Studies of induced respiratory pollenosis in the dog

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Studies of induced respiratory pollenosis in the dog
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Thesis:
Thesis (Ph.D.)--University of Florida, 1979.
Bibliography:
Bibliography: leaves 88-99.
Statement of Responsibility:
by Robert Earl Faith.
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Typescript.
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Vita.

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STUDIES OF INDUCED
RESPIRATORY POLLENOSIS IN THE
DOG












By

ROBERT EARL FAITH, JR.


A/DISSERTATION PRESENTED TO
THE UNIVERSITY
IN PARTIAL FULFILLMENT OF
DEGREE OF DOCTOR


THE GRADUATE COUNCIL OF
OF FLORIDA
THE REQUIREMENTS FOR THE
OF PHILOSOPHY


UNIVERSITY OF FLORIDA
1979














This dissertation is dedicated to Parker A. Small, Jr., who has

exhibited the patience of a saint and to Carol, someone who has a very

high regard for education.















ACKNOWLEDGMENTS

The author wishes to express his deep appreciation and thanks to

Dr. Parker A. Small, Jr., and Dr. Jack R. Hessler, who guided him through-

out the course of this investigation. Deep appreciation and thanks are

also extended to Drs. R. B. Crandall, G. E. Gifford, E. M. Hoffman,

R. H. Waldeman and H. J. Wittig for helpful and stimulating discussion

throughout the inception and performance of this investigation.

This investigation was supported by USPHS grants 5-F02-RR52193,

AI-07713 and HL-13749-05.















TABLE OF CONTENTS

PAGE
LIST OF TABLES .................................................. v

LIST OF FIGURES ................................................. vi

ABSTRACT ........................................................ x

INTRODUCTION .................................................... 1

MATERIALS AND METHODS ........................................ 9

Animals ...................................................... 9
Allergens .................................................... 9
Sensitizing Regimen .......................................... 10
Pulmonary Function Evaluation ................................ 10
Methods of Challenge ......................................... 11
Sample Collections ............................................ 14
Prausnitz-Kustner Reactions .................................. 15
Antisera ..................................................... 15
Determination of Sage Pollen Extract Binding Activity ....... 16
Quantitation of Rabbit Immunoglobulin ........................ 16
Fractionation of SPE ......................................... 16
Electrophoretic Analysis of SPE .............................. 16
Determination of Protein Nitrogen Content of SPE ............. 17

RESULTS ........................................................ 18

Response to Bronchial Challenge with Histamine ............... 18
Induction of Sensitivity to Sage Pollen ...................... 18
Observations with Passive Antibody in Neonatal Animals ....... 42
Partial Characterization of the Serum Mediator of Sensitivity. 47
Use of the Model System to Investigate the Role of Specific
Passive Antibody in Bronchial Response ....................... 47
Use of the Model System to Investigate Regeneration Time of
Skin Reactivity ......................................... .... 64
Fractionation of the Allergen (SPE) .......................... 65

DISCUSSION ...................................................... 75

REFERENCES ...................................................... 88

BIOGRAPHICAL SKETCH .............................................. 100















LIST OF TABLES


TABLE I ............................................... .....Page 34
RESULTS OF SKIN TESTS AND PK REACTIONS OBTAINED FOR NEONATAL
DOGS SENSITIZED TO PRAIRIE SAGE POLLEN

TABLE II .............. .............. ....................Page 35
RESULTS OF SKIN TESTS, PK REACTIONS AND BRONCHIAL CHALLENGE
OBTAINED IN ADULT DOGS SENSITIZED TO PRAIRIE SAGE POLLEN

TABLE III .................................................. Page 48
EFFECT OF ADSORPTION WITH ANTI-HUMAN IgE ON PK TITERS OF DOG
ANTI-SPE SERUM

TABLE IV .................................................... Page 66
SKIN SENSITIVITY OF SENSITIZED DOGS TO SAGE POLLEN EXTRACT AND
ANTI-IgE

TABLE V .............................................. Page 67
SKIN REACTIONS TO SAGE POLLEN EXTRACT AND ANTI-IgE IN DOGS
WHOSE SKIN SITES HAVE BEEN PREVIOUSLY REACTED WITH EITHER
SAGE POLLEN EXTRACT OR ANTI-IgE















LIST OF FIGURES


Figure 1 ......................................................... Page 13
Typical polygraph tracing from which physiologic data was
obtained. Of the 5 parameters monitored, 3, tidal volume, peak
expiratory flow rate, and respiratory rate may be read directly
from the chart.

Figure 2 ......................................................... Page 20
Response of a normal dog to bronchial challenge with 1 mg hist-
amine. A represents changes in parameters of respiratory
function. = respiratory resistance; 0 = tidal volume;
Z = dynamic compliance; A = peak expiratory flow rate;
U = respiratory rate. B represents changes in arterial
blood gases. = pCO2 and A = p02.

Figure 3 ......................................................... Page 22
Response of a normal dog to bronchial challenge with 1 mg hist-
amine. A represents changes in parameters of respiratory func-
tions. B represents changes in arterial blood gases. Symbols
as in figure 2.

Figure 4 ......................................................... Page 24
Response of a normal dog to bronchial challenge with 50 mg hist-
amine. A represents changes in parameters of respiratory func-
tion. B represents changes in arterial blood gases. Symbols
as in figure 2.

Figure 5 ......................................................... Page 26
Response of a normal dog to bronchial challenge with 50 mg hist-
amine. A represents changes in parameters of respiratory func-
tion. B represents changes in arterial blood gases. Symbols
as in figure 2.

Figure 6 ......................................................... Page 28
Response of a normal dog to multiple bronchial challenge with
histamine. The initial challenge was performed with 1 mg hist-
amine and the subsequent challenge with 3 mg histamine. A re-
presents changes in parameters of respiratory function. B re-
presents changes in arterial blood gases. Symbols as in figure
2.

Figure 7 ......................................................... Page 30
Response of a normal dog to multiple bronchial challenge with
histamine. The initial challenge was performed with 10 mg hist-
amine and the subsequent challenge with 15 mg histamine. A re-
presents changes in parameters of respiratory function. B repre-
sents changes in arterial blood gases. Symbols as in figure 2.







Figure 8 ........................................................ Page 32
Response of a normal dog to multiple bronchial challenge with
histamine. The initial challenge was performed with 50 mg
histamine and the subsequent challenges with 100, 50 and 20 mg
histamine. A represents changes in parameters of respiratory
functions. B represents changes in arterial blood gases.
Symbols as in figure 2.


Figure 9 .......................................................
Response of 2 minimally responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. *= respiratory resistance;
0 = tidal volume; A = dynamic compliance; A = peak
expiratory flow rate; and B = respiratory rate.

Figure 10 ......................................................
Response of 2 medially responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. Symbols as figure 9.


Page 37





Page 39


Figure 11 ...........
Response of a highly
with 4.75 mg protein


responsive dog to bronchial challenge
nitrogen SPE. Symbols as in figure 9.


Figure 12 ......................................................
Correlation of bronchial sensitivity to SPE with skin sensi-
tivity to SPE. S0= individual animal and = mean bronchial
response as a given skin sensitivity level. The overall
trend was significant at the p<0.10 level.


Page


Page 44


Figure 13 ....................................................... Page
Response of animal RW490 to bronchial challenge with 4.75 mg
protein nitrogen SPE. = respiratory resistance; 0 =
tidal volume; ] = dynamic compliance; A = peak expiratory
flow rate; and = respiratory rate.


Figure 14 ......................................................
Response of animal YW383 to bronchial challenge with 4.75 mg
protein nitrogen SPE on day 0 (A) and on day 14 (B) 2 hours
after administration of approximately 590 mg of dog anti-
SPE. Symbols as in figure 13.


Page 51


Resting pulmonary function values were:
Ra Vt Cd PEFR RR
1.27 0.59 0.15 1.81 5
1.40 0.68 0.25 2.10 7

Figure 15 ...................................................... Page 53
Temporal design of experiment to investigate the effect of
passive antibody on the bronchial response to inspired
allergen.


Figure 16 .....................................................
Response of animal G437 to bronchial challenge with 4.75 mg
protein nitrogen SPE on day 0 (A) and on day 14 (B) 2 hours
after administration of approximately 430 mg of rabbit anti-SPE


. Page 55







(a 1:30 dilution bound 50% of labeled SPE). Symbols as in
figure 13.

Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 0.51 0.30 0.62 0.97 8
B 0.43 0.43 0.48 1.06 10

Figure 17 .................................................... Page 57
Response of animal G422 to bronchial challenge with 4.75 mg
protein nitrogen SPE on day 0 (A), day 14 (B), 2 hours after
administration of 65 ml (approximately 550 mg) of a solution
of rabbit anti-SPE (a 1:30 dilution bound 50% of labeled SPE),
day 28 (C), day 42 (D), and day 56 (E), 2 hours after admin-
istration of 50 ml (approximately 475 mg) of a solution of
dog anti-SPE (a 1:40 dilution bound 50% of labeled SPE).
Symbols as in figure 13.

Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 1.54 0.49 0.15 1.60 13
B 1.16 0.37 0.19 1.48 20
C 1.59 0.57 0.24 1.46 8
D 1.42 0.33 0.14 1.09 18
E 1.64 0.42 0.29 0.73 14

Figure 18 .................................................... Page 59
Response of animal YW383 to bronchial challenge with 4.75 mg
protein nitrogen SPE on day 0 (A), day 14 (B), 2 hours after
administration of 62 ml (approximately 530 mg) of a solution
of rabbit anti-SPE (a 1:30 dilution bound 50% of labeled SPE),
day 28 (C), day 42 (D), day 56 (E), 2 hours after administra-
tion of 46 ml (approximately 430 mg) of a solution of dog anti-
SPE (a 1:40 dilution bound 50% of labeled SPE) and day 176 (F).
Symbols as in figure 13.

Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 1.03 0.45 0.18 1.96 8
B 0.72 0.60 0.26 2.02 24
C 1.19 0.45 0.40 1.58 8
D 1.07 0.52 0.25 1.72 9
E 1.18 0.67 0.24 1.15 12
F 1.49 0.46 0.22 0.96 12

Figure 19 .................................................... Page 61
Response of animal RB396 to bronchial challenge with 4.75 mg
protein nitrogen SPE on day 0 (A), day 14 (B), 2 hours after
administration of 79 ml (approximately 670 mg) of a solution
of rabbit anti-SPE (a 1:30 dilution bound 50% of labeled SPE),
day 28 (C), day 42 (D), day 56 (E), 2 hours after administra-
tion of 60 ml (approximately 570 mg) of a solution of dog anti-
SPE (a 1:40 dilution bound 50% of labeled SPE) and day 176 (F).
Symbols as in figure 13.


viii








Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 1.02 0.54 0.46 0.95 16
B 1.78 0.55 0.25 0.97 9
C 1.60 0.68 0.19 1.57 8
D 1.07 0.50 0.33 1.49 20
E 1.34 0.65 0.23 1.53 15
F 1.59 0.75 0.45 1.13 18

Figure 20 .................................................... Page 63
Response of animal G433 to bronchial challenge with 4.75 mg
protein nitrogen SPE on day 0 (A) and day 14 (B), 2 hours
after administration of 65 ml (approximately 600 mg) of a
solution of normal rabbit IgG. Symbols as in figure 13.

Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 0.65 0.54 0.29 1.37 5
B 1.70 0.40 0.18 1.30 26

Figure 21 .................................................... Page 70
Elution profile of sage pollen extract chromatographed on
DEAE-cellulose. After the initial pass-through peak was
eluted with the equilibiating buffer (0.015 M tris) the
column was eluted with a linear gradient (the starting
buffer was 0.015 M tris the elution buffer was 0.4M Nacl
in 0.015 M tris).

Figure 22 ........................................... ............ Page 72
Disc electrophoresis patterns of whole sage pollen extract and
fractions of SPE eluted from DEAE-cellulose. A whole sage
pollen extract, B first peak off DEAE, C second peak off DEAE,
D ascending first 4 of third peak off DEAE, E ascending second
\ of third peak off DEAE, F descending third k of third peak
off DEAE, H trough between third and fourth peaks off DEAE,
and I fourth peak off DEAE.

Figure 23 .................................................... Page 74
Elution profile of sage pollen extract chromatographed on
Sephedex G-25.














Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in
Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy


STUDIES OF INDUCED RESPIRATORY
POLLENOSIS IN THE DOG

By

Robert Earl Faith, Jr.

June, 1979

Chairman: Parker A. Small, Jr., M. D.
Major Department: Immunology and Medical Microbiology

This investigation resulted in the development of an animal model

for the study of respiratory allergic phenomena. A technique for

inducing hypersensitivity to prairie sage pollen (Artemisis gnopheles)

in the dog was developed. The allergen induced skin reactivity in most

dogs and respiratory hypersensitivity in 7 of the 17 animals tested.

As skin sensitivity increased bronchial sensitivity tended to increase

also. The respiratory hypersensitivity resembled naturally occurring

respiratory allergies in both man and dog. The route of allergy induc-

tion resembles the natural route and it is felt that this system pro-

vides a useful model for the study of respiratory allergies.

This model system was used to investigate several aspects of al-

lergic phenomena. Animals in which respiratory hypersensitivity was

induced were used to investigate the role of passive "blocking" anti-

body in respiratory allergy. It was observed that passive antibody will

greatly inhibit the respiratory response to inspired allergen while







completely inhibiting the cutaneous response.

The time required for regeneration of reactivity in skin sites

initially reacted with sage pollen extract of anti-IgE was investigated

by hypersensitive dogs. It was observed that the time required for

regeneration of target organ reactivity was dependent upon the initial

degree of sensitivity of the animal.

Initial, partial characterization of this allergen system was per-

formed. The allergen was found to be divisable into four populations

by anion exchange chromatography or by molecular seive chromatography.

Analytical polyacrylamide gel electrophoresis revealed the allergen to

be composed of at least nine components. The allergen system was found

to be not greatly different from other pollen allergen systems which

have been described.















INTRODUCTION


Respiratory allergies constitute a significant health problem affect-

ing a large portion of the population (1,2). Of the respiratory aller-

gies, asthma is perhaps the most compromising. It is estimated that

active asthma afflicts approximately 4% of the American population and

another 3% have had it previously (3). Although the annual mortality

from asthma is relatively small (about 4000 deaths/year) the disease

accounts for 5% of all chronic disabilities.

Asthma places a large burden on our health care system. Statistics

for the year 1967 (3) illustrate the extent of this burden. In 1967

there were 1,078,000 days of hospital care for asthmatics at a cost of

$83 million and there were 10,181,000 physician visits at a cost of

$81 million. The total direct cost of asthma in 1967 was $243 million.

In addition to the direct effects of asthma, the disease resulted in

17.5 thousand man years lost from work and house keeping in 1967. The

total cost, direct plus indirect, came to a staggering $515 million.

Allergic phenomena have been investigated extensively both in man

and in various animal species, and while there have been a number of

animal models of immediate hypersensitivity described, ranging from

mice to monkeys, no ideal model system has yet been described. The

most popular species for the study of allergic phenomena appear to be

the mouse, the rat, the guinea pig, the rabbit, non-human primates and

the dog.

In many respects mice serve nicely as biologic models. They are







small and relatively inexpensive to obtain and keep. In addition,

inbred strains allow for the study of large genetically uniform pop-

ulations. The immune system of the mouse is perhaps more fully studied

and better understood than that of any other animal. Given these facts,

it would appear that the mouse would lend itself to the study of al-

lergic disease.

Indeed, there have been a number of studies of immediate hypersensi-

tivity responses carried out in mice and mice have been shown to possess

an IgE-like homocytotropic antibody (4,5) as well as the 7S y skin

fixing antibody known for some time (4-9). The discovery of mouse IgE

enhances the usefulness of mice in the studies of allergic disease.

However, there are several disadvantages in the mouse model system when

applied to respiratory allergies. Many of the studies on immediate

hypersensitivity in the mouse have involved antigens other than natural

allergens administered by parenteral routes (not the natural route of

sensitization in respiratory allergy), and the technology does not pre-

sently exist to follow parameters of respiratory function following

bronchial challenge in the mouse. Even considering these drawbacks,

several recent studies have shown the usefulness of the mouse in study-

ing certain facets of respiratory allergic disease. It has recently

been shown that mice can be sensitized to natural aeroallergens by the

parenteral route (10) and can be sensitized by the respiratory route

both to protein antigens (11) and pollen allergens (12). Perhaps the

most interesting of these studies is that of Chang and Gottshall (12)

who induced systemic sensitivity to ragweed pollen by injecting mice

with pertussis vaccine or infecting mice with live pertussis organisms

followed by a series of aerosol treatments with ragweed pollen. One









of these aerosol treatments consisted of simply housing mice in an

environment naturally contaminated with ragweed pollen. Several other

recent studies indicate two areas where mice could be very useful in

the study of respiratory allergies. The first of these two areas

involves specific inhibition or suppression of the formation of re-

aginic antibodies to specific allergens. Several recent studies have

shown promise in this area (13-19). Secondly, mice seem especially

well suited for use in studying the genetics of allergic disease

(20,21).

Rats have been used in some laboratories in recent years to study

immediate hypersensitivity phenomena. The rat offers many of the same

advantages as the mouse as a biomedical model. The main differences

are that the rat is larger than the mouse, there are not as many inbred

strains of rats as mice and the immune system of the rat is not as

thourghly investigated as is that of the mouse.

In recent studies rats have been shown to be able to mount immediate

hypersensitive responses to a number of antigens (including natural

allergens) and the mediating class of antibody has been shown to

resemble IgE (22-24). In addition Von Hout and Johnson (25) induced

homocytotropic antibodies in rats by aerosol exposure to bovine serum

albumin (BSA) in conjunction with i.p. injection of Bordetella pertussis

vaccine. Not only may the rat be sensitized by the respiratory route

but the technology now exists to measure parameters of respiratory

physiology following antigen challenge in the rat (26-38). These studies

have employed intravenous challenge but it should not be too difficult

to devise a method of bronchial challenge. To date, the rat has lent

itself primarily to the study of three areas of allergic disease. These








are the effect of pharmacologic agents on the allergic response (26),

the induction of tolerance or the suppression of the IgE antibody

response to specific antigens (29-32) and studies on the genetic control

of reagin synthesis (33).

The guinea pig was perhaps the first animal model to be used to

study respiratory hypersensitivity, being used for this purpose as early

as 1917 (34). In early experiments it was shown that guinea pigs could

be sensitized by the inhalation of allergens such as horse dander and

pollen (34-36). Ratner (35) showed the induction of asthma-like

reactions in guinea pigs by inhalation of dry pollen. More recently

Popa, Douglas and Bouhuys (37) have shown positive respiratory responses

in guinea pigs sensitized to egg albumin. Other studies have shown that

guinea pigs may be sensitized to various protein antigens and nematode

parasite antigens (38-41). The mediating antibody class in the guinea

pig has been shown to be IgE (40,41). The guinea pig model has been

utilized to study several aspects of respiratory hypersensitivity

including antigen localization in the respiratory tract (39), the effect

of pharmacologic agents on the respiratory response (37,41,42) and

specific and non-specific passive desensitization (43-45).

The rabbit, long popular for immunological studies, has been utilized

to investigate several aspects of immediate hypersensitivity phenomena.

Rabbits have been shown to produce reaginic antibody in response to in-

jection with a number of proteins and hapten-protein conjugates (46-49).

This antibody has been shown to belong to the IgE class of immunoglobulin

(46-49). In addition, it has been shown that rabbits will produce reaginic

antibody in response to nasal instillation of pollen (50). The rabbit

model system has been utilized to investigate the ontogeny of the reaginic








response (51) and the suppression of reagin synthesis by passively

administered specific antibody (52).

Non-human primates have a relatively close phylogenetic relation-

ship to man and therefore are popular models for the study of many bio-

medical phenomena, including allergic responses. Non-human primates

have been shown to produce reaginic antibodies in response to both

naturally occurring and induced parasite infestations (2,3,53-55).

In addition, monkeys have been shown to produce reaginic antibodies as

a response to injection with hapten-protein conjugates (56) and pollen

extracts (57). The reaginic antibody produced in monkeys has been

shown to be of the IgE class (57-59), and so closely resembles human

IgE that it is antigenically cross-reactive with human IgE (57,59).

Non-human primates have lent themselves to the study of several

aspects of respiratory allergic disease. Monkeys actively sensitive

to parasite allergens will respond with a positive bronchial response

when challenged with aerosols of these antigens (53,55,60) thus provid-

ing a model for the study of the respiratory response. Studies have

also shown that monkeys may be sensitized for cutaneous, systemic or

respiratory responses by passive administration of either monkey or

human serum from sensitive individuals (2,55,61,62). This allows for

a model system, that in some respects, is more easily obtainable than

animals actively sensitized to parasites. In addition, when serum from

pollen sensitive humans is utilized to sensitize monkeys the antigenic

system is that normally found in human respiratory allergic disease.

These model systems have been utilized to study the effect of pharmaco-

logic agents on the allergic response (55), the changes in arterial

oxygen tension as a result of respiratory response (61,63) and the








bronchial cellular exudate following respiratory response (53).

The dog has been utilized extensively to investigate allergic

phenomena. Perhaps the most important single factor leading to the

popularity of the dog for these types of studies is the fact that

naturally occurring allergies in dogs are well documented (58,64-70) and

the fact that the dog is the only animal other than man in which atopic

disease is known to occur due to aeroallergens (55). Based on cutaneous

hypersensitivity testing dogs have been shown to have naturally occurring

sensitivities to a wide variety of allergens including pollens, danders,

feathers, house dust and insect allergens (71,72). Clinical allergic

disease has been reported in dogs caused by food allergens (58,66),

parasitic allergens (55,64,73) and pollen allergens (2,55,65,67-70,74-76).

The pollen which most commonly is reported to cause allergic disease in

dogs is that of ragweed (2,55,65,70,74,75,77).

Naturally occurring pollenosis in the dog has been extensively

studied and resembles pollenosis in man (2,65,74,78-80). The serum

mediator of the allergic reaction in the dog has been shown to be IgE

as it is in man (2,80-84). Clinically pollenosis in the dog usually

manifests itself as intensely pruritic dermatitis, conjunctivitis or

rhinitis. The animal may have one or any combination of these symptoms.

More rarely dogs may have pollen allergies which result in asthma. In

addition, dogs which have naturally occurring allergic disease, which

does not include asthma, may be induced to have asthmatic symptoms by

aerosol challenge with sufficient quantities of the offending allergen,

be it pollen (2,55,65) or parasite extract (77).

Naturally occurring allergic disease in the dog offers a model

system which has been utilized to investigate various aspects of the








symptom complex. One factor that has facilitated the use of naturally

sensitive dogs is the ability to passively transfer sensitivity from

naturally sensitive dogs to non-sensitive dogs (2,61,64,74,78). This

model systems naturallyy sensitive dogs or passively sensitized dogs)

has been utilized to investigate the physiology of the allergic re-

sponse cutaneouss, systemic and respiratory) (2,55,61,64,65,73,74,77,

85), the effect of pharmacologic agents on the allergic response (55,65),

changes in arterial oxygen tension as a result of allergic responses

(63) and the clinical management of the disease state (68,70).

In addition to naturally allergic dogs, another source of dogs

with immediate hypersensitivity exists. A number of studies have been

carried out which show that dogs may be induced to produce reaginic

antibody by the injection of various antigens including proteins (86,

87), hapten-protein conjugates (84,86,88) and pollen allergars (89-92).

It has also been shown that atopic dogs can be sensiti>ed to a hapten

by aerosol exposure.to a hapten-pollen conjugate (93). The reaginic

antibody induced in-these studies resembles that occurring in natural

allergies, has been shown to be IgE (84,86,87,89,91,93) and can be

passively transferred to normal dogs (84,86,87,89,93). Challenge

studies in the induced hypersensitive dog have had mixed results.

Systemic (i.v.) and respiratory challenge resulted in negative responses

in the studies of Arkins et al. (89) and Sunthonpalin et al. (91). Dhali-

wal et al. obtained positive systemic responses in dogs with induced sen-

sitivity to ragweed pollen following i.v. challenge with ragweed pollen

extract (92). Finally, Kepron et al. (88) produced positive respiratory

responses following bronchoprovocation in dogs with induced sensitivity

to 2,4-dinitrobenzene. This model system of induced hypersensitivity








has been utilized to study the ontogeny of the allergic immune response

(88).

The animal is not the only important part of the model system. It

was also necessary to give careful consideration to the allergen to be

used in the system. In studying reaginic responses in various animal

species a number of antigens have been utilized including proteins such

as BSA and haptens such as DNP. A limited number of studies have emp-

loyed naturally occurring allergens. It was felt that in developing a

model system, which would simulate naturally occurring allergies, a

natural allergen should be employed. The allergen chosen for these

studies was the pollen of prairie sage (sage pollen). This pollen

was chosen because it is strongly antigenic (94) and natural sensitivity

to it in the dog has been demonstrated (70).

These studies report the development of a model system in which the

induction of skin and bronchial sensitivity to pollen was accomplished.

This model system was utilized to study the role of "blocking" antibody

in respiratory allergies and it was found that passively administered

antibody suppressed the respiratory response to allergen challenge. The

allergen system used was partially characterized and was found to pos-

sibly be somewhat more restricted in its component makeup than other

pollen systems which have been studied.















MATERIALS AND METHODS


Animals

Neonatal (10 days to 6 weeks old at the initiation of study) and

adult mongrel dogs were purchased from, and housed by, the Health Center

Animal Research Department of the University of Florida, Gainesville,

Florida. All of the adult dogs utilized in this study were skin tested

with prairie sage pollen extract before being placed in the study.

Only skin test negative animals were used. Approximately 70% of the

animals tested were skin test negative.

Allergens

The pollen of prairie sage (Artemisia gnopheles), referred to hence-

forth as sage pollen, was chosen for these studies because it is strong-

ly antigenic (94) and is responsible for naturally occurring sensitivity

in dogs (70). Pollen was purchased initially from International Biologics

Inc., Bethany, Oklahoma. The final respiratory challenge was carried

out using pollen from Greer Laboratories, Inc., Lenoir, North Carolina.

The latter product appeared more homogeneous than the former when

examined microscopically. Pollen suspension used in sensitizing treat-

ments was made immediately prior to use. This was done to avoid extrac-

tion of water soluble components from the pollen.

For the majority of experiments, pollen extract was produced by

extracting pollen with phosphate buffered saline (PBS). In initial

studies, pollen was extracted by the method of Coca (95) as described

by Phillips (96). Before use in bronchial challenge this extract was







dialyzed extensively against PBS to remove phenol.

Sensitizing Regimen

The animals were sensitized as shown in Table I and II with sage

pollen in the following manner. Both adult and neonatal animals were

divided as follows: 5 adults and 3 neonates received no treatment: 5

10
adults and 5 neonates received 1.3 x 10 Bordetella pertussis organisms/

treatment subcutaneously (SC) twice weekly for 3 weeks, 3 adults and 4

neonates received pollen suspended in normal saline intranasaly (0.1 to

0.4 mg pollen/treatment/animal) and B. pertussis (1.3 x 10 organisms/

treatment/animal) SC twice weekly for 3 weeks; and 15 adults and 4

neonates received pollen suspended in normal saline intranasaly (0.1 to

0.4 mg pollen/treatment/animal twice weekly for 3 weeks.

Pulmonary Function Evaluation

Resistance of the respiratory airways, dynamic compliance of the

lungs, tidal volume and peak expiratory flow rates were the parameters

used to evaluate pulmonary functions.

The dogs were anesthesized with sodium pentobarbital (Burns-Biotec

Laboratories, Inc., Oakland, CA), intubated and placed in ventral recum-

bancy after which an esophagel balloon catheter was positioned at a point

where the pressure was the most negative. The following parameters were

monitored on a polygraph recorder (Brush Accuchart, Gould, Inc., Cleve-

land, OH): 1) air flow at the end of the tracheal tube with a Fleisch

pneumotachograph and a differential pressure transducer (Stalham PM

285TC); 2) tidal volume by integration of the respiratory flow rates;

and 3) trans-airway pressure (difference between esophagel and tracheal

tube pressure) with a differential pressure transducer.

Respiratory resistance was calculated according to the method of








Amdur and Mead (97) in which the trans-airway pressure differences (P)

at isovolume points (approximately mid volume) during inspiration and

expiration is divided by the sum of the air flow at these two points

(figure 1):

Resistance = P3 P4 cm H20

Vi + V2 L/sec

Dynamic compliance was calculated by dividing the difference in

volume between the 2 points where air flow was zero by the difference

in pressure at these points (figure 1):

Compliance = V2 Vi L

P2 P1 cm H20

Respiratory function was evaluated prior to bronchial challenge and

at 5 to 10 minute intervals, for up to approximately 30 minutes follow-

ing challenge. Values for respiratory resistance, dynamic compliance,

tidal volume and peak expiratory flow rates were determined by averag-

ing calculations made from 15 consecutive respiratory cycles during each

evaluation period. The animals lungs were hyperinflated prior to each

evaluation period in order to prevent atelectasis and maintain a base-

line.

Respiratory resistance was the parameter utilized to determine

whether or not an animal was sensitive. An increase in respiratory

resistance greater than 35% was arbitrarily taken to indicate a posi-

tive bronchial response. Respiratory resistance never increased more

than 8% when normal dogs were challenged with SPE.

Methods of Challenge

One week after the last sensitizing treatment the animals were




































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tested for skin reactivity with varying dilutions of SPE. The animals

were bronchially challenged within 2 weeks after the last sensitizing

treatment.

Skin tests were performed by injection of 0.1 ml of varying dilutions

(950, 95, 9.5 and 0.95 pg protein nitrogen/ml) of SPE intradermally

followed by 1.5 ml of a 1% solution of Evans blue i.v. Skin sites were

observed for blueing at 15 and 30 minutes post-injection. Any skin

sites showing a blueing reaction with a diameter greater than 5mm was

scored as a positive reaction.

Bronchial challenge was accomplished by delivering 1 ml of challenge

material over a 5 minute period through a Bird micro-nebulizer connected

to the endotracheal tube and driven by a Bird Mark VIII respirator

actuated by a pressurized gas mixture of 5% C02, 20% 02 and 75% Nz.

Peak inspiratory pressure during nebulization was 25cm H20 and the rate

was approximately 30 breaths/minute. The respirator and nebulizer were-

removed before recording of physiologic parameters. The animals were

initially challenged with PBS as a control, followed by challenge with

varying concentrations of histamine (Histamine Hcl, Fisher Scientific

Co., Pittsburgh, PA) or approximately 4.75 mg protein nitrogen of SPE.

Sample Collections

Blood samples for serum harvest were collected by femoral veni-

puncture immediately prior to skin testing. Arterial blood samples,

for blood gas determinations, were collected anaerobically via an

intracath inserted percutaneously into the femoral artery. Arterial

blood P02, and PCO2 and pH were determined with a blood gas analyzer

(model 113 Blood Gas Analyzer, Instrumentation Laboratory, Inc.,

Lexington, MA).







Nasal wash samples were collected by washing the nasal passage with

approximately 30 ml of normal saline. These samples were concentrated

10X by vacuum dialysis.

Both serum and nasal wash samples were stored at -200C until used.

Prausnitz-Kustner Reactions

Prausnitz-Kustner reactions (P.K. reactions) were performed in the

skin of normal dogs using undiluted serum and concentrated (10X) nasal

wash fluid. These reactions were run in duplicate in 2 normal dogs (i.e.,

4 skin reactions/sample). The hair was clipped from the ventrolateral

skin and 0.1 ml of the sample to be tested was injected intradermally

into skin sites. Forty-eight hours later (78) these skin sites were

challenged with 0.1 ml of a solution of SPE containing 95 pg protein

nitrogen/ml followed by 1.5 ml of a 1% solution of Evans blue i.v.

Challenged sites were read at 15 and 30 minutes post injection. Sites

showing blueing at a diameter greater than 5mm were scored as positive.

Antisera

Equine antiserum to human epsilon chain (anti-IgE) was purchased

from Kallestad Laboratories, Inc., Chaska, MN. This antisera cross

reacts with canine IgE as shown by producing skin reactions in normal

dogs at dilutions as high as 1:2048 and as reported by Halliwell, Swartz-

man and Rockey (81). Antisera to sage pollen extract was produced both

in rabbits and dogs. Rabbit antisera were induced by injecting rabbits

in multiple subcutaneous sites with approximately 1 mg protein nitrogen

of SPE emulsified in complete Freund's adjuvant. These animals were

boosted at monthly intervals with the same antigen preparation. Canine

antisera were produced by injecting adult mongrel dogs in multiple

subcutaneous sites with approximately 2 mg protein nitrogen of SPE









emulsified in incomplete Freund's adjuvant. These animals were boosted

with the same antigen at approximately 14 day intervals.

Determination of Sage Pollen Extract Binding Activity

The antibody activity in the rabbit and dog anti-SPE was determined

125 125
by the technique of Lidd and Farr (98) using I-labeled SPE. I-SPE

was obtained by trace labeling SPE by the chloramine T method as des-

125
cribed by McConahey and Dixon (99) with carrier-free I (New England

Nuclear, Boston, MA).

Quantitation of Rabbit Immrunoglobulin

Rabbit IgG was measured using radial-immunodiffusion as described

by Mancini, Carbonora and Heremans (100) utilizing goat anti-rabbit

IgG purchased from Microbiological Associates, Bethesda, MD.

Fractionation of SPE

SPE was fractionated by anion exchange chromatography using DEAE

(diethylamionethyl) cellulose (Whatman DE 32, H. Reeve Angel Inc.,

Clifton, NJ) columns and by molecular seive chromatography utilizing

Sephedex-G25 (Pharmacia Fine Chemicals Inc., Piscataway, NJ) columns.

DEAE-cellulose chromatography was performed utilizing columns equili-

brated with 0.015 molar Tris, pH 8.2. These columns were eluted with a

linear sodium chloride gradient. Molecular seive chromatography was

performed in downward flow columns equilibrated with 0.15 molar sodium

chloride, 0.015 molar Tris, pH 7.4.

Electrophoretic Analysis of SPE

Analytical disc electrophoresis was performed on whole SPE and

DEAE-cellulose chromatographic fractions of SPE. Electrophoresis was

carried out on 15% polyacrylamide gels in the presence of 0.1% Triton

X-100 (101).





17


Determination of Protein Nitrogen Content of SPE

The protein of SPE was determined by the Nessler technique using

commercial Nessler's reagent (Fisher Scientific Co., Pittsburgh, Pa).














RESULTS


Response to Bronchial Challenge with Histamine

In order to gain insight into the bronchial sensitivity of dogs to

histamine, to determine the approximate changes in respiratory functions

required to produce changes in arterial blood gases and to evaluate the

bronchial challenge monitoring system, normal dogs were bronchially

challenged with varying concentrations of histamine. Figures 2 and 3

depict the respiratory response of 2 normal dogs to bronchial challenge

with 1 mg histamine. Both animals responded to this challenge with

increases in respiratory resistance (100% and 150% respectively) and

decreases in dynamic compliance. These changes were accompanied by

decreases in arterial P02 and increases in arterial PCO2.

Figures 4 and 5 illustrate the responses of 2 normal dogs to bron-

chial challenge with 50 mg histamine. This challenge resulted in severe

changes in all respiratory functions measured as well as a pronounced

decrease in arterial P02 and increase in PCO2. While the changes in

respiratory function were pronounced they were, with the exception of

dynamic compliance, only about 15 minutes in duration.

In addition to a single bronchial challenge with histamine, 3

normal dogs were challenged with multiple challenges of histamine

(figures 6-8). When these multiple challenges were performed the res-

piratory response was prolonged and pronounced.




























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Induction of Sensitivity to Sage Pollen

The initial goal of these studies was to produce pollen hypersensi-

tivity in dogs by a natural route.

Preliminary uncontrolled studies showed that 5 out of 8 neonates

and 19 out of 21 adult dogs converted from negative to positive skin

tests following intranasal pollen instillation twice weekly for 3 weeks.

An additional group of animals received Bordetella pertussis at the same

time as the intranasal pollen and 14 out of 25 neonates and 16 out of

19 adult dogs converted from negative to positive skin tests. These

results were sufficiently encouraging to be followed by controlled

experiments.

Table I shows the results obtained in neonatal animals. All 8 of

the animals given pollen became positive to skin test, while none of

the control animals became positive. All but 1 of skin test posi-

tive animals had PK positive serum. This animal (367) was only weakly

positive to skin test. Nasal wash PK's were uniformly negative in

contrast to the observation of Patterson, et al. (77) in dogs sensitive

to ascaris but in agreement with their observations in ragweed sensitive

dogs (77).

Table II shows results obtained with adult dogs. As can be seen,

the majority of animals that underwent sensitizing treatment became

sensitive to SPE when measured by skin reactivity or serum PK positivity.

Again, all concentrated nasal wash samples were negative in transferring

skin reactivity. In addition to inducing skin and serum PK positivity

the sensitizing treatments resulted in bronchial sensitivity in 7 of

the 13 animals positive by skin and PK tests. This bronchial sensi-

tivity ranged from a minimal sensitivity to a fairly high degree of

sensitivity. Figures 9-11 illustrate dogs with varying degrees of

bronchial sensitivity.





34















TABLE I




RESULTS OF SKIN TESTS AND PK REACTIONS OBTAINED FOR NEONATAL


DOGS SENSITIZED TO ?RAIRIE SAGE POLLEN


Treatment


Skin
Test


PK
Serum


?K
Nasal Wash


None


Borderella pertussis
Sub Q





Pollen Suspension
Intranasaly plus
B. pertussis Sub Q



Pollen Suspension
Intranasaly


neg.
neg.
neg.
neg.
neg.

pos.
pos.
pos.
pos.


neg.
neg.
neg.
neg.
neg.

pos.
pos.
pos.
pos.


pos. pos.
pos. neg.
pos. pos.
pos. pos.


a.) Any challenged site showing less than 5 mm diameter blueing at 15 and
30 minutes post challenge was scored as negative, sites showing blueing
5 mm or larger in diameter were scored as positive.

b.) Any ?K site showing less than 5mm diameter blueing at 15 and 30 minutes
post challenge was scored as negative, sites showing blueing 5 mnm or
larger in diameter were scored as positive.


Animal
Number


neg.
neg.
neg.

neg.
neg.
neg.
neg.
neg.

neg.
neg.
neg.
neg.

neg.
neg.
neg.
nag.




















TABLE II

RESULTS OF SIKIN TESTS, P< REArCTIOC;S, AN-D SOSC;LAL C2a a ;.
OBTAINED IN ADULT DOGS SESSITIZED TO PRAIRIE SAGE ?OLaLE


Animal Skin
Number Test


Treatment


None


Bordetella certcssis
Sub Q


Pollen Suspension'
Intranasaly plus
B. pertussis Sub Q

Pollen Suspension
Intranasaly


Ski
Test

neg.
neg.
negi,
95 g
neg.

9.5,g
neg.
neg.

neg.
CeZ.


9.5 Lg
9.5 tg
neg.

0.95ug
0.95ug
9513
0.95ug
0.95vg

neg.
0.95ug
0.95vg
0.095lig
0.95yg
95ug

0.95ug
0.95Ug
0.95Ug


P ?K c Sronchiald
Serun S.'. Challenge


neg.
neg.
neg.
neg.


neg.
neg.
neg.

neg.
neg.


neg.
neg.
neg.

reg.

neg.
neg.
neg.

ceg.


pos. res.
pos. neg.
neg. nag.


YW3113
YW3103
R32
YW399
7Y3S8

YW3128
YW393
Y'd381
RB341.
YW3119

Ga316
YW325
RB311

Yr432 8
YW3104
GW311
YW383
G437
G433
G428
G422
0427
RB395
C429
RB2115
81326
G431
G424


neg.
reg.
neg.
neg.
N. D.
S.D.




N.D.
X.3.

S.D.

S.3.
S.D.


neg.
neg.
neg.
neg.
neg.

naeg.
neg.
neg.
nes.
neg.

neg.
54
neg.

433
neg.
neg.
108
47
133
N.D.
52
neg.
9&
neg.
neg.

neg.
neg.
neg.


.a. Skin test results prior to animal being placed on study.
b. Skin test results post sensitizing trea:sent.
c. Results of PK reactions with nasal wash samples.
d. Percent increase in respiratory resistance iolioving bronchial challenge.
e. Any challenged site showing less than 5 nm diameter blueing aC 15 and 30 minutes
post challenge was scored as negative.
f. Any PK site showing less than 5 = dia=ecer blueing at 15 and 30 =inuces -ost
challenge was scored as negative, sites showing blueing 5 =s or larger ia
diameter were scored as positive.
g. Lowest concentration in jg protein nitrogen/skin case of 3?S giving positive
skin test.
h. N1.D. indicates not done.
i. Positive skin tests in this column were obtained in animals whic -ere sensitized
as reonates and included here in an effort to obtain bronchiall- :osi--:e animals.


d
neg.
neg.
neg.
neg.


pos.
neg.
neg.
pos.
pos.
pos.
neg.
pos.
pos.
pos.
pos.
neg.
pos.
pos.
pos.


neg.
neg.
neg.
neg.
neg.

neg.
neg.
neg.

neg.
neg.
neg.
neg.
neg.
neg.
neg.
neg.
neg.
0.095pig
neg.
neg.
9.5ug
neg.
neg.



























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Response of a highly responsive dog to bronchial
challenge with 4.75 mg protein nitrogen SPE.
= respiratory resistance; 0= tidal volume;
A = dynamic compliance; A = peak expiratory
flow rate; and U = respiratory rate.


Figure 11































E


Ioo
100 P
I


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20 a
0i


Minutes post bronchial challenge








One interesting observation made here was the correlation between

skin sensitivity and bronchial sensitivity (figure 12). As the degree

of skin sensitivity increased the probability of bronchial sensitivity

also increased. This trend was significant at the p<0.1 level.

Observations with Passive Antibody in Neonatal Animals

Six neonatal animals were used to investigate the possible effect

of antibody on sensitization. These animals were divided into 2 groups

of 3 each. One group was given the regular sensitizing treatment while

the other group was given passive canine aniti-SPE antibody (1 dose

weekly for 3 weeks, at a level calculated to give the animals a serum

titer capable of binding 50% of a labeled antigen with undilute serum,

1 ml of serum could bind approximately 100 pg SPE) in addition to the

regular sensitizing treatment. One week after the last sensitizing

treatment the animals were skin tested. At this time only 1 animal

from each group was found to be skin test positive.

Approximately 3 months later the animals were again subjected to a

sensitizing regimen with pollen. This time the passive antibody was

omitted. At the end of the sensitizing regimen the animals were rested

for 1 month and then bronchially challenged. The animal which initially

had been skin test positive after being given passive antibody and pollen

was extremely sensitive to bronchial challenge as can be seen in figure

13. When challenged at this time this animal underwent extreme respira-

tory distress. It exhibited extremely rapid and labored breathing. The

animal was treated with epinephrine intravenously and isoproteranol by

nebulization and was maintained on a respirator for several hours. Had

this treatment not been performed the animal would almost certainly have

died as a result of bronchial challenge.



























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Figure 13


Response of animal RW490 to bronchial challenge with
4.75 mg protein nitrogen SPE. = respiratory resis-
tance; 0 = tidal volume; O = dynamic compliance;
A = peak expiratory flow rate; and U = respiratory
rate.


























600 \ -150

---- -125 c'

400 1001\

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2 200 50 -

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o 508

a -25 -

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-75

-100

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This animal was allowed to rest (no further treatment given) for

approximately 10 months. At the end of this time the animal was bron-

chially challenged and found to be negative to this challenge. Ap-

proximately 5 months later it was resensitized by nasal instillation

of pollen suspension twice weekly for 3 weeks. Approximately 6 weeks

later this animal was bronchially challenged and found to be positive

to about the same extent as it had been previously.

Partial Characterization of the Serum Mediator of Sensitivity

Serum samples with positive PK activity were treated in several

ways to partially characterize the mediator of these reactions. Treat-

ment of these serum samples with 2-mercaptoethanol or heating at 560 C

for 4 hours abolished PK activity. In addition, passage of positive

PK serum samples through immunoadsorbent columns (equine anti-human

IgE linked to polyacrylamide beads) greatly reduced the PK titer of

these sera (Table III).

Use of the Model System to Investigate the Role of Specific Passive

Antibody in Bronchial Response

Once having obtained animals positive to bronchial challenge it

was possible to investigate what role specific serum antibody against

the inciting agent might play. Initially 1 animal positive to bronchial

challenge (YW383) was given i.v. 62 ml of a solution containing ap-

proximately 10 mg/ml protein derived from 75 ml of anti-SPE dog serum

as a 33% ammonium sulfate fraction. A 1:30 dilution of this solution

bound 50% of labelled antigen and the amount given to the animal was

calculated to give the animal a serum allergen binding capacity of

50% with undilute serum. Fourteen days after the previous challenge

and 2 hours after administration of passive antibody the animal was





48


Animal
YW325
VW328
YW383
RB490
G433


TABLE III

EFFECT OF ADSORPTION WITH ANTI-HUMAN IgE ON ?K
TITERS OF DOG ANTI-SPE SERUM

?. K. Titersa
Preadsorption Postadsoropionb
512c 3
2048 256
1024 32
4096 256
512 16


a. Titers shown are the mean of at least four determinations of each
sample and are recorded as the reciprocal of the highest twofold
dilution giving a positive reaction.

b. Serum samples were adsorbed by passage through a column of anti-
human IgE covalently bound to polyacrylamide beads.

c. Any challenged site showing 5 am or greater diameter blueing at
15 and 30 minutes post challenge was scored as positive.







bronchially challenged with SPE. As can be seen in figure 14, the

passive antibody greatly inhibited the changes induced by the allergen.

Based on these findings the following study was designed. Anti-

bodies raised in both rabbits and dogs were used to study their effect

on bronchial challenge. It was felt that the use of heterologous and

homologous antisera would allow an animal to be given passive anti-

body twice within a reasonable period of time.

The studies were conducted as shown in figure 15 and involved

1 control and 4 experimental dogs. Figures 16, 17, (a,b & c), 18

(a,b & c) and 19 (a,b & c) illustrate the effect of passive rabbit

antibody on the bronchial response in sensitive animals. As can be

seen this passive antibody greatly reduced the effect of the allergen.

Skin tests of all 4 animals immediately prior to bronchial challenge

(2 hours post passive antibody) were negative. The animals shown in

figures 18c and 19c had regained partial bronchial positivity 2 weeks

post passive rabbit antibody while the animal depicted in 17c was

still negative at this time. All 3 of these animals had between 20 and

55% of the rabbit passive antibody remaining at the time of this chal-

lenge as judged by radialimmunodiffusion. Two weeks later (4 weeks

post passive antibody) these animals regained full bronchial sensitivity

(see figures 17d, 18d and 19d). These animals had also fully regained

their skin sensitivity at this time.

Since it is conceivable that the observed inhibition could have

been a result of rabbit serum and not rabbit antibody to pollen,

normal rabbit serum was given to 1 dog. Figure 20 illustrates the

bronchial response of this animal 2 weeks before and 2 hours after

i.v. injection of a 33% ammonium sulfate fraction of normal rabbit






































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Response of animal G422 to bronchial challenge with
4.75 mg protein nitrogen SPE on day 0 (A), day 14 (B),
2 hours after administration of 65 ml (approximately
550 mg) of a solution of rabbit anti-SPE (a 1:30
dilution bound 50% of labeled SPE), day 28 (C), day
42 (D), and day 56 (E), 2 hours after administration
of 50 ml (approximately 475 mg) of a solution of dog
anti-SPE (a 1:40 dilution bound 50% of labeled SPE).
* = respiratory resistance; 0 = tidal volume;
D = dynamic compliance; A = peak expiratory flow
rate; and U = respiratory rate.


Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 1.54 0.49 0.15 1.60 13
B 1.16 0.37 0.19 1.48 20
C 1.59 0.57 0.24 1.46 8
D 1.43 0.33 0.14 1.09 18
E 1.64 0.42 0.29 0.73 14


Figure 17






































10 20 30



















Figure 18


Response of animal YW383 to bronchial challenge with
4.75 mg protein nitrogen SPE on day 0 (A), day 14 (B),
2 hours after administration of 62 ml (approximately
530 mg) of a solution of rabbit anti-SPE (a 1:30 di-
lution bound 50% of labeled SPE), day 28 (C), day 42
(D), day 56 (E), 2 hours after administration of 46
ml (approximately 430 mg) of a solution of dog anti-
SPE (a 1:40 dilution bound 50% of labeled SPE) and
day 176 (F). = respiratory resistance; 0 = tidal
volume; E = dynamic compliance; A = peak expiratory
flow rate; and U = respiratory rate.


Resting pulmonary function values were:
RA Vt Cd PEFR RR
A 1.03 0.45 0.18 1.96 8
B 0.72 0.60 0.26 2.02 24
C 1.19 0.45 0.40 1.58 8
D 1.07 0.52 0.25 1.72 9
E 1.18 0.67 0.24 1.15 12
F 1.49 0.46 0.22 0.96 12





59












A. B.
Day 0 Day 14
100 2 hrs. post rab. ab.
75
50 -50
25 -25

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25 C. D .c
1 Day 28 Day 42 E
3 100r
> 75-
50- -50
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I E. FC
o Day 56 Day 176
100- 2 hrs. post dog ab.
75-
50- -50
25- -25

-25-


0 10 20 30 0 10 20 30
Minutes post bronchial
challenge


























Response of animal RB396 to bronchial challenge with
4.75 mg protein nitrogen SPE on day 0 (A), day 14 (B),
2 hours after administration of 79 ml (approximately
670 mg) of a solution of rabbit anti-SPE (a 1:30
dilution bound 50% of labeled SPE), day 28 (C), day
42 (D), day 56 (E), 2 hours after administration of
60 ml (approximately 570mg) of a solution of dog
anti-SPE (a 1:40 dilution bound 50% of labeled SPE)
and day 176 (F). *)= respiratory resistance;
o = tidal volume; D = dynamic compliance; A =
peak expiraotry flow rate; and I = respiratory rate.


Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 1.02 0.54 0.46 0.95 16
B 1.78 0.55 0.25 0.97 9
C 1.60 0.68 0.19 1.57 8
D 1.07 0.50 0.33 1.49 20
E 1.34 0.65 0.23 1.53 15
F 1.59 0.75 0.45 1.13 18


Figure 19





61









00 A. B.
75 Day 0 Day 14
50 2 hrs. post rab. ab. 50
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Minutes post bronchial
challenge




























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serum equivalent to the amount of protein received by the animals

which were given passive antibody. This treatment had little if any

effect on the bronchial response of this animal.

Two weeks after the animals had regained bronchial sensitivity

they were given passive dog anti-SPE antibody and challenged bronchially

2 hours later. These animals were given an amount of 33% ammonium

sulfate fraction of dog antibody calculated to give the animal a serum

binding capacity for labeled allergen of 50% with undilute serum.

Figures 17e, 18e and 19e show that dog passive antibody greatly reduced

the bronchial response of the animals when challenged with allergen.

Figures 18f and 19f show that in both animals tested (the only two

survivors) full bronchial sensitivity was recovered by the time they

were next challenged.

These animals were allowed to rest (no further treatment given) for

approximately 1 year. At the end of this time they were bronchially

challenged and found to be negative to this challenge. Approximately

5 months later they were resensitized by nasal instillation of pollen

suspension twice weekly for 3 weeks. Approximately 6 weeks later they

were bronchially challenged and found to be positive to approximately

the same extent that they had been previously.

Use of the Model System to Investigate Regeneration Time of Skin

Reactivity

It is well known that allergens induce the degranulation of sensi-

tized mast cells, causing the release of vasoactive compounds, when they

react with specific antibody fixed to the surface of the mast cells. In

addition it has been shown (102,103) that anti-IgE will induce degranu-

lation of mast cells. Therefore, anti-IgE as well as specific antigen








was utilized to react skin sites in this study.

Initially the 3 animals utilized in this study were skin tested

with varying dilutions of sage pollen extract and anti-IgE to determine

the optimal dilution for reacting the skin sites to be utilized for

determination of regeneration time of reactivity. The results of this

determination are indicated in Table IV. Based on data presented in

this table, skin sites on the right sides of the animals were reacted

with SPE at a concentration of 0.095 mg PN/ml and sites on the left

sides with anti-IgE at a 1:8 dilution. The 3 animals utilized included

one highly sensitive to SPE, one weakly sensitive and the other moder-

ately sensitive.

Following the reaction of skin sites with either SPE or anti-IgE

the same sites and control sites were challenged (2/test/material/

challenge time) with SPE (0.095 mg PN/ml) and anti-IgE (1:8 dilution)

at 12 hours, 24 hours and 24 hour intervals thereafter. In addition,

serum samples were drawn just prior to challenge and used in PK

reactions. As indicated in Table V the highly sensitive animal was

positive to skin test at 12 hours post triggering of skin sites and

remained positive thereafter. The moderately sensitive animal regained

skin sensitivity by 48 hours post triggering of skin sites while the

weakly sensitive animal did not regain skin reactivity until 96 hours

post triggering of skin sites. PK reactivity paralled recovery of skin

sensitivity.

Fractionation of the Allergen (SPE)

In order to gain some insight into the complexity of the allergen

system utilized in these studies, SPE was subjected to fractionation

by anion exchange and molecular seive chromatography. Initially SPE




















SKIN SENSITIVITY OF SENSITIZED DOGS


TABLE IV

TO SAGE POLLEN


EXTRACT AND ANTI-IgE


TEST MIATERIAL
SPE
0.95 mg ?%N/Ml
0.095 mg PN/ml
0.0095 mg ?N/ml
0.00095 ng ?N/ml


Anti-IgE
undilute
2b

4
8
16
32_
64
128


rW383


G356


YW325


20 18 12a 12 12 14


* Extracting
buffer

P. K. Reactions

a. Skin test results
test site.


0 0 0 0


are reported as the diameter


0 0


of blueing


in =n at the


b. The strengths of the anti-IgE used are shown as the reciprocal of the
dilution used.
c. Just prior to testing, serum samples were collected from the animals and
utilized for P. K. reactions in the skin of normal dogs.


































+++ II+ 1 111


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was fractionated by means of anion exchange chromatography on DEAE-

cellulose. When eluted with a linear NaCI gradient the SPE gave

four major fractions (figure 21), a pass through fraction and 3 eluted

fractions. These fractions as well as whole SPE were subjected to

electrophoretic analysis on polyacrylamide gels in the presence of

0.1% Triton X-100. The results of this analysis (figure 22) revealed

SPE to have at least nine components.

Molecular seive chromatogrphy of whole SPE on Sephedex G-25

revealed SPE to have 4 major size distributions of components (figure

23). The first peak off of G-25 was an excluded peak and could there-

fore have been composed of more than one size population. To investi-

gate this possibility concentrated material from this peak was chromato-

graphed on Sephedex G-50. This material migrated through G-50 as a

single, sharp, included peak indicating a single population of molecular

size.

































C .) l




0 PJ 4
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t *IC


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0 0 4* -


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DISCUSSION

The results of the studies reported here show that dogs may be

sensitized to pollen allergens by the respiratory route. The sensiti-

zation route used simulated the natural route of sensitization and the

resulting sensitivity resembled that which occurs naturally. In

addition, the allergen employed was a naturally occurring allergen.

Two possible differences in this model system and the naturally oc-

curring allergic disease are that fairly large doses of allergen were

used to induce sensitivity (i.e., 0.1-0.4 mg/animal/treatment), and the

sensitizing regimen led to sensitivity after only 3 weeks of exposure.

It is felt that this model system may be more useful than some previously

described models for investigating certain facets of the allergic

phenomena such as the differences) between individuals that do and do

not become sensitized; (1) as judged by skin reactions, and (2) as judged

by respiratory reactions. It may also be useful in dissecting the

events which lead to sensitization.

Once the animal species and the allergen were chosen for development

of the model system, one of the initial considerations was whether the

method of Amdur and Mead (97) could be used to monitor and quantitate

changes in respiratory function in the dog as a result of bronchial

challenge. To accomplish this, normal dogs were bronchially challenged

with histamine and parameters of respiratory function were followed.

Challenge of normal dogs with 1 mg aerosolized histamine resulted in

moderate but significant increases in respiratory resistance. This may







indicate that the canine bronchial tree is somewhat less sensitive to

histamine than is that of other animals, at least it is less sensitive

than is the bronchial tree of monkeys. Patterson and Talbot (104)

observed similar changes in rhesus monkeys with much smaller challenge

doses of histamine. When the histamine challenge dose was increased to

50 mg or was given as multiple challenges the changes in respiratory

function were severe. These results showed the technique of Andur and

Mead to be very useful in monitoring the respiratory physiology of the

dog.

The histamine challenges of normal dogs also allowed for the investi-

gation of the correlation of the changes in arterial blood gases with

changes in respiratory resistance. Even the moderate changes in respir-

atory function observed in dogs challenged with 1 mg histamine resulted

in significant changes in arterial p02 and pC02. These changes range

from a 30% to 60% decrease in p02 and pCO2, indicating the animals

were clinically compromised. The results obtained here are similar to

changes in arterial blood gases observed by Patterson and Harris (63)

and Booth et al. (73) following bronchial ascaris challenge in ascaris

sensitive dogs. These results allowed for an indication that arterial

blood gases would change with changes in respiratory function seen in

dogs with induced bronchial sensitivity to sage pollen.

The initial goal of this study was to produce dogs with respiratory

sensitivity. The main desire here was that the model system simulate

the natural state in man, both in route of sensitization and in develop-

ment of respiratory sensitivity. Since man is sensitized to pollens by

inhalation the dogs used here were given whole pollen by nasal instil-

lation. Initial attempts were made to deliver pollen as an aerosol

in a closed chamber. This technique proved to be unsuccessful. The









fact that the pollen was suspended in saline deviates from the natural

state in man but allowed for a manageable means of dosing the dogs and

also allowed for some degree of quantitation of the dose delivered.

After being subjected to the sensitizing regimen 100% of neonates and

87% of adult animals converted from negative to positive skin test.

In addition, a high percentage of these animals (87% of neonates and

82% of adults) developed sufficient quantities of serum reagin to

passively transfer skin sensitivity as demonstrated in PK reactions.

The number of animals which became bronchially sensitive following the

sensitizing treatments was not as high as those sensitive to skin test

or as judged by PK reactivity. Approximately 40% of the adult animals

became positive to bronchial challenge neonatess were not tested for

bronchial sensitivity). This rate of production is satisfactory as it

allows for a readily available source of animals with respiratory hyper-

sensitivity to be utilized in studies of allergic disease.

One interesting observation in these studies was the positive

correlation between the degree of cutaneous and bronchial sensitivity.

As cutaneous sensitivity increased bronchial sensitivity tended to

increase.

In those animals which developed respiratory allergy as a result of

this sensitizing regimen, the respiratory response to challenge was not

often severe when viewed clinically. Based on the histamine challenge

studies in this investigation and bronchial ascaris challenges in ascaris

sensitive dogs (63,73) changes in arterial blood gases probably occurred

in dogs sensitive to sage pollen following bronchial challenge. There

are at least two possible explanations for the somewhat low bronchial

response of dogs. The lung of the dog contains fewer mast cells than









do some other organs of the dog (105) which might account for a reduced

response in this organ. However, this is probably not the case as the

lung histamine content/gram tissue of the dog and man are very similar

(105,106). The other possibility is that the dogs lungs are not as

sensitive to histamine as are the lungs of some other species such as

primates. Indeed the studies of Patterson and Talbot (104) indicate

that this may be true. Regardless, the respiratory response in the

dog is great enough to be quantitated and used in investigations of

factors which suppress respiratory responses.

The canine model of respiratory allergy described here has advan-

tages over previously described canine models. The advantages over

using animals with naturally occurring allergy are that animals with

induced hypersensitivity are more readily available in significant

numbers than are dogs with naturally occurring allergies, and by

actively inducing the hypersensitive state the population of allergic

animals are all sensitive to the same allergen. The advantages of this

model over passively sensitized dogs are the same as for dogs with

naturally occurring allergies. There are several advantages of this

model system to other actively induced hypersensitivity model systems.

Most of the other actively induced systems result only in cutaneous

and/or systemic hypersensitivity whereas this system results in respira-

tory sensitivity. The model system described here utilized a method for

induction of sensitivity which simulates the natural model of induction

of respiratory allergies whereas other actively induced model systems

rely on perenteral injection of allergen for induction of hypersensitivity.,

In addition to bronchial sensitivity, this model system is similar

to naturally occurring human respiratory allergy in several ways. One








major factor which shows canine allergy to be similar to human allergy

is the class of antibody which mediates the reactions. IgE mediates

allergic reactions in man. The antibody class mediating allergic

reactions in the dog has been shown to be IgE (80-84). Kessler et al.

(85) have shown rabbit anti-canine IgE will cause asthma-like reactions

in dogs challenged with aerosols in reversed PK-type reactions. These

authors also show dog reaginic activity to be eliminated by heating

at 560C for 4 hours and by treatment with 2-mercaptoethanol as is

human reaginic activity (107,107). The serum mediator observed in

these studies had the same properties.

Another similarity between the canine respiratory hypersensitive

response and human respiratory allergy is the pharmacological mediators

of the response. Histamine is certainly involved in the response in

both species. In man other mediators such as slow-reacting substance

of anaphylaxis (SRS-A) are also involved in the response (109).

Bronchial challenges with SPE in dogs sensitive to sage pollen tended

to result in reactions which were more prolonged that those resulting

from histamine challenge. This would be consistent with mediators other

than, or in addition to, histamine being involved in this response.

This possibility is borne out by a recent report which describes an

SRS-A-like substance in dog lung (110).

Even in its negative aspects the canine model system has similar-

ities to human allergy. It was stated in the materials and methods

section that approximately 30% of apparently normal dogs skin tested

with SPE, in the process of choosing animals for this study, were found

to be skin test positive. It is not known if these animals were bronch-

ially positive. An analogous situation exists in man. There are








several reports of positive skin tests with a number of challenges in

normal non-atopic people (111-113). The percentage of positivity ranged

from 1.6% to 38.6% for individual allergens. Some normal individuals

had sufficiently high reaginic antibody levels in their serum to pas-

sively sensitize the skin of non-reactive individuals (113). Other

studies have shown that normal non-atopic people can be sensitized to

various allergens by injection of the allergen (114-116). Broncho-

provocation testing was not carried out in these individuals but they

manifested no clinical symptoms during pollen seasons. There are ob-

viously differences between individuals which do and do not develop

allergic disease. Perhaps the canine model system described here could

be useful in gaining insight into these differences.

Once a successful model system had been developed it was used to

investigate several areas of allergic responses that were of interest.

The area of primary interest was the effect of specific "blocking"

antibody on the development of sensitization in the neonatal animal

and on the bronchial allergic response in the adult animal.

There is evidence that feedback inhibition plays a role in regulation

of the immune response. The suppression of the formation of both IgM

and IgG antibodies has been shown to occur when specific passive anti-

body is administered at approximately the same time as antigen injection

(117,118). More recently it has been shown that reagin synthesis is

suppressed in rabbits by sufficient levels of preexisting 7S antibody

(119) or passively administered specific antibody (52). These findings

led to the investigation of the effect of specific passive antibody

on sensitization to pollen in the neonatal dog. Neonatal animals were

utilized for this study for two reasons. First, neonatal rabbits have







been shown to preferentially produce reagins in response to immuniza-

tion with BSA (51), an observation which has now been confirmed in mice

(120) and dogs (88). Secondly neonatal animals being smaller than adults

require smaller amounts of passive antibody to achieve significant

serum titers.

The results of these investigations with neonatal animals are very

difficult to interpret. While only 1 of 3 animals given passive antibody

developed skin sensitivity as a result of passive antibody the same

observation was made in animals not given passive antibody. The neonatal

animal which did develop skin sensitivity after passive antibody and

pollen treatment later developed a strong bronchial response after a

second sensitizing regimen of pollen treatments. This result is not

presently understood. Perhaps this animal was genetically predisposed

to allergic disease or perhaps other factors were responsible for the

observed result. In any case further investigation is necessary to

explain this observation. No insight was gained as to whether specific

passive "blocking" antibody would suppress the respiratory sensitization

in the dog.

The adult, bronchially sensitive dog allowed investigation into the

question of the effect of "blocking" antibody on the respiratory allergic

response. The classical explanation of desensitization resulting from

immunotherapy is the induction of "blocking" antibodies. There is de-

bate in the literature in regard to the role of "blocking" antibody in

allergic states. Much of the available information has been derived

from the study of clinical immunotherapy. Clear-cut answers are often

lacking for a variety of reasons: (1) the data are often subjective

(121-128); (2) many of the studies deal with upper respiratory disease








rather than lower respiratory disease (121-124,126,127,129,130); and (3)

the active desensitization is doing more than merely producing "blocking"

antibody, such as reducing leukocyte sensitivity to allergen induced

histamine release and possible causing allergen binding free IgE thus

blocking mast sensitization, and hence the relative role of "blocking"

antibody is unclear (131-136).

In the study reported here the effect of intravenous "blocking"

antibody on respiratory challenge was investigated. Previous investi-

gations have shown that passive antibody will inhibit respiratory as

well as systemic responses to allergens given intravenously (77,78,137,

138). This finding is to be expected in view of the fact that the

allergen was introduced into the same fluid compartment (intravascular)

as the passive antibody and hence was readily available for binding by

antibody. However, antibodies introduced into the circulation by inject-

tion or active synthesis may be able to bind allergen introduced via

the respiratory route and block its induction of degranulation of sensi-

tized mast cells. In a study which combined both intravenous passive

antibody administration and respiratory route challenge, the authors

state that their results were "suggestive but not conclusive" (137).

Investigations have shown that a portion of the IgG present in respira-

tory secretions originates from serum by transudation (139-142). In dogs

immunized by the intravenous route specific antibody (143,144) and anti-

body producing cells (145) have been shown to appear in bronchoalveolar

spaces. In view of these studies it seems reasonable to assume that if

specific antibody is introduced into circulation in sufficient quantities

enough may find its way to respiratory secretions to block hypersensi-

tivity reactions due to inhaled allergens. The studies reported here









clearly indicate that passive "blocking" antibody will inhibit hyper-

sensitivity reactions in the dog cutaneouss as well as bronchial).

Both heterologous and homologous antisera abolished skin reactivity in

sensitive animals. This is in agreement with earlier findings of others

(77,78,137,138). Most importantly, passive antibody, both hetero-

logous and homologous, greatly suppressed (in some cases completely

abolished) the respiratory allergic response to inspired allergen.

These results demonstrate conclusively that "blocking" antibody will

inhibit the respiratory allergic reaction due to inspired allergen in

the dog.

The canine model of allergic reactions was utilized to gain some

insight into the time required for mast cells or skin sites to regain

reactivity after being induced to release mediators either by specific

antigen or anti-IgE. This area of investigation has relevance in the

syndrome of drug allergies. Some individuals suffer from allergies to

drugs which are utilized to maintain a normal physiologic state (such as

insulin in the case of the diabetic). There have been a number of

cases of insulin allergy in diabetes reported (146,147). In treating

a patient with a drug to which he is allergic it is important to

eliminate the allergic reaction. One approach to this problem is the

process of acute desensitization. When undertaking acute desensitiza-

tion it would be of interest to have an indication of how long it takes

a target organ to regenerate its ability to react to the allergen once

it has been reacted. The model system developed in the study reported

here was used to determine how rapidly skin reactivity is regenerated

in the skin of hypersensitive dogs after skin sites have been initially


reacted.








The results indicate that highly sensitive animals regain sensitivity

very rapidly after reactive cells have been reacted while weakly sensi-

tive animals have a prolonged recovery time of reactivity. Two interest-

ing and unexpected results of this study are shown in Table V. These

are that skin sites in the highly sensitive animal (YW 383) triggered

with anti-IgE took longer to regenerate reactivity than those triggered

with specific antigen and in the two less sensitive animals the initial

treatment of the skin sites rendered normal as well as treated sites unre-

active to SPE as well as anti-IgE. These phenomena may be explained in

several ways. In the case of the highly sensitive animals it may be

that the anti-IgE caused degranulation of all of the mast cells at the

sites whereas the pollen extract fired only those cells with specific

antibody attached. If this is true, mast cells in the sites reacted

initially with anti-IgE might have to regenerate their granules as

well as bind specific antibody to their surfaces. Sites reacted ini-

tially with SPE might have unreacted mast cells remaining which would

simply have to bind specific antibody to their surfaces possibly allowing

for a more rapid recovery of reactivity. In the case of the less sensi-

tive animals, where reactivity of normal skin sites returned at the same

time as in reacted skin sites, it may be that excess pollen extract

and/or anti-IgE entered circulation and inactivated normal skin sites

by systemic desensitization. This is certainly not inconceivable con-

sidering that in each animal 40 skin sites were reacted with anti-IgE

and 40 skin sites were reacted with SPE. The fact that PK activity of

serum tended to return in parallel with skin site reactivity lends

strength to this assumption.

Osler (148,149) has described the steps in the allergic response as








immunoglobulin fixation to target cells, antigenic induction of confor-

mational change in the cell bound immunoglobulin; activation of enzy-

matic systems, and enhanced release of vasoactive compounds. In acute

desensitization the final 3 steps of this scheme occur. In maintenance

of the desensitized state ideally the first step of the scheme is the

one that should be blocked. The data presented here indicate that the

procedure required to block this step will vary with the state of sensi-

tivity of the individual involved.

The final aspect of these studies was an initial characterization

of the pollen allergen utilized. Only the non-dialyzable fraction of

SPE was studied. This limitation was made because this was the portion

of SPE utilized for provocation throughout these studies. When sub-

jected to molecular seive chromatography SPE was shown to have 4 major

size populations. SPE was also shown to have 4 major populations based

on charge when chromatographed by ionexchange chromatography. When

analyzed by analytical disc electrophoresis on 15% polyacrylamide gels

in the presence of 0.1% Triton X-100 SPE was shown to have at least 9

components. Triton X-100 was employed in these studies because initial

electrophorectic analysis without Triton X-100 resulted in poor gel pene-

tration of SPE. It was felt that this problem might be due to aggregation

of SPE components and that a detergent system might help to overcome this

problem. Triton X-100 was chosen as the detergent because it is nonionic

and causes relatively little denaturation of proteins (101).

Some other pollen systems have been shown to have large numbers of

antigenic components. Timothy pollen has at least 18 to 28 antigenic

components (150-152), cocksfoot pollen has at least 18 antigenic com-

ponents (150-153) and birch pollen has at least 8 antigenic components









(154). Identifying pollen extract components by antigenic number is not

comparable to physiocochemical identification by chromatography and elec-

trophoresis and the different methods may lead to identification of

different numbers of components. Based on chromatographic separation

timothy and birch pollens have more components than sage pollen.

Timothy pollen extract can be separated into as many as 16 fractions

by anion-exchange chromatography (150) and birch pollen extract may

be separated into as many as 7 fractions by molecular seive chromato-

graphy and 8 fractions by anion-exchange chromatography (154). The fact

that these two pollen extracts have a larger number of chromatographic

fractions than sage pollen may be due to the fact that these extracts

were produced by methods that homogenized the pollen (releasing more

components) rather than a real difference in the number of components.

In any case sage pollen extract does not appear to be greatly different

than other pollen extracts, although it may be somewhat more limited

in its component make up.

In conclusion the studies reported here have shown that the dog

can be a useful model system for the study of respiratory allergies.

A model system was developed in which a natural allergen was employed,

the natural route of sensitization was employed and respiratory sensi-

tivity resulted. The model system was shown to be useful in the study

of the role of blocking antibody and the regeneration time of target

organ sensitivity. This model system should prove useful in the inves-

tigation of other aspects of allergic disease such as factors leading

to sensitivity. The allergen system was shown to be not greatly dif-.

ferent from some other pollen allergen systems which have been studied.

This model system should allow for an increase in the knowledge of al-





87


lergic phenomena.

A significant portion of these studies has been previously published

(155).















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