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This is to certify that the
thesis entitled

CHARACTERIZATION OF IMMUNE RESPONSE TO SESAME AND
THE EFFECT OF BOILING AND BAKING ON SESAME
ALLERGENICITY IN A MURINE MODEL

presented by

LALITHA NAVULURI

has been accepted towards fulfillment
of the requirements for the

 

 

 

 

Master of degree in Food Science and Human
Science A“ Nutrition
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Date

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CHARACTERIZATION OF IMMUNE RESPONSE TO SESAME AND
THE EFFECT OF BOILING AND BAKING ON SESAME ALLERGENICITY
IN A MURINE MODEL

By

LALITHA NAVULURI

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Food Science & Human nutrition

2006

ABSTRACT
CHARACTERIZATION OF IMMUNE RESPONSE TO SESAME AND
THE EFFECT OF BOILING AND BAKING ON SESAME ALLERGENICITY
IN A MURINE MODEL
By
LALITHA NAVULURI
Food allergy, including sesame allergy, is a growing and significant international
public health problem. The mechanism of sesame allergy is not completely known and a
mouse model to study sesame allergy is unavailable at present. In this study, efforts were
made to address these problems, by comparing the immune response of sesame with
vanilla (a non-allergenic food). Furthermore, a pilot study was conducted to test the
effect of boiling and baking on sesame allergenicity in a mouse model. Two specific
hypotheses were tested. 1) Whereas sesame triggers Type-2 associated immune response
(i.e., increased specific IgE, IgGl antibody levels and eosinophilia), vanilla elicits Type-
1 associated immune response (i.e., elevated specific IgG2a) in mice. 2) Boiling as well
as baking reduces allergenicity of sesame in mouse model.

We found that exposure of mice to sesame via intraperitoneal (i.p.) or
epicutaneous route resulted in significantly elevated specific IgE, IgGl as well as IgG2a
antibody responses relative to vanilla. Exposure of mice to sesame, but not vanilla,
resulted in significant eosinophilia only in the i.p. route of sensitization. These patterns
of response were dependent on route, dose and strain of mice used. Boiling and baking
significantly reduced the allergenicity of sesame to varying extents depending on the

strain of the mice used and the method of processing.

(Ded'icated' to my lbving flusfianJ
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My (fear son
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iii

ACKNOWLEDGEMENTS

The work was accomplished with the help and support of many individuals. First
I must thank my major advisor, Dr. Venu Gangur for his guidance and continuous moral
and financial support throughout M.S program. He showed me different ways to
approach a research problem and the need to be persistent to accomplish any goal. I
would like to thank the members of my thesis committee, Dr. John Linz and Dr. Kirk
Dolan for their enthusiastic support and guidance. Besides my advisors, I would like to
thank my lab members Dr. Hanem Mahamood (Post-Doc), Neil Birmingham, Sridhar
Samineni and Caleb Kelly for helping me with my research work and analysis of data.
My special thanks to my friend, Sitaram Parvathaneni, for being there whenever I needed
him. I would like to thank my well-wisher, Mrs. Harsha Gangur for encouraging me all
the time. Last but not least, I thank my family for being supportive and helping me

achieve my goal.

iv

TABLE OF CONTENTS

List of Tables ........................................................................
List of Figures ........................................................................
Abbreviations ........................................................................
Chapter 1: Introduction ...........................................................
Chapter 2: Review of literature ..................................................
Chapter 3: Materials & Methods ................................................
Chapter 4: Results ..................................................................
Chapter 5: Discussion .............................................................

List of References ...................................................................

Page
vii
Viii

xiii

34
49
95

102

LIST OF TABLES

Table 1 Functions of different antibody isotypes .............................. 8

Table 2 Common manifestations of IgE mediated food allergy ............... 10
Table 3 Prevalence of food allergies in United States .......................... 11
Table 4 Commonly allergenic foods in USA, Canada and European union. 13
Table 5 Uses of Sesame in Food, Pharmaceutical and Cosmetic Industries. 16
Table 6 Global Distribution of Reported Sesame Allergy ..................... 18
Table 7 Sesame Allergy: Pattern of Clinical Symptoms. . . . . . . . . . . .. .. ........ 23
Table 8 Sesame Seed Allergens: Chemical Nature .............................. 27
Table 9 Animal models for allergenic foods .................................... 29
Table 10 Effect of processing on allergenicity of foods ......................... 31
Table 11 Allergenic vs. non- allergenic foods comparative study in mice. . 97

vi

Figure 1
Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

LIST OF FIGURES

Classification of adverse reactions to foods. . . . . . . .. .
Global trend of sesame allergy between 1950 and 2003

Experimental protocol for intraperitoneal route of
immunization of mice ........................................

Experimental protocol for epicutaneous route of
immunization of mice .........................................

Experimental protocol for sesame processing .............
Food specific IgE antibody levels in the plasma of
C57B1/6 mice (I/P) ............................................

Food specific IgGl antibody levels in the plasma of
C57Bl/6 mice (I/P) ............................................

Food specific IgE antibody levels in the plasma of Balb/c
mice (l/P) .............................................

Food specific IgGl antibody levels in the plasma of
Balb/c mice (I/P) ..............................................

Food specific IgE antibody levels in the plasma of Balb/c
mice (E/C) ............................................

Food specific IgGl antibody levels in the plasma of
Balb/c mice (E/C) .............................................

Food specific IgG2a antibody levels in the plasma of
C57Bl/6 mice (I/P) .............................................

Food specific IgGZa antibody levels in the plasma of
Balb/c mice (I/P) .............................................

Food specific IgG2a antibody levels in the plasma of
Balb/c mice (E/C) .............................................

Food specific IgG2b antibody levels in the plasma of
C57Bl/6 mice (I/P) .............................................

vii

17

43

47

50

51

56

6O

61

62

64

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27
Figure 28

Figure 29

Figure 30 (a)

Figure 30 (b)

Food specific IgG3 antibody levels in the plasma of
C57Bl/6 mice (I/P) ............................................

Food specific IgM antibody levels in the plasma of
C57Bl/6 mice (I/P) ............................................

Food specific IgA antibody levels in the plasma of
C57B1/6 mice (I/P) ............................................

Food specific IgG2b antibody levels in the plasma of
Balb/c mice (I/P) .............................................

Food specific IgG3 antibody levels in the plasma of
Balb/c mice (I/P) .............................................

Food specific IgM antibody levels in the plasma of
Balb/c mice (I/P) .............................................

Food specific IgA antibody levels in the plasma of Balb/c
mice (I/P) .............................................

Food specific IgG2b antibody levels in the plasma of
Balb/c mice (E/C) .............................................

Food specific IgG3 antibody levels in the plasma of
Balb/c mice (E/C) .............................................

Food specific IgM antibody levels in the plasma of
Balb/c mice (E/C) .............................................

Food specific IgA antibody levels in the plasma of Balb/c
mice (E/C) .............................................

Blood eosinophil profile in C57 Bl/6 strain mice (I/P). ..
Blood eosinophil profile in Balb/c strain mice (I/P) ......
Blood eosinophil profile in Balb/c strain mice (E/C). . ..

Binding of unboiled and boiled sesame seeds to specific
IgE in the plasma of C57BL/6 mice ........................

Binding of unboiled and boiled sesame seeds to specific
IgE in the plasma of C57BL/6 mice expressed as
percentage .......................................................

viii

65

66

67

68

70

71

73

75

76

Figure 30 (c)

Figure 30 ((1)

Figure 31 (a)

Figure 31 (b)

Figure 31 (c)

Figure 31 (d)

Figure 32 (a)

Figure 32 (b)

Figure 32 (c)

Figure 32 (d)

Figure 33 (a)

Figure 33 (b)

Figure 33 (c)

Figure 33 (d)

Binding of unboiled and boiled sesame seeds to specific
IgGl in the plasma of C57BL/6 mice ......................

Binding of unboiled and boiled sesame seeds to specific
IgGl in the plasma of C57BL/6 mice expressed as
percentage .......................................................

Binding of unboiled and boiled sesame seeds to specific
IgE in the plasma of Balb/c mice ............................

Binding of unboiled and boiled sesame seeds to specific
IgE in the plasma of Balb/c mice expressed as
percentage .........................................................

Binding of unboiled and boiled sesame seeds to specific
IgGl in the plasma of Balb/c mice ...........................

Binding of unboiled and boiled sesame seeds to specific
IgGl in the plasma of Balb/c mice expressed as
percentage ........................................................

Binding of unboiled and boiled sesame seeds to specific
IgE in the plasma of ASW mice ..............................

Binding of unboiled and boiled sesame seeds to specific
IgE in the plasma of ASW mice expressed as percentage

Binding of unboiled and boiled sesame seeds to specific
IgGl in the plasma of ASW mice ...........................

Binding of unboiled and boiled sesame seeds to specific
IgGl in the plasma of ASW mice expressed as
percentage .......................................................

Binding of unboiled and boiled sesame extract to specific
IgE in the plasma of Balb/c mice ............................

Binding of unboiled and boiled sesame extract to specific
IgE in the plasma of Balb/c mice expressed as percentage

Binding of unboiled and boiled sesame extract to specific
IgGl in the plasma of Balb/c mice ............................

Binding of unboiled and boiled sesame extract to specific

IgGl in the plasma of Balb/c mice expressed as
percentage .........................................................

ix

84

84

85

85

85

85

86

86

86

86

89

89

89_

89

Figure 34 (a)

Figure 34 (b)

Figure 34 (c)

Figure 34 ((1)

Figure 35 (a)

Figure 35 (b)

Figure 35 (c)

Figure 35 ((1)

Figure 36 (a)

Figure 36 (b)

Figure 36 (c)

Figure 36 (d)

Binding of unboiled and boiled sesame extract to specific
IgE in the plasma of ASW mice ...............................

Binding of unboiled and boiled sesame extract to specific
IgE in the plasma of ASW mice expressed as percentage

Binding of unboiled and boiled sesame extract to specific
IgGl in the plasma of ASW mice .............................

Binding of unboiled and boiled sesame extract to specific
IgGl in the plasma of ASW mice expressed as
percentage .........................................................

Binding of raw and baked sesame to specific IgE in the
plasma of Balb/c mice ..........................................

Binding of raw and baked sesame to specific IgE in the
plasma of Balb/c mice expressed as percentage ............

Binding of raw and baked sesame to specific IgGl in the
plasma of Balb/c mice ..........................................

Binding of raw and baked sesame to specific IgGl in the
plasma of Balb/c mice expressed as percentage ............

Binding of raw and baked sesame to specific IgE in the
plasma of ASW mice ...........................................

Binding of raw and baked sesame to specific IgE in the
plasma of ASW mice expressed as percentage .............

Binding of raw and baked sesame to specific IgGl in the
plasma of ASW mice ..........................................

Binding of raw and baked sesame to specific IgGl in the
plasma of ASW mice expressed as percentage .............

90

90

90

9O

93

93

94

94

94

94

LIST OF ABBREVIATIONS

DLC Differential Leukocyte Count

E/C Epicutaneous route of administration
ELISA Enzyme Linked Immunosorbent Assay
I/P Intraperitoneal route of administration
Ig Immunoglobulin

IL Interleukin

MHC Major histocompatibility complex

Th T helper lymphocyte

xi

CHAPTER 1: INTRODUCTION

Allergy or immediate hypersensitive reaction is an abnormal response of the
immune system to substances that are normally harmless such as pollen, foods, drugs
etc., Food allergy is an immune -mediated adverse reaction to food that occurs when the
immune system identifies a normally harmless food as damaging to the body and
responds defensively to one or more specific proteins in that food. Food allergy is usually

expressed as an immediate hypersensitive reaction.

Hypersensitive reaction is an inappropriate response of the immune system to
antigens. There are 4 types of hypersensitivity reactions classified as Type-I to Type-IV
[J aneway, 2005]. The Type I reaction is the immediate type of reaction and most food
allergies belong to this class. These are mediated by the IgE class of antibodies produced
against foods. They are expressed clinically very quickly with dramatic symptoms such
as skin rashes, hives, asthma etc. These are diagnosed by skin prick testing and serum
screening for specific IgE antibodies. There is evidence for the type IV hypersensitivity
in some cases of food allergy such as gluten enteropathy (or Celiac disease), which is
mediated by a non-IgE mechanism involving T cells and monocytes called the delayed

hypersensitive reaction [Sampson, 1995; Macdonald TT, 2005].

Recent epidemiological studies reveal that up to 4% of the American population is
affected with food allergies, with 6% of children below 3 years age [Bock, 1987;

Sicherer, et a1. 2004]. F ood-related allergic reactions account for approximately 30,000

emergency department visits each year, and 150-200 deaths in USA [Sampson, 2003;

Sampson, 2004].

The basic mechanism of food allergy involves an initial sensitization phase
followed by an effector phase [Sampson HA, 1996]. The sensitization phase involves first
encounter of the body to a particular allergen. When the culprit food enters the body for
the first time, the B-lyrnphocytes (the lymphocytes that undergo maturation in bone
marrow and are primarily responsible for antibody production and differentiation) in the
body produce specific IgE antibody against that allergen. These antibodies enter the
circulation and bind to mast cells. When the body is exposed to that particular allergen
for the second time, the effector phase is precipitated that is, the preformed IgE antibody
on the mast cell binds to the allergen. Cross linking of allergen with at least 2 IgE
antibodies results in the disruption of the mast cells releasing chemical mediators that are

responsible for the symptoms of allergy [Sampson HA, 1996].

Symptoms of food allergy may begin within minutes to a few hours after ingestion
of food. Symptoms differ widely ranging from skin reaction (hives, eczema, itching,
swelling) to gastrointestinal symptoms (nausea, vomiting, diarrhea, cramps), respiratory
symptoms (tightness of throat, difficult breathing, wheezing, sneezing, asthma, rhinitis,
laryngeal edema) and systemic symptoms like lowered blood pressure and anaphylactic
shock in highly susceptible individuals that can be potentially fatal [Sampson, 2003;

Lemke, 1994; Sampson HA, et a1. 1992].

Besides a direct impact (such as high risk of fatality and medical costs),
food allergy has an indirect impact with food product recalls due to suspected cross
contamination with allergens. There is around 70% increase in food recalls due to
allergens over a 10 years period according to Dr. Kenneth F alci, (Director,
CFSAN) with 120 Class I food product recalls in the year 1997 [US Food and Drug

Administration].

In addition, there is growing concern that genetically modified foods might
be allergenic [Celec P, et al. 2005]. Thus, with the increasing use of genetically
modified (GM) foods that express novel proteins, there is growing concern that
novel proteins expressed may have the potential for causing allergy. In addition,
there is increasing use of herbs both for culinary and therapeutic purposes and the
quest for filnctional foods (foods that have health benefits beyond the nutritive
value). Therefore, there is a real need for predicting allergenicity of GM foods.
Studying the activation of immune system by allergenic vs. non-allergenic foods

might provide distinguishing immune markers.

Although any food has the potential to cause allergy in sensitized
individuals, certain foods are reported to be more common causes of allergy than
others. These are called red-flag foods [US Food and Drug Administration] and are
responsible for almost 90% of the allergic reactions [Hefle, et al. 1996]. Red-flag
foods include peanut, treenuts, cow milk, chicken egg, wheat, soy, fish and shell

fish. Sesame is one of the commonly allergenic foods according to the Canadian

and European union list of red flag foods [Health Canada and the Canadian Food
Inspection Agency; Goodwin]. Even though the USFDA list of red flag foods does
not contain sesame, sesame allergy is a growing concern in this country as well
[Gangur, et a1. 2005]. Furthermore, it was recently reported that the prevalence of
sesame allergy may be increasing globally during the past 50 years [Gangur, et a1.
2005]. Thus, a suitable animal model of sesame allergy is desirable to study the
mechanisms of this disease as well to develop effective preventive/therapeutic
methods. However, currently a suitable animal model for sesame is unavailable and

development of such a model is one of the focii of this study.

Even though extensive research is ongoing about allergenic foods, still the answer
to the question “why some foods cause allergy while others don’t?” is unclear. In
particular very little is known about the differences in the mechanism of activation of the
immune system by allergenic vs. non-allergenic foods-«two pathogenically distinct food
types. In this study efforts were made to address this problem, by comparing the immune
responses against a model allergenic food (sesame) and a model rarely/non-allergenic
food (vanilla) in a mouse model. Besides, positive outcome from this study will lead to a

sesame allergy mouse model for further studies.

There is growing concern that processing of foods might be contributing to
the increased incidence of food allergies [Mondoulet, et al. 2005]. It has been
established that allergenicity of some foods such as peanuts, egg, milk etc, can be

altered by subjecting them to various processing methods (Table 8). However,

although sesame has gained widespread use in the food industry, we are not aware
of previous reports testing the effect of processing on sesame allergenicity.
Therefore, as a second part of this study, we examined the impact of boiling and
baking , two of the commonly used processing methods, on sesame allergenicity in

mouse model.

In this study, two specific hypotheses were tested: 1) whereas sesame triggers a
Type-2 associated immune response (that is, increased specific IgE, IgGl antibody levels
and eosinophilia), vanilla elicits a Type-1 associated immune response (that is, elevated
specific IgG2a) in mice. 2) Boiling as well as baking reduces allergenicity of sesame in a

mouse model.

There were three aims designed to test these hypotheses: 1) to characterize the
antibody isotype responses in mice exposed to sesame or vanilla via intraperitoneal and
epicutaneous routes; 2) to study the peripheral blood eosinophilia (elevated eosinophil
levels) in these mice; and 3) to study the effect of boiling and baking on sesame

allergenicity using hyper-immune serum from sesame sensitized mice.

CHAPTER 2: REVIEW OF LITERATURE

In this section, I have reviewed the literature in the following format. I present a
summary of the literature on food allergy and follow this with a summary of the literature
on sesame allergy, the focus of this study. In addition, I present a general review on the
effect of food processing on allergenicity, since the effect of processing on sesame

allergenicity is currently unclear.

Food allergy: a serious and potentially fatal adverse reaction to food

The adverse reactions to food have been classified as toxic and non- toxic. Non-
toxic reactions are further classified as immune- mediated and non- immune mediated. In
this classification, food allergy falls under immune mediated, non-toxic adverse
reactions. The food allergy is again classified in to two types. IgE- mediated and non-IgE
/cell mediated reactions (Figure l) [Bruijnzeel-Koomen, et al. 1995]

ADVERSE REACTIONS TO FOOD

TOXIC NON- TOXIC

/\

IMMUNE MEDIATED NON IMMUNE MEDIATED
(FOOD ALLERGY) (FOOD INTOLERANCE)

/\

IgE NON- IgE

 

l l l

ENZYMATIC PHARMACOLOGICAL UNDEFINED

Figure 1 Classification of adverse reactions to foods

Food allergy is also designated as a food induced hypersensitive reaction. A
hypersensitive reaction is an inappropriate response of the immune system to antigens.
According to Gel and Comb’s classification of hypersensitivities, there are four types of
hypersensitive reactions Type-I to Type-IV [J aneway, 2005]. Type I hypersensitivity is
an immediate reaction mediated by IgE antibody against a soluble antigen. IgE antibody
binds to mast cells and basophils, causing their degranulation and the release of various
chemical mediators like histamine, which results in onset of different disease symptoms.

Most food allergies and airways allergies belong to this class.

In Type H hypersensitive reactions IgG and IgM antibodies play major role. The
antigen is a non-soluble one. Antibodies bind to the fixed antigen and activate the
complement system, which in turn initiates the release of chemical mediators that are
responsible for the disease. Examples include autoimmune hemolytic anemia, rheumatic
fever, drug allergies etc. [Solensky, 2006 ] It is unclear if this mechanism plays a role in

food allergy.

In type III hypersensitivities, IgG and IgM bind to circulating antigens (Ag)
forming "immune" complexes (IC). IC develop into an extremely large mesh work with
Ag bridges, which are usually cleared by binding to complement. Failure of normal
clearance mechanisms will lead to disease. E.g., systemic lupus erythematosus. Similar to
Type-H hypersensitivity, it is not known whether this mechanism may play role in food

allergies.

Type IV reactions are delayed type hypersensitive reactions, mediated by CD4 +

T cells and require intact antigen presenting cells. Examples include contact dermatitis

and some food allergies such as gluten enteropathy (or Celiac disease) [Sampson, 1995;

Macdonald TT, 2005].

In this study different antibody isotypes and eosinophil responses were used as

readouts for measuring the immune response against sesame and vanilla in mice. The

functions of different antibody isotypes in humans is tabulated below (Table 1).

Table 1. Functions of different antibody isotypes in humans

 

 

 

 

 

 

 

Functional activity IgE IgG] IgG2 IgG3 IgM IgA
Neutralization - ++ ++ ++ + ++
Opsonization - +++ + ++ + +
Sensitization for - ++ - ++ - -
killing

by NK cells

Sensitization of +++ + - + - -
mast cells

Activates - ++ + +++ +++ +
complement system

 

 

 

 

 

 

 

 

(Source: Janeway, 2005)

IgE, commonly known as reaginic or (homocytotropic) antibody, is an isotype of

Ig responsible for Type I Hypersensitivity. IgG is the major class of Ig in serum. In mice,

4 subclasses are designated; IgGl , IgGZa , IgG2b and IgG3 (Source: Janeway, 2005).

IgG is the only class of Ig which is able to cross the placenta in humans and other

primates. In rodents it is provided via yolk sack. IgM is the first isotype of antibody

synthesized during the primary humoral response. IgA protects GI tract and perhaps
respiratory tract from pathogens and is synthesized by mucosal epithelial cells (Source:

Janeway, 2005).

Eosinophils are a type of blood leucocytes which migrate to the place of
inflammation and release chemical mediators which contribute to the symptoms of
allergy. They are usually found in large numbers at the site of allergic inflammation

(Source: Janeway, 2005).

Siggificance of food allergy:

Food allergy has both direct and indirect influences on the socioeconomic
status of the world. Food allergy is a major food safety issue with direct impacts like
high risk of fatality, work days lost and medical costs (Miles.s, et al, 2005). The
indirect impacts include economic losses due to food product recalls due to suspected
cross contamination with allergens. Allergenic food recall activity increased from an
average of 35 per year at the beginning of the last decade to an average of 90 per year
during the last four years of the same decade according to Dr. Kenneth Falci,
Director, CFSAN, USFDA. (Source:http://www.cfsan.fda.gov/~dms/alrgawar.html)

In addition to this, changing dietary habits, increasing prevalence of food
allergies, growing concerns regarding the potential allergenicity of genetically
modified foods (Gizzarelli, et a1, 2006) suggests a need to study extensively about

food allergy and its mechanism.

Clinical symptoms of food allergy

Symptoms of food allergy may appear within minutes after ingestion of food or it
may take a few hours (sampson, 2005). In the latter case, the late onset of symptoms
makes the diagnosis and identification of the culprit food difficult. Symptoms differ
widely affecting more than one system in the body and not following a fixed sequence of
appearance. These symptoms range from skin reaction (hives, eczema, itching, swelling)
to gastrointestinal symptoms (nausea, vomiting, diarrhea, cramps), respiratory symptoms
(tightness of throat, difficult breathing, wheezing, sneezing, asthma, rhinitis, laryngeal
edema) (sampson, 2005).. Systemic symptoms like lowered blood pressure and
anaphylactic shock can be potentially fatal in highly susceptible individuals [Sampson
HA, et al. 1992]. Usually the reaction to food allergy starts with itching but this may vary
among affected individuals. The most common manifestations of food allergy are listed

in Table 2 [Sampson, 2003, Lemke, et al. 1994].

Table 2 Common manifestations of IgE mediated food allergy

 

 

Type of symptoms Manifestation
Gastrointestinal symptoms Nausea, Vomiting, Diarrhea, Abdominal
pain
Cutaneous symptoms Urticaria, Eczema or Dermatitis,

Angioedema, pruritis

Respiratory symptoms Rhinitis, Asthma, Laryngeal edema
Other symptoms Anaphylactic shock, hypotension, Oral
allergy syndrome

 

 

 

10

Food allergy has been reported to affect up to 4% of the US population with 6-8%
of children below 4 years age [Bock, 1987; Sicherer, et a1. 2004]. Infants are more
commonly affected than adults. Children tend to outgrow food allergies, especially
allergies to cows' milk, eggs, and soybeans [Bock, 1987]. That may be providing one
potential reason why the prevalence of food allergies in adults is considerably lower than
in infants. Some food allergies are more prevalent than the others due to reasons like
relatively more consumption, variations in the method of cooking, the form of food in
which it is consumed, etc. Prevalence of some common food allergies is given in Table 3

[Sampson, 2004].

Table 3 Prevalence of food allergies in United States (source: Sampson, 2004)

 

 

Food Young children Adults
Milk 3.5% 0.3%
Egg 1 .3% 0.2%
Peanut 0.8% 0.6%
Tree nuts 0.2% 0.5%
Fish 0. 1% 0.4%
Shellfish 0. 1% p 2.0%
Overall 6% 3.7%

 

Although any food has the potential to cause a hypersensitive reaction in a
sensitized individual, most food allergies (approximately 90%) are caused by 8 major
food types. Accordingly, the US Food and Drug Administration (FDA) initiative on the
food allergen awareness efforts, currently focus on the 8 foods that are most frequently
implicated in serious allergic reactions, including milk, eggs, fish, wheat, tree nuts,

legumes (particularly, peanuts and soybeans), crustaceans, and mollusks [US Food and

11

Drug Administration]. However, in November 2003, a new directive passed into
European Commission (EC) law (2003/89 EC) includes 11 foods in addition to sulfites
on the EC allergen labeling list including celery, crustaceans, egg, fish, gluten, milk,
mustard, peanuts, sesame, soy, and tree nuts [Goodwin, 2004]. Health Canada and the
Canadian Food Inspection Agency have jointly identified 10 foods including peanuts, tree
nuts, soy, milk, eggs, fish, crustaceans and shellfish, sesame seeds, sulfites, and wheat in
addition to sulfites and these 10 foods are primarily responsible for approximately 90%

of severe adverse food reactions among the Canadian population. (Table 4)

12

Table 4 Common allergenic foods in the USA, canada and european union

 

 

Country Regulatory agency Commonly allergenic foods
USA USFDA Wheat, Cow milk
Chicken egg
Fish, Shellfish
Peanut

Tree nuts, Soy

Canada CFIA peanuts
tree nuts
sesame seeds
milk, eggs, fish
crustaceans (crab, crayfish, lobster and
shrimp)
shellfish (clams, mussels, oysters,
scallops), soy, wheat
sulphites

Europe EF SA cow's milk, celery
cereals containing glutein
eggs, fish, peanuts
tree nuts, soy
crustacean (crab, crayfish, lobster and
shrimps)
sesame, mustard
Sulphites

 

USFDA - United States Foods and Drugs Administration;

CFIA — Canadian Food Inspection Agency;

EFSA — European food safety authority
Sources: 1. http://www.fda.gov/ora/complianceiref/cpg/cpgod/cpgSS5-
250.htrn#allergies

2. http://www.inspectiongc.ca/english/fssa/labeti/allerg/allergeshtml
3. http://www.efsa.eu.int/science/nda/nda opinions/341 en.html

Thus, sesame is included in the allergen list of both the EC and Canadian Food
Inspection Agency but not in the allergen list of the FDA. Recent years have seen

tremendous progress in the research and public awareness on peanut allergy [Sampson, et

13

al. 2004]. In contrast, both research and public awareness on sesame allergy have been

limited [Perkins, 2001; Perkins, 1996; Perkins, 1998; Sampson, 2004].

In the following section, the review of the literature has focused on addressing the
following questions: Is sesame allergy globally prevalent? What is the spectrum of
clinical symptoms and available clinical immunology data? Does sesame encompass
more than one type of immune-mediated disease? What is the chemical nature of sesame
allergens and their immune cross-reactivity with other foods? The following review of
literature has been published in the official journal of the American College of Allergy,
Asthma & Immunology--Annals of Allergy, Asthma & Immunology [Gangur, et al.

20051

Sesame seed and oil: growing use in food, pharmaceutical, and cosmetic industries

It has been reported that sesame cultivation for human use is traceable to 2450 BC
in Babylon [Kagi, et al. 1993]. The family Pedaliaceae to which sesame belongs contains
at least 18 species, most of which are indigenous to Africa and Asia [Kagi, et al. 1993].
Among these, Sesamum indicum, originally from India, is widely cultivated in many
countries, including India, the Middle East, the United States, China, and other Asian and
Latin American countries. It is a perennial herbaceous plant that grows up tol m in height
with pink or bluish white flowers [Kagi, et al. 1993]. Sesame seeds are available in 3
different colors—white, brown, and black. They are used either directly, as crushed seed,
or as a paste. They are used in food either directly or after hulling. Use of sesame seed

paste (tahini) is common in the Mediterranean diet [Dalal, et al. 2002; Kagi, et al. 1993].

14

In India, sesame seed is mixed with jaggery or sugar and rolled into cakes or balls and
eaten as a confectionary. Sesame seed is becoming a favorite gamishing item in Western
fast food industries. It is widely used in the baking industry, where there are reports of
occupational allergy (asthma and urticaria- a condition in which red, itchy, and swollen
areas appear on the skin) to sesame involving bakers [Alday, et al. 1996; Keskinen, et al.

1991] (Table 5).

Sesame seeds contain approximately 50% to 60% oil. Although sesame seeds are
most frequently used in the food industry, sesame oil is used in the food (e.g., in salad
dressings in Oriental, Chinese, and South American cuisines), pharmaceutical, and
cosmetic industries. Sesame oil displays many desirable properties, such as stability,
neutral, inert, non- irritating, non- viscous and heat resistant effects [Kagi, et a1. 1993].
There is a report claiming that sesame oil is less antigenic compared with cottonseed or
peanut oil [Torsney, et al. 1964]. Thus, it has been used in the preparation of
intramuscular injections, plasters, liniments, ointments, capsules, soaps, lipsticks, and
emulsions and in the formulation of suspensions and ophthalmic preparations. Liniment
that contains sesame oil (linimentum zinci oxydi oleosum) has been used in the treatment
of eczema and leg ulcers. A Chinese ointment for burn wounds that contains 60% sesame
oil (shiunkoh) has been used for centuries in Japan and China [Kubo, et a1. 1986].
Notably, sesame oil in injections, ointrnents, and cosmetics has been reported to cause
contact allergic dermatitis [Alday, et al. 1996; Asero, et al. 1999; Pecquet, et al. 1998;

Perkins, et al. 1998; Phy, et al. 2003].

15

Table 5. Uses of Sesame Seed and Oil in Food, Pharmaceutical and Cosmetic Industries
(Source: Gangur, et al. 2005)

 

 

Type of Application Examples
Food Industry Bakery foods: Falafel burger buns, Hamburger buns
buns, Pizza, Wafers, Bread sticks, Bread
crumbs, Pastries, Pretzels, Italian bread,
Bagels, Biscuits, Crackers, Biscuits,
Bread, Sandwich, Cookies
Other foods: Halvah, Tahini (sesame seed paste), Salad

Pharmaceutical Industry

Cosmetic Industry

dressing, Simit (Turkish Pretzel), Candy,
Soups, Dressed chicken, Sauce, Gluten
free foods (for celiac patients), Ground
seed (almond paste substitute), Sesame
paste noodles, Chocolate, Protein bars,
Ice cream cones coated with sesame,
Margarine

Ointrnents (e. g. Shiunkoh, linimentum zinci oxydi
oleosum), Intra muscular injections (e.g., progesterone)
Plasters, Liniments, Capsules, Emulsions, Ophthalmic

Preparations

Lipstick (e.g. L’Oreal), Body oil (e. g. Laboratire Vichy,
France), Moisturizing Cream (e.g. L’Oreal), Soaps

Sesame allergy: an emerging global epidemic?

The prevalence of sesame allergy in the global population is unknown. However,

there has been a significant increase in the number of reports of hypersensitivity to

sesame since the first report from the United States in 1950 (Figure 2). Subsequent

reports are mostly from developed countries, including the United States, Australia, many

European countries, and Asia (Israel and Japan) (Table 6).

16

7////A Published Reports

- Subjects Affected

mtoaom 85:93.”. *0 ngaz

15

u 9 6 3 0

 

V////////////////////////////4

7///////////////////////////////////////

 

   

 

 

1000 :
100 —

£88m 85:2... 5 8§E< 8.8.35 .o .3532

03

2000

9

ow
O

1 950-59 1960-69 197049 1 980-89 199

Figure 2 Global trend of sesame allergy between 1950 and 2003

(Source: Gangur, et al. 2005)

17

Table 6 Global Distribution of Reported Sesame Allergy (Source: Gangur, et al. 2005)

 

 

 

Continent Age of Subjects (Years) Total References
(Countries) 0-4 5-14 15-20 2 21 Number
of
Subjects
Afflicted
*
Asia 47 8 3 6 64 [Dalal, et al. 2003;
(Israel and Kubo, et al. 1986;
Japan) Levy, et al. 2001;
Wolff, et al. 2003]
Australia ---531--- 1 532 [Phan, et al. 2003;
Sporik, et al. 1996]
Europe 4 10 4 56 107 [Alday, et al. 1996;
(England, Asero, et al. 1999;
Finland, Kagi, et al. 1993;
France, Keskinen, et a1. 1991;
Germany, Pecquet, et al. 1998;
Italy, Eberlein-Konig, et al.
Netherlands, 1995; Ewan, et al.
Spain, 1996; Fremont, et al.
Switzerland) 2002; Kolopp-Sarda,
et al. 1997; Pajno, et
al. 2000; Pastorello, et
al. 2001]
North 6 12 2 12 32 [Phy, et al. 2003;
America Torsney, et al. 1964,
(United States) Beyer, et al. 2002;
Chiu, et al. 1991;
Malish, et al. 1981;
Rubenstein, 1950;
Uvitsky, et a1. 1951]
Total Number 57** 30** 9 75 735
of Subjects
Mined

 

*Total may be larger than the sum of age groups since it also includes subjects with

unspecified age

"Excludes 531 subjects from Australia (where age range was 0-14 yrs.)

18

An epidemiological study on immediate hypersensitivity (IHS) to foods among
Australian children revealed that sesame was fourth most prevalence (0.42% prevalence),
following egg (3.2%), milk (2%), and peanut (1.9%). Sensitivity to sesame was more common
than that to any single tree nut studied [Sporik, et al. 1996]. In the United Kingdom, report of a
completed government research project in 1996 estimated an overall prevalence of severe

allergic reactions to sesame in the general population of 1 in 2,000 (0.05%).

A recent study on food allergy among Israeli children (n =9,070) found food
allergy prevalence to be 1.7%, with sesame the third most common food causing
sensitization (0.18% prevalence), following egg (0.5%) and cow’s milk (0.3%). Sesame
sensitization was more prevalent than that of peanut (0.04%) [Dalal, et al. 2002].
Furthermore, sesame was second only to cow’s milk as a leading cause of anaphylaxis
[Dalal, et al. 2003; Dalal, et al. 2002]. Based on the finding that sesame is a major cause
of severe IgE-mediated food allergic reactions among infants and young children in
Israel, these authors suggest that sesame allergy is a matter of geography [Dalal, et al.
2002] because of the widespread use of sesame paste in the Middle Eastern diet, with
consequent high levels of allergen exposure. Interestingly, although the use of sesame in
food is common in India, no published reports exist of sesame allergy from this country.
This observation parallels the situation of peanut allergy in Southeast Asia and China.
Whether this pattern is due to lack of reporting or true absence of disease due to
difference in the method of food preparation or late introduction of sesame in the diet of

infants remains unclear.

l9

While studying the prevalence of sesame allergy, it is useful to distinguish sesame
allergy from sesame sensitization. Most articles reviewed herein studied sesame allergy
based on immediate clinical reactions following exposure to sesame. One study examined
only sesame sensitization based on skin prick test (SPT) but did not study sesame disease
[Sporik, et al. 1996]. Available published data on sesame allergy suggest a potential trend
of global epidemic of sesame allergy, although formal evidence from studies on global

prevalence is lacking.

Hypersensitivity to sesame: clinical and immunologic features

Analysis of pooled clinical data on sesame allergy from all published reports is
presented in Tables 6 and 7. Sesame allergy occurs in individuals of all ages from
infancy to adulthood. Interestingly, sesame allergy appears to be relatively less frequent
in the pubertal age group (ages, 15—20 years). It is unclear from the literature whether
allergy reported among adults is simply due to the persistence of the condition or newer
sensitization when the individual became an adult. However, given the age distribution

(Table 6), it seems likely that adult patients are newly sensitized.

There is only 1 report of 3 children outgrowing sesame allergy in Israel [Dalal, et
a1. 2003; Dalal, et al. 2002]. Since shellfish, tree nut, and most peanut allergies are rarely
outgrown and are common among adults, it is possible that sesame allergy might belong

to the same category as these [Sampson, 2004].

20

Analyses of clinical presentation of sesame allergy revealed that clinical
symptoms varied from subject to subject, and they included erythema, conjunctivitis,
atopic dermatitis, contact dermatitis, allergic asthma, rhinitis, systemic anaphylaxis,
urticaria, gastrointestinal symptoms, angioedema, dyspnea, pruritus, and oral allergy
syndrome (Table 7). Notably, systemic anaphylaxis was commonly reported, suggesting
the serious nature of the allergic reaction. Contact dermatitis to sesame has been
predominantly attributed to the use of sesame seed oil [Hayakawa, et al. 1987; Kubo, et
al. 1986; Stem, et al. 1998; van-Dijk, et al. 1973]. These studies confirmed contact
hypersensitivity by patch testing (read at 48 to 72 hours) with sesame oil or its
components. There is one report of contact allergy associated with a positive SPT result,
although this study did not use patch testing. Conjunctivitis was less often reported. The
type of symptom was independent of the age group of patients or reporting country (data
not shown). Due to incomplete information on sex in the studies, it was not possible to

test the role of sex in reported sesame allergy.

In most cases, sesame allergy was elicited following exposure to sesame in food
or therapeutics or cosmetics or due to occupation (among bakers). There was an unusual
case of hypersensitivity to sesame oil following daily intramuscular injection of
progesterone in sesame oil to a patient following in vitro fertilization and embryo transfer
[Phy, 2003]. This patient exhibited IHS, as evidenced by a positive SPT result to sesame

oil, in addition to pulmonary compromise and marked eosinophilia.

21

Analyses of clinical immunology data on sesame allergy reports provided a clear
pattern of underlying immune basis for sesame allergy (Table 7). Thus, most reported
sesame allergy may be classified into at least 2 major categories: ( 1) 11-18 associated with
a positive SPT result and/or specific IgE antibodies in the blood and (2) delayed
hypersensitivity (DHS) associated with positive patch test results with sesame oil. In this
group, no data were available for SPT or IgE, except in 1 case IgE was tested and found
negative. In addition to these 2 major groups, there was a minor group with only 4 cases
of IHS associated with negative SPT results and negative specific IgE antibodies in the
blood. Many symptoms in this category overlapped with the first category. The

underlying mechanisms in this category are unclear.

22

Table 7 Sesame Allergy: Pattern of Clinical Symptoms (Source: Gangur, et al. 2005)

 

Clinic N o. of Clinical Symptomsal Reference

a1.5“bl'tcntsa5 1) A N SAUGAE
Testr cts «

 

 

ng
Positi 2 + + + [Vocks, et al.
ve 1993]
SP7? 1 + + + [Uvitsky, et
and/ al. 1951]
or IgE 23 + + + + + + + [Dalal, et al.
2003]
1 + + + + [Keskinen, et
al. 1991]
1 + + + [Alday, et al.
1996]
8 + + + + + + [Kanny, et al.
1996]
531 + + + [Sporik, et al.
1996]
1 + + + + [J ames, et al.
1991]
9 + + + + + + [Kagi, et al.
1993]
10 + + + + + [Levy, et al.
2001]
12 + + + + + [Morisset, et
al. 2003]
3 + + + + [Malish, et al.
1981]
l + + + + [Asero, et al.
1999]
2 + + [Phan, et al.
2003]
12 + + + + + [Kolopp-
Sarda, et al.
1997]
10 + + + + + [Pastorello, et
al. 2001]
6 + + + [Fremont, et
al. 2002]
l + + + + [Chiu, et al.
1991]
28 + + + + + + + [Wolff, et al.

23

Negat
ive
SPT
and
IgE

Positi
ve
Patch
Test
(0:1)d

20

15

1

14

+++

+ +

+

2003]
[Beyer, et al.
2002]
[Torsney, et
al. 1964]
[Rubenstein,
1950]
[Pecquet, et
al. 1998]

[Stem, et al.
1998]
[Phy, et a1.
2003]
[Pajno, et al.
2000]
[Eberlein-
Konig, et a1.
1995]

[Hayakawa,
et al. 1987]
[van-Dijk, et
al. 1973]
[Kubo, et al.
1986]
[Malten, et
aL 1973]

2‘CD: Contact Dermatitis, AD: Atopic Dermatitis or Eczema, D: Dyspnea, A: Allergic
Asthma, N: Nasal Symptoms Such as Rhinitis, SA: Systemic Anaphylaxis, U: Urticaria,

G: Gastrointestinal Symptoms, AE: Angioedema
bSPT: Skin Prick Test
cNo data for IgE

dNo data for SPT and IgE, except in Kubo Y (1986), where IgE tested negative

In summary, most reported sesame allergies belong to either IHS to sesame

proteins linked to an underlying IgE antibody response (classic Gell and Coombs type I

hypersensitivity reaction) or DHS to sesame oil caused by the classic cell-mediated

immune response (Gell and Coombs type IV hypersensitivity reaction).

24

Sesame allergens: chemical and immune properties

Studies on characterization of sesame allergens suggest that sesame allergens
belong to 2 broad categories based on their chemical nature and the type of
hypersensitivity they elicit. The first category includes proteins or glycoproteins, which
have a wide range of molecular mass (7—78 kDa); this suggests that sesame seed contains
multiple allergens and sesame proteins are linked to mainly IHS reactions mediated by
IgE antibodies. The second category is lignin-like molecules in sesame oil; the 3
chemical allergens identified are sesarnol, sesarnin, and sesamolin, which are all reported
to be unsaponifiable fractions of sesame oil (Table 8). Although sesame seeds are
available in 3 colors (white, brown, and black) and show quantitative differences in
protein content (e.g., white seeds have more protein than black seeds, 182 vs. 28 mg/ g),

all 3 varieties have been reported to trigger allergic reactions [Fremont, et al. 2002].

Although an international nomenclature committee on allergens has designated 3
proteins in sesame as allergens (Ses i I [9 kDa, 2S albumin], Ses i 2 [7 kDa, ZS albumin],
and Ses i 3 [45 kDa, 78 vicilin], available evidence suggests the existence of many more
IgE-binding proteins in sesame seed (Table 8). Both Ses i 2 and Ses i 3 are seed storage
proteins, and Ses i 2 is the major soluble protein (constitutes approximately 25% of total
protein content). Ses i 3, a 78 vicilin type globulin of S indicum, shows 80% homology
with a major peanut allergen (Ara h I) and shares IgE-binding epitope with this peanut
allergen. There are reports of cross reactivity among allergens in sesame and allergens in
other foods, including hazelnut, rye, kiwi, poppy seed, black walnut, cashew, macadamia,

pistachio, and peanuts [Asero, et al. 1999; Beyer, et al. 2002; Hlywka, et al. 2000; Vocks,

25

et al. 1993] (Table 8). Given the general suggestion in the field of cross reactivity
between nuts [Sicherer, et a1. 2001], it might be prudent for sesame allergic patients to
avoid these cross-reacting foods in addition to sesame, although clinical relevance of
such cross reactivity remains to be established. Furthermore, the general rule for food

allergy is to expose children to a food challenge to avoid unusual elimination diets.

All reported contact allergic dermatitis to sesame has been shown to be caused
by 3 sesame oil components: sesamol, sesarnin, and sesamolin (Table 8). Sesame oil used
for pharmaceutical purposes contains approximately 0.002% of sesamol and sesamolin
and 0.27% of sesarnin. It is reported that the relatively stable nature of sesame oil is due
to sesamol, an antioxidant [Neering, et al. 1975]. However, others dispute the presence of
sesamol in sesame oil [Hayakawa, et al. 1987]. These authors also report that the key
allergenic structure of sesame oil is present only in sesamolin and sesamin. The following
structure was suggested to be responsible for allergenicity: tetrahydro-l-(3, 4

(methylenedioxy) phenyl) 1H, 3H-furo (3, 4—C) furan [Hayakawa, et al. 1987].

26

Table 8 Sesame Seed Allergens: Chemical Nature (Source: Gangur, et al. 2005)

 

 

Nature Allergen Name/Molecular Shares IgE Reference
of Weight (kDa) Epitope
Allergen With:
Protein ~20, ~50 H, R, K, PS [Vocks, et al. 1993]
BW, BN, C, [Hlywka, et al. 2000]
H, M, P
14, 25, 30 [Kolopp-Sarda, et al.
1997]
14 [Alday, et al. 1996]
9 (2 S albumin: major [Pastorello, et al. 2001]
allergen; Ses i I), 30, 14.4,
38.7, 43, 18, 53
7 (2 S albumin major PN (Ara h [Beyer, et al. 2002]
soluble protein; Ses i 2), 9, 1)
20, 25, 29, 32, 34, 45 (7 S
vicilin-type globulin; Ses i
3), 52, 78
14 (2 S albumin precursor; [Wolff, et al. 2003]
major allergen), 20-25, 30-
35, 40
12, 13, 22, 23.5, 32, 34, 36, [Fremont, et al. 2002]
47, 53, 57.5
10, 12, 15-20, 25, 30-67 PS (10-12 [Asero, et al. 1999]
kDa)
Oil Sesamin/Sesamolin/Sesamol [Neering, et al. 1975]
(unsaponi Sesamin/Sesamolin [Hayakawa, et al. 1987]
fiable
lignan
like
molecule)
Sesamin/Sesamolin [Kubo, et al. 1986]

 

Abbreviations: H: Hazelnut, R: Rye, K: Kiwi, PS: Poppy Seed, BW: Black Walnut, BN:
Brazil Nut, C: Cashew, M: Macadamia, P: Pistachio, PN: Peanut

Animal models of food allergy: an overview

In the recent past, enormous efforts have been undertaken to develop animal

models of food allergy using mouse, rat, pig, guinea pig, rabbit or dog as model species

27

[Adel-Patient, et al. 2003; Bassan, et al. 2002; Dearrnan, et a1. 2001; Helm, et al. 2002;
Knippels, et al. 1998; Miller, et al. 1999; Piacentini, et al. 2003; Teuber, et al. 2002].
These models have been summarized in the Table 9. Consequently, at present, there are
animal models for many food allergies including peanut, hazelnut, milk, egg, soy, wheat,
fish [Adel-Patient, et a1. 2005; Adel-Patient, et al. 2003; Birmingham, et al. 2005;
Frick, et a1. 2005; Knippels et al., 1998; Kroghsbo, et al. 2003; Lee, et al. 2004;
Untersmayr, et al. 2003]. In contrast to this, currently there are no animal models
reported to study sesame allergy. Although a mouse model to study sesame allergy is
desirable, currently it is unclear whether sesame can elicit IgE antibody response in mice
as it does in humans. Therefore, one goal of the current study was to characterize the

immune response to sesame in mice.

There is a concern that exposure of mice to any food may induce an allergic
response (Kimber, et al. 2003). In this study by characterizing the immune response to

sesame and vanilla, efforts were made to test this concern in our model.

In the present study three different strains of mice were used to represent the
genetic variation and differences in the levels of response to food allergy. ASW strain of
mice are high- IgE responder mice strains while C57 BL/6 strain is a moderate to low

responder.

28

Table 9 Animal models for allergenic foods

 

 

.EQQdm___Animal model Route Reference
Cow BN rat Intraperitoneal [Miller, et a], 1999]
milk BALB/c mouse Intraperitoneal [Adel-Patient K et a]. 2003 ]
C3H/Hej mouse Intra gastric L' et 1 1999’
Dog Subcutaneous [ If a ' ]
Guinea pig Oral [Frrck, et al. 2005]
DEA/2 mouse Oral [Piacentini et al., 2003]
NMRI mouse Subcutaneous [Ito, et a1, 1997]
Intrapemma‘ [Poulsen, et al. 1990]
Egg BN rat Oral [Knippels, et al. 1998]
BALB/c mouse lntra gastric [Lee et a1 2004]
Guinea pig Oral ’ '
New Zealand rabbit Oral [Marokko, et al' 1991]
[Bassan, et al. 2002]
Peanut BN rat Oral [Knippels, et a], 2003]
BALB/c mouse Intraperitoneal [Adel-Patient et a]. 2005]
1““ dcm' [Betts, et al. 2004]
C3H/Hej mouse Oral .
Subcutaneous [L1, Ct 31. 2000]
Intraperitoneal [Li, et al. 2003]
[Pons, et al. 2004]
Dog Subcutaneous
_ , [Teuber, et al. 2002]
Neonatal swrne Intraperitoneal
[Helm et al. 2002]
Tree nuts BALB/c mouse Oral [Scholl, et a1, 2005]
Dog SuPcutaneous [Teuber, et a1, 2002]
BALB/c mouse Epicutaneous [Birmingham], et al 2005]
Fish BALB/c mouse lntra gastric route [Untersmayr, et a], 2003]
Soy BALB/c mouse Oral [Kroghsbo, et a]. 2003]
Dog Subcutaneous [Teuber, et al. 2002]
Wheat Dog Subcutaneous [Frick, et al. 2005]

 

29

Role of processing in food allergy

It has been established that allergenicity of some foods can be altered by
subjecting them to various processing methods [Alvarez-Alvarez, et a1. 2005; Chung, et
a1. 2003]. An allergen is typically a protein or glycoprotein. The structural features of an
allergen influence its immunological properties. Allergenic reaction occurs when a
specific IgE antibody binds to a special structural moiety of the allergen called an
epitope, which is a group of amino acids. When an allergenic food is subjected to heat/
mechanical/ chemical/ enzymatic treatment, significant alterations in protein structure
occur. First, loss of tertiary structure occurs followed by secondary structure loss (55-
70°c), cleavage of disulphide bonds (70-80°c), formation of new intra/ inter molecular

interactions, disulphide bond rearrangement (80-90°c) and the formation of aggregates

(90-100°c) [Davis, et al. 1998]. Besides these physical modifications, chemical
modifications like maillard reaction [Maleki, et al. 2000] may also occur leading to a
disorganized structure. Changes in the allergen protein structure results in the disruption
of these epitopes or exposure of new epitopes resulting in either a decrease or increase in
the allergenicity. In some cases new epitopes are formed turning a non allergenic food in
to an allergenic food. For example pecan nut [Malanin, et al. 1995]. Some foods do not

respond to the above treatments as their allergenic proteins are heat stable (casein, egg,

fish etc.) [Besler, et al. 2001].

We are not aware of any studies on the influence of processing on sesame

allergenicity. We review here the literature on the effect of processing on other food

types and we summarize those observations in the Table 10. Other studies were done

30

testing the other allergenic foods by subjecting them to different processing methods like

boiling, baking, autoclaving, microwaving, roasting, enzymatic treatment, cooking,

blanching etc. Some of these treatments increased the allergenicity (roasting on peanut)

while some decreased (boiling on peanut) and others exhibited no influence at all

(mango, celery, almond).

Table 10 Effect of processing on allergenicity of foods

 

 

No Food Processing method Effect on Reference
Allergenicity
1 Peanut Roasting (350°c/20min) Increased [Chung, et a].
2003]
Maturation Increased
Curing at 77°C Increased [Chung, et al.
2003]
Roasting ( 170°c/20min) Increased
Boiling (100°c/20min) Decreased
- o Decreased
Frying (120 c)
[Beyer, et al. 2001]
Boiling Decreased
[Mondoulet, et al.
2005]
2 Hazelnut Roasting (140°c/40min) Decreased [Hansen, et a].
2003]
3 Almond Roasting
Autoclaving No effect [Venkatachalam et
Blanching al. 2002]
Microwave heating
4 Celery Celery spice No effect [Baumepweber’ et
Cooked (110°c/15min) No effect al. 2002]

31

Cereals- wheat, rye,
barley and oats flour

Wheat

Milk whey proteins

Milk casein

Potato

Rice

Beef

Chicken egg

Lupine

Heat

Enzymatic
fragmentation

Heat
Enzyme hydrolysis

Heat
Enzyme hydrolysis

Heat

Incubated at 37°C for

30-120 min with a 10%
miso (condiment)
solution

Heat 1 00°C

Homogenization
Freeze drying

Peptic treatment

Homogenization
Freeze drying

Heat (90°c/30min)

Boiling( 60min)
autoclaving

-121 degrees C, 18 atm,
up to 20 min

32

Decreased

Decreased

Decreased

Decreased

No effect

Decreased

Decreased

Decreased

Decreased
Decreased

Decreased

Decreased
Decreased

Decreased

Decreased

Decreased

[Varjonen, et al.
1996]

[Watanabe, et al.
2000]

[Lee, 1992]

[Koppelman, et al.
2002]

[Izumi, et al. 2000]

[Fiocchi, et al.
1998]

[Fiocchi, et al.
1995]

[Quirce, et al.
2001]

[Alvarez-Alvarez, et
al. 2005]

12

13

14

Soy

Mango

Peach

-138 degrees C, 2.56
atrn, up to 30 min)

microwave heating (30
min)

extrusion cooking

Protease treatment Decreased
Technological No effect
processing

chemical lye peeling and Decreased
ultrafiltration

33

[Yamanishi, et al.
1996]

[Dube, et al. 2004]

[Brenna, et al.
2000]

CHAPTER 3: MATERIALS AND METHODS

I. Materials:

A. Equipment:

ELISA reader (Bio-Tek, Vermont), SDS apparatus (Bio-Rad, USA 300 ul).

B. Chemicals and Reagents:

Blood smear staining reagents are from Hema 3* manual staining system
which includes a fixative, solution I and solution H (Fischer Diagnostics, VA, USA).
Chemicals and reagents used for ELISA are biotin labelled secondary antibodies (BD
biosciences, Pharrningen, San Diego, CA, USA), SAAP - Alkaline phosphatase
conjugated streptavidin (Jackson Immunolaboratories), Substrate — Alkaline phosphatase
— (Sigma Diagnostics Inc), 1.5M NaCl, 0.001M NaHzPO4,0.1 M NazHPO4, 2% NaN3
(J .T.Baker, NJ, USA). Reagents prepared for ELISA wereIOX PBS (NaHzPO4.HzO,
NazHPO4, NaCl), Wash buffer (10XPBS, Tween 20, 2%NaN3), Dilution buffer
(10XPBS, Tween 20, 2%NaN3, BSA), Blocking buffer (10XPBS, 2%NaN3, BSA), 5%
gelatin blocking buffer (Gelatin), Coating buffer (NazCO3, NaHCOg, 2%NaN3, BSA),

Substrate buffer (2M MgClz.6HZO, Di ethnolamine, pH- 9.8)

Chemicals used in the Lowry-Folin assay were 0.1N NaOH, 0.5% CuSo4.5H20,

1% sodium citrate, 2% NazCO3 (J .T. Baker, NJ, USA), BSA (Intergen, NY, USA)

Reagents used were 0.1N NaOH, Folin reagent (Sigma — Aldrich, MO, USA), Solution A

34

(0.5% CuSO4.5HzO + 1% sodium citrate), Solution B (2% NaZCO3 in 0.1N NaOH),

Solution C (1 ml sol. A+ 50 ml sol. B).

All chemicals and reagents used for SDS — PAGE were from Bio-Rad, USA.

They were 30% Degassed Acrylamide/Bis (Acrylamide, N’N’-bis-methylene-
acrylamide), 1.5M Tris—HCI (Tris base, pH-8.8), 0.5M Tris-HCl (Tris base pH-6.8).
Reagents prepared were sample buffer (0.5M Tris-HCl, glycerol, 10% SDS, 0.5%
bromophenol blue), 10X running buffer (Tris base, Glucine, SDS); Separating/resolving
gel (11% Monomer solution {30% Degassed Acrylamide /Bis, 1.5M Tris-HCl}, 10% w/v
SDS, 10% APS {Ammonium per sulphate}, TEMED), Stacking gel (11% Monomer
solution {30% degassed Acrylamide/ Bis, 0.5M Tris-HCl, 10% w/v SDS}, 10% APS,
TEMED).

Chemicals and reagents used for alum preparation were 10% Al K(SO4)2.12 H20,
0.2% Phenol Red, 2N NaOH, 0.15 M NaCl. Mechanically hulled sesame seeds were
purchased from Arrowhead Mills, TX, USA; Commercial food extracts from Greer
Laboratories, NC, USA; Baked sesame foods like sesame crisp bread (WASA, Germany)
and Aunt Millie’s classic sesame hamburger buns (IN, USA) were purchased from

Meijer, Lansing, USA.

C. Software:
For reading ELISA plates, KC4 version 3.1 (BIO-TEK instruments, Vermont,
USA) software was used; for plotting graphs, Slide Write Plus version 6.1 (Advanced

Graphics Software, Inc., California, USA) was used; for computing statistics of the

35

experimental data and to evaluate significance, GraphPad software (GraphPad software.

Inc., San Diego, CA) was used.

11. Animals:
All mice used in these studies were purchased from Jackson Laboratories (Bar
Harbor, Maine, USA). All animals were female and 6 weeks old when they arrived. All

procedures involving mice were in accordance with Michigan State University policies.

Management:

Animals were housed in metal cages layered with wood shavings, 3-4 mice per
cage with a 12 hour light/dark cycle and were provided with ad libitum water and
standard pelleted food. Foods used in the experiments were not part of the diet. Mice
were given 1-week rest before starting the experiment with the intention of letting them

adjust to the new environment.

III. Methods:
A. Sterilization:

Sterilization of the equipment like pipettes, was done by spraying them with
90% ethyl alcohol. Surgical instruments, pipette tips and test tubes sterilization was done
by autoclaving at 120°C for 30 minutes. Solution sterilization was done using membrane

filters.

B. Preparation of alum and antigen solution for injections:

36

Alum adj uvant was prepared using standard procedure optimized in Dr.
Gangur’s laboratory. Just before injections, commercial extracts of sesame and vanilla,
with known protein concentrations, were opened in a tissue culture laminar flow hood
and required volume of solution was taken out using a sterile pipette and added to freshly

prepared alum to make solutions of specific concentration.

C. Immunization technigues:

a. Intraperitoneal route of immunization:
The antigen solution was prepared in alum and was drawn in to a 5 ml syringe.
Mouse was taken out of the cage with its tail and held on the back of neck. Mouse was
turned with the abdomen facing upwards and shaken twice gently in such a way that the
body organs move forward, away from the site of injection. On an imaginary line joining
the top of the two femur bones and a little right to the center or midline, a no.2 gauze
needle was inserted halfway through and 0.5 ml of solution was injected after aspiration.

The animal was put back in the cage.

b. Epicutaneous route of immunization:

The antigen solution was prepared in saline. Mouse was taken out of the cage
with its tail. Hair on its back had been clipped. Mouse was held with its skin on the back
of the neck and placed in a perforated plastic tube with anesthetic gauze. After anesthesia,
mouse was taken out and 100ul of antigen solution was applied on the clear back with a

pipette tip and let dry. A patch was applied around its body covering the site of the

37

application using a latex free, non-stick plastic bandage. Animal was put back in the

cage.

D. H_andling and Bleeding Technige:

The mouse hind foot bleeding protocol was developed by MSU veterinarian
of ULAR, Dr. Susan Stein. Briefly, mouse was allowed to warm under a light for few
minutes to facilitate vasodilatation. A 50ml plastic tube was taken and holes were made
in it. The mouse was picked from the cage by base of its tail and quickly moved to
tabletop. Mouse was restrained by grasping the skin between the ears, placed in the
perforated plastic tube and its hind leg was extended. Petroleum jelly was applied with a
cotton swab to get a clear view of the dorsal vein and to avoid instant blood clotting. Vein
was punctured with a 25 gauze needle and blood was collected in to a 300ul heparinized
micro-capillary tube (Microvette, Germany). Pressure was applied to the puncture site

with gauze to stop the blood flow and the mouse was put back in the cage.

E. Antibody analysis by Indirect ELISA:

The protocol used has been described earlier [Birmingham 2003] Enzyme-
linked immunosorbent assay (ELISA) was optimized for each of the food extracts
taking into account the background activity (that is, all reagents added except for the
sample). All reagents were used at a final volume of 50 til/well except for blocking
buffer that was used at 75 til/well. Washing was done with 200 ul/well using an

automatic ELISA washer (Dynex Technologies, Ultra-wash Plus). Each food extract

38

was analyzed for protein content by the Lowry's method and then used in ELISA for
coating at concentrations ranging from 10 to 5000 ug/ml. Briefly, ELISA plates (96-
well EIA/RIA plate, 96-well easy washTM, high binding, Corning, New York) were
coated with food extracts diluted in carbonate buffer (0.05 M, pH 9.6) and incubated
at 4 °C, overnight. Unbound extract was discarded and the plates were blocked in
PBS at 37 °C. For IgE assay, blocking was performed with 5% gelatin. After washing
(0.05% Tween 20 in PBS), serum samples were added at various twofold dilutions
from 1:20 to 1:640 or in some experiments at 1:30 to 1:61 ,440 in dilution buffer
(0.085% BSA, 0.05% Tween 20 in PBS). Following incubation, plates were washed
and a biotin-labeled anti-mouse IgGl, lgGZa, IgGZb, IgG, IgA, IgM or IgE antibody
added. After incubation, plates were washed and streptavidin alkaline phosphatase
conjugate was added at 1:4000 (in dilution buffer). Subsequently, plates were washed
and p-nitro-phenyl phosphate (PNPP) substrate added (1 Tablet per 5 ml substrate
buffer, according to manufacturer's instruction; Sigma). Reactions were allowed to
develop at room temperature in the dark and absorbance measured in a microplate
reader using dual mode with wavelengths at 405 nm (peak) minus 690 nm
(background) (Microplate ELISA Reader, SoftMax program, Molecular Devices).
According to manufacturer's instructions, dual mode provides more accurate
measurements since it adjusts the reading for background interference (Personal
communication, Technical Services, Molecular Devices). All plates included negative
controls (no mouse serum sample background and no antigen coating control) and a
positive internal control (a reference mouse serum sample containing known levels of

food-specific IgE antibody).

39

F. Slide preparatiorLLd staining protocol:
Ten ul of blood was drawn into a pipette from the dorsal vein of the foot and
dropped on one edge of a slide. Another slide was aligned at 45° angle to this slide and
the blood drop was spread in to a smear to the other edge. It was made sure that the slide

used for spreading does not have serrated edges. Slide was let dry at room temperature.

The HEMA-3 manual staining system was used for differential staining of
the slides. A slide was dipped in HEMA 3 fixative (methyl alcohol) for 5 seconds and
taken out. Excess solution was tapped off the slide and it was dipped in HEMA 3 solution
I (eosin stain) for 5 seconds. After draining off the excess solution, slide was dipped in
HEMA 3 solution H (methylene blue) for 5 seconds and rinsed in double distilled water.

The slide was air dried.

G. Differential Leukyocyte Count (DLC) protocol:

A drop of oil was placed on the smear. Using the oil immersion objective
lens, cells were counted following the “Battlement method” that is, counting 3 fields
along the horizontal edge of smear followed by two fields downwards towards the
center of the smear, then 2 fields side wards in a horizontal direction and finally 2
fields out wards, away from the center to reach the edge of the smear again. (Atlas of
Veterinary Hematology: Blood and Bone Marrow of Domestic Animals by John W
Harvey) A total of 200 cells were counted per slide and the numbers were expressed

as percent values.

40

H. Boiling protocol:

In the first approach, the seeds to be boiled were placed in sieve tea
infusers, which were labeled. The infusers were tied to a plastic rod with cotton strings.
The boiling container was filled with water and valves were turned open. Once the
water started boiling, the tea infusers with seeds were dipped into the water and the
timer was turned on. At specified time points the infusers were removed and cooled
rapidly on ice. In the second approach where the pre made extract was boiled, the
extracts were placed in plastic tubes and sealed with Parafilrn. These tubes were tied to
the bars of a metal container, which in turn was dipped into a water bath. At specified

time points, the tubes were removed and cooled rapidly on ice.

I. Harvesting sesame seeds from baked foods:
Sesame seeds from baked foods were scrapped off using a forceps and

collected. Extracts were made using 1X PBS after grinding in mortar.

J. Lowry-Folin method of protein estimation:
The protocol kindly provided by Dr. Winie Chiang (Dr. Gale Strasburg’s

lab, MSU) was used.

K. Statistical analysis:
All statistics on the data were performed using GraphPad software available

online (www.graphpad.com). For comparison of two groups and finding p values, the

41

paired t-test was used. For comparison of multiple groups, one-way ANOVA was used.

The statistical significance level was set at p<0.05.

Experimental design:

EXPERIMENT I (Invivo study)

The purpose of this experiment is to address first and second aims, that are to
characterize the differential antibody response and blood leukocyte profile against sesame
and vanilla in mice. Two different routes of administration were used to expose the mice
to sesame and vanilla. i) intraperitoneal injections, a common method of allergen
administration in the literature; ii) epicutaneous application, a more physiologically

relevant method of exposure, without the use of any adjuvant.

EXPERIMENT l A (Intraperitoneal route of sensitization of mice)
Approach:

Mice were bled 3 days prior to initial sensitization and then injected with
commercial food extract in alum base or alum alone Intraperitoneally. Alum was used as
an adjuvant. The mice were bled 8 days after injection and blood was collected in to
heparinized tubes. Blood smears were prepared on glass slides in the ratio of 2 slides per
mouse. 3 days rest were given to the mice before the second injection. This procedure
was repeated till all the mice received 3 injections. The plasma was separated by
centrifugation and analyzed for different Food - specific antibody levels (IgE, IgGl,
IgG2b, IgG3, IgM, IgA) using indirect ELISA. Blood smears were stained by using a

pre optimized HEMA 3 manual staining system and subjected to differential leukocyte

42

count (neutrophils, eosinophils, basophils, monocytes and lymphocytes) using oil

immersion microscopy. Protocol is illustrated in Figure 3.

sesame
g..,,-g_ . Antibody
Or ‘P ”‘1“ . ’ Bleeding " analysis +
_ I/P with alum DLC
vanilla

 

2 doses-100, 1000ug/mouse

N = 3-4 per group

2 strains of mice — C57Bl/6, Preb|eed 1st R 2nd R 3rd R
Balb/c

 

 

 

Figure 3. Experimental protocol for intra peritoneal route of
immunization of mice. Prebleed= bleeding prior to sensitization, 1"t R=
bleeding after first exposure, 2"° R= bleeding after second exposure,
3“ R= bleeding after third exposure, l/P = intra peritoneal

EXPERIMENT I B (Epicutaneous route of sensitization of mice)

Approach:

Mice were bled 3 days prior to initial sensitization and hair on their back was
clipped using a size 40-hair clipper. Commercial food extracts in saline or saline alone
was applied on the back of the mice depending on the group and covered with a patch on
day 1. After 4 days the patch was taken off (that is,) and on day 5 and the mice were bled

at a pre determined time point. Two blood smears were prepared per mouse. Six similar

43

applications of food extract in saline or saline alone were done and mice were bled after
each application at specified time points. Blood was collected in 200 ul heparinized blood
collection tubes. Later the tubes were centrifuged and the separated plasma was collected
and stored at —80°c. Blood smears were prepared. Blood smears were stained and
subjected to differential leukocyte count similar to the first experiment. Food - specific
antibody levels in plasma were measured using an indirect ELISA technique. The

protocol is described schematically in Figure 4.

Sesame 5'1; Patch on AntlbOdY
- ~' analysis +
Or —> —* Patch off "P DLC
_ Epicutaneous ,
vanilla Bleeding

application with

saline

Pre 1*"t R .......... 6‘“ R

 

3 doses-5, 50, 500 ug/mouse
N = 3-4 per group

 

 

strain of mice — Balb/c

 

Figure 4 Experimental protocol for epicutaneous route of
immunization of mice. Pre= bleeding prior to sensitization, 1" R=
bleeding after first exposure, 6"1 R= bleeding after sixth exposure

44

EXPERIMENT H (Processing experiment)

Purpose of this experiment was to study the third aim that is, effect of
processing methods on the allergenicity of sesame (ability of sesame to bind to sesame-
specific IgE antibody and induce allergy) and immunogenicity (ability of sesame to bind

to sesame- specific IgGl).

Under this experiment H, 3 different methods were utilized.

1) Boiling seeds in sieved containers through which water can freely move and
seeds are always in direct contact with water. 2) Making extracts from sesame seeds and
boiling them in sterile, sealed, 15ml plastic tubes in such a way that the boiling water
cannot come in direct contact with the extracts. This way we can avoid protein leaching
fi'om seeds into the water. 3) Collecting sesame seeds from baked foods (burger buns,

crisp bread, sesame sticks) and making an extract.

EXPERIMENT H A (Boiling sesame seeds in sieved containers)

Equal weights of mechanically hulled sesame seeds were placed in 4 sieved
metal containers (tea infusers) and labelled as unboiled, 3 minutes boiled, 30 minutes
boiled, 60 minutes boiled. The unboiled seeds were kept aside and other strainers with
seeds were dipped in boiling water at the same time and each one was taken out at the
specified time. The seeds were subjected to rapid cooling on ice. Then the seeds were
ground separately using mortar and pestle and extracted in 1X PBS (phosphate buffer
saline). The extracts were subjected to centrifugation at 4000 RPM for 10 minutes. The

supematants were separated from the sediment and oil fraction by repeated centrifugation

45

and stored in 15 ml sterile, plastic tubes at —20 0c. Protein concentration was determined
by using the Lowry- Folin assay. An indirect ELISA technique was used to test the
binding of these extracts to sesame specific antibodies (IgE, IgGl) in the plasma of mice
that were exposed to sesame. Plasma collected from the Experiment I A and I B was used

for this purpose.

EXPERIMENT H B (Boiling the extracts in plastic tubes)

Sesame seeds were ground in a mortar and extracted in 1x PBS. The extract was
centrifuged repeatedly at 4000 RPM until a clear supernatant free of oil fraction and
sediment was obtained. Equal quantities of this supernatant was aliquoted in 4 different
15ml plastic tubes and labelled as unboiled, 3 minutes boiled, 30 minutes boiled and 60
minutes boiled. Except for the unboiled fraction, the other three tubes were dipped in
boiling water for the specified times. After removing from boiling water, they were
cooled rapidly on ice and subjected to indirect ELISA to test the binding of these extracts

similar to the experiment H A.

EXPERIMENT II C (Baking experiment)

Sesame crisp bread and sesame burger buns bun, 2 baked foods with sesame,
were purchased. Sesame seeds on the surface of these foods were collected. They were
ground and extracted in 1X PBS. After repeated centrifugation at 4000 RPM, clear

supematants were collected and tested for their allergenicity similar to H A and H B.

46

Schematic representation of protocols for all the three experiments is shown

 

 

below in Figure 5.
II A
Sesame Boiling for Grind & Centrifuge _
seeds ‘P 30min and + extract in "’ 8‘ collect
60min 1X PBS Supernatants
II B . -
- . Borltng for
Sesame —> Grind & _, Centrifuge +30min and -—>
extract in & collect
seeds 60min
1X PBS Supernatant
t
H C l d' t ELISA
n trec
Collecting Grind & .
sesame extract Centrifuge With sesame +ve
8‘ collect —> lasma from
seeds from —’ in 1X " P
Supernatant mice
baked foods PBS

 

 

 

Figure 5 Experimental protocol for sesame processing.

The percentage of binding of sesame to specific IgE was calculated using the
following formula. The value of the highest binding of unboiled sesame (HB) (that is, at
lowest dilution of plasma) was taken as 100 and the percentage of binding (PB) was
calculated at all other dilutions of plasma (TB) for both the boiled extracts using the

formula; PBI= (TBIX 100)/ HB. TB] refers to the binding of a boiled extract at a specific

47

dilution of plasma. The mean of these percentages was calculated to express the

approximate average percentage of binding of each boiled extract.

48

CHAPTER 4: RESULTS

1. Characterization of specific antibody isotype responses to sesame and vanilla in a
mouse model

Mice were evaluated for the levels of sesame and vanilla specific antibody
isotypes in the plasma before and after every exposure to food. Two methods of food
sensitization were employed: intraperitoneal (i.p.) and epicutaneous. For i.p. sensitization,
C57Bl/6 and Balb/c strains of mice were used. For epicutaneous method, only Balb/c
strain was used. The following section describes the questions addressed and the results

obtained from these experiments.

1.1 : Sesame elicited enhanced Type-2 associated antibody responses relative to vanilla

in an intraperitopeal mogel of sensitization
Groups of C57Bl/6 mice were injected with sesame and vanilla intraperitoneally

as described in methods section. Sesame specific and vanilla specific Type-2 associated
antibodies (that is, IgE and IgG1) were measured during primary (day 8, after first
injection), secondary (day 7, after 2nd injection) and tertiary (day 5 and day 40, after 3 rd
injection) immune responses. Pre-sensitization plasma (that is, plasma of the mice before
exposure to food did not show significant antibody levels. Following injections, there was
significant increase in the antibody levels in comparison with the pre immunization levels
in the mice injected with sesame at both the doses of 100 ug/mouse and 1000 ug/mouse
(P<0.05). In contrast, there were no detectable antibodies for vanilla in vanilla injected
mice. There was a significant difference (P<0.05) between peak IgE and IgG] levels of

sesame 100 ug/mouse group and same response of vanilla 100 ug/mouse group and

49

(P<0.05) between sesame 1000 ug/mouse group and vanilla 1000 ug/mouse group. Both
the antibody isotype levels changed significantly with the dose (P<0.05) (i.e.) mice that
received 1000 ug sesame /mouse showed relatively higher IgE levels than those with 100
ug/mouse; the group with 100 ug sesame /mouse showed higher lgGl levels than 1000

ug/mouse. The data are shown in Figures 6 and 7 respectively.

We then examined the antibody response in Balb/c strain of mice. As evident, they
also exhibited similar pattern in the antibody profiles, that is, mice that were exposed to
sesame intraperitoneally developed high plasma IgE and IgG] levels following exposure to
the food (P<0.05) while those mice that were exposed to vanilla did not develop these
antibodies. Pre immune plasma was negative for Food - specific IgE and IgG1 antibodies in
both the groups. There was significant difference between the levels of both antibody
isotypes when compared between sesame treated group and vanilla treated group (P<0.05).

The results are displayed in Figures 8 and 9 respectively.

50

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1.2: Sesame elicited enhanced Type-2 associated antibody responses relative to vanilla
in epicutaneous model of sensitization

Pre immunization plasma was negative for Food - specific IgE and IgG1
antibodies in all the groups of Balb/c mice that were exposed to the foods epicutaneously.
Sesame treated mice showed a dose dependent elevation of plasma IgE and IgG1 levels
in comparison with vanilla treated mice. Mice that received 5 ug and 50 ug sesame
showed no significant change in IgE levels. But those mice that received 500 ug sesame
showed a significant increase in IgE (P<0.05). Similarly plasma IgGl levels did not vary
in 5 ug sesame/ mouse group while the mice that received 50, SOOug sesame showed a
significant rise in the specific IgGl levels. Vanilla did not induce any significant change

in specific IgE, IgGl levels. The results are shown in Figures 10 and 11 respectively.

55

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57

1.2 : Sesame elicited enhanced Type-1 associated antibody responses relative to

vanilla in intraperitoneal model of sensitization

The plasma of C57Bl/6 strain mice that received sesame and vanilla
intraperitoneally, was screened for Food - specific Type-l associated antibody, that is,
IgGZa, during primary (day 8), secondary (day 7) and tertiary (day 5, day 40) immune
responses. Pre-sensitization plasma (pre bleed) did not have significant antibody levels.
Following injections, sesame exhibited a dose dependent elevated IgGZa antibody levels
while vanilla did not. There was significant difference between the levels of IgGZa in
sesame 100 and vanilla 100 groups (at peak response) (P<0.05). The data are graphically
represented in Figure 12. Balb/c mice that were exposed to sesame intraperitoneally
developed high plasma IgG2a levels while those mice that were exposed to vanilla
showed no change in the levels. Pre immune plasma was negative for Food - specific
IgGZa in both the groups. There was significant difference between the IgGZa levels of
mice that received 100 ug sesame at peak response and that of mice that received 100 ug

of vanilla (P<0.05). The results are displayed in Figure 13.

1.3 : Sesame elicited enhanced Type-1 associated antibody responses relative to vanilla

in epicutaneous model of sensitization

Balb/c mice that were exposed to sesame and vanilla through an epicutaneous
route showed a similar dose- dependent pattern with elevated plasma IgGZa levels in
sesame treated mice with a significant difference in comparison with vanilla treated mice

(P<0.05) between peak response of IgG2a at 50, SOOug/mouse dose while mice that

58

received 5 ug sesame showed no significant elevation. Control group mice that received

saline alone, showed no Food - specific IgGZa. The results are portrayed in Figure 14.

59

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62

1.4 : Sesame and vanilla diffeifl eliciting other antibody isotypes in mice in
Mritgeal method of sensitization
C57Bl/6 strain of mice that were injected with sesame and vanilla
intraperitoneally, were screened for other Food - specific antibodies, that is, IgG2b, IgG3,
IgM and IgA, during primary (d8), secondary (d7) and tertiary (d5, d40) immune

responses. Pre sensitization plasma (pre bleed) did not have significant antibody levels.

Following injections, sesame triggered elevated IgG2b and IgM antibody levels
while vanilla did none. The difference was significant. However there was no significant
change in the plasma levels of specific IgG3 and IgA in all the groups. The data are

graphically represented in Figures 15, 16, 17 and 18.

Mice belonging to Balb/c strain upon intraperitoneal exposure to the food
exhibited similar pattern in the antibody profiles, that is, mice that were exposed to
sesame through intraperitoneal route developed high plasma IgGZb, IgGB and IgM levels
while the mice that were exposed to vanilla showed no significant change in the levels of
these four antibody isotypes. Pre immune plasma was negative for these Food - specific
antibodies. Plasma IgA levels did not vary significantly in both the groups. The results

are displayed in Figures 19, 20, 21 and 22 respectively.

63

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71

1.5 : Sesame and vanilla differ Ln eliciting other antibody isotypes in mice in
epicutaneous method of sensitization

Balb/c mice that were exposed to sesame and vanilla through epicutaneous route
showed significantly different antibody levels in the plasma. Sesame treated mice showed
elevated plasma IgGZb, IgG3 (P<0.05) levels in comparison with vanilla treated mice.
However, the IgGZb, IgG3 antibody response to sesame was dose influenced. The mice
that were given SOOug sesame /mouse showed peak levels of IgG2b, IgG3 when
compared with the other mice that received 5 ug sesame/mouse or 50 ug sesame/mouse.
IgM and IgA levels did not vary much in all the groups. Vanilla showed higher plasma
IgM levels than sesame even though there was no change in the levels before and after
immunization. Control group mice that received saline alone, showed no significant Food
- specific IgGZb and IgA with minor levels of IgG3 and IgM without significant variation
in the levels from prior to and after sensitization. The results are portrayed in Figures 23,

24, 25 and 26 respectively.

72

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76

2. Characterization of peripheral blood eosinophilia profile in sesame vs. vanilla
sensitized mice
2.1 : Sesame but not vanilla induce eosinon hilia in mice in intranertitoneal model of
sensitization
Peripheral blood leukocyte analysis of C57Bl/6 mice that received sesame or vanilla
intraperitoneally revealed that there is relatively more increase in eosinophil counts post
exposure to sesame at both the doses with a 5% rise at 100 ug/ mouse dose (P<0.05) and a
9% rise at 1000 ug/ mouse dose (P<0.05) while vanilla influenced less eosinophilia with a
1.5% increase at both the doses. The peak eosinophil counts precipitated by sesame at both
100 and 1000 ug/mouse dose were significantly high in comparison with those of vanilla
(P<0.05). The food-induced eosinophilia exhibited a significant dose dependent response.

The results are displayed in Figure 27.

Balb/c mice that were injected with sesame at 100 ug/mouse intraperitoneally
exhibited a similar pattern with a 6.5% increase in the plasma eosinophil levels (p< 0.05)

while those that received vanilla showed non-significant change in the levels. (Figure 28)

2.2 : Neither sesame nor vanilla induce eosinonhilia in mice in enicutaneous model of

sensitization
Balb/c mice that were exposed epicutaneously to sesame or vanilla revealed no
significant eosinophilia response at any of the doses tested. Data are graphically

represented in Figure 29.

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3. Characterization of influence of processing on sesame allergenicity

3.1: Boiling sesaw seeds reduces sesame aller enici that is bindin of I E
antibody to sesame)

IgE is the hall mark of allergy. An allergen causes an immune response, which can be
measured by elevation of IgE levels. The extracts of sesame prepared from boiled and
unboiled sesame seeds were evaluated for their allergenic properties by testing their
binding to sesame specific IgE in the plasma of mice exposed to sesame. Three different

strains of mice were used.

There was significant difference in binding to specific IgE in the plasma of
C57Bl/6 mice by unboiled sesame in comparison with the sesame boiled for 30 minutes
and that for 60 minutes (P<0.05). The binding of sesame boiled for 30 minutes was
significantly higher than that of 60minutes (P<0.05). (Figure 30 (a)). The binding was
decreased by 78% after boiling the sesame seeds for 30 minutes and by 69% after 60

minutes boiling. (Figure 30 (b))

A similar pattern was observed when the plasma of sesame sensitized Balb/c mice
was used, that is the binding of sesame to specific IgE was significantly decreased with
boiling (P<0.05) and sesame boiled for 30 minutes showed relatively more binding to

specific IgE than the sesame boiled for 60 minutes (P<0.05) (Figure 31 (a)).

81

Sesame lost 17% of its IgE binding ability with 30 minutes boiling and 61%

upon 60 minutes boiling. (Figure 31 (b))

When sesame specific IgE positive plasma from ASW mice was used, the results
exhibited similar pattern as that to Balb/c mice, that is, binding ability significantly
decreased with boiling (P<0.05) and 30 minutes boiled sesame had relatively more
binding ability than the 60 minute boiled sesame (P<0.05). (Figure 32 (a)). Sesame lost
31% of binding to specific IgE after boiling for 30 minutes and upon continued boiling

for 60 minutes the loss of binding was increased to 33%. (Figure 32 (b))

3.2 : Boiling sesame seeds reduces sesame immunogenicity (that is, binding of 1ng
antibody to sesame)

An immunogen causes an immune response which can be measured by
elevation in any Ig levels. The extracts of sesame prepared from boiled and unboiled
sesame seeds were evaluated for their immunogenic properties by testing their binding to

sesame specific IgGl in the plasma of mice exposed to sesame.

There was no significant difference in binding to specific IgGl in the plasma of
C57Bl/6 mice by unboiled sesame in comparison with the sesame boiled for 30 minutes
and that for 60 minutes (P<0.05 at lowest dilution of plasma). The binding of sesame boiled
for 30 minutes was significantly lower than that of 60minutes (P<0.05). (Figure 30 (c))
The binding was decreased by 21% after boiling the sesame seeds for 30 minutes and by

22% after 60 minutes boiling. (Figure 30 (d))

82

A similar pattern was observed when the plasma of sesame sensitized Balb/c
mice was used with the exception that sesame boiled for 30 minutes showed relatively
more binding to specific IgGl than the sesame boiled for 60 minutes (P<0.05)(Figure 31

(c)). Sesame lost 15% of its IgGl binding ability with 30 minutes boiling and 30% upon 60

minutes boiling. (Figure 31 (d))

When sesame specific IgGl positive plasma from ASW mice was used, the results
exhibited similar pattern as that involving the Balb/c mice, that is, binding ability
significantly decreased with boiling (P<0.05) and 30 minutes boiled sesame had relatively
more binding ability than the 60 minute boiled sesame (P<0.05) (Figure 32 (c)). Sesame
lost 3% of binding to specific IgGl after boiling for 30 minutes and upon continued boiling

for 60 minutes the loss of binding was increased to 13%. (Figure 32 (d))

83

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86

3.3 : Boiling sesame extract reduces sesame allergenicig (that is, binding of IgE
antibody to sesame)

The extracts of sesame prepared beforehand and subjected to boiling were
evaluated for their allergenic properties by teSting their binding to sesame specific IgE in
the plasma of mice exposed to sesame. Two different strains of mice were used.

In Balb/c model, the binding of sesame to specific IgE was significantly decreased with
boiling (P<0.05 at lowest dilution of plasma) (Figure 33 (a)). Sesame lost 29% of its IgE

binding ability with 30 minutes boiling and 15% upon 60 minutes boiling. (Figure 33 (b)).

When sesame specific IgE positive plasma from ASW mice was used, similar
results were found (Figure 34 (a)) with sesame loosing 32% of binding to specific IgE
after boiling for 30 minutes and upon continued boiling for 60 minutes the loss of binding

was decreased to 25%. (Figure 34 (b)).

3.4 : Boiling sesame extract reduces sesame immunogenicity (that is, binding of
IgG] antibody to sesame)

The extracts of sesame prepared beforehand and subjected to boiling were evaluated
for their immunogenic properties by testing their binding to sesame specific IgGl in the
plasma of mice exposed to sesame (Figure 33 (c)). In Balb/c model, sesame lost 13% of its

IgGl binding ability with 30 minutes boiling and 12% upon 60 minutes boiling. (Figure 33

(d))-

When sesame specific IgGl positive plasma from ASW mice was used, sesame
showed slightly reduced IgGl (Figure 34 (c)) with a loss of only 4% of binding to specific
IgGl after boiling for 30 minutes and upon continued boiling for 60 minutes the loss of

binding was decreased to 2%. (Figure 34 (d)).

88

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3.5 : Baking reduces sesame allergenicity (that is, binding of IgE antibody to sesame)

When the plasma of sesame sensitized Balb/c mice was used raw sesame showed
relatively more binding to specific IgE than the sesame collected from crisp bread and
burger buns (P<0.05 at lowest dilution of plasma). The binding of sesame to specific IgE
has significantly decreased with baking (P<0.05) (Figure 35 (a)). Sesame from crisp bread
showed 10% less IgE binding ability and sesame from burger buns showed 58% less IgE

binding ability when compared with the raw sesame. (Figure 35 (b))

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exhibited a similar pattern as that involving the Balb/c mice, that is, IgE binding ability
significantly decreased with baking (P<0.05 at lowest dilution of plasma) However, sesame
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(Figure 36 (a)). Sesame from crisp bread showed 47% less IgE binding ability and sesame
from burger buns showed 29% less IgE binding ability when compared with the raw

sesame. (Figure 36 (b))

3.6 : Baking reduces sesame antigenicig (that is, binding of IgG1 antibody to

sesame

When the plasma of sesame sensitized Balb/c mice was used raw sesame showed
relatively more binding to specific IgGl than the sesame collected from crisp bread and
burger buns (P<0.05 at lowest dilution of plasma). The binding of sesame to specific IgGl

was significantly decreased with baking (P<0.05) (Figure 35 (c)).

91

Sesame from crisp bread showed 26% less IgGl binding ability and sesame from
burger buns showed 20% less IgGl binding ability when compared with the raw sesame.

(Figure 35 (d)).

When sesame specific IgE positive plasma from ASW mice was used, the results
exhibited a similar pattern as that involving the Balb/c mice, that is, IgGl binding ability
significantly decreased with baking (P<0.05 at lowest dilution of plasma). Sesame from
crisp bread showed relatively less binding ability than that from burger buns (P<0.05).
(Figure 36 (c)). Sesame from crisp bread showed 21% less IgGl binding ability and
sesame from burger buns showed 4% less IgGl binding ability when compared with the

raw sesame. (Figure 36 (d)).

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94

CHAPTER 5: DISCUSSION

This study was undertaken with the following questions and rationale: (i) Does
sesame elicit a detectable immune response in mice? If so, one could characterize the
allergic immune responses such as IgE and eosinophilia and use the mouse model to
develop a sesame allergy disease model in future studies; (ii) Can we identify specific
immune markers to distinguish the activation of immune system by the allergenic food
(sesame) from that of a model non-allergenic food (vanilla) in terms of the antibody
response and blood leukocyte profile in mouse model. If so, in future studies, these
markers could be tested for application in predicting allergenic potential of novel foods;
(iii) Is it feasible to use the mouse system to test the effect of food processing on
allergenicity? If so one could plan future studies to compare human vs. mouse systems to

test the validity of using the mouse model for such studies.

There are several important findings from this study: (i) Sesame seed can elicit
sesame binding IgE antibody response in mice. This ability of sesame seed is an intrinsic
property because IgE response was demonstrable in two strains of mice with different
MHC haplotype by two different routes of exposure that is, transdermal exposure to
sesame without the use of adjuvant and intraperitoneal exposure with the use of alum
adjuvant; (ii) sesame was more potent in eliciting both Type-1 as well as Type-2
associated immune response compared to vanilla independent of host strain used; (iii)
Sesame also elicited a significant eosinophilia response only in the intraperitoneal model

of sensitization but not in epicutaneous model; and (iv) boiling and baking appear to

95

reduce allergenicity as well as immunogenicity, although the relative effect was

dependent on which strain of mouse was used in the study.

Among other antibody isotypes tested, sesame triggered the plasma IgG3
antibody response in Balb/c strain of mice but not C57Bl/6 strain. On the other hand IgM
antibodies elicited by sesame was dependent upon the route of exposure to sesame, that
is, increased IgM levels were seen in both the strains by only Intraperitoneal route of
exposure to sesame but not by the epicutaneous route. There was no detectable plasma
IgA antibody response by sesame or vanilla independent of the strain of the mice and
route of exposure to the food type. Thus, in most of the cases, sesame was found to be

more immunogenic than vanilla in this study.

Sesame was also superior in eliciting another hallmark of allergic response——
peripheral blood eosinophilia. Thus, intraperitoneal exposure to sesame but not vanilla
elicited significant eosinophilia in both C57Bl/6 and Balb/c mice. However epicutaneous
exposure of mice to sesame failed to induce such a change. It is unclear at present, the
reason for this difference. However, it is possible that as opposed to epicutaneous method
of sensitization, systemic injection might elicit a local inflammatory response that might
help sesame to promote eosinophilia. However, vanilla when injected did not promote
eosinophilia. This suggests that systemic injection of only allergenic foods might promote
eosinophilia. It remains to be seen if this readout along with IgE response might be used

to predict allergenic potential of novel foods.

96

There are few previous studies that compared the immune response of allergenic

vs. non-allergenic food types. These are summarized in Table 11.

Table 11 Allergenic vs. non- allergenic foods comparative study in mice

 

 

Allergenic food Non allergenic Animal Research Reference

Food model & Findings

route of
exposure

Ovalbumin Potato agglutinin Balb/c —- I/P IgE, IgG in [Dearman, et al.
(egg) without allergenic foods 2003 ; Deannan,
BSA (bovine adjuvant IgG but no IgE et al. 2003]
serum albumin) in non allergenic
Peanut foods
agglutinin
chicken eggs, coffee, sweet C57BL/6- No [Birmingham et
peanuts, potatoes, carrots, I/P -with distinguishing of al. 2002]
almonds, white potatoes, adjuvant allergenicity by
filberts- cherries, lettuce, Specific IgGl
hazelnuts, and spinach
walnuts,
soybeans, and
wheat
Peanut Potato protein Balb/c — I/P IgE, IgG in [Atherton, et al.
agglutinin containing acid without allergenic foods 2002]
Ovalbumin phosphatase adj uvant IgG but no IgE
(e88) enzymatic in non allergenic

activity (PPE) foods
Peanut Potato acid Balb/c -ora1 IgE, IgG in [Dearman, et al.
agglutinin phosphatase gavage allergenic foods 2001]
Ovalbumin (PAP) IgG but no IgE
(egg) in non allergenic

foods

 

97

None of the above studies involve sesame or vanilla. We have studied all the six
antibody isotypes in this model while the previous studies only looked at IgE and IgG.
Moreover, eosinophilia response has not been studied in any of these studies.
Furthermore, we are not aware of previous studies comparing immune response to
allergenic vs. non-allergenic food following epicutaneous exposure to foods in the
absence of adjuvants—a novel feature of our approach. This might be useful, because
with this adjuvant-free approach, one could eliminate the non-specific effects caused by

adjuvants in the system.

While this study has demonstrated that both hallmarks of allergic response (IgE
and eosinophilia) are inducible in mouse model using sesame seeds, mechanisms of their
induction remain to be studied. Based on the literature, it is possible to hypothesize that
sesame elicits elevated IgE antibody response via activation of IL-4, a critical cytokine
required for antibody class switching to IgE isotype; it elicits eosinophilia via activation
of IL-5, a critical cytokine required for eosinophilia response. Follow-up studies
conducted in our laboratory have provided evidence in support of these hypotheses
(Appendix-II). However, use of IL-4 and IL-5 gene knockout mice might be employed to

test this hypothesis further.

A major limitation of this study was that we did not use oral method of exposure
for studying immune response. This decision was based on the evidence in the literature
and the data from our laboratory that oral exposure to foods in mice results in oral

tolerance rather than detectable antibody response [Chase, 1946; Mizumachi K, 2002].

98

Therefore, we had no choice but to go for systemic injections and epicutaneous exposure
as methods of food exposure. As evident from this study and other studies from our lab,

significant immune responses could be elicited by these methods.

A second objective of this study was to conduct a pilot study testing the effect of
boiling and baking on sesame allergenicity. The rationale was that, if the processing
techniques were to exhibit such influence on sesame allergenicity then further studies
could be conducted involving humans and extending this knowledge towards preparation

of non/hypo-allergenic sesame containing food products.

There are 3 important findings from this preliminary study. (i) Boiling sesame
seeds for 30minutes and 60minutes lead to significant loss of allergenic nature of sesame
as evident by its binding to specific IgE. This was confirmed by similar results with the
use of sesame specific IgE and IgG1 positive plasma from three different strains of mice;
(ii) Boiling sesame extract in tubes in order to avoid the leaching of antigen also showed
similar effects; (iii) Baked sesame seeds from crisp bread and burger buns showed
reduced allergenicity. However there were quantitative differences between plasma of
different strains of mice suggesting that IgE antibody produced by different individuals

might differ in their relative ability to bind to processed sesame allergens.

Although the mechanism of reduction in allergenicity is unclear at present, it is

possible that processing alters the structure of allergenic proteins in such a way that the

IgE binding epitopes loose their structural integrity [Gruber P, et al. 2004; Maleki, et al.

99

2000]. This effect may not be specific to the IgE binding epitope alone. Because in most
cases there were reductions in IgGl binding as well suggesting global alteration in

protein structure.

A major limitation of this second part of the study was that we measured
allergenicity based on in vitro binding to IgE by ELISA. It is noteworthy that more
studies need to be done to further confirm these results using IgE inhibition assays as
well as in vivo testing of allergenicity by exposing mice to processed sesame seeds and
test their ability to elicit IgE responses. These types of studies have been done in human
system but not in mouse models because they typically need large volumes of plasma
(typically 5 to 10 ml) that is expensive to get from mouse models. Thus, firture studies
including studies with plasma samples of sesame allergic patients are necessary to draw

final conclusions on the effect of baking and boiling on sesame allergenicity.

In summary: (i) Characterization of immune response to sesame in mice should help
in developing a mouse model of sesame allergy; (ii) The pilot study on the effect of boiling
and baking on sesame allergenicity in mice suggest the need of future studies
using blood samples from human sesame allergic patients; and (iii) this study has also

contributed to an improved understanding of the activation of immune system by a

model allergenic vs. a model non-allergenic food.

100

FUTURE DIRECTIONS

As the next step in this research, there are several future studies that may be

conducted as suggested below:

(i) Test the impact of sesame sensitization on sesame specific IL-4, and IL-5 cytokine
responses in the spleen to understand the mechanisms of IgE and eosinophilia responses
in this model. My hypothesis would be that sesame but not vanilla elicits a systemic IL-4
and IL-5 response in the mouse model. Furthermore, the role of chemokines like eotaxin,

which play a key role in eosinophil recruitment, may also be studied.

(ii) Based on the results obtained, future studies to develop a sesame allergy disease
model may be conducted. My hypothesis would be that sesame but not vanilla exposure
results in allergy in mice. (iii) Future studies may be conducted using a large panel of
allergenic foods vs. a large panel of non-allergenic foods and a set of novel foods such as
GM (genetically modified) foods, to test whether IgE response, and eosinophilia response
might be able to help to predict allergenic potential of novel foods; (iv) Effect of
processing on sesame allergenicity needs to be confirmed by additional studies such as
IgE inhibition and in viva testing as well as testing serum samples form sesame allergic

patients.

101

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