalants (Figs. 12 and 13). One uterine muscle rcnctd to the scllsitizing dust but to none of tlw inhalants tctstcd.
HAMPTO.\’
.2x1)
STt-I . I Ad: f
ANTIGENIC
SUNNAKY
--
Pros
INJECIWI
TESTING (10,000
WIT~I
OF REACIWK~ 50,000
ANTIGEN KNITS)
UNITS SO.
BY
DA1.E:
OF
SEIUES
C:
AWN-PHECIPIT.\TED
OF I:TEKI TESTED
Feathers Cat dander Dog dander Human dander Cottonseed Orris Horse dander Pyrethrum Tobacco Silk Wool
C-DUST NO.
0 0
1
0
:
0 0 0 0 0 0 8 0 0 0
1 2 1
i 0 0 0 0 3 3 1
Ragweed Cotton linters Glue JUte” C-dust B-dust D-dust containe~l
EXTKACT
OF POSITIVE REACl-IONS
4 3 3 4
1 0
dlternaricc illonilia albicans Nwor Aspergillus IIormodendrum Pentillipvm Dcmatium
*Jute
123
TL.T
IV
TABLE
GUINEA
STUDIES
too
small
a quantity
of protein
Fig.
for
analyses.
Testing
strength
1 C.C.
10.
C-dust extract prQduced a contraction and neutralized the uterine muscle to D-dust extract (sensitizing antigen) in two experiments (Fig. 14). B-Dust extract produced a contraction and neutralized to D-dust extract in one experiment (Fig. 15).
124
THE
JOURNAL
OF
ALI,ERGT
DISCUSSION
Humau dander, although we of the antigens for guinea pigs in house dust, was not the only autigeuic factor in dust extract other than the common inhalants, since, in four of the fire experiments in
Fig.
12.
which human dander was the only inhalant eausiug muscular contraction, dust extract produced a contraction after the muscle was shown to be neutralized to human dander. There was no evidence of a histamine-like substance in the human dander extract or the dust extracts.
HAMPTON
AND
STULL:
ANTIGENIC
STUDIES
BY
DALE
TEST
125
In order to determine that human dander was antigenic for guinea suspension of human dander Pigs, 50,000 units of alum-precipitated were injected into each of four guinea pigs. The uteri of two guinea pigs were reactive to the dander extract. B-dust produced a positive reaction in the only instance in which it was tested on a uterine muscle, sensitive to human dander, but it did not completely neutralize to this dander extract in 10,000 unit strength (Fig. 16).
Fig.
14.
In the experiments in which a different dust extract produced a reaction prior to testing with the sensitizing dust extract, the uterine
‘l’lllt
JOI’RNAI,
Fig.
012
.II,I,ER(;T
15.
muscle was c+ompletcly ncwtralizerl ill all of twelre hstalices to the sensitizing dust. #Six of this proup were st,rili.cht c~~oss-,lerltralization c*xperiments.
HAMPTON
AND
STULL:
ANTIGENIC
STUDIES
BY
DALE
TEST
127
In the experiments in which the sensitizing dust extract produced a reaction prior to testing with another dust. extract., t.he uterine muscle was completely neutralized in twelve of thirteen instances to another dust. Six were straight cross-neut.ralization experiments. In one experiment (guinea pig 23) in which a uterine horn was sensitized by D-dust .extract, C-dust. extract produced a small contraction after the muscle was shown to be neutralized to D-dust.
Fig.
16.
In carrying out Dale tests in the past, using antigens other than dust, it has been an unusual occurrence for one uterine horn to be sensitive and the ot.her insensitive. It must be pointed out, however, that in this study wit.h house dust, the two uterine horns frequently failed to respond alike. For examples, in series A, horn No. 1 of guinea pig 15 (Fig. 3) reacted to feathers while horn No. 2 did not, and horn No. 2 of guinea pig 7 reacted to dust extract while horn No. 1 failed to react? although in each instance the horns gave the normal response to histamine. In fact, in series A, in six of the ten experiments, the two ut.erine horns of the respective guinea pigs failed to react similarly. This was the case in series B, C, and D (guinea pig 33) in a small number of inst.ances. However, the respective horns were not previously tested with exactly the same inhalant antigens. It is interesting to note that the two horns reacted similarly in the cross-neutralization experiments in which only dust extracts were used. This suggested that the dust extracts might contain minute quantities of various inhalant antigens, and, although the inhalants themselves did not protluce a muscular contraction, they may have neutralized the smooth
128
THE
JOURNAL
OF
ALLEROY
muscle antibodies. Consequently, the failure of dust extract to produce a contraction may have been due t.o the accumulative neutralizing activity of the several inhalants. This might suggest that the only antigens in dust. extract were the inhalants which were tested. That this was not true, however, was shown by the many experiments itr which dust extract produced positive Dale tests after t.he uterine muscle was shown to be neutralized to the reacting inha1ant.s. On the other hand, it is possible that inhalant ext.racts cont.ain traces of house dust, and it may thus be the accumulative effect. of the dust factor in the various inhalants t.hat. brings about neutralization without producing contractions. The presence of 0.4 per cent phenol in the inhalant extracts was another possible cause of inhibiting reactions after multiple tests. This was very unlikely in view of the many instances in which the muscles were tested with many inhalants and subsequently reacted to dust extract as well as to histamine. One might assume that multiple sensitization occurred with such a composite antigen as house dust, but why, in some instances, one horn was found sensitized to one antigen, while the ot.her horn wa.s apparently sensitized to a different one, is not explained in the present study. The “nonspecific t.oxic factor” producing anaphylactoicl symptoms in gross anaphylaxis, described by Coulson and Stevens, was not observed in these experimends in which the Dale test was used. However, their extracts were not said to be dialyzed, wherea.s the ext.racts used in this study mere dialyzed. Precipitin tests were performed with the dust extract used for sensitization in sera from guinea pigs 25 (D-dust), 2i (D-dust)? 22 (Cdust), 26 (C-dust), 21 (B-dust)? and 28 (B-dust), and the sera failed to show precipibins. SUMMARY
Twenty-eight of 31 guinea pigs , given a single intraperitoneal injection of purified alum-precipitated house dust extract from four different sources, were found sensitive by the Dale t.est. Positive Dale tests were obta.ined with feabher extracat in 10 of %I experiments, with dog dander in 4 of 20 experiments, and with cat dander in 4 of 20 experiments. Cottonseed, cotton linters, kapok, raw silk, orris, glue, l)yrethrum, wool, tobacco, ragweed, jute, horse dander, and the molds, Alterw~k. Aspergillus, Yztcor, Non,ilia. albicans,, Denmtiwn~., Penicillium, and Hornzodendrunc, failed to produce positive Dale tests in a total of 147 tests on dust-sensitized guinea pigs. Uterine muscles, sensitized by dust. extract, gave positive Dale reactions to dust extracts after the muscles were nantralised to certain rea.ct.ing inhalants in 13 of 18 instances.
HAMPTON
AND STULL:
ANTIGENIC
STUDIES BY DALE TEST
129
With the use of 3 dust extracts, 2 prepared with dusts from individual sources (B-dust and C-dust) and one from several sources (D-dust), neutralization to the sensitizing dust extract was effected by one or by the other two dusts in all of the 12 experiments. The sensit.izing dust extract neutralized to one of the other dust extracts in 12 of 13 experiments. Human dander extract gave a positive Dale test in 8 of 12 uteri sensitized by dust extract, but completely neutralized to dust extract in only one instance. Alum-precipitated human dander extract sensitized 2 of 4 guinea pigs injected, and one of t.hese two was sensitive to dust extract. House dust extract contained an antigen other than that found in the known inhalants tested, and this antigen was common to three different dust extracts as determined by the Dale test.
1. Hampton, S. F., Stull, A., and Cooke, R. A.: Antigenie Studies by the Dale Test (Abstract), J. ALLERC+Y 10: 465, 1939. 2. Cooke, R. A.: Studies in Hypersensitiveness : IV. New Etiologic Factors in Bronchial Asthma, J. Immunol. 7: 147, 1922. 3. Ramsdell, 8. G., and Walzer, hf.: On Antigens; Antigenie Properties of Glue, Wheat and House Dust, J. Immunol. 14: 207, 1927. Sensitization of Guinea Pigs to Cotton Liters 4. Coulson, E. J., and Stevens, H.: and House Dust Extracts, Proc. Sot, Exper. Biol. & Med. 40: 457, 1939. 5. Cooke, R. A., and Stull, A.: The Preparation and Standardization of Pollen Extract for the Treatment of Hay Fever, J. ALLERQY 4: 87, 1933. 6. Hampton, 5. F., Stull, A., and Cooke, R. A.: Antigenie Studies by the Dale Test : I. The Antigenic Relationship of Certain Pollens, J. ALLERGY 10: 417, 1939. The Protein Nitrogen Unit for 7. Stull, A., Cooke, R. A., and Glidden, M.: Standardization of Inhalant Extracts for Diagnosis and Treatment of Allergic Diseases, J. ALLEK~Y 8: 454, 1937.