Citation
Studies of induced respiratory pollenosis in the dog

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Title:
Studies of induced respiratory pollenosis in the dog
Creator:
Faith, Robert Earl, 1942-
Publication Date:
Language:
English
Physical Description:
xi, 100 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Allergens ( jstor )
Allergies ( jstor )
Antibodies ( jstor )
Antigens ( jstor )
Dogs ( jstor )
Histamines ( jstor )
Pollen ( jstor )
Rabbits ( jstor )
Skin ( jstor )
Skin tests ( jstor )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF ( mesh )
Dogs ( mesh )
Immunology and Medical Microbiology thesis Ph.D ( mesh )
Respiratory Hypersensitivity ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1979.
Bibliography:
Bibliography: leaves 88-99.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Earl Faith.

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University of Florida
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University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
000896508 ( ALEPH )
25605343 ( OCLC )
AEK5138 ( NOTIS )

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Full Text












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




STUDIES OF INDUCED
RESPIRATORY POLLENOSIS IN THE
DOG
By
ROBERT EARL FAITH, JR.
A'DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR 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. Kessler, 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.
iii


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
iv


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
v


LIST OF FIGURES
Figure 1
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
Response of a normal dog to bronchial challenge with 1 mg hist
amine. A represents changes in parameters of respiratory
function. = respiratory resistance; O = tidal volume;
A = dynamic compliance; A = peak expiratory flow rate;
= respiratory rate. B represents changes in arterial
blood gases. = pC02 and A = p2 .
Figure 3
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
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
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
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
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.
vi
Page 13
Page 20
Page 22
Page 24
Page 26
Page 28
Page 30


Page 32
Figure 8
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 Page 37
Response of 2 minimally responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. = respiratory resistance;
O = tidal volume; A = dynamic compliance; A = peak
expiratory flow rate; and H = respiratory rate.
Figure 10 Page 39
Response of 2 medially responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. Symbols as figure 9.
Figure 11 Page 41
Response of a highly responsive dog to bronchial challenge
with 4.75 mg protein nitrogen SPE. Symbols as in figure 9.
Figure 12 Page 44
Correlation of bronchial sensitivity to SPE with skin sensi
tivity to SPE. = individual animal and = mean bronchial
response as a given skin sensitivity level. The overall
trend was significant at the p<0.10 level.
Figure 13 Page 46
Response of animal RW490 to bronchial challenge with 4.75 mg
protein nitrogen SPE. = respiratory resistance; O =
tidal volume; = dynamic compliance; A = peak expiratory
flow rate; and El = respiratory rate.
Figure 14 Page 51
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.
Resting pulmonary function values were:
Ra Vt Cd PEFR
1.27 0.59 0.15 1.81
1.40 0.68 0.25 2.10
RR
5
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 h of third peak off DEAE, E ascending second
\ of third peak off DEAE, F descending third \ 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.
IX


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
x


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.
xi


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
1


2
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
l
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


3
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 (25-38). 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


4
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


5
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


6
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


7
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 (naturally sensitive dogs or passively sensitized dogs)
has been utilized to investigate the physiology of the allergic re
sponse (cutaneous, systemic and respiratory) (2,55,61,64,65,73,74,77,
85), the effect of pharmacologic agents on the allergic response (55,65),
changes ir 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 allergsrs (89-92).
It has also been shown that atopic dogs can be sensitized 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 ejt _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 ej: al. (88) produced positive respiratory
responses following bronchoprovocation in dogs with induced sensitivity
to 2,4-dinitrobenzene. This model system of induced hypersensitivity


8
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 Biologies
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
9


10
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
i o
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
i o
0.4 mg pollen/treatment/animal) and jB. 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


11
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 Pi* 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 Pi 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


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 1




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 yg 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% CO2, 20% O2 and 75% N2.
Peak inspiratory pressure during nebulization was 25cm H2O 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 PO2, and PCO2 and pH were determined with a blood gas analyzer
(model 113 Blood Gas Analyzer, Instrumentation Laboratory, Inc.,
Lexington, MA).


15
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 -20C 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 yg 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


16
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-
1 2 5
cribed by McConahey and Dixon (99) with carrier-free I (New England
Nuclear, Boston, MA).
Quantitation of Rabbit Immunoglobulin
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
chlorida, 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 Kessler'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 PO2 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 PO2 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.
18


Figure 2
Response of a normal dog to bronchial challenge with 1 mg hist
amine. A represents changes in parameters of respiratory
function. = respiratory resistance; O = tidal volume; A =
namic compliance; A= peak expiratory flow rate; Q = respira
tory rate. B represents changes in arterial blood gases. Q
pC2 and =p02.


% Change in respiratory functions
I __
O-^jcnK) i\) ui -si O
OaiOcnouiOuiO
03


Figure 3
Response of a normal dog to bronchial challenge with 1 mg hist
amine. A represents changes in parameters of respiratory func
tion. = respiratory resistance; O = tidal volume; A =
dynamic compliance; A = peak expiratory flow rate; SI = res
piratory rate. B represents changes in arterial blood gases.
= pC2 and A = pC>2.


o
o
% Change in respiratory functions
-!jiro k> ui ~sj 6 F3
uiOoiOuiOiOaiO
Respiratory rate
% Change in arterial blood gases
ZZ


Figure 4
Response of a normal dog to bronchial challenge with 50 mg hist
amine. A represents changes in parameters of respiratory func
tion. Q = respiratory resistance; O = tidal volume; A = dy
namic compliance; = peak expiratory flow rate; Q = res
piratory rate. B represents changes in arterial blood gases.
Q = pC2 and = p2.


% Change in respiratory functions
o Respiratory rate
ZT
a
n


Figure 5
Response of a normal dog to bronchial challenge with 50 mg hist
amine. A represents changes in parameters of respiratory func
tion. = respiratory resistance; 0= tidal volume; A =
dynamic compliance; A = peak expiratory flow rate; S3 = res
piratory rate. B represents changes in arterial blood gases.
9 = pC2 and A = p02.


% Change in respiratory functions
9Z


Figure 6
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. =
respiratory resistance; O = tidal volume; A = dynamic comp
liance; A = peak expiratory flow rate; £2 = respiratory rate.
B represents changes in arterial blood gases. = pC02 and
A = p02 .


Post Bronchial Challenge (min.)
% Change in respiratory function
j- - ro i\)
o o ai o 01 o ai
SZ


Figure 7
Response of a normal dog to multiple bronchial challenge with
histamine. The initial challenge was performed with 10 mg
histamine and the subsequent challenge with 15 mg histamine.
A represents changes in parameters of respiratory function.
@ = respiratory resistance; O = tidal volume; A = dynamic
compliance; A = peak expiratory flow rate; Si = respiratory
rate. B represents changes in arterial blood gases. Q = pC2
and A = p2.


Post Bronchial Challenge(min)
O
O
% Change in respiratory functions
>i U] N>
01 o ui o
00
o
o
o
o
05
o
o
00
o
o
o
o
o
IN) W cn 05
o o O o o o
Respiratory rate
0£


Figure 8
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. 9 = respiratory resistance; O = tidal volume;
A = dynamic compliance; = peak expiratory flow rate;
£2 = respiratory rate. B represents changes in arterial
blood gases. @ = pC2 and A= p02.


% Change in respiratory functions
ZZ


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, jet 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
TA3LE I
RESULTS OF SKIN TESTS AND ?K REACTIONS OBTAINED FOR NEONATAL
DOGS SENSITIZED TO PRAIRIE SAGE POLLEN
Animal
Treatment
Skin
PK
PK
Number
Test
Serum
Nasal Nash
363
None
a
neg.
b
neg.
neg.
365
neg.
neg.
neg.
366
neg.
neg.
neg.
318
Bordetella oertussis
neg.
neg.
neg.
321
Sub Q
neg.
neg.
neg.
322
neg.
neg.
neg.
323
neg.
neg.
neg.
326
neg.
neg.
neg.
317
Pollen Suspension
pos.
pos.
neg.
320
Intranasaiy plus
pos.
pos.
neg.
324
B. oertussis Sub Q
pos.
pos.
neg.
327
pos.
pos.
neg.
364
Pollen Suspension
pos.
pos.
neg.
367
Intranasaiy
pos.
neg.
neg.
363
pos.
pos.
neg.
369
pos.
pos.
neg.
a.) Any challenged site showing less chan 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 cm or
larger in diameter were scored as positive.


35
TASLZ II
RESULTS OF SKIN TESTS, PK REACTIONS, AND BRONCHIAL r-V '
OBTAINED IN ADULT DOGS SENSITIZED TO PRAIRIE SAGE POLLEN
Animal
Skin
Skicb
PK
?R
Bronchial
Number
Test
Test
Serum
S.W.C
Challenge
TO 113
d
neg.
None
neg.
e
neg.
neg.
neg.
TO 10 3
neg.
neg.
neg.
neg.
neg.
R32
neg.
neg. c
neg.
neg.
neg.
YW399
neg.
95 Ug1-
neg.
r.eg.
neg.
TOSS
neg.
neg.
neg.
neg.
TO 128
neg.
Bordetalla pertussis
9 5 -g
neg.
neg.
neg.
TW393
neg.
Sub Q
neg.
r.eg.
neg.
neg.
Yy381
neg.
neg.
r.eg.
neg.
neg.
RB341
neg.
neg.
neg.
neg.
neg.
TO 119
neg.
neg.
neg.
r.eg.
neg.
GU316
neg.
Pollen Suspension
9.5 Ug
pos.
r.eg.
neg.
TO25
neg.
Intranasaly plus
9.5ug
pos.
neg.
54
RB311
neg.
B. pertussis Sub Q
neg.
neg.
neg.
neg.
TO23
neg.
Pollen Suspension
0.95ug
pos.
neg.
433
TO 104
neg.
Intranasaly
0.95Ug
neg.
neg.
neg.
GW311
neg.
93Ug
neg.
neg.
neg.
TO83
neg.
0.9 5 Ug
pos.
ae3.
108
G437
neg.
0.95ug
pos.
N.D."'
47
G433
neg.
0.95ug
pos.
N.D.
133
G428
neg.
neg.
neg.
N.D.
N.D.
G422
neg.
0.95ug
pos.
N.D.
52
G427
neg. .
0.95US
pos.
N.D.
neg.
RB39S
0.095Ug
0.095Ug
pos.
N.D.
94
C429
neg.
0.95Ug
pos.
N.D.
neg.
RB2115
neg.
95Ug
neg.
N.D.
neg.
BW326
9 .Sug1
0.95ug
pos.
N.D.
neg.
G431
neg.
0.95ug
pos.
N.D.
neg.
G424
r.eg.
0.95ug
pos.
N.D.
neg.
a. Skin test results prior to animal being placed on study.
b. Skin test results post sensitizing treatment.
c. Results of PK reactions with nasal wash sanles.
d. Percent increase in respiratory resistance following bronchial challenge.
e. Any challenged sice showing less than 5 era diacecer blueing at 15 and 30 minutes
post challenge was scored as negative.
f. Any ?K site showing less than 5 zm diameter blueing at 15 and 3C minutes post
challenge was scored as negative, sites showing blueing 5 m or larger in
diameter were scored as positive.
2. Lowest concentration in ug protein nitrogen/skin test of SPE giving positive
skin test.
h. M.D. indicates not done.
i. Positive skin tests in this column were obtained in animals which were sensitized
as reonates and included here in an effort to obtain bronchially positive animals.


Response of 2 minimally responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. Q = respiratory resistance;
O = tidal volume; A = dynamic compliance; A = peak expira
tory flow rate; and Q = respiratory rate.
Figure 9


% change in respiratory function
LZ


Figure 10 Response of 2 medially responsive dogs to bronchial challenge
with A.75 mg protein nitrogen SPE. = respiratory resistance;
O = tidal volume; A = dynamic compliance; A = peak expira
tory flow rate; and S3 = respiratory rate.


o
Respiratory rate(breaths/min.)


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 E3 = respiratory rate.
Figure 11


Minutes post bronchial challenge
% change in respiratory functions
j. ro
o cn o o
300H


42
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 yg 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.


Correlation of bronchial sensitivity to SPE with skin sensi
tivity to SPE. 9 = individual animal and = mean bronchial
response at a given skin sensitivity level. The overall trend
was significant at the p<0.10 level.
Figure 12


% Increase In Respiratory Resistance
Following Bronchial Challenge
c/>
O
§
IS
e



Figure 13 Response of animal RW490 to bronchial challenge with
4.75 mg protein nitrogen SPE. = respiratory resis
tance; O = tidal volume; = dynamic compliance;
= peak expiratory flow rate; and El = respiratory
rate.


% change in respiratory functions
i i r\) .p> CT> 00 O
O S l N o o O O O
-O'
O'


47
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 56C
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 I 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


TABLE III
48
EFFECT OF ADSORPTION WITH ANTI-HUMAN IgE ON ?K
TITERS OF DOG ANTI-SPE SERUM
?. K. Titers3
Animal
Preadsorption
Postadsorp
TW325
512
3
YW323
2043
256
YW383
1024
32
RB4S0
4096
256
G433
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 mm or greater diameter blueing at
15 and 30 minutes post challenge was scored as positive.


49
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


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.
= respiratory resistance; O = tidal volume; = dynam
ic compliance; = peak expiratory flow rate; and Ei = re
spiratory rate.
Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 1.27 0.59 0.15 1.81 5 '
B 1.40 0.68 0.25 2.10 7




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


Day of
Study o
14 28 42 56
// 1
176
Bronchial Passive Bronchial Bronchial Passive
challenge ab. challenge challenge ab.
(rabbit) (dog)
followed by followed by
bronchial bronchial
challenge challenge
Bronchial
challenge


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 (a 1:30 dilution bound 50% of labeled SPE). = res
piratory resistance; O = tidal volume; = dynamic com
pliance; ^ = peak expiratory flow rate; and 0 = respira
tory rate.
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


% change in respiratory functions
0
Respiratory rate (breath/min.)


Figure 17 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; O = tidal volume;
D = dynamic compliance; A = peak expiratory flow
rate; and El = 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


change in respiratory functions
57
Minutes post bronchial
chai lenge


Figure 13
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; O = tidal
volume; = dynamic compliance; A. = peak expiratory
flow rate; and El = 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


% change in respiratory functions
59
chai lenge


Figure 19 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) 0= respiratory resistance;
O = tidal volume; CD dynamic compliance; A =
peak expiraotry flow rate; and EH = 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


% change in respiratory functions
61
chai lenge
Respiratory rate (breaths/min.)


Figure 20 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. Q = respiratory resistance
O = tidal volume; = dynamic compliance; A = peak
expiratory flow rate; and Si = respiratory rate.
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


% change in respiratory functions
50
25
0
10 20 30 0 10 20 30
Minutes post bronchial challenge
0
Respiratory rate (breaths/min.)


64
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


65
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


66
TABLE IV
SKIN SENSITIVITY OF
SENSITIZED DOGS
TO SAGE POLLEN
EXTRAC
T AND ANTI-IgE
TEST MATERIAL
SPE
YW383
G356
YW3
25
0.95 mg PN/ml
20
18
12a
12
12
14
0.095 mg PN/ml
13
17
10
9
5
7
0.0095 mg PN/ml
11
13
5
6
0
0
0.00095 mg PN/ml
5
7
0
0
0
0
Anti-IgE
undilute
14
13
12
13
10
11
2b
12
13
11
11
11
10
4
12
12
10
11
11
10
8
11
12
9
11
10
8
16
12
10
/
6
9
8
32_
12
11
0
0
7
7
64
9
7
0
0
0
0
128
0
0
0
0
0
0
Extracting
buffer
0
0
0
0
0
0
c
?. K. Reactions + +
a. Skin test results are reported as the diameter of blueing in mm at the
test site.
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 ?. K. reactions in the skin of normal dogs.


TABLE V
SKIN IUACTIONS TO SAGE TOU.EN EXTRACT AND ANTi-IgE IN DOCS WHOSE SKIN SITES HAVE SEEN PREVIOUSLY REACTED
WITH EITHER SAGE POLLEN EXTRACT OR ANTl-IgE
Anima1
Time I*ost
SPE
Reacted
Side**
Anti-IgE
Reacted Side*3
Con t ro1C
P.
K.d
Number
Reaction1*
SPE
f
anti-
-lgEK
SPE
nnti-
lgE
SPE
anti
-IgE
YW 383
12 hours
18
14
16
14
0
0
0
0
17
15
16
19
+
YW383
24 hours
15
16
14
15
14
12
11
13
15
16
15
13
+
YW383
48 hours
13
12
14
15
12
12
12
13
13
12
14
15
+
C356
12 hours
d'1
0
0
0
0
0
U
0
0
0
0
0
0356
24 hours
0
0
0
0
0
0
0
0
U
0
0
0
-
0356
48 hours
8
11
y
12
12
12
10
1 1
11
10
14
1 1
+
YW 325
12 hours
0
0

U
0
0
0


0
0
0
_
YW 325
24 hours
0
0

0
0
u
0
0
0
0
0
0
-
YW 32 5
48 hours
0
0
0
0
0
0
0
0
0
0
0
0
-
YW 325
72 hours
0
0
0
0
0
0
0
0
u
0
0
0
-
YW325
96 hours
6
7
11
8
7
5
y
11
7
7
ID
8
-
a. Kl^lil aide akin altea were Initially reacted with sage pollen extract,
h. Left side skin sites were initially reacted with antl-lgE.
c. Control skin sites were sites not reacted with either sage pollen extract or antl-lgE before challenge.
d. P. K. reactions were run in the skin of normal dogs using serum samples collected from the experimental
dogs at the time of skin challenges.
e. Time post reaction is that after skin sites were Initially reacted with either sage pollen extract
or antl-lgE.
f. Sage pollen extract used was 0.0J5 mg.
g. Antl-lgE used was at a 1:8 dilution.
h. Skin test results are recorded as mm of blueing.


68
was fractionated by means of anion exchange chromatography on DEAE-
cellulose. Wien eluted with a linear NaCl 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.


Figure 21 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 0.4 M Nacl
in 0.015 M tris).


Optical density (280mju)
Conductivity (juMHOSxlO3)


Figure 22 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 h of third peak off DEAE; E, ascending second
\ of third peak off DEAE: F, descending third h, of third peak
off DEAE; H, trough between third and fourth peaks off DEAE;
and I, fourth peak off DEAE.


72


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


4.8
4.0-
*
E
O
GO
00
co
c
TJ
O
O
CL
O
32-
2.4-
E 16
0.8
20
60 80
Effluent fraction
120
140


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 difference(s) 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
75


76
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 pC02, 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


77
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 (neonates 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


78
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 thah 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


79
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 e_t 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 56C 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


80
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


81
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


82
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


83
clearly indicate that passive "blocking" antibody will inhibit hyper
sensitivity reactions in the dog (cutaneous 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-lgE 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-lgE. These phenomena may be explained in
several ways. In the case of the highly sensitive animals it may be
that the anti-lgE 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-lgE 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-lgE 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-lgE
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.
Osier (148,149) has described the steps in the allergic response as


85
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


86
(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 shoxjn 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).


REFERENCES
1. Caplin, I.: The Allergic Asthmatic, Charles C. Thomas, Springfield,
Ill., 1968.
2. Patterson, R.: Laboratory models of reaginic allergy, Prog. Allergy,
13, 332-407, 1969.
3. Respiratory Diseases: Task Force Report on Problems, Research
Approaches, Needs; The Lung Program, National Heart and Lung
Institute, DHEW Publication No. (NIH) 73-432, 1972.
4. Clausen, C. R., Munoz, J. and Bergman, R. K.: A reaginic type of
antibody stimulated by extracts of Bordetella pertussis in inbred
strains of mice, J, Immunol., 104, 312-319, 1970.
5. Prouvost-Danon, A. and Binaghi, R.: Reaginic antibody in adult
and young mice. Production and biologic properties, Int. Arch.
Allergy Appl. Immunol., 38, 648-656, 1970.
6. Nussenweig, R. S., Merryman, C. and Benacerraf, B.: Electrophor
etic separation and properties of mouse anti-hapten antibodies in
volved in passive cutaneous anaphylaxis and passive hemolysis,
J. Exp. Med., 120, 315-328, 1964.
7. Mota, I. and Peioxoto, J. M.: A skin sensitizing and thermolabile
antibody in the mouse, Life Sci., 5, 1723-1728, 1966.
8. McCamish, J.: A heat-labile skin sensitizing activity of mouse
serum, Nature, 214, 1228-1229, 1967.
9. Prouvost-Danon, A., Peioxoto, J. and Queiroz, J.: Antigen induced
histamine release from peritoneal mast cells of mice production
reagin-like antibody, Immunol., 15, 271-286, 1968.
10. Fairchild, S. S. and Malley, A.: Induction of mouse homocytotropic
antibodies to timothy pollen antigens, J. Immunol., 115, 446-449,
1975.
11. Gerbrandy, J. L. F. and Bienenstock, J.: Kinetics and localization
of IgE tetanus antibody response in mice immunized by the intra
tracheal, intraperitoneal and subcutaneous routes, Immunol., 31,
913-919, 1976.
12. Chang, I. C. and Gottshall, R. Y.: Sensitization to ragweed pollen
in Bordetella pertussis-infected or vaccine-injected mice, J.
Allergy Clin. Immunol., 54, 20-24, 1974.
88


Full Text
UNIV ERSITYOIFFL OR |D A
3 1262 08554 5209


STUDIES OF INDUCED
RESPIRATORY POLLENOSIS IN THE
DOG
By
ROBERT EARL FAITH, JR.
A'DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR 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. Kessler, 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.
iii

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
iv

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
v

LIST OF FIGURES
Figure 1
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
Response of a normal dog to bronchial challenge with 1 mg hist¬
amine. A represents changes in parameters of respiratory
function. O = respiratory resistance; O = tidal volume;
A = dynamic compliance; A = peak expiratory flow rate;
â–  = respiratory rate. B represents changes in arterial
blood gases. © = pC02 and A = pÜ2 .
Figure 3
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
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
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
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
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.
vi
Page 13
Page 20
Page 22
Page 24
Page 26
Page 28
Page 30

Page 32
Figure 8
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 Page 37
Response of 2 minimally responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. © = respiratory resistance;
O = tidal volume; A = dynamic compliance; A = peak
expiratory flow rate; and El = respiratory rate.
Figure 10 Page 39
Response of 2 medially responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. Symbols as figure 9.
Figure 11 Page 41
Response of a highly responsive dog to bronchial challenge
with 4.75 mg protein nitrogen SPE. Symbols as in figure 9.
Figure 12 Page 44
Correlation of bronchial sensitivity to SPE with skin sensi¬
tivity to SPE. © = individual animal and ★= mean bronchial
response as a given skin sensitivity level. The overall
trend was significant at the p<0.10 level.
Figure 13 Page 46
Response of animal RW490 to bronchial challenge with 4.75 mg
protein nitrogen SPE. © = respiratory resistance; O =
tidal volume; â–¡ = dynamic compliance; A = peak expiratory
flow rate; and El = 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.
Resting pulmonary function values were:
Ra Vt Cd PEFR
1.27 0.59 0.15 1.81
1.40 0.68 0.25 2.10
RR
5
7
Page 51
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 h, of third peak off DEAE, E ascending second
\ of third peak off DEAE, F descending third \ 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.
xx

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
x

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.
xi

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
1

2
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
l
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

3
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 (25-38). 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

4
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

5
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

6
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

7
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 (naturally sensitive dogs or passively sensitized dogs)
has been utilized to investigate the physiology of the allergic re¬
sponse (cutaneous, systemic and respiratory) (2,55,61,64,65,73,74,77,
85), the effect of pharmacologic agents on the allergic response (55,65),
changes ir 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 allergsrs (89-92).
It has also been shown that atopic dogs can be sensitized 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 ejt _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 ej: al. (88) produced positive respiratory
responses following bronchoprovocation in dogs with induced sensitivity
to 2,4-dinitrobenzene. This model system of induced hypersensitivity

8
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 Biologies
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
9

10
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
i o
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
i o
0.4 mg pollen/treatment/animal) and 1$. 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

11
Aiudur 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 - Pi 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

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 1


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 yg 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% CO2, 20% O2 and 75% N2.
Peak inspiratory pressure during nebulization was 25cm H2O 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 PO2, and PCO2 and pH were determined with a blood gas analyzer
(model 113 Blood Gas Analyzer, Instrumentation Laboratory, Inc.,
Lexington, MA).

15
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 -20°C 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 yg 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

16
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-
1 2 5
cribed by McConahey and Dixon (99) with carrier-free I (New England
Nuclear, Boston, MA).
Quantitation of Rabbit Immunoglobulin
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
chlorida, 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 Kessler'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 PO2 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 PO2 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.
18

Figure 2
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; A =
namic compliance; A= peak expiratory flow rate; Q = respira
tory rate. B represents changes in arterial blood gases. Q
pCÜ2 and ▲ =p02.

% Change in respiratory functions
I __
O-^jcnrb i>o ui -si o
OaiOcnouiOuiO
03

Figure 3
Response of a normal dog to bronchial challenge with 1 mg hist
amine. A represents changes in parameters of respiratory func
tion. © = respiratory resistance; O = tidal volume; A =
dynamic compliance; A = peak expiratory flow rate; £3 = res¬
piratory rate. B represents changes in arterial blood gases.
© = pCÜ2 and A = pC>2.

o
o
% Change in respiratory functions
Ñ cji i\> k> ui ~sj 6 F3 üi
uiOoiOoiOüiOaiO
Respiratory rate
% Change in arterial blood gases
ZZ

Figure 4
Response of a normal dog to bronchial challenge with 50 mg hist¬
amine. A represents changes in parameters of respiratory func¬
tion. O = respiratory resistance; O = tidal volume; A = dy¬
namic compliance; ▲ = peak expiratory flow rate; Q = res¬
piratory rate. B represents changes in arterial blood gases.
Q = pCÜ2 and ▲ = pÜ2.

% Change in respiratory functions
o Respiratory rate
ZT
a
n

Figure 5
Response of a normal dog to bronchial challenge with 50 mg hist¬
amine. A represents changes in parameters of respiratory func¬
tion. ©= respiratory resistance; 0= tidal volume; A =
dynamic compliance; A = peak expiratory flow rate; O = res¬
piratory rate. B represents changes in arterial blood gases.
9 = pCÜ2 and A = p02.

% Change in respiratory functions
9Z

Figure 6
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. Q =
respiratory resistance; O = tidal volume; A = dynamic comp¬
liance; A = peak expiratory flow rate; £2 = respiratory rate.
B represents changes in arterial blood gases. © = pC02 and
A = p02 .

Post Bronchial Challenge (min.)
% Change in respiratory function
j- - - ro i\)
o oí ai o 01 o ai
SZ

Figure 7
Response of a normal dog to multiple bronchial challenge with
histamine. The initial challenge was performed with 10 mg
histamine and the subsequent challenge with 15 mg histamine.
A represents changes in parameters of respiratory function.
© = respiratory resistance; O = tidal volume; A = dynamic
compliance; A = peak expiratory flow rate; Si = respiratory
rate. B represents changes in arterial blood gases. O = pCÜ2
and A = pÜ2.

Post Bronchial Challenge(min)
o
O
% Change in respiratory functions
â– Nl W N
ui O U1 o
ro
O
O
o
o
05
o
o
03
o
o
o
o
o
- m w oi m
o o O o o o
Respiratory rate
0£

Figure 8
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. 9 = respiratory resistance; O = tidal volume;
A = dynamic compliance; â–² = peak expiratory flow rate;
â–¡ = respiratory rate. B represents changes in arterial
blood gases. @ = pCÜ2 and A= p02.

% Change in respiratory functions
ZZ

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, jet 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
TA3LE I
RESULTS OF SKIN TESTS AND ?K REACTIONS OBTAINED FOR NEONATAL
DOGS SENSITIZED TO PRAIRIE SAGE POLLEN
Animal
Treatment
Skin
PK
PK
Number
Test
Serum
Nasal Nash
363
None
a
neg.
b
neg.
neg.
365
neg.
neg.
neg.
366
neg.
neg.
neg.
318
Bordetella oertussis
neg.
neg.
neg.
321
Sub Q
neg.
neg.
neg.
322
neg.
neg.
neg.
323
neg.
neg.
neg.
326
neg.
neg.
neg.
317
Pollen Suspension
pos.
pos.
neg.
320
Intranasaiy plus
pos.
pos.
neg.
324
B. oertussis Sub Q
pos.
pos.
neg.
327
pos.
pos.
neg.
364
Pollen Suspension
pos.
pos.
neg.
367
Intranasaiy
pos.
neg.
neg.
363
pos.
pos.
neg.
369
pos.
pos.
neg.
a.) Any challenged site showing less chan 5 mm diameter blueing at 15 and
30 minutes post challenge was scored as negative, sices 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 mm or
larger in diameter were scored as positive.

35
TASLZ II
RESULTS OF SKIN TESTS, PK REACTIONS, AND BRONCHIAL CEALLENG
OBTAINED IN ADULT DOGS SENSITIZED TO PRAIRIE SAGE POLLEN
Animal
Skin
Skicb
PK
PR
Bronchial
Number
Test
Test
Serum
S.W.C
Cnallenge
W3113
d
neg.
None
neg.
e
neg.
neg.
neg.
YW3103
neg.
neg.
neg.
neg.
neg.
R32
neg.
neg. c
neg.
neg.
neg.
YW399
neg.
95 Ug1-
neg.
neg.
neg.
YW3S3
neg.
neg.
neg.
neg.
W3128
neg.
Bordetella pertussis
9.5ug
neg.
neg.
neg.
TW393
neg.
Sub Q
neg.
r.eg.
neg.
neg.
YW381
neg.
neg.
r.eg.
neg.
neg.
RB341
neg.
neg.
neg.
neg.
neg.
YW3119
neg.
neg.
neg.
r.eg.
neg.
GU316
neg.
Pollen Suspension
9.5 Ug
DOS.
neg.
neg.
YW325
neg.
Intranasaly plus
9.5ug
pos.
aeg.
54
RB311
neg.
B. pertussis Sub Q
neg.
neg.
neg.
neg.
YV323
neg.
Pollen Suspension
0.95ug
pos.
neg.
433
YW3104
neg.
Intranasaly
0.95Ug
neg.
neg.
neg.
GW311
neg.
95Ug
neg.
neg.
neg.
YW383
neg.
0.9 5 Ug
pos.
ae3.
108
G437
neg.
0.95ug
pos.
N.D."'
47
G433
neg.
0.95ug
pos.
N.D.
133
G428
neg.
neg.
neg.
N.D.
N.D.
G422
neg.
0.95ug
pos.
N.D.
52
G427
neg. .
0.95US
pos.
N.D.
neg.
RB39S
0.095Ug
0.095Ug
pos.
N.D.
94
C429
neg.
0.95Ug
pos.
N.D.
neg.
RB2115
neg.
95Wg
neg.
N.D.
neg.
BH326
9 .Sug1
0,95ug
pos.
N.D.
neg.
G431
neg.
0.95ug
pos.
N.D.
neg.
G424
r.eg.
0.95ug
pos.
N.D.
neg.
a. Skin test results prior to animal being placed on stud'/.
b. Skin test results post sensitizing treatment.
c. Results or PK reactions with nasal wash sanóles.
d. Percent increase in respiratory resistance following bronchial challenge.
e. Any challenged sice showing less than 5 era diacecer blueing at 15 and 30 ninutes
post challenge was scored as negative.
f. Any ?K site showing less than 5 zm diameter blueing at 15 and 3C minutas post
challenge was scored as negative, sites showing blueing 5 m or larger in
diameter were scored as positive.
g. Lowest concentration in ug protein nitrogen/skin test of 3?E giving positive
skin test.
h. M.D. indicates not done.
i. Positive skin tests in this column were obtained in animals which were sensitized
as reonates and included here in an effort to obtain bronchiaily positive animals.

Response of 2 minimally responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. Q = respiratory resistance;
O = tidal volume; A = dynamic compliance; A. = peak expira¬
tory flow rate; and Q = respiratory rate.
Figure 9

% change in respiratory function
LZ

Figure 10 Response of 2 medially responsive dogs to bronchial challenge
with 4.75 mg protein nitrogen SPE. © = respiratory resistance;
O = tidal volume; A = dynamic compliance; A = peak expira¬
tory flow rate; and S3 = respiratory rate.

o
Respiratory rate(breaths/min.)

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 El = respiratory rate.
Figure 11

Minutes post bronchial challenge
% change in respiratory functions
j- , — ro
o cn o o
300H

42
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 yg 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.

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

% Increase In Respiratory Resistance
Following Bronchial Challenge
c/>
O
§
IS
e

Figure 13 Response of animal RW490 to bronchial challenge with
4.75 mg protein nitrogen SPE. © = respiratory resis¬
tance; O = tidal volume; â–¡ = dynamic compliance;
▲ = peak expiratory flow rate; and £1 = respiratory
rate.

% change in respiratory functions
O'

47
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 56°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

TABLE III
48
EFFECT OF ADSORPTION WITH ANTI-HUMAN IgE ON ?K
TITERS OF DOG ANTI-SPS SERUM
?. K. Titers3
Animal
Preadsorption
Postadsorp
YW325
512°
3
YW323
2043
256
YV383
1024
32
RB490
4096
256
G433
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 mm or greater diameter blueing at
15 and 30 minutes post challenge was scored as positive.

49
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

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.
© = respiratory resistance; O = tidal volume; □ = dynam¬
ic compliance; ▲ = peak expiratory flow rate; and H = re¬
spiratory rate.
Resting pulmonary function values were:
Ra Vt Cd PEFR RR
A 1.27 0.59 0.15 1.81 5 '
B 1.40 0.68 0.25 2.10 7

75
50
25
O
25
50
â– 75
Minutes post bronchial challenge

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

Day of
Study o
14 28 42 56
// 1
176
Bronchial Passive Bronchial Bronchial Passive
challenge ab. challenge challenge ab.
(rabbit) (dog)
followed by followed by
bronchial bronchial
challenge challenge
Bronchial
challenge

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 (a 1:30 dilution bound 50% of labeled SPE). © = res¬
piratory resistance; O = tidal volume; □ = dynamic com¬
pliance; ^ = peak expiratory flow rate; and 0 = respira¬
tory rate.
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

Minutes post bronchial challenge
% change in respiratory functions
Respiratory rate (breath/min.)

Figure 17 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; O = tidal volume;
D = dynamic compliance; A = peak expiratory flow
rate; and El = 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

change in respiratory functions
57
Minutes post bronchial
chai lenge

Figure 13
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; O = tidal
volume; â–¡ = dynamic compliance; A = peak expiratory
flow rate; and SI = 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

% change in respiratory functions
59
chai lenge

Figure 19 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) . 0= respiratory resistance;
O = tidal volume; CD — dynamic compliance; A =
peak expiraotry flow rate; and ESI = 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

% change in respiratory functions
61
chai lenge
Respiratory rate (breaths/min.)

Figure 20 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. Q = respiratory resistance
O = tidal volume; â–¡ = dynamic compliance; A = peak
expiratory flow rate; and Si = respiratory rate.
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

% change in respiratory functions
50
25
0
10 20 30 0 10 20 30
Minutes post bronchial challenge
0
Respiratory rate (breaths/min.)

64
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

65
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

66
TABLE IV
SKIN SENSITIVITY OF
SENSITIZED DOGS
TO SAGE POLLEN
EXTRAC
T AND ANTI-IgE
TEST MATERIAL
SPE
YW383
G356
YW3
25
0.95 mg PN/ml
20
18
12a
12
12
14
0.095 mg PN/ml
13
17
10
9
5
7
0.0095 mg PN/ml
11
13
5
6
0
0
0.00095 mg PN/ml
5
7
0
0
0
0
Anti-IgE
undilute
14
13
12
13
10
11
2b
12
13
11
11
11
10
4
12
12
10
11
11
10
8
11
12
9
11
10
8
16
12
10
*»
1
6
9
8
32_
12
11
0
0
7
7
64
9
7
0
0
0
0
128
0
0
0
0
0
0
Extracting
buffer
0
0
0
0
0
0
c
?. K. Reactions + + -
a. Skin test results are reported as the diameter of blueing in toa at the
test site.
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 ?. K. reactions in the skin of normal dogs.

TABLE V
SKIN REACTIONS TO SAGE TOU.EN EXTRACT AND ANTl-IgE IN DOCS WHOSE SKIN SITES HAVE SEEN PREVIOUSLY REACTED
WITH EITHER SAGE POLLEN EXTRACT OR ANTI-IgE
Anima1
Time Post
SPE
Reacted
Side*1
Anti-lgE
Reacted Side*3
Con t ro1C
P.
K.d
Number
Reaction12
SPE
f
anti-
-lgEK
SPE
nnti-
lgE
SPE
anti
-IgE
YW383
12 hours
18
14
16
14
0
0
0
0
17
15
16
19
+
YW383
24 hours
15
16
14
15
14
12
11
13
15
16
15
13
+
YW383
48 hours
13
12
14
15
12
12
12
13
13
12
14
15
+
0356
12 hours
o'*
0
0
0
0
0
U
0
0
Ü
0
0
0356
24 hours
0
0
0
0
0
0
0
0
U
0
0
0
-
0356
48 hours
8
11
y
12
12
12
10
1 1
11
10
14
1 1
+
YW 125
12 hours
0
0
o
U
0
0
0
Ü
Ü
0
0
0
_
YW 125
24 hours
0
0
Ü
0
0
U
0
0
0
0
0
0
-
YW 32 5
48 hours
0
0
0
0
0
0
0
0
0
0
0
0
-
YW 12.5
72 hours
0
0
0
0
0
0
0
0
u
0
0
0
-
YW325
96 hours
6
7
11
8
7
5
y
11
7
7
ID
8
-
a. Kl^lil aide akin altes were Initially reacted with sage pollen extract.
L. Left side skin sites were initially reacted with anti-lgE.
c. Control skin sites were sites not reacted with either sage pollen extract or anti-lgE before challenge.
d. P. K. reactions were run in the skin of normal dogs using serum samples collected from the experimental
dogs at the time of skin challenges.
e. Time post reaction is that after skin sites were Initially reacted with either sage pollen extract
or anti-lgE.
I. Sage pollen extract used was 0.0‘J5 mg.
g. Anti-lgE used was at a 1:8 dilution.
h. Skin test results are recorded as mm of blueing.

68
was fractionated by means of anion exchange chromatography on DEAE-
cellulose. Wien eluted with a linear NaCl 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.

Figure 21 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 0.4 M Nacl
in 0.015 M tris).

Optical density (280mju)
Conductivity (juMHOSxlO3)

Figure 22 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 h of third peak off DEAE; E, ascending second
\ of third peak off DEAE: F, descending third h, of third peak
off DEAE; H, trough between third and fourth peaks off DEAE;
and I, fourth peak off DEAE.

72

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

4.8
4.0-
*
E
O
GO
CM
co
c.
TJ
"5
O
CL
O
32-
2.4
E 16
0.8
20
60 80
Effluent fraction
120
140

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 difference(s) 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
75

76
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 pC02, 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 ad. (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

77
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 (neonates 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

78
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 thah 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

79
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 e_t 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 56°C 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

80
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

81
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

82
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

83
clearly indicate that passive "blocking" antibody will inhibit hyper¬
sensitivity reactions in the dog (cutaneous 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-lgE 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-lgE. These phenomena may be explained in
several ways. In the case of the highly sensitive animals it may be
that the anti-lgE 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-lgE 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-lgE 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-lgE
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.
Osier (148,149) has described the steps in the allergic response as

85
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

86
(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 shoxjn 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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53, 530-544, 1977.

BIOGRAPHICAL SKETCH
Robert Earl Faith, Jr., was born March 7, 1942, in El Paso, Texas.
His public school education was received in El Paso where, in January,
1960, he graduated from Stephen F. Austin High School. He received his
undergraduate training at Texas Technological College and the University
of Texas at El Paso. In June, 1965, he was awarded a Bachelor of
Science degree with a Biological Science major and a Chemistry minor by
the University of Texas at El Paso. He then entered Texas A and M
University where he studied Veterinary Medicine. In August, 1968, he
was awarded the degree of Doctor of Veterinary Medicine. In September,
1968, he entered the University of Florida where he pursued training in
laboratory animal medicine in the Division of Comparative Medicine and
graduate studies in the Department of Immunology and Medical Microbiology.
In August, 1971, he was awarded the degree Master of Science. He contin¬
ued his graduate studies in the Department of Immunology and Medical
Microbiology through June, 1974.
In July, 1974, he joined the National Institute of Environmental
Health Sciences where his responsibilities included research in immuno-
toxicology and laboratory animal medicine. In January, 1978, he became
Director of the Biomedical Research Center of Oral Roberts University
School of Medicine.
100

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the/degree oj
Doctor of Philosophy.
Parker A. Small, Jr./
Chairman
Professor of Immunology
and Medical Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Professor and Chairman of
Immunology and Medical Microbiology
enneth I. Berns
I certify that I have read this study and that in my opinion
conforms to acceptable standards of scholary presentation and is
adequate, in scope and quality, as a dissertation for the degree
Doctor of Philosophy. ^ v
it
fully
of
Richard B. Crandall
Professor of Immunology and
Medical Microbiology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality,
Doctor of Philosophy.
as dissertation for the degree of
rue'/ 1
Richard E. W. Halliwell
Associate Professor and Chairman
Department of Medical Sciences
College of Veterinary Medicine
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy. .
Marc Jaeger
Professor of Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy. ./> j
( Jío-yíQ Alvin F. Moreland
Professor of Comparative

This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
June 1979
Dean, College of Medicine
Dean, GradúaSchool

UNIVERSITY .^ FLORIDA
3 1262 08554 5209



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