Prevention of homocytotropic antibody formation and anaphylactic sensitization by prefeeding antigen

Prevention of homocytotropic antibody formation and anaphylactic sensitization by prefeeding antigen M. F. David, M.D. Winnipeg, Manitoba,

Canada

Studies were conducted in rats to determine whether pre-feeding antigen can prevent the development of Bordetella pertussisinduced homocytotropic antibody (HCA) formation and anaphylactic sensitization to the fed antigen. DA rats fed ragweed for a minimum of 2 wk and Sprague-Dawley rats fed horse serum for at least 4 wk demonstrated specific unresponsiveness to anaphylactic sensitization as measured by their inability to form HCA and their normotensive response to intravenous challenge with antigen. This state was more easily attained when feeding of antigen was started at the age of 30 days than at 90 or 150 days, and resistance to anaphylactic sensitization could be maintained by continued feeding of antigen. However, if feeding was discontinued for 2 mo or longer, the rats regained their ability to become anaphylactically sensitized to the fed antigen. The results suggest that induction of anaphylactic sensitization can be prevented by pre-feeding antigen.

States of immediate hypersensitivity in man presenting as allergic rhinitis or extrinsic asthma are presumed to be spontaneously occurring conditions in genetically predisposed individuals. That these can be preventable conditions is an approach that has not been taken in their management. Animal models of immediate hypersensitivity biologically akin to human state of allergy confirm the presence of a genetic predisposition to anaphylactic sensitization.’ They demonstrate as well, however, that certain modes of antigen presentation to the animal, such as the dose utilized or the concomitant use of specific adjuvants, have an important determining influence on the induction of anaphylactic sensitization.2* 3

From the Winnipeg Clinic and Departmentof Paediatrics,University of Manitoba. Supported by grants from the Medical Research Council of Canada, the Winnipeg Clinic ResearchInstitute, the Children’s Hospital of Winnipeg ResearchFoundation Inc., and Mr. A. Cohen and Sony of Canada. Preliminary reports of this study were presented at the Thirty-first Annual Meeting of the American Academy of Allergy, San Diego, Calif., February, 1975 (J. ALLERGYCLIN. IMMUNOL. 55: 135, 1975)and the Ninth International Congressof Allergology, Buenos Aires, Oct. 24-30, 1976. Received for publication March 24, 1977. Accepted for publication June 27, 1977. Reprint request to: Dr. M. F. David, 425 St. Mary Ave., Winnipeg, Manitoba, R3C ON2. Vol. 60, No. 3, pp. 180-187

As early as 1911, Wells and Osborne4 have observed that guinea pigs fed a cornmeal diet before parenteral sensitization with zein protein did not die upon a subsequent challenge with zein. This interesting observation, however, has not been investigated further and has had no effect on the direction of management of atopic conditions. In 1946, Chase5 rendered normal adult guinea pigs highly refractory to delayed hypersensitivity reactions to dinitrochlorobenzine painted on the skin by pre-feeding the animals the hapten. Thomas and Parrott reported in 1974 that the ability to mount humoral antibody response to bovine serum albumin (BSA) was significantly lowered in rats fed BSA. Initial presentation of antigen through the gastrointestinal tract therefore appears to alter significantly the response of the organism to a subsequent exposure. The present experiments were designed to expand on earlier observations to determine whether the induction of homocytotropic antibody (HCA) formation and of anaphylactic sensitization to ragweed and horse serum could be prevented by pre-feeding the antigens. MATERIALS Animals

AND METHODS

Inbred male DA rats raised at the University of Manitoba, Central Animal Care House and random-bred male Sprague-Dawley rats bred at the Dentistry Animal House of the Health Sciences Centre were used.

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Homocvtotropic

Antigens

Commercially prepared aqueous extract I : IO of giant ragweed (Ambrosia hifidu, containing 91,000 PNU/ml or approximately 0.90 mg of protein nitrogen/ml, Lot No. C574758). and timothy (Phleum pratense containing 40,000 PNU/ml or approximately 0.40 mg of protein nitrogen/ml, Lot No. K4624506) were brought from HollisterStier Laboratories, Spokane, Wash. Horse serum (Lot No. 843 I I) containing 77 mg of protein per milliliter, as determined by the Lowry method, was bought from Microbiological Associates, Inc., Bethesda, Md. Formalin-inactivated f?. permsis vaccine containing 40 x IO” phase I organisms/milliliter (Lot No. 1001) was purchased from Connaught Laboratories, Ltd., Willowdale, Ontario, Canada.

Sensitization Rats were divided into two groups, control and antigenfed. Both groups had free access to food (Teklad rat diet, Teklad Mills. Winfield, Iowa) and water. The antigen-fed group received, in addition, 0.5 ml of either aqueous giant ragweed extract (containing approximately 0.45 mg of protein) or timothy extract (containing approximately 0.20 mg of protein) or horse serum(containing approximately 40 mg of protein) for 5 days a week, for 1 to 20 wk via a feeding needle. At appropriate times, and in antigen-fed groups at least 72 hr after the last feeding, each group was given one of the following sensitization treatments: (1) B. perkwis phase I organisms intraperitoneally, 0.01 ml/gm body weight; (2) B. pertussis vaccine intraperitoneally and antigen (0.005 rnl/gm body weight of horse serum or giant ragweed extract) in the footpads; or (3) saline intraperitoneally and antigen in the footpads. Sera from 2 ml blood was collected 10 days after sensitization and tested for HCA by 48-hr passive cutaneous anaphylaxis (PCA) on normal DA rats. Two to 5 days later the rats were challenged intravenously with specific antigen to detect the presence of systemic reactivity as measured by hypotension (anaphylaxis).

Passive cutaneous anaphylaxis (PCA) PCA reactionswere done on normal DA rats. The dorsal surface of the body was shaved. Sera and serum dilutions of 0. I ml aliquots were injected intradermally about one inch apart, and approximately 48 hr later the challenging antigen (I ml of I : t 0 aqueous giant ragweed extract containing approximately 0.098 mg of protein or 1: IO horse serum containing approximately 8 mg of protein) was injected intravenously via the jugular vein followed immediately by I ml of 17~ Evans blue dye in normal saline. The rat was sacrificed 20 min later by ether anesthesia, and the skin of the dorsum was dissected to expose the undersurface where reaction demonstrated by bluing of the sites could be measured more accurately. PCA reaction was considered positive if the diameter of the bluing was 5 mm or more. In

titration of HCA activity, the reciprocal of the highest dilution of serum giving a 5 mm bluing was taken as the end point.

antibody formation and anaphylactic sensitization

181

Anaphylaxis Twelve to fifteen days after sensitization, the rat was anesthetizedwith pentobarbital (3 mg/ 100gm body weight) with ether supplementation. A tracheostomy was established and a tracheal cannula was inserted to ensure a free airway. A carotid artery and a jugular vein were cannulated. The catheter to the jugular vein was utilized for introducing antigens; the one to the carotid artery was connected to a pressure transducer (Statham transducer Model P23Db) for continuous monitoring of blood pressure on a Beckman Dynograph (R4 I 1) recorder. Sterile challenging solutions (normal saline, aqueous giant ragweed extract with approximately 0.90 mg of protein/milliliter, and horse serum with approximately 77 mg of protein/milliliter) were warmed to 37” C and given in a I-ml dose. A positive response was exhibited as a drop in blood pressure within I to 2 min after antigen injection, which lasted over 30 min. At the end of the experiment all rats were sacrificed by ether anesthesia.

lmmunodiffusion To determine grossly whether significant amounts of horse serum were absorbed by feeding, rats fed horse serum for 4 wk were bled under ether anesthesia on the last day of feeding of approximately I ml of blood, and the serum was separated. The amount of horse antigen in the serum was determined by simple radial immunodiffusion on microscope slides utilizing Mancini’s technique.’ Clean slides were pre-coated with 0.5% agarose (Induviose A45 L’industrie Biologique Francaise) in double distilled water, unto which was layered 3.0 ml of I .5% agarose in Verona] buffer pH 8.4 containing a final concentration of 2% goat antihorse serum (GIBCO 0851717). Two-millimeter wells were punched in the gel and were loaded with approximately 7 ~1 of varying dilutions of horse serum, Lot No. 843 I I, which served as the standard, and sera from control nonsensitized and horse serum-fed rats. These were allowed to diffuse in a moist chamber at 37” C for 7 days. They were then washed in several changes of buffer for 48 hr. blotted dry, and then stained with amido black. The diffusion of the test sera was read against the standard.

RESULTS Homocytotropic

antibody

formation

(HCA)

In control DA and Sprague-Dawley rats, specific HCA were consistently induced when giant ragweed extract or horse serum was administered concurrently with B. pertussis. The results are shown in Tables I and II. The antibody titers ranged from those demonstrated only when full serum was transferred and up to 1 : 160 serum dilution. The antibodies were reproducibly passively transferred to the skin of normal DA recipients and found to persist at the site without a significant decrease in titer for as long as 15 days. Preheating serum to 56” C for 30 min to 4 hr before transfer abolished its capacity to confer cutaneous reactivity to normal skin. The 48-hr PCA reaction

182 David

J. ALLERGY

TABLE I. Prevention

of HCA formation

Pre-sensitization antigen feeding

and anaphylaxis

Sensitization treatment

to ragweed

CLIN. IMMUNOL. SEPTEMBER 1977

in DA rats by pre-feeding Shock with

intravenous

ragweed challenge

48-hr PCA reaction

Saline

Horse serum

o/5 O/IO 10/l 1”

O/5 o/4 O/8

O/5 o/4

O/8

O/S o/4 8/8

12/12tt

O/S

S/5

o/5

o/10 o/12

o/5 o/4

015 o/4

015 o/4

015 O/8

o/5 o/5

015 015

o/5 o/5

5/51§

o/5

515

O/5

o/5

o/4

o/4

o/4

Ragweed

Control None None None

None Pertussis Pertussis and ragweed Pertussis and horse serum Ragweed Horse serum

None None None Antigen-fed group Ragweed Ragweed Ragweed Ragweed

Pertussis Pertussis and ragweed Pertussis and horse serum Ragweed

Numerator denotesnumber of animals giving a positive reaction and the denominator, the total number of animals tested. *Titers of positive reactions: 2, 10, 10, 10, 10, 10, 20, 40, 40, 160. -FChallengedwith horse serum. *Titers: 10, 20, 20, 40, 80, 80, 80, 160, 160, 160, 160, 160. §Titers: 20, 20, 40, 80, 160.

TABLE II. Prevention

of HCA and anaphylaxis

to horse

serum

in Sprague-Dawley

rats by feeding

Shock with

challenge

horse serum Pre-sensitization antigen feeding

Sensitization treatment

intravenous

48-hr PCA reaction

Saline

None Pertussis Pertussis and horse serum Pertussis and ragweed Horse serum Ragweed

015 O/IO 12115”

015 o/4 o/15

o/5 o/4 o/15

o/5 o/4 15/15

9/l 1tS

O/6

616

O/6

o/10 o/5

o/4

o/4

o/4

Pertussis Pertussis and horse serum Pertussis and ragweed Horse serum Pertussis and horse serum

O/IO o/19

O/6 0119

O/6 o/19

O/6 0119

4/5tg

o/4

414

o/4

O/8 5/5**

o/4

o/4

o/4

Ragweed

Horse serum

Control None None None None None None Antigen-fed group Horse serum Horse serum Horse serum Horse serum Timothy

Numerator denotesnumber of animals giving a positive reaction and the denominator, the total number of animals tested *Titers: 1, 1, 1, 5, 5, 10, 10, 10, 40, 40, 80, 80. tchallenged with ragweed. $Titers: 1, 1, 2, 2, 2, 10, 10, 20, 80. $Titers: 1, 1, 20, 20. **Titers: 5, 5, 10, 10, 10.

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Homocytotropic

2oofSaline7

i-

WJ

-

Horse Serum7

antibody

formation

I

and anaphylactic

sensitization

Ragweed

183

1

I

E

i

::o, : 0)

a’ -d

t

-

t

t

t

t t

I 067

t

200 100

-

-

ocI

.I.

zoo-

; +

loo-

2

_^

I oAf

0

-

0.D

-

-mr

t

t

v t

. t

t

4/ t

5 min.

FIG. 1. Blood pressure response to intravenous antigen challenge in DA rats. Rats received one B, of the following sensitization treatments 12 to 15 days earlier. A, B. pertussis intraperitoneally; ragweed extract in the footpads; C, B. pertussis intraperitoneally and ragweed in the footpads; and ragweed in the footpads but fed ragweed extract 14 days 0, B. pertussis intraperitoneally prior to sensitization. Arrows indicate the time of intravenous injections of isotonic saline, horse serum, or ragweed.

abolished by intraperitoneal administration of diphenhydramine hydrochloride (0.5 mg/lOO gm body weight) or by intragastric administration of cyproheptadine hydrochloride (4 mg) via a feeding tube 30 min and 1% hr, respectively, before antigen challenge. The biologic activities of the induced HCA are thus not unlike those of IgE antibodies. Ragweed extract or horse serum alone injected in the footpads did not trigger the production of detectable HCA. Rats fed antigen adequately prior to sensitization could not be primed by B. pertussis vaccine to produce HCA. DA rats fed ragweed for 2 to 4 wk failed to develop HCA to ragweed but formed HCA to horse serum. However, rats fed only for one to two days became sensitized and formed HCA to ragweed extract. Sprague-Dawley rats pre-fed horse serum for 4 to 20 wk did not form HCA to horse serum but formed HC.4 to ragweed. Rats fed timothy for one month and sensitized to horse serum formed HCA to horse serum. Thus, the unresponsiveness induced by pre-feeding antigen is specific for fed antigen. Feeding of antigen and subsequent parenteral injection of antigen alone or feeding of antigen and subsequent intraperitoneal injection of B. pertussis failed to induce HCA in serum.

was

Anaphylaxis To determine the actual state of reactivity of animals to antigen and the relevance of the presence of HCA in the serum, the rats were challenged with antigen intravenously 12 to 15 days after sensitization. The results of such challenge in the experimental

animals are shown in Tables I and II. Control rats which received either ragweed or horse serum with B. pertussis vaccine developed hypotension upon specific antigen challenge, as seen in Figs. 1 and 2. The drop in blood pressure started within 1 min after antigen injection, and the lowest blood pressure levels were reached in 7 to 15 min. The drop from initial mean blood pressure ranged from 35% to 90%. None of the rats showed signs of recovery of blood pressure 30 to 60 min after antigen challenge, with the exception of two rats (1 DA and 1 SD) of 20 sensitized to horse serum, which showed partial recovery of blood pressure to 93% and 90%) respectively, of initial mean blood pressure. Six rats (3 of 20 sensitized to horse serum and 3 of 14 sensitized to ragweed) died within 10 min after intravenous antigen challenge. Sensitization with ragweed extract and horse serum alone did not induce hypotension upon challenge with specific antigen. DA rats pre-fed ragweed extract for 2 to 4 wk before sensitization with B. pertussis and ragweed did not develop hypotension with ragweed challenge. If feeding was for only 1 to 2 days, sensitization occurred. Rats rendered unresponsive to ragweed could be made anaphylactically sensitized to horse serum. Similarly, Sprague-Dawley rats fed horse serum for 4 wk prior to parenteral sensitization with horse serum and pertussis did not demonstrate any change in blood pressure upon horse serum challenge. On the other hand, such animals could be made exquisitely sensitive to ragweed (Fig. 2). Rats fed ragweed extract or horse serum and in-

184

David

J. ALLERGY

I

3

Ragweed

I

-Horse

CLIN. IMMUNOL. SEPTEMBER 1977

Serum-

01 c 1

28 200 r 100 4

1-1

0I D t

100

-

200o I E t

FIG. 2. Blood pressure response to intravenous antigen challenge received one of the following sensitization treatments 12 to 15 intraperitoneally; B, B. pertussis intraperironeally and horse serum in footpads; D, fed horse serum for 4 wk before sensitization with and horse serum in the footpads; E, fed horse serum for 4 wk pertussis intraperitoneally and ragweed extract in the footpads. intravenous injection of isotonic saline, horse serum, or ragweed.

TABLE III. Effect feeding

of age at which

is started

formation

on homocytotropic

horse

serum antibody

and anaphylaxis Age at onset of feeding of horse serum

Experimental

condition

Factors affecting the induction and maintenance of unresponsiveness to anaphylactic sensitization

2mo

5mo

o/11 o/10

O/6

O/6

616

416

For the following experiments, Sprague-Dawley rats were used and the antigen utilized was horse serum.

8/10* lO/lO

6/10t 8/8

2/4$

Age when feeding

Controls PCA Anaphylaxis

parenteral sensitization with antigen. Levels detected by immunodiffusion in 2% goat antihorse serum in 1.5% agarose ranged from 50 to 150 pg/ml in 7 out of 10 sera. No horse antigen was seen in normal sera.

1 mo

Horse serum-fed PCA Anaphylaxis

in Sprague-Dawley rats. Rats days earlier: A, B. pertussis in footpads; C, horse serum 8. pertussis intraperitoneally before sensitization with B. Arrows indicate the time of

414

was started

jetted subsequently with B. pertussis alone or antigen alone did not demonstrate shock upon specific antigen challenge.

Rats fed horse serum at the age of 30 days for 4 wk (0.5 ml/day, 5 days/wk) and then sensitized with 8. pertussis intraperitoneally and horse serum in the footpads consistently became unresponsive to horse serum as shown by their inability to form HCA and failure to develop hypotension upon challenge with horse serum. If feeding was started at the age of 2 mo or 5 mo for a period of 4 wk, although no HCA were detected, anaphylactic sensitivity as demonstrated by hypotension on intravenous challenge was induced in these animals (Table III).

lmmunodiffusion

Duration

The presence of horse serum was tested for grossly in 10 sera obtained after feeding for 4 wk and prior to

Thirty-day-old rats were fed 0.5 ml per day 5 days a week for 4, 8, 12, or 20 wk and then sensitized 3

Feeding was for 4 wk and sensitization 3 days after the last feeding. Controls were tested at comparable ages. Numerator denotes the number of animals giving a positive reaction and the denominator, the total number of animals tested. *Titers: 1, 5, 5, 5, 10, 10, 10, 40. tTiters: 1, 1, 40, 40, 80, 80. *Titers: 1, 20.

of feeding

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TABLE IV. Effect of duration

of feeding

185

antibody formation and anaphylactic sensitization

of horse serum on homocytotropic

antibody

formation

and anaphylaxis Duration Experimental

condition

1 day/wk*

of feeding

x 4

of horse serum prior to sensitization

4 wk*

8wk

12wk

20 wk

o/23 o/9

018 O/4

014 O/4

o/2 012

6110 s/s

3/4$

214

212

414

Horse serum-jed

PCA Anaphylaxis

2/12t 9/l 1

Controls PCA Anaphylaxis

S/IO 10/10

Feeding was started at the age of 30 days. Sensitization was done at the age of 60 days (*) or 3 days after the last feeding. Controls are the same as shown in Table III. Numerator denotes the number of animals giving a positive reaction and denominator, the number of animals tested. TTiters: I, IO. $-Titers: I, 5. 5.

days after the last feeding with the pertussis and horse serum. As seen in Table IV, complete unresponsiveness attained after 4 wk of feeding is maintained with continued feeding. An additional group of 30-day-old rats were fed 2.5 ml of horse serum in one day, once a week for 4 wk. In contrast to the rats fed horse serum almost daily for 4 wk, these rats did not achieve complete unresponsiveness, although the total antigen consumed was the same as that of the daily fed rats. Compared to controls, however, there is a decrease of sensitivity, in that only 2 out of 12 showed HCA response as compared to 8 out of 10 of controls. This suggests that unresponsiveness attained by prefeeding can be of varying degrees and can best be achieved by more frequent feeding. The antigen-fed rats (in Tables III and IV not including rats fed starting at the age of 5 mo) which showed hypotension on horse serum challenge were different from the control rats in that: (1) the percent drop of mean blood pressure (45.2 t SE 2.2, n = 15) was significantly (p < 0.001) less than the control rats (68.3 t SE 3.9, n = 15); and (2) these rats had a partial recovery of blood pressure within 30 to 60 min after horse serum challenge. The rats fed for 4 wk starting at the age of 5 mo behaved similarly to the control rats. The percent drop of mean blood pressure was 67.8 ? SE 5.3 (n = 4) with no signs of recovery within 60 min. While all rats showing HCA by positive PCA reaction manifested shock on direct challenge, some rats without detectable levels of HCA became hypotensive upon direct antigen challenge. This was noted, however, only in rats which received the horse serum simultaneously with B. pertussis. Antigen alone did not induce this state of exquisite sensitivity. Intravenous antigen challenge, therefore, is a more sensitive gauge of the state of animal sensitivity, especially where levels of HCA are low. This might be due to

TABLE V. Effect of cessation feeding on homocytotropic anaphylaxis

of horse serum antibody and

Time of sensitization after feeding is stopped Experimental condition

3 days

1 mo

2mo

3mo

Horse serum-fed 0111 O/IO

PCA Anaphylaxis

O/8

2l8*

l/5"

O/8

616

S/5

6110 8/8

314 212

214-k 4l4i

Controls PCA Anaphylaxis

8110 loll0

Feeding was started at the age of 30 days for 4 wk and time of sensitization was as shown above. Controls are the same as shown in Table III, except the data for 2 mo. Numerator denotes the number of animals giving a positive reaction and the denominator, the total number of animals tested. *Titers = 1. tAge of rats is 6 mo.

the increased sensitivity of the animal to histamine effects following the administration of B. pertussis.8 Effect of terminating

feeding

In rats fed starting at the age of 30 days for 4 wk, feeding of horse serum was stopped and their ability to become anaphylactically sensitized to horse serum was tested 3 days, 1, 2, and 3 mo later. Unresponsiveness was maintained without further feeding for 1 mo, but ability to become anaphylactitally sensitized was regained in all animals tested 2 mo later. Percent drop in mean blood pressure was 53.7 -+ SE 2.6 (n = lo), significantly (p < 0.05) different from the controls. In addition, in contrast to controls those rats showed partial recovery in blood pressure within 60 min. HCA formation, however, was present only in 20% of the rats after 2 mo (Table V).

188 David

DISCUSSION How an organism copes with the antigenic bombardment it receives from the gastrointestinal tract daily is not known. It appears, nevertheless, that initial presentation of antigen to an organism via feeding markedly alters the immunologic response of the organism to that specific antigen.4-6 Our experiments extend earlier observations and demonstrate that the induction of HCA formation and of anaphylactic sensitization to complex soluble antigens such as ragweed and horse serum can be prevented specifically by pre-feeding antigen. There is ample evidences-” that antigen given orally stimulates an immunologic response primarily of the IgA type. Walker, Isselbacher, and Blockl*, l3 further demonstrated that such specific antibody on the mucosal surface complexes rapidly with subsequently administered antigen, enhancing antigen breakdown and diminishing uptake by the small intestine. The almost universal IgE response to parasitic infestations has been suggested to result in a localized anaphylaxis allowing the access to parasites of other immunoglobulins and immunocytes facilitating worm expulsion. 14,l5 Antibody responses in the gastrointestinal tract, therefore, appear to protect an organism by facilitating expulsion of extraneous substances which are potentially harmful to the organism. Despite this mechanism for clearing excess antigens, significant amounts of functionally intact macromolecules gain access systemically. 16* l7 Furthermore, during inflammatory disease processes in the gut, this absorption of antigenically intact molecules is enhanced.is The Kupffer cells of the liver may play a significant role in the trapping and processing of these gut-derived antigens.ls, 2o These cells take up antigen avidly, especially if complexed with antibody,*l but, unlike other macrophages of the reticuloendothelial system, do not seem to process this antigen for antibody synthesis.** Blockade of the Kupffer cells by carbon particles23 or by disease24was shown to lead to increased antibody production. Presumably this is because of the impairment of hepatic clearance of immunogenic molecules and increased availability of these to other lymphoid organs.*l The normal functioning of the liver, therefore, appears to be of prime importance to the organism in allowing it to deal with antigens in a manner least deleterious to itself. One of the factors shown in our experiments to have a critical effect on attaining unresponsiveness is the age when feeding is started. Unresponsiveness could be attained after 4 wk of feeding if this was started at the age of 1 mo, but rats fed starting at the

J. ALLERGY

CLIN. IMMUNOL. SEPTEMBER 1977

age of 2 mo or 5 mo sensitized anaphylactically, although showing no detectable HCA antibody. In the newborn, there is not only an enhanced selective absorption of specific antibody molecules through the w t 25, 26 but also an increased permeability to macromolecules in general. 27The ease of inducing unresponsiveness to anaphylactic sensitization by starting feeding antigen to the younger animal may be due to a persisting relative permeability of the gut to antigen, allowing concentrations tolerogenic to reactive cells to build up,** or to an increased capacity of the Kupffer cells in the younger animal in rendering antigens nonimmunogenic, or to the persistence of suppressor substances in such animals.2s, 3o In the unresponsive rats, the lymphoid cells capable of reacting and inducing anaphylactic sensitivity are not deleted, since, if feeding is stopped for 2 mo, the animals regain their ability to be made anaphylactically sensitized to the same antigen. On the other hand, as long as antigen is continued to be fed no sensitization can be induced. Results also suggest that the administration of small but frequent doses of the antigen leads to a more complete state of unresponsiveness than the feeding of large amounts at longer intervals. The continuous presence of antigen in the organism in one form or another was not specifically looked for in our experiments but may be a prerequisite for unresponsiveness. These are compatible with current ideas of tolerance, that induced unresponsiveness to foreign antigens is the result of a delicate balance in the complex interaction of antigen, reactive cells (T, B, and macrophages),31, 32and the products of such interactions33, 34favoring unresponsiveness. While our data demonstrate merely the lack of stimulation of IgE-class antibodies, by pre-feeding antigen, Thomas and Parrott have shown that feeding BSA leads to a decreased ability of the animal subsequently immunized parenterally to form humoral antibodies to BSA. The present studies clearly demonstrate that the prevention of immediate hypersensitivity states is possible by pre-feeding antigen. Although some factors that have a bearing on the phenomenon have been delineated, the feasibility of attaining unresponsiveness in the adult and in the anaphylactically sensitized animal remains to be investigated. The potential of feeding antigen as a prophylactic measure in the management of human allergic diseases cannot be ignored. I express my appreciation to Mr. Brian Neal and Mrs. May Tasconafor their technical assistance.

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REFERENCES I. Levine, E., and Vaz, N. M.: Effect of combination of inbred strain, antigen dose in immune responsiveness and reagin production in the mouse, Int. Arch. Allergy. 39:156, 1970. 2. Jarrett, E. E., Haig, D. M., McDougall, W., and McNulty, E.: Rat IgE production. II Primary and booster reagin antibody responses following intradermal or oral immunization. Immunology 30:67 1, 1976. 3. Bazin, H , and Platteau, B.: Production of circulatory reaginic (IgE) antibodies by oral administration of ovalbumin in rats, Immunology 30:679, 1976. 4. Wells, H. G., and Osborne, T. B.: The biological reactions of the vegetable proteins, J. Infect. Dis. 8:77, 191 I. 5. Chase, M. W.: Inhibition of experimental drug allergy by prior feeding of the sensitizing agent, Proc. Sot. Exp. Biol. (N. Y.) 61:257, 1946. 6. Thomas. H. C., and Parrott, M. V.: The induction of tolerance to a soluble protein antigen by oral administration, Immunology 27:631, 1974. 7. Heremans, J.: Antigen titration by simple radial immunodiffusion in plates, in Williams, C. A., and Chase, N. W., editors; Methods in immunology and immunochemistry, New York, 197 I, Academic Press, Inc., vol. 3, p. 213. 8. Malkiel, S., and Hargis, B. J.: Histamine sensitivity and anaphylaxis in pertussis vaccinated rat, Proc. Sot. Exp. Biol. Med. 81:689, 1952. 9. Ogra, P. L., Karzon, D. T., Righthand, F., and MacGillivary, M.: lmmunoglobulin response in serum and secretions after immunization with live and inactivated polio vaccine and natural infection, N. Engl. J. Med. 279~893, 1968. IO. Crabbe, P. A., Nash, I). R., Bazin, H., Eyssen, H., and Heremans, J. F.: Antibodies of the IgA type in intestinal plasma cells of germfree mice after oral or parenteral immunization with ferritin, J. Exp. Med. 130~723, 1969. Il. Ogra, P. L., and Karzon, D. T.: Distribution of polio virus antibody in serum, nasopharynx and alimentary tract following segmental immunization of lower alimentary tract with polio vaccine, J. Immunol. 102:1423, 1969. 12. Walker, W. A., Isselbacher, K. J., and Block, K. J.: Immunologic control of soluble protein absorption from the small intestine A gut surface phenomen, Am. J. Clin. Nutr. 27: 1434, 1974. 13. Walker, W. A., Isselbacher, K. J., and Block, K. J.: Intestinal uptake of macromolecules. III. Studies on the mechanism by which immunization interferes with antigen uptake, J. Immunol. I15:854, 1975. 14. Barth, E. E. E., Jarrett, W. F. H., and Urquhart, G. M.: Studies on the mechanism of self-cure reaction in rats infected with Nipposrrungyius bruziliensis, Immunoiogy 10~459, 1966. 15. Ogilvie, B. M., and Love, R. J.: Cooperation between antibodies and cells in immunity to a nematode parasite, Transplant. Rev. 19:147, 1974. 16. Walker, W. A., Cornell, R., Davenport, L. M., and Isselbather, K. J.: Marcromolecular absorption: Mechanism of horseradish peroxidase uptake and transport in adult and neonatal rat intestine, J. Cell. BioI. 54: 195, 1972. 17. Rotheberg, R. M., Kraft, S. C., Farr, R. S., Kriebel, G. W., and Goldberg, S. S.: Local immunologic responses to ingested

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

3 I.

32.

33. 34.

formation

and anaphylactic

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