anaphylaxis, and the relations and immunity.*...

THE NATURE OF ANAPHYLAXIS, AND THE RELATIONS

BETWEEN ANAPHYLAXIS AND IMMUNITY.*

RICHARD WEIL, M.D.(From the Department of Experimental Therapeutics, Cornell Medical School.)

In spite of the striking difference between the mani-festations of anaphylaxis and of immunity, there are manyfacts which indicate that they are closely related phenomena.A guinea-pig, by virtue of a single injection of an alienproteid, becomes hypersensitive towards that proteid, but, byfrequent repetition of the same, becomes immune thereto.An immunized guinea-pig, on the other hand, possesses aserum which, when injected even in minute amounts into anormal guinea-pig, renders the latter highly hypersensitiveto the specific antigen in question.

There have been two important theories in which theattempt has been made to unify the phenomena of anaphy-laxis and of immunity, and to explain them upon a singlebasis. Both of these theories have been largely developedby Friedberger, although other workers have contributedmaterially to their advancement. The first of these, in pointof time, maintains that the anaphylactic reaction is intracellu-lar; the second, which has now very largely displaced theformer in the literature, -the last article in which Friedber-ger9 accepted and advocated the cellular theory havingappeared in I909,- maintains that the reaction is entirelyhumoral. The difference between these two conceptions isfundamental, and the determination of the correct view is offirst importance for the whole subject of immunity. Each ofthese theories will now be briefly considered.

The Cellular Theory. -Ehrlich's theory of immunity isbased upon the belief that the cells of the body respond tothe stimulus of foreign proteid by the over-production ofspecific immune substances. These immune substances are

*Received for publication Feb. 2, I9I3.(497)

WEIL.

thrown off and circulate in the blood as " immune bodies."After a single injection of a foreign proteid, however, certainanimals, notably the guinea-pig, instead of becomingimmune become hypersensitive. It is assumed, in accord-ance with the terms of Ehrlich's theory, that in these ani-mals the cells have retained their immune bodies as " sessilereceptors" (Besredka,2' 3 Friedberger9), and that the antigen,upon re-injection, becomes anchored by these cells, and soproduces toxic or anaphylactic symptoms. Besredka modi-fied this conception in the sense that the cells, which in hisview chiefly enter into the anaphylactic reaction, namely,the brain cells, have not produced their sessile receptors, buthave taken them up out of the blood. When animals arepassively sensitized by the injection of immune serum, it isfound that a certain interval (of about six hours) mustelapse before these animals become anaphylactic; the injec-tion of antigen during this " incubation period " produceseither no symptoms or only slight ones. This phenomenonwas interpreted by Otto and, following him, by Friedemann,Friedberger, and others, as indicating that the antibodiespassively introduced became effective in producing hypersen-sitization only when they had become anchored by the bodycells, and that the " incubation period " represented the timenecessary to effect this fixation.As a corollary to these views, it was assumed (Friedber-

ger) that the function of the circulating immune bodies inan immunized animal was simply to bind antigen when intro-duced, and so to protect the body cells, with their fixedreceptors, from attack.

This theory, which at first sight seems reasonable andattractive, has now practically been abandoned in Germany.Friedemann, in his review of anaphylaxis (Jahresberichtiiber die Ergebrisse der Immunitatsforschung, published inI91I), and Friedberger, in a similar review (DeutscheKlinik, I91i), voice the judgment of the entire Germanschool, in finally rejecting this conception. They maintainthat, under certain conditions, the incubation period, charac-teristic of passive hypersensitization, can be abolished, and,

498

THE NATURE OF ANAPHYLAXIS.

consequently, that cellular fixation is an unnecessary assump-tion. Furthermore, it was found impossible (Friedberger 11)to verify the necessary corollary indicated above, that anexcess of free immune bodies in the circulation effectivelyprotects the sensitized animal. These objections will be con-sidered in the body of this article.

The Humoral Theory. -The humoral theory is basedvery largely upon the observation of Friedemann'3 (p. 598)that guinea-pigs may be instantaneously poisoned, with allthe symptoms of anaphylaxis, by means of an injection of amixture of antigen and of immune serum, which has beenincubated over a brief period. It is therefore assumed thatthis phenomenon represents the type of all anaphylacticreactions. This theory maintains the unity of the antibodiesconcerned in both processes- anaphylaxis and immunity-and correlates the difference in effects simply with thedifference in the amounts of antibody present in the blood.The function of antibody aided by complement is supposedto be that of a proteolytic ferment. This conception originatedwith, and has been largely developed, by Vaughan24 and hisschool. When only small amounts of antibody are present, asafter a single injection, the degradation of the proteid intro-duced in a second injection is incomplete, and toxic proteosesare produced; hence, the manifestations of anaphylaxis.When large amounts are present, as after repeated injections,the degradation of the foreign proteid, upon injection, advancesrapidly to a much further point, resulting in the production ofnon-toxic end-products; hence, the absence of symptoms,characteristic of immunity. There are many facts thatappear to support this theory, and, consequently, it has metwith very general acceptance. The chief difficulty encount-ered by this view is the " incubation period," which unques-tionably accompanies the typical experiment in passivesensitization. In order to obviate this difficulty, it isassumed that the incubation period represents a temporarycondition of anti-anaphylaxis, due to the simultaneousintroduction of the traces of antigen which are supposed to

499

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persist in all immune sera. The validity of this assumptionwill be discussed in a later part of this article.

It must be apparent from the preceding brief analysisthat the chief grounds of objection to the cellular theory arebased upon: (i), certain experiments which seemed to indi-cate that the " incubation period " of passive sensitization isan accidental phenomenon which may be abolished byappropriate procedures; (2), experiments which indicatethat the intravenous injection of immune serum does notprotect sensitized animals against anaphylactic shock uponthe subsequent introduction of the antigen.

These objections will now be critically and experimentallyconsidered.

The " Incubation Period " of Passive Senitization. -Ashas already been stated, Otto 19 showed that passive sensitiza-tion becomes effective only after the lapse of a period oftwenty-four hours. This fact was confirmed by observationsof Rosenau and Anderson, and since then by other authors.

It was shown by Richet, 1907, that passive sensitization toactino-congestin may occur within one to two hours. Doerrand Russ7 showed that guinea-pigs could be passively sensitizedso as to present mild symptoms upon the injection of antigenafter from one to two hours -severe symptoms after fourhours. These observations, and others of a similar nature,demonstrated that instead of twenty-four hours, one hourmight suffice for a mild degree of sensitization, and fourhours for an extreme degree. They did not, however,abolish the " incubation period," and it was entirely con-ceivable that the cells could " bind " sufficient immune bodywithin four hours, or even less, to render the animalhypersensitive.The literature, however, teems with the assertion that the

separate, but simultaneous, injection of antigen and of theappropriate immune serum has been shown to produce im-mediate anaphylactic shock. Thus, Anderson and Frost'(p. 7) state: "On the other hand, it has been reported by

500

THE NATURE OF ANAPIIYLAXIS.

several observers, Richet,20 Doerr and Russ,7 Friedberger,10' 11Biedl and Kraus,4 that they have at last succeeded in producingimmediate anaphylactic reactions in fresh guinea-pigs byinjecting suitable mixtures of antigen and anaphylactic anti-body." This assertion has been so frequently repeated thatit has come to be almost an axiom in anaphylactic literature.Additional authorities are cited by Friedemann and by Fried-berger in their summaries of anaphylaxis, and the same con-clusions are drawn from their work. If, however, the originalarticles, from which these deductions are drawn, be criticallyexamined, the whole fabric of proof becomes more thandoubtful.

Weil-Halle and Lemaire,2' 26 who are often cited in this con-nection, in I908 made simultaneous injections of immunerabbit serum and of horse serum into normal guinea-pigs.In one series, they caused the death of the pigs in from eightto fifteen days- in a second series, in about eleven days. Inan earlier paper, they described similar experiments, in whichagain death occurred in only one instance, in less than fourdays. Evidently, it is questionable whether such resultscan be considered at all analogous with anaphylactic death.

Richet,21 in I909, prepared a mixture of crepitin and of theserum of a dog immunized thereto. After incubating fortwenty minutes, he injected this mixture into a normal dog,which at once evinced " anaphylactic " symptoms.

20 itharRichet, in I909, in the article specifically referred to byAnderson and Frost, did not show that the injection of" suitable mixtures of antigen and of anaphylactic antibodyproduce anaphylactic shock." What he did actually show,but in the article previously referred to, was that such mixtures,after a period of incubation, became toxic for dogs. Thetwo statements are fundamentally different, and the confusionof this fact has produced the assumption that in passiveanaphylaxis the incubation period is simply an unessential,accidental phenomenon.

Doerr and Russ,7 cited to the contrary by Anderson andFrost, state specifically (pp. I89 and I90) that by no com-bination of immune serum and of antigen was it possible to

50I

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induce anaphylactic symptomns in the guinea-pig. Theyconclude that an incubation period invariably precedessensitization.

Biedl and Kraus cite experiments which indicate thatmixtures of very large quantities of immune serum and ofantigen, previously mixed in vitro, may produce immediate"symptoms " in normal guinea-pigs, although in no case didanaphylactic death supervene. The separate injection ofthe two factors, even within a period of ten minutes, producedno symptoms in their experiments as protocolled. Theystate, but without any experimental evidence whatever, thatthe simultaneous, but separate, injection .of immune serumand of antigen could be shown to be active (" wirksam ").Here again, in the absence of a single protocol, and in viewof the well-known " activity " of immune sera alone, wheninjected intravenously into normal guinea-pigs, it seems mostunjustifiable to draw any deduction invalidating the occur-rence of the incubation period.

Friedberger, in the second article cited by Anderson andFrost (the first article containing nothing germane to thesubject), describes a series of experiments with "anaphyla-toxin," which is a hypothetical substance, produced byincubation of immune body and of antigen.

It must be quite evident from the above citations that inno single instance in the literature has it been satisfactorilyshown that passive sensitization can occur without the lapseof an incubation period. What has, however, been verydefinitely shown is that appropriate mixtures of antibodyand of antigen, after a variable period of incubation, mayproduce immediate anaphylactic symptoms, and even death,when injected into normal guinea-pigs. It has been demon-strated that the mixture of antigen and antibody undergoesdigestion during incubation, and the degradation products ofthe proteids so produced are believed to be.the toxic factors.The hypothetical toxin so produced has been called"anaphylatoxin " by the Germans, " apotoxin," by Richet.Now, it may be admitted without further discussion that

"anaphylatoxin " produces toxic symptoms in guinea-pigs.

502


The same symptoms may be produced by degradationproducts of proteids produced by purely chemical, asdistinguished from biological methods; such, for example,as peptones (Biedl and Kraus5), or B. imidoazolylethylam-inochlorhydrate (Dale and Laidlaw6).

Indeed, it has been shown by Dold8 that guinea-pigsmay be thrown into typical anaphylactic shock by the injec-tion of solutions of their own body proteid. It is evident,therefore, that the symptoms of anaphylaxis are not at allcharacteristic of that special condition in the guinea-pig, butmay be induced by many other forms of intoxication. Itis therefore not an entirely warranted assumption that"anaphylatoxin" is the specific toxin which actually pro-duces the characteristic symptoms in an anaphylactic experi-ment. But even if this assumption be granted, nothing isthereby added to the weight of probability in favor of thehumoral theory. If a toxic substance results from the unionof antigen and of antibody, it is just as reasonable to believethat this same toxic substance is formed within the cell bythe action of the anchored antibodies, as that it is formed inthe blood, by the action of the same bodies in the circula-tion. In either case, it might be expected to produce thesymptoms which accompany the injection of " anaphyla-toxin," as prepared by the incubation of antigen andantibody in vitro.

It seemed important to verify experimentally the concep-tion that an incubation period necessarily accompaniespassive sensitization. According to the terms of thehumoral theory, it ought to be possible, by properly gaug-ing the amounts, to introduce into a normal guinea-pigsuch a combination of antigen and of antibody (of foreignproteid and of serum from an animal immunized thereto),as at once to precipitate the toxic phenomena of anaphy-laxis. An experiment was therefore planned in such amanner that a series of animals received a wide range ofcombinations of these two factors - antigen and antibodybeing given simultaneously; in another series, the same com-binations were employed, but the injections of antibody andof antigen were separated by a time interval. In the

503

WEIL.

former case anaphylaxis failed to supervene; in the latter, itinvariably occurred.

These facts are illustrated in Tables I. and II.

TABLE I.Showing that the simultaneous introduction of antibody and antigen

is ineffective.

Guinea-pigs.

I...................

2...................

3..................

4...................

5...................

6...................

7....... .........

8...................

9...................

Immune Serum 328.

0.I CC.

0.1 i

0.1

0.5 "

0.5 "

0.5 "

1.0 "

1.0 iI.0

Immune serum 328 was obtained from a rabbit, immunized by repeated intra-venous injections against horse serum, and sensitized guinea-pigs in amounts ofi cubic centimeter when introduced intraperitoneally.

TABLE II.Showing that the same amounts of anti-body and of antigen used in Table I.

produce anaphylactic death when a proper interval elapses between the twoinjections.

Guinea-pig. Immune Serum 32S. Two Weeks Later Results.Horse Serum.

I.......... . o. I CC. 0.01 CC. t

2 ...................- 0.1 " I.0 " t

3 ................... 0.5 0.1 " t

4-................... 05 . " t5 ................... 1.0 " 0.5 " t

6 ................... .0 " I.0 t

t indicates immediate anaphylactic death.

Results.

No symptoms.

Malaise.

No symptoms.

Malaise.

No symptoms.

is GI

Horse Serum.

0.01 CC.

0.1 i

I.0 1

0.01 "

0.1 1

1.0 it

O.OI "

0.1 "

0.1 "

504


In Table I. the guinea-pigs were injected with varyingamounts of horse serum and of serum derived from a guinea-pig highly immunized to horse serum. The immune serumwas in these experiments injected into one jugular and thehorse serum immediately thereafter into the opposite vein.In none of the animals were anaphylactic symptoms induced;in those which received one cubic centimeter of horse serumthere was slight evidence of discomfort. In Table II. theguinea-pigs received an injection of immune serum derivedfrom the same animal that furnished the serum used in theprevious set of experiments. After an interval of forty-eight hours they received an intravenous injection of horseserum. In every case death resulted immediately with con-vulsions. If the amounts of antigen and of antibodyemployed in the two sets of experiments be compared, itwill be found that they exactly coincide. These two sets ofexperiments, taken together, demonstrate that some changemust occur in the injected guinea-pig before it becomessensitized; in other words, that in addition to the relativeamounts of antibody and of antigen that enter into thereaction, there must be some additional factor which condi-tions the production of anaphylactic shock.

In order to determine the nature of this factor, the incuba-tion period above described was submitted to furtherinvestigation. It seemed possible that this interval might beoccupied by the induction of changes in the introducedimmune bodies, of such a nature as to insure the productionof anaphylactic shock upon the final injection of the antigen.In order to test this hypothesis, a series of guinea-pigswas passively sensitized by the injection of immune serum.These guinea-pigs were then bled to death, and the totalrecovered amount of the serum from each one was theninjected severally into normal pigs. The duration of theincubation period was then tested in the latter, and it wasfound that it had not undergone any notable diminution.The results of this experiment do not coincide with thoseof Biedl and Kraus (l.c., pp. i i9 and 120).

505

506 XWEIL.

TABLE III.To show that qualitative changes do not occur in immune serum by passage

through the normal animal.

G.P. Horse Serum o.z cc. Result.Intravenously.

I . 0.5 cc. 269, to-1 After I hour. No symptoms.gether with 5

2 . . gucic. norsinal S 3 hours. Moderate symptoms.2guinea-pig se-rum, intraperi-

3 . j toneally ...... J " 6 " Convulsions. t

4 ... ( [Bled to death. Injected into No. 7, i.p.'icc. SerUm 2691I

5 .... intraIeritoneally. After 24 hours. . " " 8, "

6 .... J L.. . s 9,

7. Received 6cc. of 4... Horse serum O.I CC. No symptoms.after Ij hours.

8 ... " 64 cc. of 5- Horse serum O.I cc. Mild symptoms. No con-after 3i hours. vulsions, recovered.

9 ... " 5 cc. of 6... Horse serum O. cc. Convulsions. tafter 6 hours.

Guinea-pig 269 was highly immunized to horse serum. It yielded a serum ofwhich . cubic centimeter was sufficient to sensitize normal guinea-pigs. In pigsI, 2, and 3, five cubic centimeters of normal serum were added to the one cubiccentimeter of immune serum in order that the total amounts of the intraperi-toneal injections might approximate those in 7, 8, and 9.

In view of the failure to demonstrate any such qualitativechange in the immune bodies, a further experiment wasundertaken, in order to determine whether there is anyquantitative alteration in the immune bodies during theincubation period. This experiment consisted in injectingtwo duplicate sets, of three pigs each, with graded amountsof immune serum. The pigs of the first set were bled afterfive to seven minutes, those of the second after twenty-fourhours. The serum of each animal was then injected intra-peritoneally into a normal guinea-pig, and the latter weretested by an intravenous injection of .I cubic centimeterof horse serum after an interval of sixteen hours. Theresults are shown in Table IV. a. It seems perfectly clear

THE NATURE OF ANAPHYLAXIS. 507

from this table that there had been a marked diminution inthe amount of the circulating immune bodies during theinterval of twenty-four hours (cf. Levin 16).

TABLE IV. a.To show the quantitative diminution ofimmune serum introduced into the

normal guinea-pig.

Guinea-pig.

I.............

2.............

3...........

4............

5............

6.............

7............

8.............

9.......

10 .............

II.............

12.............

Serum Guinea-pig 269.

0.2 CC., i.V.

0.2 " "

0.4 it ..

0-4 4. Is

0.8 " "

o.8 " "

After I6 hours O.I cc. horseserum, intravenously.

After I 6 hours o. I cc. horseserum, intravenously.

After I 6 hours O.I cc. horseserum, intravenously.

After i6 hours O.I cc. horseserum, intravenously.



Bled in 5 minutes. Defibrinatedblood, 6 cc., i.p., to 7.

Bled in 24 hours. Defibrinatedblood, 8 cc., i.p., to 8.

Bled in 7 minutes. Defibrinatedblood, 5 cc., i.p., to 9.

Bled in 24 hours. Defibrinatedblood, 7 cc., i.p., to 10.

Bled in 4 minutes. Defibrinatedblood, 9 cc., i.p., to II.

Bled in 24 hours. Defibrinatedblood, 6& cc., i.p., to 12.

Mild symptoms.

No symptoms.

Convulsions, recovered.

Very mild symptoms.

Convulsions. t

No convulsions, sligbtly paretic.

The serum of Guinea-pig 269 sensitized in amounts of o. I cubic centimeter (i.v.,means intravenously; i.p., intraperitoneally).

2

WEIL.

The same facts are somewhat differently illustrated inTable IV. b.

TABLE IV. b.

Received, i.v., 0.3 cc. serumRabbit 204.

Received, i.v., o.6Rabbit 204.

Received, i.p., o.6Rabbit 204.

cc. serum

cc. serum

4..... ... Received, i.p., I.O cc. serum

Rabbit 204.

5..................

6..................

After 48 hours, 0.3 cc. horseserum, i.v.

After 48 hours, 0.3 cc. horseserum, i.v.

Bled in 5 minutes. Serum2.5 CC., i.P., to 5.

Bled in 5 minutes. Serum2.5 cc., i.p., to 6.

Bled in 48 hours. Serum2 Cc., i.P., to 7.

Bled in 48 hours. Serum2.5 cc., i.p., to 8.

Very sick.

Convulsions. t 2 minutes.

7 .... .............. After 48 hours, 0.3 cc. horse No symptoms.serum, i.v.

8. After 48 hours, 0.3serum. i.v.

cc. horse Moderate symptoms.

The question arises as to the fate of the immune bodies,which had disappeared from the circulation. It seems pos-

sible to conclude either that they had been excreted by thekidneys, or by the intestinal tract, or that they stillremained in the body, but had passed out of the circulationand were anchored by the body cells.The former view does not seem a priori to be the prob-

able truth. It is known from observation in the humanclinic that very large quantities of homologous serum can beintroduced intravenously by transfusion, without inducingany albuminuria (cf. Hektoen,15 p. I83).

But, fortunately, it was possible to test this hypothesisexperimentally. There were at hand four guinea-pigs whichhad been sensitized passively seventy days previously by theinjection of graded (2, 4, 6 and 8 cubic centimeters)

Guinea-pig.

I..................

2..................

3...... .........

508


amounts of immune serum. The two which had receivedthe smaller amounts were tested by the injection of .I cubiccentimeter of horse serum intravenously; both died in typi-cal convulsions. The two which had received the largeramounts were bled to death, and their serum injected intotwo normal guinea-pigs, which were similarly tested after aninterval of sixteen hours. The latter failed to manifest thesymptoms of anaphylaxis.

TABLE V.

7To show persistence of anaphylaxis in spite of disappearance of antibodyjromserum in passive sensitization.

Guinea-Pig. IX.2s. XI1.2. Result.

I. 0.2 cc. Serum 6o8, i.p. 0.4 cc. horse serum, i.v. t 2 minutes.

2 . 0.4 * "a, o.8 " " . " t at once.

3. o.6 " " " " Bled. Serum injected,i.p., into 5.

4 . o.8 " " "I " Bled. Serum injected,i.p., into 6.

XII.2 XII.3.

5 ......... Received serum of 3. Horse serum, o.5 cc. i.v. Mild symptoms.

6 ......... "SI SI 4. " " No symptoms.

Serum 6o8 was derived from a guinea-pig highly immunized to horse serumand was found to sensitize normal guinea-pigs in amounts of .I cubic centimeterwhen injected intraperitoneally.

It is therefore quite clear that the foreign immune bodiesdo persist in the guinea-pig sensitized with homologousserum over a period of at least seventy days. Passivesensitization was found by Otto to persist for as long as sixtydays. On the other hand, they have disappeared from theserum. The conclusion seems permissible that they hadbeen taken up, and held by the body cells.

In this connection it may be pertinent to cite certain

WEIL.

observations made by Schultz, 22,23 which seem to demon-strate by means of physiological methods, the presence ofantibodies in the cells in anaphylaxis. The material usedby him was derived from actively anaphylactic animals, but,inasmuch as there are no demonstrable differences betweenactive and passive anaphylaxis, the observation is directly inline with the conclusions drawn from the foregoing experi-ments. Schultz removed pieces of smooth muscle fromsensitized guinea-pigs, and submitted them to the action ofhorse serum. The contraction curves of these muscles wereregistered, and compared with curves produced by musclestaken from normal guinea-pigs under similar conditions.The contractions produced by the former showed a strikingincrease over those produced by the latter. Schultz'sobservation indicates the specific alteration of the body cellsin anaphylaxis, and therefore strengthens the conclusion -drawn from the experiments previously described - thatthe body cells " fix" immune substances introduced in pas-sive sensitization. In order to secure definite and positiveconfirmation of this view, the attempt was made to demon-strate the presence of specific antibodies in the body cellsby the methods of complement-fixation and of precipitinformation. These experiments failed to give the expectedresults. Inasmuch as their recital would occupy a largeamount of space, and would fail to advance the understand-ing of anaphylaxis, they will not be reported in this place.

Since it has been shown that " fixation " of the immunesubstances, which occurs during the latent period of passivesensitization, is the necessary preliminary to the inductionof anaphylactic shock, the conclusion seemed unavoidable.that the reaction that marks the introduction of antigen mustnecessarily take place in the body cells. The union ofantigen and antibody within the circulating blood wouldappear, to judge from the results in Table I., to be entirelydevoid of toxic effects.

The Immnunization of Sensitized Guinea-pigs. -In thesecond part of this investigation the conclusion reached

510


above was accepted as a valid working hypothesis. Thisconclusion coincides in part with the view advanced in 1907,by Besredka, that the symptoms of active anaphylaxis aredue to the interaction between the anchored receptors(" sensibilisin") in the cells and the introduced antigen.It differs entirely from the theory subsequently announcedby Friedberger, and now very generally accepted, that thereaction takes place in the circulating blood. Furthermore,it makes no assumption as to the nature of the reactionbetween antigen and antibody, which is believed by theFriedberger school to result in proteolysis of the former.

Following the line of thought indicated by the precedingconclusion, the relationship between anaphylaxis and immu-nity was interpreted in the terms of a view which Ehrlichlong ago maintained of immunity in general (Ehrlich,Experimental Researches on Spec. Therapeutics, HarbenLectures, I907, p. 14). In the immune animal, upon thelines of this theory, the immune bodies are present in suchlarge amount in the circulating blood that the antigen, wheninjected, is at once totally occupied by them, and, hence, theantibody present in the cells is absolutely protected fromattack. The immune animal is believed to possess " fixed "

or sessile antibodies, exactly like the hypersensitive animal;it differs from the latter, however, in possessing an enormousnumber of free, circulating antibodies which effectivelyprotect those in the cells from attack. This view was atone time accepted by Friedberger, but is now practicallyabandoned by the German school. In order to test thevalidity of this theory, the following series of experimentswas planned, which, in part, led to the confirmation, in partto the modification, of this belief.By way of preliminary to these experiments, the theory of

cellular absorption, or fixation, of introduced immune bodieswas submitted to still further verification. It was assumedthat the capacity of the body cells of the guinea-pig toabsorb foreign proteid must be limited. Hence, if thenormal guinea-pig could be saturated, to use a chemicalanalogy, with normal rabbit serum it might be possible to

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inject the animal subsequently with immune rabbit serumwithout inducing the condition of hypersensitization. Themanner in which the experiment progressed dictated asecondary modification, whereby the attempt was made todisplace the fixed rabbit immune bodies in a passivelysensitized guinea-pig by means of subsequent injection ofnormal rabbit serum.The results of these experiments are summarized in

Table VI.:

TABLE VI.

To show interference with sensitization by heterologous immune serum throughprevious saturation with normalheterologous serum.

Guinea-pig 890, XII.nI, subcutaneously, four cubic centimeters of normalrabbit serum. XII.12, received intraperitoneally, .3 cubic centimeter of serumderived from Rabbit 208, highly immunized to horse serum. (Of this serum,.I cubic centimeter sufficed to render a normal untreated guinea-pig hypersensi-tive, see Guinea-pig 856, below.) XII.13, received .3 cubic centimeter horseserum intravenously; died at once. In this animal, therefore, the single previousinjection of normal rabbit serum failed to protect.

Guinea-pig 892, XII.Ii, three and one-half cubic centimeters normal rabbitserum, subcutaneously. XII.3, eight cubic centimeters normal rabbit serum,subcutaneously. XII.14, five cubic centimeters normal rabbit serum, subcuta-neously. XII.i5, five cubic centimeters normal rabbit serum, subcutaneously, at10.45. At II.45, .3 cubic centimeter serum of Rabbit 204, intraperitoneally.(Of this serum, .i cubic centimeter injected intraperitoneally, sufficed to make anormal, untreated guinea-pig hypersensitive, see Guinea-pig 85o, below.)XII.i6, .5 cubic centimeter horse serum, intravenously. No symptoms. (In thisguinea-pig the subcutaneous tissues on XII.i6 were somewhat edematous, owingto the large amount of normal rabbit serum introduced.) The animal failed tobecome hypersensitive, as shown above.

Guinea-pig 856 received .I Serum 208, intraperitoneally. After 24 hours,horse serum, intravenously, .I cubic centimeter. t

Guinea-pig 85o received .i Serum 204, intraperitoneally. After 24 hours,horse serum, intravenously, .oi cubic centimeter. t

(The results obtained in Guinea-pigs 890 and 892 wererepeated in other anitnals, but additional protocols wouldadd nothing essential to the data contained in the precedingtable.)

These results, which are entirely in harmony with theassumption which dictated the experiment, seemed to sustainthe theory, herein maintained, as to the fixation of antibodies

51I2.


by the cells and the dependence of the anaphylactic reactionupon this fixation. (The possibility of displacing the fixedantibody is indicated in Guinea-pig 897, and has been verifiedin other animals, but, although a very interesting fact, playsno further part in these experiments.)

If immunity is due to the presence of excess of free anti-bodies, two results ought necessarily to follow: In the firstplace, it ought to be possible to make a passively sensitizedanimal immune, simply by introducing an excess of anti-bodies into its blood immediately before making the injectionof the antigen. (If a longer time were allowed to elapsebetween the protective and the toxic injections, the object ofthe experiment would be defeated, since the free immunebodies would be removed in part from the blood, as shownin Table IV.) In the second place, it ought to be possible,by properly regulated procedures, to prove that guinea-pigsimmunized against a foreign proteid are also potentiallyhypersensitive thereto; in other words, that their cellspossess anchored antibodies, or " sessile receptors."

Friedberger1' undertook, some years ago, to demonstratethe truth of the cellular theory of anaphylaxis by means ofthe former of the above methods. He was the first toattempt to protect sensitized animals by means of the injec-tion of immune rabbit serum, previous to the injection of theantigen. Although in some instances death was postponedfor several hours, he failed in these experiments to save anyof his guinea-pigs from a fatal result, and this too, in spite ofthe fact that he used very large amounts of immune serum,and barely more than the ininimal lethal dose of antigen.Friedberger (p. 667) used two cubic centimeters and twoand one-half cubic centimeters of immune rabbit serum. Inview of the fact that he has apparently failed to titrate theserum, the number of immune units, or of " minimal sensi-tizing doses," contained in these doses cannot be determined.Evidently, these results are so unsatisfactory that they wouldseem to overthrow, rather than to support, the view thatimmune substances in circulation protect the sessile recep-tors. As an actual fact in the very same paper in which

5 I 3

WEIL.

Friedberger reports these experiments, he states that hefinds himself compelled to abandon his previous conceptionson this subject, and then propounds a new theory, accordingto the terms of which the protection afforded a sensitizedanimal, by means of the injection of an immune serum,depends upon the fact that "the remnant of antigen con-tained by these sera makes the sensitized animals anti- anaphy-lactic."

Into a discussion of Friedberger's views upon this latterquestion, I shall not now enter in extenso.

In brief, the following points may be noticed. Thetheory of antianaphylaxis has been invoked by Friedbergerto explain not only the delay, or " incubation period,"incident to passive sensitization, but also the protectionwhich might be afforded to sensitized animals by means ofthe injection of immune serum. This explanation postulatesthe invariable presence of sufficient remnants of antigen in.1 cubic centimeter of immune serum to produce antiana-phylaxis. It postulates that these remnants are invariablyand immediately destroyed in the blood of the normalguinea-pig upon injection, in spite of the fact, shown inTable III. of this paper, that these same traces of antigenmust be assumed to persist,-and not to be destroyed, in orderto account for the re-occurrence of the incubation phase in asecond series of guinea-pigs sensitized with the blood of thefirst series. It assumes that the guinea-pig is at once madeantianaphylactic by a sensitizing injection, whereas there isno experimental ground for believing that an animal can bemade antianaphylactic without previous sensitization; and,even if this is admitted to be plausible, there is no analogyfor the belief that antianaphylaxis can be induced in suchfashion as to be absolute even against massive doses ofantigen for one or two hours, and then disappear so com-pletely that doses of .oI cubic centimeter at once provefatal. Moreover, it is inexplicable that the injection of . Icubic centimeter of immune rabbit serum should suffice,according to Friedberger's view, to produce antianaphylaxis(the incubation period) in passive sensitization, whereas

5 I4


thirty times the same dose becomes necessary to make thesame guinea-pig antianaphylactic, when the same immunerabbit's serum is used to protect it, after passive sensitizationhas been accomplished (see Table IX.). If .I cubic centi-meter of immune serum contains enough antigen to makethe guinea-pig antianaphylactic at one time, why shouldthree cubic centimeters be necessary at another? Thehumoral theory is thus supported by a series of additionalhypotheses, entirely unproven, and not satisfactorily con-sistent with one another.

Evidently the crux of the entire question lies in the deter-mination of the facts concerning the degree of protectionafforded to sensitized animals by the introduction of immuneserum. If it is no greater than is indicated by Friedberger'sexperiments, one is almost forced to conclude that themechanism of such protection must be different in kind, differ-ing so enormously as it does in degree from that of immunityin general. In this case, one might entertain Friedberger'shypothesis, that immune serum protects by inducing anti-anaphylaxis through the remnant of antigen contained by it.If, on the other hand, it is possible to demonstrate thatimmune serum can really protect sensitized animals against ahigh multiple of the minimal lethal dose, Friedberger'spresent view becomes entirely superfluous. If, moreover, itcan be shown that the degree of protection afforded isroughly proportional to the amount of immune seruminjected, anti-anaphylaxis is no longer a reasonable factorin the explanation.

Friedberger's experiments have not been repeated by anyother workers to my knowliedge.The protection of passively sensitized guinea-pigs, by

means of the introduction of large amounts of immunebody, offered apparently a fairlysimple problem. Neverthe-less, the successful issue of this experiment waited upon alarge number of futile trials. Owing to the relative easewith which large amounts of immune rabbit serum could beobtained, the experiments were originally carried out withthis material.

5 I 5

WEIL.

In Table VII. are indicated the results of one of the earlierexperiments. Rabbit 204 was bled on October fifteenth andfound to sensitize guinea-pigs when injected intraperitoneally,in amounts of .i cubic centimeter. Smaller amounts werenot tested. Thirteen guinea-pigs were then injected intra-peritoneally with double this quantity,- .2 cubic centime-ter. The minimal amount of horse serum, when injectedintravenously, necessary to kill these animals was thendetermined, and found to be .005 cubic centimeter. In theremaining animals of the series, three in number, the protec-tive effect of five to ten (minimal?) sensitizing doses of rabbitserum was determined. The injection of immune serum wasmade into the jugular of one side, and that of the horseserum into the opposite jugular five minutes later. Guinea-pig No. io shows that five (minimal?) sensitizing doses ofimmune rabbit serum failed to protect against double thelethal dose of horse serum. Guinea-pigs Nos. II and I2show that ten times the said sensitizing dose protectedagainst the minimal lethal dose, although one of them diedafter thirty-six hours.

5 I6


TABLE VII.To showprotection against anaphylactic death by injection ofimmune serum.

Guinea-pig. October iS.

0.2 cc. serum ofimmune RabbitNo. 204, injectedintraperitoneally.

October x6, Horse SerumInjected.

0.05

0.2

0.01

0.005

0.005

0-005

0.003

CC., 1.V.

is it

0.0025 " "

0.0025 " "

0.5 cc. 204, i.v.

0o.o0 cc. H.S., i.v.

1.O CC. 204, i.V.f 0.005 cc. H.S., i.v.

1.0 CC. 204, i.V..o005 cc. H.S., i.v.

1.0 CC. 204, i.V.

Results.

Anaphylactic death.

It S

de

S e v e r e convulsions,recovery.

Anaphylactic death.

Paretic.

Anaphylactic death.

Slight symptoms.Died in 24 hours.

Mild symptoms.

Died before introduc-tion of horse serum.

H.S. means horse serum; i.v., intravenously.

The result of this experiment is certainly somewhat disap-pointing, inasmuch as the protective value of the free immunebodies seems very low. Evidently, if the theory is correct,much larger quantities of immune body must be in circula-tion in the immune animal than those here introduced.And, as an actual fact, it is easily demonstrable that such isthe case. An analysis of all the sera obtained from theimmune guinea-pigs used in these experiments indicatesthat they sensitized frequently, in amounts never greater than.1 cubic centimeter (smaller amounts were not tested).Hence it is clear that an immune guinea-pig may have incirculation hundreds of sensitizing doses.

I.,

2.

3.4.5.6.

7.

8.

9.

10.

II.

12.

13.

5 I 7

518 WEIL.

In another experiment, therefore, as shown in Table VIII.,larger amounts of immune serum were injected prophylacti-cally.

TABLE VIII. (As Table VII.)

Guinea-pig. Rabbit Serum, 208. On next Day. Results.

I.......... 0.1 cc., i.p. O.I cc. horse serum, i.v. Anaphylactic death.

2 ....... 0.2 " " 0.03 s' "C is 64 St i

3 ......... 0.2 St " 0.01 " " " is {C i

4 ....... .. 0.2 " " 0.01 " " " " c S

5...... 0.2 is " 1.0 CC. 208.50.02 " horse serum.

3.5 cc. serum, 204. No symptoms.6 ....... 0.4 to O.I " horse serum, i.v.

1.0 " " " i.P. "c

1,1,f .0 cc serum, 204. is it

7 * -C .... . ..0.2" " horse serum. Mild "

8 ....... O.I cc. serum, 0.01 " " " Anaphylactic death.204, i.p.

The technic followed in this, and in all similar experiments, was as follows:The sensitized guinea-pig being tied on the Friedberger board, both jugularsexposed. Into one jugular is inserted a platinum iridlium needle, bent at a rightangle. By means of the Record syringe 2 to 3 cubic centimeters of blood arenow slowly withdrawn, the assistant compressing the vein at the root of the neck.The syringe is now carefully detached, leaving the needle in place, and anotherRecord syringe, containing the desired amount of serum to be injected, is attached.This injection being slowly made, the needle is withdrawn, and the vessel clampedand ligated. The second vein can now be utilized for the injection of horseserum, either immediately or after an interval. Should there be some mishap inthe preparation of either vein the intracardiac route may still be utilized.The preliminary withdrawal of blood does not appear to be essential to the

success of the experiment, but was adopted in cases where large amounts of serumwere injected, in order to obviate the possible effects of overburdening the heart.

In this series the sera derived from Rabbits 204 and 208were used; either one sensitized in amounts of .I cubic cen-timeter. It will be seen that here again one cubic centimeterfailed to protect effectively, whereas three and three and one-half cubic centimeters (thirty and thirty-five sensitizing doses)


protected against ten toxic doses. It is probable that theminimal lethal dose was actually lower than here arbitrarilyassumed, to judge by Table VII., and that these animals wereactually protected against about twenty lethal doses. In afinal experiment, summarized in Table [X., a more completeseries of tests was performed, in order to determine themaximal protective effects of large doses of immune body.Moreover, normal rabbit serum was also injected into anumber of sensitized guinea-pigs by way of control, in orderthat the mere introduction of foreign serum, aside from itsimmune properties, might not be interpreted as a possiblefactor in the results.

TABLE IX. (As Table VII.)

G . . Rabbit After 48 Hours. Results.Serum 204.

I 0.3cc.,ip. 0.1 cc.horse serum, i.v. Anaphylactic death.

2.. i is s 0.02 " " I Ss Si "9

., .. f, 5 Normal rabbit serum, 1.5 cc, i.v. go s

3 * Horse serum, O.0I cc., i.v. St s

; w, ~, 5 Normal rabbit serum, 2 cc., i.v. i i

4..* * Horse serum, O.I cc., i.v. 9

X, ~, 5 Normal rabbit serum, 2 CC., i.v. t

5 Horse serum, 0.05 cc., i.v.

6 {. .. , 5 Normal rabbit serum, 2 CC., i.v.9

6. X Horse serum, 0.02 CC., i.V.

Js CX I Rabbit serum 208, I cc., i.v.7 * * Horse serum, o. I CC., i.V.

8 .. * .. I Rabbit serum 208, I cc., i.v.. . Horse serum, 0.05 cc., i.v.

9C, .X * J Rabbit serum 208, 1.5 CC., i.v.9 it* is SI

Horse serum, 0.2 CC., i.v.

lO .. * . { Rabbit serum 208, 1.5 cc., i.v. Very mild symptoms.Horse serum, 0.15 cc., i.v.

.. s, .. 5 Rabbit serum 208, I.5 cc., i.v. it St it

I I . . X Horse serum, O.I5 cc., i.v. " id d

12 .. 4 . { Rabbit serum 208, 1.5 cc., i.v. Severe symptoms.Horse serum, O.I cc., i.v. Recovered.

WEIL.

Serum of Rabbit 204, sensitized in amounts of .I cubic centimeter, when givenintraperitoneally. Likewise, serum of Rabbit 208 sensitized in amounts of .05cubic centimeter.

In this experiment guinea-pigs weighing about 350 grams were used. This isadvisable, inasmuch as the smaller animals - e.g., those weighing I 5O grams, suchas were used in many other experiments - often die immediately upon theintroduction of even I cubic centimeter of immune rabbit serum intravenously.Furthermore, in this experiment the serum of immune Rabbit 208 was usedimmediately after it had been drawn. This is also an important factor in theseexperiments, inasmuch as the immune serum deteriorates extremely rapidly,having noticeably less sensitizing doses per unit even after a few hours. Theserum may be titrated as to its sensitizing value by giving graduated d6ses tonormal pigs, and testing the results on the succeeding day. Another possibleplan is to make prelininary phlebotomies of 2 or 3 cubic centimeters on theimmune rabbits, and test this serum quantitatively on guinea-pigs. If found to beof high value, i.e., if .I or .05 cubic centimeter are sufficient to sensitize, therabbit may be thoroughly bled on the succeeding day, the serum now being usedin the immunizing experiments. This latter serum, however, must again be testedas to its titer. In any case, it is absolutely essential in these experiments to titratethe immune serum. The injection of large amounts of immune serum, as inFriedberger's experiments, may fail to protect sensitized animals; but unless the" value " of that serum is definitely known, no conclusions can be drawn.

Analysis of the table shows that in guinea-pigs sensitizedwith .3 cubic centimeter of- the immune rabbit serum thelethal dose of horse serum was as low as .o0 cubic centi-meter. Normal rabbit serum, in amounts of one and one-half and two cubic centimeters, failed to protect suchguinea-pigs against this dose of horse serum, as also againstsomewhat larger doses. Immune rabbit serum, however,when used in quantities of thirty sensitizing doses, protectedagainst fifteen lethal doses of horse serum. Twenty sensi-tizing doses, however, failed to protect against ten, or evenfive, lethal doses of horse serum.The results of this experiment indicate conclusively that

an important function of .immune bodies circulating in theserum is to afford protection to the animal against anaphy-lactic shock. They show that a large excess of immunebodies must be in circulation in order to accomplish thisfunction. They also prove that the presence of normalrabbit serum fails to produce any such result.

This conclusion having been reached, it remained to carryit one step further, and to determine whether the same

520


conditions obtain when the serum of an immune guinea-piginstead of that of an immune rabbit is used in giving pro-

tection. In this case it is possible to make use of activelysensitized animals, and to determine at the same timewhether guinea-pigs prepared in this way can be protectedin the same manner as those passively sensitized. In per-

forming this experiment again, a series of mishaps were

encountered, which may be passed over, inasmuch as theywere of a technical nature. In Table X. are summarizedthe outcome of the most successful series, in which resultssimilar to those described for the rabbit's serum were

obtained.

TABLE X.Actively sensitizedguinea-pigs protected by injections ofimmune serum.

After 14 Days.

Horse serum, O.I cc., i.v.

" " 0.05 cc., i.v.

St cc 0.01 Cc., i.v.

Normal guinea-pig serum, I cc., i.v.

Horse serum, 0.05 cc., i.v.

Normal guinea-pig serum, 2 CC., i.V.Horse serum, 0.05 cc., i.v.

Normal guinea-pig serum, 1.5 cc., i.V.Horse serum, 0.02 CC., i.V.

Guinea-pig serum 812, 1.5 cc., i.v.Horse serum, 0.05 cc., i.V.

Guinea-pig serum 812, 2 CC., i.V.Horse serum, O.I cc., i.v.

Guinea-pig serum 84I, 2 CC., i.v.

Horse serum, O.I cc., i.v.

Guinea-pig serum 84I, 0.5 cc., i.v.Horse serum, 0.02 CC., i.V.

Result.

Anaphylactic death.

it is

.s 66

Severe symptoms.Recovery.

Mild symptoms.

Anaphylactic death.

It is clear from this table that the serum of immunized

Guinea-pig.

1.

2.

3.

4.

Sensitized5- by intra-

p e r i to-neal in-

6. jection

ofo. I cc.

horse se-rum.

8.

9.

IO.

Guinea-pigs 812 and 84I yielded serum which sensitized normal guinea-pigs inamounts of .I cubic centimeter. Smaller quantities were not tested.

52I1

is It

69 46

WEIL.

guinea-pigs is as effective in affording protection to sensi-tized animals as is that of immunized rabbits. Moreover,it is clear that actively sensitized animals can be protectedin the same manner as those passively sensitized.At this point it will be convenient to discuss certain of the

features of active anaphylaxis. It is well known, owing tothe experiments of Otto, that actively sensitized guinea-pigscontain in their serum free antibodies. This is proven bythe fact that when such guinea-pigs are bled, and their serumis injected into normal guinea-pigs, the latter become hyper-sensitive to the proteid in question. According to the cur-rent theory, these free antibodies are the etiological factorsin the induction of anaphylactic shock, both in the originalactively sensitized guinea-pigs, and in those passively sensi-tized by the serum of the former. According to the theoryadvocated in this paper the free antibodies in the activelysensitized pigs represent an imperfectly developed immunity.If this theory be correct, it should be possible to show thatthese immune bodies are present in amounts far too small toprotect the sessile antibodies, it having previously beenshown that thirty or more minimal sensitizing doses arerequired for this purpose.As an actual fact, it can be shown that the blood of such

actively sensitized pigs contains regularly extremely smallamounts of antibody, probably not more than one to threeminimal sensitizing doses. This is clearly demonstrated bythe fact that it has never been possible in my experience tosensitize passively with one-half of the total quantity of theblood removed by exsanguination from an actively hyper-sensitive guinea-pig; indeed, the total quantity itself notinfrequently fails to sensitize. This amount of free anti-body, however, although ineffective in protecting the sensi-tized pigs, is sufficient when absorbed by the cells of thenormal guinea-pig injected therewith to sensitize the lattertowards the specific proteid involved.A very interesting problem here presents itself. If it is

true that the immune bodies in the circulation protect thecells, why should it be necessary to give doses so very much

522


larger than those which have been anchored? In otherwords, why should the minimal protective dose be thirty orforty times as great as the sensitizing dose? It is difficult atthe present moment to offer a solution of this problem.The first explanation which suggested itself was that suf-ficient immune body " got by" the serum, when large dosesof antigen were injected, and reached the body cells beforeneutralization could occur. The results obtained in TableVII., however, do not support this view, inasmuch as fivetimes the sensitizing dose failed to protect the animal fromimmediate death upon the injection of only twice the mini-mal lethal dose. The only other solution appears to lie inthe assumption of an increased " avidity " of the anchoredantibodies, as compared with the free antibodies, for anti-gen. This view, which is in harmony with a conclusionpreviously reached by Dr. William Elser, in connection withthe study of agglutinins, appears highly plausible, and issupported by a large array of experiments, made in connec-tion with another problem, which Elser has not yet pub-lished. The cause of this increased avidity is as yetundetermined. Hektoen'5 (p. i6i) has reached a similarview.With this, the first undertaking, namely, the protection of

the anaphylactic animal by means of immune serum, may beconsidered completed. A considerable number of experi-ments was performed, in which the protective serum,derived from immune rabbits, was injected intravenously, andthe subsequent dose of antigen was administered intraperi-toneally. It was expected by this means to obtain muchmore striking evidences of protection than by the experi-ments previously described. On the contrary, it was foundthat the effects were even less marked. A number of unsus-pected factors were discovered which complicated the results.These experiments will therefore not be detailed here.

Anaphylaxis in "Immune Animals." -The second prob-lem which was undertaken involved the demonstration of thepresence of latent or " masked " anaphylaxis in immunized

523

WEIL.

animals. According to the theory herein advocated allimmune animals have cells loaded with sessile antibodies;these, however, are protected from attack by injected pro-teid through the presence of large amounts of free antibody.Evidently the simplest and most obvious mode of approach-ing the problem would be to deprive immune animals oftheir blood, and then to inject the antigen. The methods oftransfusion and of exsanguination are at hand for this pur-pose, but-it was deemed wiser, on technical grounds, not toattempt them. Manwaring 17X 18 has performed experiments ofthis sort with dogs, and de Waele 2 with rabbits; the resultspartially support the cellular theory. If it were possible toprepare animals by immunization in such a manner as to avoida high degree of immunity, it might still be possible to provethat these animals were at the same time both immune andhypersensitive. Animals with a low degree of immunity canbe prepared either by making an inadequate number ofprevious injections, or by allowing a considerable interval toelapse after the last injection given. The latter type ofanimal was tested in a considerable series, and most of themreacted in a manner more or less characteristic of anaphy-laxis. A striking result was given by Guinea-pig 289, whichhad received seven injections of horse serum between Junefourteenth and October eighteenth. On December sixteenth,after an interval of two months, this animal received anintravenous injection of one cubic centimeter of horse serum.The animal died immediately in convulsions, whereas normalpigs of the same size (six hundred grams) withstood thesame injection without symptoms. Death was obtained inone other instance, and extremely severe symptoms, followedby recovery, supervened in two others.

Another group of guinea-pigs was prepared by givingonly two injections of horse serum. According to a currentnomenclature such animals are "antianaphylactic." Aftertwenty-four days it was found that two of these animals were" immune " to horse serum, inasmuch as they failed to showsymptoms on the injection of two and one-half and fourcubic centimeters intraperitoneally. On the other hand,

524


they died with typical anaphylactic symptoms on intrave-nous injection. These results are shown in Nos. 3 to 6 ofTable XI. Number i of the same table shows that on thefourth day after the second injection the animal was highlyhypersensitive, but recovered. Number 2 shows that twenty-one days after the third injection the animal was anaphy-lactic. The interpretation of the results obtained in Nos. 3and 5 is, clearly, that there were sufficient free immunebodies to neutralize large doses of antigen, if slowlyabsorbed -as from the peritoneal cavity, but not enough toprotect the sessile bodies in case the antigen were introducedin bulk -as intravenously.

TABLE XI.Anaphylaxis in immunizedguinea-.pigs.

Guinea-pigs.

I ...... F Oct. 29, I cc. horse serum,i.v. Convulsions, recov-ered.

F OCt. 29, 0.1 cc. followed by0.4 cc. horse serum, i.v.

2 .. No symptoms.Oct. 2I, re- Oct. 25, re- Nov. 19,0.5 cc. horse serum,

ceived 0.75 ceived 0.5 1i.V. tcc o.e~cc. horse ~Nv 8 .cc. horse srmr a pserum,i t r a p erito- i.p. No symptoms.

4........ neally. neally. Nov. 18, 0.5 cc. horse serum,i.v. t

5......... Nov. I9, 4 cc. horse serum,i.p. No symptoms.

6 ......Nov. 19,0.3 cc. horse serum,L. J L J i.v.

These experiments seem to demonstrate, therefore, thatimmunized animals are also potentially anaphylactic. Inthe terms of the theory herein supported their body cellscontain sessile receptors, or anchored antibodies, in sufficientnumber to produce an anaphylactic reaction, but are pro-tected by the free antibodies of the serum.

525

WEIL.

Summary of Experiments.- J. It has been impossibleto produce anaphylactic shock in guinea-pigs by injectingantigen and antibody simultaneously. For sensitization tooccur, an interval of time must elapse between these twoinjections.

2. No qualitative changes have been shown to take placein the introduced immune bodies during this interval.

3. Quantitatively, it has been shown that there is amarked diminution in the circulating antibodies in the bloodduring this interval.

4. It has been shown that in spite of the disappearanceof the antibodies from the blood, they persist in the body,as is shown by the persistence of the induced anaphylacticstate.

5. By previously saturating the guinea-pig with normalrabbit serum, it has been possible to prevent sensitization bymeans of immune rabbit serum.

6. Guinea-pigs that had been either actively or passivelysensitized were protected against anaphylactic shock byintroducing into their blood large amounts of immune body.

7. Guinea-pigs that had been immunized, in the popularacceptation of that term; by the frequently repeated injec-tion of antigen, were shown to be potentially anaphylactic.

CONCLUSIONS.

i. Anaphylaxis is due to the reaction between specificantibodies present in the cells and the introduced antigen.

2. In passive sensitization, the body cells absorb theintroduced antibodies from the blood, and the animal is thusmade anaphylactic.

3. The function of immune bodies present in the serumis to neutralize the introduced antigen, and so to protect thebody cells.

4. The anaphylactic animal regularly contains in his cir-culation an insufficient quantity of antibodies to protect hisbody cells.

5. The immunized animal is potentially anaphylactic.

526


His body cells possess anchored immune bodies, but are pro-tected by those in circulation.

6. Exactly the same antibodies are present in anaphy-laxis as in immunity. In the former they predominate in thecells- in the latter, in the serum.

[My sincere thanks are due to Dr. Famulener, of the New York CityBoard of Health, who throughout my work has kept me generously sup-plied with horse serum.]

BIBLIOGRAPHY.

i. Anderson and Frost. Studies upon anaphylaxis. Hygienic Lab.,Bull. No. 64, June, I91O.

2. Besredka. C. R. Soc. de Biol., 1907, lXiii, 294.3. Besredka and Steinhardt. Ann. de l'Inst. Pasteur, 1907, XXi, 384.4. Biedl and Kraus. Ztschrft. f. Immunitaetsforschung, I9I0, iv,

I 15.

5. Bie,dl and Kraus. Ztschrft. f. Physiologie, 19I0, XXiV, 258.6. Dale and Laidlaw. Journ. of Physiology, .190, xli, 3I8.7. Doerr and Russ. Ztschrft. f. Immunitaetsforschung, i909, iii 8i.8. Dold. Ztschrft. f. Immunitaetsforschung, I9II, X, 53.9. Friedberger. Ztschrft. f. Immunitaetsforschung, 1909, ii, 208.

Io. Friedberger. Ztschrft. f. Immunitaetsforschung, I909, iii, 692.iI. Friedberger. Ztschrft. f. Immunitaetsforschung, I910, iv, 636.12. Friedberger and Castelli. Ztschrft. f. Immunitaetsforschung, 19I0,

vi, I79.13. Friedemann. Ztschrft. f. Immunitaetsforschung, I909, ii, 591.14. Gay and Southard. Journ. of Med. Research, 1907, XVi, 143.I5. Hektoen. Harvey Lectures, I909-I9g0, 150.i6. Levin. Ztschrft. f. Immunitaetsforschung, 1909, i, 3.17. Manwaring. Ztschrft. f. Immunitaetsforschung, 19II, Viii, I.i8. Manwaring. Ztschrft. f. Immunitaetsforschung, i9I1, Viii, 589.I9. Otto. Muench. med. Wochenschrift, 1907, I665.20. Richet. C. R. Soc. de Biol., I909, lxvi, 8io.21. Richet. C. R. Soc. de Biol., I909, lxvi, I005.22. Schultz. Hygienic Lab., Bull. No. 8o, 19I2.23. Schultz. Journ. of Pharm. and Exp. Therap., 1910, 549.24. Vaughan and Wheeler. Journ. of Inf. Dis., I907, iv, 476.25. Weill-Halle and Lemaire. C. R. Soc. de Biol., I907, lxiii, 748.26. Weill-Halle and Lemaire. C. R. Soc. de Biol., I908, lxv, iii.27. de Waele. Ztschrft. f. Immunitaetsforschung, 1912, XV, 193.

anaphylaxis, and the relations and immunity.*...

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