ALTERNATE ALLERGY TESTING FOR EOSINOPHILIC …users.wpi.edu/~sahobson/Pdfsused/STEMThesisFinalCopy.pdfHobson 6 Abstract Eosinophilic Esophagitis (EoE) is a chronic, antigen-driven

ALTERNATE ALLERGY TESTING FOR EOSINOPHILIC ESOPHAGITIS (EOE) Sydney Hobson

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Table of Contents

ACKNOWLEDGEMENTS ........................................................................................................................... 4

ABSTRACT ............................................................................................................................................... 6

LITERATURE REVIEW ............................................................................................................................... 7 EOSINOPHILIC ESOPHAGITIS ....................................................................................................................... 7 ANTIGEN RECOGNITION ............................................................................................................................ 7

Eosinophils ...................................................................................................................................... 7 Other Important Cells ...................................................................................................................... 8

ALLERGY TESTING .................................................................................................................................... 9 Skin Prick Tests ................................................................................................................................ 9 Patch Testing ................................................................................................................................ 10 Blood Tests .................................................................................................................................... 10 Accuracy of Allergy Testing Methods for Eosinophilic Esophagitis .................................................. 11 Difference Between Typical Allergies and EoE Allergies .................................................................. 11

MORE ACCURATE TREATMENT OPTION ...................................................................................................... 12 Restrictive Diets and Frequent Endoscopies ................................................................................... 12 Disadvantages of the Elimination Diets and Endoscopies ............................................................... 14

CYTOKINES AND SALIVA ........................................................................................................................... 14 “Eosinophilic Esophagitis Related Cytokines in Saliva: Characterization and Methodological Considerations” ............................................................................................................................. 14 Role of Cytokines in Eosinophilic Esophagitis .................................................................................. 15 Specific Cytokines and Their Roles in Eosinophilic Esophagitis ........................................................ 15

RESEARCH PLAN .................................................................................................................................... 17 RESEARCHABLE QUESTION ....................................................................................................................... 17 HYPOTHESIS ......................................................................................................................................... 17 PROCEDURE OVERVIEW .......................................................................................................................... 17

MATERIALS AND METHODS .................................................................................................................. 18 MATERIALS FOR TEST #1 ......................................................................................................................... 18 PROCEDURE FOR TEST #1 ........................................................................................................................ 19 MATERIALS FOR TEST #2 ......................................................................................................................... 21 PROCEDURE FOR TEST #2 ........................................................................................................................ 22

RESULTS ................................................................................................................................................ 24 TEST #1 ............................................................................................................................................... 24 TEST #2 ............................................................................................................................................... 26

CONCLUSIONS ....................................................................................................................................... 30

REFERENCES .......................................................................................................................................... 32

APPENDIX ............................................................................................................................................. 34 APPENDIX A: LIMITATIONS & ASSUMPTIONS ............................................................................................... 34 APPENDIX B: TEST #1 ............................................................................................................................. 34

Preparation for Test #1 .................................................................................................................. 34 Well Plate Division Test #1 ............................................................................................................. 37 Well Plate Standards Test #1 ......................................................................................................... 37 Well Plate Division test #1 ............................................................................................................. 37

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Aspiration and Washing Directions Test #1 .................................................................................... 38 APPENDIX C: TEST #2 ............................................................................................................................. 39

Well Plate Design Test #2 .............................................................................................................. 39 Aspiration and Washing Directions Test #2: ................................................................................... 41

APPENDIX D: ........................................................................................................................................ 41 Raw Data Test #1 .......................................................................................................................... 41 Fixed Data Test #1 ......................................................................................................................... 47

APPENDIX E: ......................................................................................................................................... 48 Raw Data Test #2 .......................................................................................................................... 48 Fixed Data Test #2 ......................................................................................................................... 52

APPENDIX F: ......................................................................................................................................... 54 Analysis Test #1 Raw Data ............................................................................................................. 54 Analysis Test #1 Fixed Data ........................................................................................................... 56 Analysis Test #2 Raw Data ............................................................................................................. 57 Analysis Test #2 Fixed Data ........................................................................................................... 61

APPENDIX G: NOTES .............................................................................................................................. 64 Do We Know What Causes Eosinophilic Esophagitis? A Mechanistic Update .................................. 64 Food Allergy Testing in Eosinophilic Esophagitis: What the Gastroenterologist Needs to Know ...... 66 Eosinophilic Esophagitis From an Allergy Perspective: How to Optimally Pursue Allergy Testing & Dietary Modification in the Adult Population ................................................................................. 67 Eosinophilic esophagitis: A clinicopathological review.................................................................... 73 Biology in Focus – 35.1 .................................................................................................................. 83 Biology in Focus – 35.2 .................................................................................................................. 89 Eosinophilic Esophagitis................................................................................................................. 94 Allergic Reactions .......................................................................................................................... 97 What are Cytokines ....................................................................................................................... 99 Allergy skin test ........................................................................................................................... 100 Food Patch Testing ...................................................................................................................... 102 Skin Prick Tests ............................................................................................................................ 103 Blood tests .................................................................................................................................. 105 Alternate Food Allergy Tests to Avoid .......................................................................................... 106 Eosinophilic Esophagitis Related Cytokines in Saliva: Characterization and Methodological Considerations ............................................................................................................................ 107 Eosinophilic Esophagitis............................................................................................................... 109 Promising Modalities to Identify and Monitor Eosinophilic Esophagitis ........................................ 111 Sandwich ELISA ........................................................................................................................... 112 A striking local esophageal cytokine expression profile in eosinophilic esophagitis ....................... 113 T-helper 2 Cytokines, Transforming Growth Factor B(beta)1, and Eosinophil Products Induce Fibrogenesis and Alter Muscle Motility in Patients with Eosinophilic Esophagitis.......................... 114 Understanding eosinophilic esophagitis: the cellular and molecular mechanisms of an emerging disease ........................................................................................................................................ 116 Approaches to determine expression of inflammatory cytokines .................................................. 119

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Acknowledgements

I would like to thank Mass Academy for providing me with the opportunity to explore

my passion for finding a possible solution for less invasive testing for a disease that my brother

has called Eosinophilic Esophagitis (EoE). I’d also like to specifically thank Mr. Barney for

believing in my project and providing me with a grant to complete additional testing. Thank

you to Ms. Curran, my STEM teacher, for inspiring me to choose a project that I’m passionate

about and challenging me to think outside the box, no matter how many obstacles arise. I’d

also like to thank Mr. Regele, my STEM advisor, for his continual interest and follow-up on my

project.

Thank you to Dr. Olive from Texas Children’s Hospital who wrote the article that inspired

my project, “Eosinophilic Esophagitis Related Cytokines in Saliva: Characterization and

Methodological Considerations,” for always responding to my emails and providing feedback on

my project. I’d also like to thank Dr. Davis and Dr. Hiremath, who worked with Dr. Olive on the

article, who also provided feedback on my testing results. A big thank you to Dr. Lee from

Boston Children’s Hospital, who specializes in EoE, for discussing my testing methodology and

results with me.

I would also like to thank two companies that I purchased my materials from,

Stallergenes Greer and Thermofisher Scientific for providing excellent customer service and

answering any questions about the materials that I was going to buy. I’d also like to thank Dr.

Manning and Elizabeth Crowley of WPI for proving me with lab space and their time to

complete my project. I’d also like to thank my friend for donating her saliva for me to use in my

project.

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I would also like to thank my parents for purchasing the ELISA kit for me. I knew I was

asking a lot as it was very expensive, but they did it in a heartbeat without thinking. They have

always believed in me with everything I do and that is something that I hold so dear in my

heart. Their positive, love and support has not only pushed me to believe in myself, but has

made me a more determined and motivated person to dream big and reach my goals.

Finally, I would like to thank the most important person in this project, my brother who

has EoE. He has inspired me to pursue a research project that attempts to tackle the most

pressing project in the EoE world, allergy testing. His strength while facing the whole process of

the disease has motivated me to try and find a cure to make sure that no one else has to go

through the lengthy and invasive process that is currently in place now. Thanks to his saliva, it’s

possible that a less invasive testing for EoE could be established. He is the strongest person

that I know and for that, I cannot thank him enough.

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Abstract Eosinophilic Esophagitis (EoE) is a chronic, antigen-driven immune disease that causes a

delayed inflammation in the esophagus, resulting in food impaction. Routine allergy testing

methods, time-consuming food elimination diets, and invasive and frequent endoscopies are

used to find the cause of the inflammation. This study involving a patient with dairy-induced

EoE and his sister, focuses on a less invasive saliva-based allergy testing method that reduces

the number of endoscopies and amount of time needed to find the cause of EoE. Saliva samples

taken from the patient and his sister were exposed to either dairy or wheat. An ELISA kit was

used to quantify the salivary concentration of IL-13 cytokines, proteins involved in EoE. For the

EoE patient, the first test showed an increase of cytokine levels in the wheat sample (p-value =

4.25*10^-08), and a decrease in the dairy sample (p-value = 1.37*10^-15). The second test

showed different trends in the data and more variable results. Therefore, additional testing

needs to be completed in order to determine if saliva is viable for EoE allergy testing.

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Literature Review Eosinophilic Esophagitis

EoE, or eosinophilic esophagitis, is a chronic, antigen-driven, immune disease that

occurs in the esophagus. The most common antigens contributing to EoE are foods: dairy, soy,

eggs, wheat, peanuts/tree nuts, and seafood (McGrowan & Platts-Mills, 2016). Scientists have

also associated EoE with a few single nucleotide polymorphisms. Studies have also correlated

seasonal aeroallergens with EoE, especially since an increased EoE diagnosis occurs during the

summer and fall months (Runge & Dellon, 2015). Many scientists currently believe the

pathogenesis of EoE has to do with all three possible contributors: food allergens, genetics, and

aeroallergens. These contributors cause a delayed inflammation of the esophagus. Patients

with EoE typically experience dysphagia (difficulty swallowing) and food impaction. Younger

children may even experience signs of refusing food and growth problems. There have also

been studies showing that 50-80% of EoE patients have other allergic diseases such as asthma,

atopic dermatitis, and allergic rhinitis or sinusitis (Runge & Dellon, 2015). About 1 in every 1000

people are diagnosed with EoE; males are affected by the disease 75% more than females. EoE

is more commonly found in Caucasians.

Antigen Recognition Eosinophils

Eosinophils (white blood cells) are thought to be the main contributors to tissue

remodeling of the esophagus. People without EoE do not have eosinophils in their esophagus

while people with EoE do. Once an antigen is recognized by cytokines and/or chemokines (cell

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signaling molecules that assist with cellular communication in immune responses), eosinophils

are sent from the blood stream to the esophageal tissue (Philpott et at., 2015). Eosinophils

then release specific mediators causing tissue damage to the esophagus and dysmotility to the

esophageal muscles. Eosinophils also produce transforming growth factor-b (TGF-b), which

activates myofibroblasts and produces fibrotic tissue in the lamina propria. This buildup in cells

and tissue causes the esophagus to inflame and results in the two most common symptoms

seen in EoE patients: dysphagia and food impaction (Philpott et al., 2015). If untreated, this

buildup of cells can result in dangerous choking episodes.

Other Important Cells

The eosinophilic infiltration can also activate the migration of mast cells (another type of

white blood cell) to the esophagus, creating even greater inflammation. Only a small number

of mast cells are found in the lamina propria of a normal esophagus. However, patients with

EoE have an increased number of mast cells in their connective tissue, intraepithelial layers, and

muscle layers of the esophagus (Philpott et al., 2015). Mast cells carry out type 1

hypersensitivity reactions. These reactions occur when an antigen comes into contact with IgE

or Immunoglobulin E (an antibody that binds to mast cells). Specific mediators are then

released by mast cells to help combat the effect of the antigen. Furthermore, scientists believe

that mast cells could be the cause of smooth muscle spasms, another symptom that EoE

patients can experience. These scientists also believe that mast cells have a role in the

remodeling process of the esophagus as they produce TGF-b which regulates connective tissue

production (H. Philpott, 2015).

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EoE patients experience an increased number of T lymphocytes (a type of white blood

cell that produces cytokines) after antigen exposure. It has been proposed that these

cytokines, specifically TH2, could be responsible for the migration of eosinophils and mast cells

into the esophageal tissue (Philpott et al., 2015). T lymphocytes are critical in figuring out the

pathogenesis of EoE because they produce cell signalers responsible for cell infiltration. In

addition, a new experiment done in mice supports the hypothesis that basophils and TSLPs

(thymic stromal lymphopoietin) could be a cause of EoE symptoms (Philpott et. al, 2015).

Because the discovery is new, more research needs to be done to confirm that the findings are

the same with humans who have EoE.

Allergy Testing

In order to determine the antigen causing the inflammation of the esophagus, doctors

first run the three most common allergy testing methods on EoE patients: skin prick tests, patch

tests, and blood tests. Because the results of these tests lack accuracy, Doctors often have to

resort to using other methods in order to correctly identify the antigen causing EoE in the

patient.

Skin Prick Tests Skin prick tests are commonly used to identify the source of many allergic reactions.

These tests are used to measure the presence of an Immunoglobulin E (IgE) antibody for an

allergen (“Allergy skin tests,” 2014). IgE antibodies trigger allergic reaction symptoms such as

hives and shortness of breath (“Allergic reactions TTR…”). When administering the test, nurses

will apply a drop of many different types of allergen extracts onto various locations on the skin.

They will then lightly prick the surface over the drops of extracts with small needles known as

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lancets. These small abrasions allow the body to react to only a small amount of the extract,

producing a less severe reaction. Two other substances, histamine and either glycerin or saline,

are also scratched onto the surface to serve as positive and negative controls. If an allergic

reaction to a substance occurs, a red, raised bump will form on the surface where the skin was

pricked (“Allergy skin tests,” 2014).

Patch Testing

Unlike skin prick tests and blood tests, patch testing is usually performed to indicate if a

delayed allergic reaction is occurring. Delayed reactions usually take a couple of days to

develop. To detect if this type of reaction is occurring, different allergen extracts are placed on

patches which are then in turn applied to the skin. After 48 hours, the patches are removed

and the skin is evaluated by a nurse or doctor. If the skin is irritated, the patient is most likely

allergic to whichever extract substance was placed on the patch in that location (“Allergy skin

tests,” 2014).

Blood Tests Like skin prick tests, blood tests are also used to measure the presence of IgE antibodies.

The difference between blood tests and skin prick tests is that blood tests take several days to

process while skin prick testing results are almost immediate. Blood tests also are not affected

by antihistamines. This allows people who cannot take skin prick tests (as they experience

intense rashes) to take blood tests. The downside to blood testing is that the severity of the

allergy cannot be determined, only the information about the presence or absence of an allergy

(“Blood tests…”).

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Accuracy of Allergy Testing Methods for Eosinophilic Esophagitis The three methods of allergy testing (skin prick tests, patch tests, and blood tests) are

not useful in detecting EoE allergens. Skin prick tests have a high false positive rating. About

50-60% of all skin prick tests and blood tests show a positive reaction to an allergen when an

allergic reaction does not occur. Although they are rare, false negatives are also able to occur

in skin prick tests (“Blood tests”). In addition, EoE does not result in an instantaneous reaction.

The reaction to allergens by EoE patients is delayed and occur over a long period of time. While

a patch test assesses for a delayed response, the test has difficulty analyzing and testing for

food allergic responses. In fact, patch testing has not even been cleared or standardized to

validate food allergies (“15 unproven methods of food…,” 2015).

Difference Between Typical Allergies and EoE Allergies

Patients with EoE have an increased level of B cells (lymphocytes that produce

antibodies) inside the esophageal mucosa. TH2 cytokines cause the B cells to produce IgE

antibodies. However, due to accuracy problems from allergy testing and unclear results from

experiments meant to reduce IgE levels, scientists think the focus for determining the

pathology of EoE should be shifted to TH2 cytokines rather than B cells or IgE antibodies

(Philpott et al., 2015). It is possible that EoE is not an IgE mediated response. Therefore, skin

prick tests and blood tests, which test for the amount of IgE, are not accurate for EoE allergy

testing. In other words, it is not preferred to rely on these methods for determining the antigen

causing EoE symptoms.

In addition, EoE and typical allergies elicit different responses. Typical allergic reactions

result in symptoms like shortness of breath, hives, or anaphylaxis (“Allergic reactions TTR…”).

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On the other hand, when a specific antigen is recognized, people with EoE experience a delayed

swelling of their esophagus caused by a cell infiltration and extra tissue creation in the

esophagus.

More Accurate Treatment Option

As standard allergy testing methods for EoE reactions are not accurate in determining

the antigen eliciting the response, most doctors recommend going on an elimination diet with

frequent endoscopies. In most cases, after the correct antigen is determined and permanently

removed from the patient’s diet, the inflammation will decrease (Philpott et al., 2015). As

these diets are highly successful, this is direct evidence that food allergens can cause EoE.

Restrictive Diets and Frequent Endoscopies

Before a food elimination diet begins, an endoscopy will be taken to determine the

number of eosinophils per high powered field or hpf (an area of specific length used as a

measurement for eosinophil quantification in EoE). If there are over fifteen eosinophils per hpf,

then EoE is definitely present. After starting the elimination diets, the goal is for the number of

eosinophils to be below fifteen. If this occurs, then the correct antigen has been discovered

and the patient is considered in remission. In order to stay in long term remission, the

eliminated food should continue to be eliminated from the patient’s diet. If it is not, the EoE

symptoms will continue to occur (McGowan & Platts-Mills, 2016).

Empiric Elimination Diet

In empiric elimination diets, the most common foods associated with typical food

allergies are avoided. After approximately six weeks of the diet, an endoscopy will be taken to

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determine if the patient is in a state of remission. If the patient is indeed in remission, each

food that was eliminated from the diet will be reintroduced back in one at a time. After each

reintroduction, an endoscopy will be taken (about six weeks after of the reintroduction) to

determine if this was the antigen causing the EoE symptoms. If this food contained the EoE

causing antigen, there would be an increase in the number of eosinophils per hpf (over fifteen)

(McGowan & Platts-Mills, 2016).

Six-Food Elimination Diet

In the six-food elimination diet, the most common foods associated with EoE are

removed from the diet: milk, soy, egg, wheat, peanuts/tree nuts, and seafood. The

methodology is similar to the empiric diet where an endoscopy is taken after six weeks on the

diet to determine if the patient is in histological remission. If the patient is in remission, each

food that was eliminated from the diet will be reintroduced one at a time. Again, after each

reintroduction, an endoscopy will be taken to determine if this was the antigen causing the EoE

symptoms. If this was the correct antigen causing EoE, there would be an increase in the

number of eosinophils per hpf. Other doctors will complete the same six food elimination diet,

but will instead remove one of the six foods from the patient’s diet one at a time, including an

endoscopy to see if the number of eosinophils have decreased (McGowan & Platts-Mills, 2016).

Four-Food Elimination Diet

This diet is almost exactly the same as the six-food elimination diet. The only difference

is that the four-food elimination diet removes the four most common foods associated with

EoE: milk, wheat, egg, soy/legumes (McGowan & Platts-Mills, 2016).

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Disadvantages of the Elimination Diets and Endoscopies

The time it takes to determine the antigen causing the EoE can take anywhere from

twelve weeks to a couple of years. Not only are endoscopies costly, but they are also invasive.

As half of EoE patients are children, the procedure can be risky due to the high number of

endoscopy repetitions, which requires going under anesthesia each time the surgery is

repeated. Additionally, the various food elimination diets can be challenging to maintain and

impractical for the parents and children. Trying to find foods that do not contain the necessary

food(s) can be tricky and very expensive. The strict diets can also be a tough to maintain

especially during school events and social events.

Cytokines and Saliva

Scientists are currently trying to look for different microbial analytes in saliva. In the

future, these analytes could potentially be used as biomarkers to noninvasively identify and

diagnose Eosinophilic Esophagitis (Bae, 2014).

“Eosinophilic Esophagitis Related Cytokines in Saliva: Characterization and Methodological Considerations” In 2014, a group of researches quantified EoE related cytokines in saliva. The

researchers used five types of cytokines that have been linked with EoE: eotaxin 3 (Eo3), thymic

stromal lymphopoietin (TSLP), interleukin 4 (IL 4), interleukin 5 (IL 5), and interleukin 13 (IL 13).

After salivary collection through passive drool (PD) and active saliva with oral swabs (OS), the

number of cytokines in the samples were quantified using magnetic high sensitivity human

multiplex assays (Hiremath et al., 2014). The amount of IL 4, IL 5, and IL 13 in the samples were

measured using the Luminex 200 platform, while the Eo3 and TSLP were measured using

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sandwich ELISA techniques. After reviewing the results, the researchers concluded that if more

testing is done, saliva could possibly be used as a noninvasive method to test for EoE (Hiremath

et al., 2014).

Table 1.1: This is information from the experiment regarding the specific salivary cytokines

related to EoE.

Role of Cytokines in Eosinophilic Esophagitis

Cytokines are cell signaling proteins that aid with cell communication throughout the

body. Cytokines are usually involved in immune responses by stimulating the movement of

cells towards sites of inflammation (“What are cytokines?”, 2017). Interleukins specifically

regulate the immune systems response to inflammation (“What are cytokines?”, 2017).

Specific Cytokines and Their Roles in Eosinophilic Esophagitis Most research points to IL-4 being responsible for initiating the TH2 response. Although

IL-4 is produced by T helper cells during inflammatory responses, the initial origin of IL-4

cytokines is unknown in atopic diseases (Mulder & Justinich, 2011).

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IL-5 acts as an eosinophil differentiation factor and activator. This means that IL-5 is one

of the cytokines responsible for activating the eosinophilic infiltrate to the esophagus. (Mulder

& Justinich, 2011).

IL-13 has been linked to stimulate both the local tissue and eosinophil inflammatory

response in EoE. IL-13 decreases esophageal epithelial cell differentiation in order to maintain

the barrier function of the esophageal mucosa. EoE patients’ biopsies also show an increase in

IL-13 mRNA levels (Mulder & Justinich, 2011).

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Research Plan Researchable Question

Will a patient with dairy-induced EoE exhibit an increase in the concentration of salivary

cytokines upon exposure to dairy source material but not wheat source material?

Hypothesis

Although the cytokine concentration in the saliva of a patient with dairy-induced EoE

will increase upon exposure to dairy source material, cytokine concentration upon exposure to

wheat source material will be unaltered.

Procedure Overview

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Figure 1.1: This is a flow chart of the procedure

Three saliva samples from an EoE patient, his sister (who does not have EoE), and

another unrelated person to the EoE patient who does not have allergies were taken. Then,

diluted dairy and wheat source material was injected the saliva samples. Dairy was used as a

positive control as the patient’s EoE is stimulated by dairy and wheat was used as a negative

control since the EoE patient is not allergic to it. Control samples were also taken where

nothing was injected into the saliva. Then, an ELISA test was run to quantify the concentration

of cytokines in the samples.

Materials and Methods Materials for Test #1 Table 2.1: Shows the materials for test #1

Name of material: Where material was obtained:

Price:

Milk, bovine source material (F235) greerlabs.com $49.89 Wheat, whole source material (F395) greerlabs.com $49.89 Distilled water Manning Lab, WPI n/a IL-13 Human ELISA kit, ultrasensitive: thermofisher.com $510.00

Hu IL-13 Standard, recombinant Hu IL-13. Contains 0.1% sodium azide

thermofisher.com n/a

Standard Diluent Buffer. Contains 0.1% sodium azide thermofisher.com n/a Antibody Coated Wells. 12 x 8 Well Strips thermofisher.com n/a Hu IL-13 Biotin Conjugate, (Biotin-labeled anti-IL-13). Contains 0.1% sodium azide


Streptavidin-HRP (100X). Contains 3.3 mM thymol thermofisher.com n/a Streptavidin-HRP Diluent. Contains 3.3 mM thymol thermofisher.com n/a Wash Buffer Concentrate (25X) thermofisher.com n/a Stabilized Chromogen, Tetramethylbenzidine (TMB) thermofisher.com n/a Stop Solution thermofisher.com n/a Plate Covers, adhesive strips thermofisher.com n/a

Various sized of pipettes Manning Lab, WPI n/a Well plate aspirator Manning Lab, WPI n/a Vacuum aspirator Manning Lab, WPI n/a

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Saliva from EoE patient n/a n/a Saliva form EoE patient’s sister n/a n/a ELISA well plate reader Manning Lab, WPI n/a Small tubes Manning Lab, WPI n/a

All of the materials indented under the “IL-13 Human ELISA kit, ultrasensitive” are included in the $510.00. Procedure for Test #1

Before going to the lab, the EoE patient and his sister each spit into two centrifuge tubes

and one glass container. Each centrifuge and glass container contained about 5 mL of saliva

each. The saliva samples were transported to the lab within two hours.

The necessary reagents were prepared before starting the procedure. After letting the

Wash Buffer Concentrate reach room temperature, 960 mL of distilled water was diluted with

40 mL of the Wash Buffer Concentrate. For more information on the dilution of the Wash

Buffer Concentrate, refer to the “Preparation for Test #1” section in appendix B. Next, the Hu

IL-13 Standard was reconstituted using Standard Diluent Buffer to create a 50 pg/mL Hu IL-13

Mix. Refer to the “Preparation for Test #1” section in appendix B for more information how to

create the reconstituted Standard. Then 0.3 mL of the Standard Diluent Buffer was added to 6

tubes labeled 25, 12.5, 6.2, 3.1, 1.6, and 0.78 pg/mL Hu IL-13. Afterwards, a serial dilution of

the standards was performed. To complete the serial dilution, see table 3.1 in the “Preparation

for Test #1” section in appendix B. The instructions in table 3.1 were followed, making sure to

mix thoroughly in between with a pipette. Moreover, the secondary antibody was prepared.

To do this 120 µL of the Streptavidin-HRP was transferred into a tube with 12 mL of Diluent.

For more information refer to the “Preparation for Test #1” section in the appendix B.

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In order to prepare the saliva samples, the powdered dairy and wheat source material

was reconstituted. To do this, each vile of source material was filled half way and shaken until

the mixture was liquefied. Then, 500 µL of the reconstituted wheat source material was

injected into a centrifuge bottle filled with 5 mL of the EoE patient’s saliva, and a centrifuge

bottle filled with 5 mL of the EoE patient’s sister’s saliva. Also, 500 µL of the reconstituted dairy

source material was injected into a centrifuge bottle filled with 5 mL of the EoE patient’s saliva,

and a centrifuge bottle filled with 5 mL of the EoE patient’s sister’s saliva.

To start the procedure, 100 µL of the standards were added to wells in rows G and H in

the 96 well plate. See the “Well Plate Standards Test #1” section in appendix B for more

information on how exactly the well plate was divided with the various standards. Then, 50 µL

of the Standard Diluent Buffer and 50 µL of the respective samples were added to each well in

rows A – F. See the “Well Plate Division Test #1” section in appendix B for more information of

how exactly the well plate was divided with the various saliva samples. At this point in the

procedure, the well plate was covered with a plate cover and incubated in room temperature

for 2 hours.

After the 2-hour incubation period, the solution was aspirated from the wells with a

vacuum aspirator. The well plate was washed 4 times. Refer to the “Aspiration and Washing

Directions Test #1” section in appendix B for more information on how to complete this step.

Then 100 µL of biotinylated Hu IL-13 Biotin Conjugate was added into each well. Afterwards,

the well plate was covered with a plate cover and incubated for 1 hour in room temperature.

After the 1-hour incubation period, the solution was aspirated from the wells with a

vacuum aspirator. The well plate was washed 4 times. Refer to the “Aspiration and Washing

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Directions Test #1” section in appendix B for more information on how to complete this step.

Next, 100 µL of Streptavidin-HRP Working Solution was added to each well. The well plate was

covered with a plate cover and incubated for 30 minutes in room temperature.

Afterwards, the solution was aspirated from the wells with a vacuum aspirator. The well

plate was washed 4 times. Refer to the “Aspiration and Washing Directions Test #1” section in

appendix B for more information on how to complete this step. Then, 100 µL of Stabilized

Chromogen was added to the wells. Next, the well plate was incubated for 30 minutes at room

temperature in the dark (the well plate was placed in a closed drawer). At this point in the

incubation period, the Stabilized Chromogen caused the wells to turn various shades of blue.

After the Incubation period, 100 µL of Stop Solution was added to each well, causing the

solution to turn from yellow to blue. Then, the 96 well plate was placed in the ELISA well plate

reader in order to quantify the results. The results were then analyzed with T-Tests.

To find the MSDS and more information on the procedure from the IL-13 Human ELISA

kit, ultrasensitive, data was obtained from Thermo Fisher Scientific.

Materials for Test #2 Table 2.2: Shows the materials for test #2

Name of material: Where material was obtained:

Price:

Milk, bovine source material (F235) greerlabs.com $49.89 Wheat, whole source material (F395) greerlabs.com $49.89 Distilled water Manning Lab, WPI n/a IL-13 Human Instant ELISA kit: thermofisher.com $499.00

1 aluminum pouch with a Microwell Plate coated with monoclonal Antibody to human IL-13, Biotin-Conjugate (anti-human IL-13 monoclonal antibody), Streptavidin-HRP and Sample Diluent, lyophilized. 2 aluminum pouches with a human IL-13 Standard curve (colored)


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2 aluminum pouches with a human IL-13 Standard curve (colored)


1 bottle (25 ml) Wash Buffer Concentrate 20x (phosphate-buffered saline with 1% Tween 20)


1 vial (12 ml) Sample Diluent (Use when an external predilution of the samples is needed)


1 vial (15 ml) Substrate Solution (tetramethyl-benzidine) thermofisher.com n/a 1 vial (15 ml) Stop Solution (1M Phosphoric acid) thermofisher.com n/a 2 Adhesive Films thermofisher.com n/a

Various sized of pipettes Manning Lab, WPI n/a Well plate aspirator Manning Lab, WPI n/a Vacuum aspirator Manning Lab, WPI n/a Saliva from EoE patient n/a n/a Saliva from EoE patient’s sister n/a n/a Saliva from a person who is not biologically related to the EoE patient and does not have allergies

n/a n/a

ELISA well plate reader Manning Lab, WPI n/a Small tubes Manning Lab, WPI n/a

All of the materials indented under the “IL-13 Human Instant ELISA kit” are included in the $499.00. Procedure for Test #2

Before going to the lab, the EoE patient, his sister, and another person who does not

have allergies and is not biologically related to the EoE patient each spit into two centrifuge

tubes and one glass container. Each centrifuge and glass container contained about 5 mL of

saliva each. The saliva samples were transported to the lab within two hours.

The necessary materials were prepared before starting the procedure. After letting the

Wash Buffer Concentrate reach room temperature, 25 mL of the Wash Buffer Concentrate was

diluted with 475 mL of distilled water. The saliva samples were prepared in the same way as

test #1. First, the powdered dairy and wheat source material were reconstituted. To do this

each vile of source material was filled half way and shaken it until it was liquefied. Then, 500 µL

of the reconstituted wheat source material was injected into a centrifuge bottle filled with 5 mL

Hobson 23

the EoE patient’s saliva, a centrifuge bottle filled with 5 mL of the EoE patient’s sister’s saliva,

and a centrifuge bottle filled with 5 mL of another person’s saliva who does not have allergies

and is unrelated to the EoE patient. Also, 500 µL of the reconstituted dairy source material was

injected into a centrifuge bottle filled with 5 mL of the EoE patient’s saliva, and a centrifuge

bottle filled with 5 mL of the EoE patient’s sister’s saliva, and a centrifuge bottle filled with 5 mL

of another person’s saliva who does not have allergies and is unrelated to the EoE patient.

To begin the procedure, distilled water was added to all standard wells. To see where

the standards were located and the specific concentrations on the well plate, see “Well Plate

Design Test #2” in appendix C. Afterwards, 100 µL of distilled water and 50 µL of each sample

were added to the sample wells. To see how this is divided up, see “Well Plate Design Test #2”

in appendix C. Then, a plate cover was placed on the well plate and incubated at room

temperature for 3 hours.

After the incubation period, the well plate was aspirated and washed 3 times with a

vacuum aspirator. Refer to the “Aspiration and Washing Directions Test #2” section in

appendix C for more information on how to aspirate and wash the wells. Then, 100 µL of TMB

Substrate Solution was added to all of the wells. The well plate was incubated for 10 minutes in

the dark (the well plate was placed in a closed drawer). After the 10 minutes, 100 µL of the

Stop Solution was added into each well. The well plate was transported to the ELISA well plate

reader within 5 minutes to quantify the results. Afterwards, T-Tests were completed on the

results.

To find the MSDS and more information on the procedure from the IL-13 Human Instant

ELISA kit, information was obtained from Thermo Fisher Scientific.

Hobson 24

Results Test #1 In order to complete the data analysis, all of the actual data points need to be

subtracted from the 0 pg/mL blank well. The 0 pg/mL blank well represents when there are

zero cytokines in the saliva solution. The data from the 0 pg/mL blank well was compromised

due to human error. The 0.78 pg/mL blank well was used instead. The raw data and the fixed

data (taking into account the subtraction of the 0.78 pg/mL blank well from all of the values)

can be found in appendix D. Also, due to human error, wells in columns 1, 2, and 12 were

compromised and not used in the data analysis.

For the standards (rows G and H), the 25 pg/mL dilution had an average absorbance of

0.736 nm. The 6.2 pg/mL dilution had an average absorbance of 0.178 nm. The 3.1 pg/mL

dilution had an average absorbance of 0.107 nm. The 0.78 pg/mL dilution had an average

absorbance of 0.038 nm. The 0 pg/mL dilution had an average absorbance of 0.028 nm.

-0.100

0.000

0.100

0.200

0.300

0.400

0.500

0.600

1 2 3 4 5 6 7 8 9 10

Abso

rban

ce (n

m)

Sample Number

Data from Patient with EoE (TEST #1)

Control (B) Wheat (B) Dairy (B)

Hobson 25

Figure 2.1: This is the graph of the absorbance levels of the salivary IL-13 cytokines from the

patient with EoE. The grey points represent the control group that just contained the saliva

from the patient with EoE. The orange points represent the saliva from the patient with EoE

that was exposed to wheat. The blue points represent the saliva from the patient with EoE that

was exposed to dairy.

A line graph was used in order to see the general patterns that each group’s data points

formed (control, wheat, and dairy). There is a general increase (D = 0.158 nm) in absorbance in

the wheat exposed saliva group compared to the control saliva group. There is a general

decrease (D = - 0.346 nm) in absorbance in the dairy exposed saliva group compared to the

control saliva group. The average absorbance for the control saliva is 0.335 nm. The average

absorbance for the wheat exposed saliva group is 0.493 nm. The average absorbance for the

dairy exposed saliva group is -0.011 nm.

-0.050

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

1 2 3 4 5 6 7 8

Abso

rban

ce (n

m)

Sample Number

Data from EoE Patient's Sister (TEST #1)

Control (M) Wheat (M) Dairy (M)

Hobson 26


patient’s sister. The grey points represent the control group that just contained the saliva from

the patient’s sister. The orange points represent the saliva from the patient’s sister that was

exposed to wheat. The blue points represent the saliva from the patient’s sister that was

exposed to dairy.


formed (control, wheat, and dairy). There is a general decrease (D = - 0.080 nm) in absorbance

in the wheat exposed saliva group compared to the control saliva group. There is a general

decrease (D = - 0.273 nm) in absorbance in the dairy exposed saliva group compared to the

control saliva group. The average absorbance for the control saliva is 0.284 nm. The average

absorbance for the wheat exposed saliva group is 0.204 nm. The average absorbance for the

dairy exposed saliva group is 0.011 nm.

Test #2

In order to complete the data analysis, all of the actual data points need to be

subtracted from the 0 pg/mL blank well. The 0 pg/mL blank well represents when there are

zero cytokines in the saliva solution. The raw data and the fixed data (taking into account the

subtraction of the blank from all of the values) can be found in appendix E. Also, due to human

error, wells D12, E12, F12, G12, H12, G11, and H11 had to be compromised so they were not

taken into account during data analysis.

For the standards (columns 1 and 2), the 200 pg/mL dilution had an average absorbance

of 2.865 nm. The 100 pg/mL dilution had an average absorbance of 2.006 nm. The 50 pg/mL

dilution had an average absorbance of 1.085 nm. The 25 pg/mL dilution had an average

Hobson 27

absorbance of 0.495 nm. The 12.25 pg/mL dilution had an average absorbance of 0.236. The

6.25 pg/mL dilution had an average absorbance of 0.123 nm. The 3.13 pg/mL dilution had an

average absorbance of 0.060 nm. The 0 pg/mL dilution had an average absorbance of 0 nm.

Figure 2.3: This is the graph of the absorbance levels of the salivary IL-13 cytokines from the EoE

patient. The grey points represent the control group that just contained the saliva from the EoE

patient. The orange points represent the saliva from the EoE patient that was exposed to

wheat. The blue points represent the saliva from the EoE patient that was exposed to dairy.


formed (control, wheat, and dairy). There is no increase or decrease (D = 0.000 nm) in

absorbance in the wheat exposed saliva group compared to the control saliva group. There is a

general increase (D = 0.016 nm) in absorbance in the dairy exposed saliva group compared to

the control saliva group. The average absorbance for the control saliva is -0.034 nm. The

-0.070-0.060-0.050-0.040-0.030-0.020-0.0100.0000.0100.020

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Sample Number

Data from EoE Patient (TEST #2)

Control (B) Dairy (B) Wheat (B)

Hobson 28

average absorbance for the wheat exposed saliva group is -0.034 nm. The average absorbance

for the dairy exposed saliva group is -0.018 nm.

Figure 2.4: This is the graph of the absorbance levels of the salivary IL-13 cytokines from the EoE

patient’s sister. The grey points represent the control group that just contained the saliva from

the patient’s sister. The orange points represent the saliva from the patient’s sister that was

exposed to wheat. The blue points represent the saliva from the patient’s sister that was

exposed to dairy.


formed (control, wheat, and dairy). There is a general decrease (D = - 0.001 nm) in absorbance

in the wheat exposed saliva group compared to the control saliva group. There is a general

increase (D = 0.021 nm) in absorbance in the dairy exposed saliva group compared to the

control saliva group. The average absorbance for the control saliva is -0.028 nm. The average

absorbance for the wheat exposed saliva group is -0.028 nm. The average absorbance for the


-0.060-0.040-0.0200.0000.0200.0400.0600.0800.1000.1200.140

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Sample Number


Control (M) Dairy (M) Wheat (M)

Hobson 29


other person unrelated to the EoE patient who has no allergies. The blue points represent the

control group that just contained the saliva from the other person. The orange points

represent the saliva from the other person that was exposed to wheat. The grey points

represent the saliva from the other person that was exposed to dairy.


formed (control, wheat, and dairy). There is a general increase (D = 0.020 nm) in absorbance in

the wheat exposed saliva group compared to the control saliva group. There is a general

increase (D = 0.034 nm) in absorbance in the dairy exposed saliva group compared to the

control saliva group. The average absorbance for the control saliva is -0.040 nm. The average

absorbance for the wheat exposed saliva group is -0.019 nm. The average absorbance for the


-0.060-0.050-0.040-0.030-0.020-0.0100.0000.0100.0200.0300.040

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Sample Number

Data from Other Person's Saliva (TEST #2)

Control O Dairy O Wheat O

Hobson 30

Conclusions

The first experiment identified a significant difference between the two control groups,

the two wheat groups, and the two dairy groups. There were also significant differences

between the control groups and the wheat groups and the control groups and the dairy groups

for the EoE patient’s saliva samples and his sister’s saliva samples.

It was hypothesized that the concentration of cytokines in the EoE patient’s saliva

exposed to dairy would increase (as dairy stimulates his EoE) and the concentration of

cytokines in his saliva samples exposed to wheat would decrease (since he is not allergic to

wheat). After the experiment was completed, it was confirmed that the opposite occurred: the

concentration of cytokines for the EoE patient’s saliva exposed to dairy experienced a general

decrease in cytokines and the saliva exposed to wheat experienced a general increase in

cytokines. After contacting experts in the field such as Dr. Lee from Boston’s Children’s

hospital, Dr. Olive, Dr. Hiremath, and Dr. Davis from the paper, “Eosinophilic Esophagitis

Related Cytokines in Saliva: Characterization and Methodological Considerations,” they

suggested that additional testing should be completed due to the significant results from the

test, regardless of the fact that the opposite occurred from what was hypothesized. Many

follow up experiments were considered, but it was ultimately decided to complete the same

test with an additional person’s saliva. This would allow for possible patterns in the data to

emerge between people the two people without EoE and the EoE patient.

After completing a second round of testing with three people’s saliva samples (the EoE

patient, the EoE patient’s sister, and the other test subject), other information about the

validity of this test was procured. The most important finding from the second test was the

Hobson 31

data from the EoE patient. After taking the averages of the data points from the EoE patient’s

control, dairy, and wheat groups, it was seen that there was a general increase in cytokines in

the saliva that was exposed to dairy and no increase or decrease in the cytokines in the saliva

that was exposed to wheat. Also, the range of absorbance for tests 1 and 2 were very different

as well. Although two different tests were used (test 1 used an ultrasensitive ELISA kit and test

2 used an instant ELISA kit), they both quantified the concentration of IL-13 cytokines in the

saliva solution. So, theoretically, the concentration of IL-13 cytokines in the saliva solutions for

the EoE patient and his sister should have come out to be the same in test 1 and 2.

Although the results were different between the tests, this shows that more research

needs to be done in order to determine why the results came out the way they did. The validity

of this method of allergy testing cannot be concluded from a sample size of two tests. In order

to determine if this method can prove to be more accurate than the 50-60% ratings of the skin

prick tests and blood tests, more saliva testing must be completed. Additional follow up

experiments should be considered such as increasing the ration of source material to distilled

water or increasing the ratio of the source material mixture to the saliva. Also, it is possible

that testing with additional negative controls besides wheat, testing with peanuts (an allergen

that induces an anaphylaxis reaction for the EoE patient) and dairy (the allergen that induced

the EoE patient’s EoE response), or testing with additional people’s saliva could give more

insight into the accuracy of this method of allergy testing. As research is continued hopefully

predictors, consistencies, and patterns will be seen in the larger sample size.

See appendix F for more information on the p-values and analysis information from test

#1 and test #2.

Hobson 32

References 15 unproven methods of food allergy testing Retrieved

from http://www.kidswithfoodallergies.org/page/unproven-methods-food-allergy-

tests.aspx

Allergic reactions TTR | AAAAI. Retrieved from https://www.aaaai.org/conditions-and-

treatments/library/at-a-glance/allergic-reactions

Allergy skin tests. Retrieved from http://www.mayoclinic.org/tests-procedures/allergy-

tests/basics/definition/prc-20014505

Bae, J. (2014). Narrative reviews. Epidemiology and Health, 36, e2014018.

doi:10.4178/epih/e2014018

Blood tests | food allergy research & education Retrieved

from https://www.foodallergy.org/life-food-allergies/food-allergy-101/diagnosis-

testing/blood-tests

Hiremath, G., Olive, A., Davis, C. M., Shulman, R. J., & Devaraj, S. (2014). 252 eosinophilic

esophagitis related cytokines in saliva: Characterization and methodological

considerations. Gastroenterology, 146(5), 60. doi:10.1016/S0016-5085(14)60211-0

IL-13 human ELISA kit, ultrasensitive - thermo fisher scientific. Retrieved

from https://www.thermofisher.com/order/catalog/product/KHC0134?SID=srch-srp-

KHC0134

Hobson 33

IL-13 human instant ELISA kit - thermo fisher scientific. Retrieved

from https://www.thermofisher.com/order/catalog/product/BMS231INST?SID=srch-srp-

BMS231INST

McGowan, E., & Platts-Mills, T. (2016). Eosinophilic esophagitis from an allergy perspective:

How to optimally pursue allergy testing & dietary modification in the adult

population. Current Gastroenterology Reports, 18(11), 1-10. doi:10.1007/s11894-016-0531-

z

Mulder, D. J., & Justinich, C. J. (2011). Understanding eosinophilic esophagitis: The cellular and

molecular mechanisms of an emerging disease. Mucosal Immunology, 4(2), 139-147.

doi:10.1038/mi.2010.88

Philpott, H., Nandurkar, S., Thien, F., Gibson, P. R., & Royce, S. G. (2015). Eosinophilic

esophagitis: A clinicopathological review.Pharmacology & Therapeutics, 146, 12-22.

doi:10.1016/j.pharmthera.2014.09.001

Runge, T., & Dellon, E. (2015). Do we know what causes eosinophilic esophagitis? A mechanistic

update. Current Gastroenterology Reports, 17(9), 1-10. doi:10.1007/s11894-015-0458-9

What are cytokines? Retrieved from https://www.news-medical.net/health/What-are-

Cytokines.aspx

Hobson 34

Appendix Appendix A: Limitations & Assumptions

1. The ELISA kits that were purchased were expensive. Budget restrictions limited how many experiments were able to be conducted.

2. Initially eosinophils wanted to be used, but due to legality issues, this idea was never implemented. However, cytokines, proteins also involved in the cell infiltration to the esophagus that EoE patients experience, were utilized.

3. Allergy extracts that are used to complete skin prick testing wanted to be used in the experiments. However, only licensed physicians are able to purchase those extracts. Instead, powdered source material was used.

4. It was assumed that the saliva samples used contained IL-13 cytokines. 5. It was assumed that the trends that were seen in the EoE patient’s results are

representative of the EoE population. 6. It was assumed that the trends that were seen in the EoE patient’s sister’s results are

representative of the population of people without allergies. 7. It was assumed that the trends that were seen in the other person participating in test

#2’s results are representative of the population of people without allergies. 8. It was assumed that the results observed from test #1 and test #2 are predicative of

future results if the two tests were to be repeated. 9. It was assumed that the ELISA kits purchased had all of the correct materials and

substances included in the kits 10. It was assumed that ELISA well plate reader used produced accurate data. 11. It was assumed that the EoE patient is not allergic to wheat. 12. It was assumed that the EoE patient’s sister is not allergic to dairy or wheat. 13. It was assumed that the other person participating in test #2 is not allergic to dairy or

wheat. Appendix B: Test #1 Preparation for Test #1 Wash Buffer Concentrate Test #1

This is information about the Wash Buffer Concentrate can be found on the thermofisher

protocol guide for the IL-13 Human ELISA Kit, Ultrasensitive (can be found on

thermofisher.com): “Allow the Wash Buffer Concentrate (25X) to reach room temperature and

mix to ensure that any precipitated salts have redissolved. Dilute 1 volume of the Wash Buffer

Hobson 35

Concentrate (25X) with 24 volumes of deionized water (e.g., 50 mL may be diluted up to 1.25

liters, 100 mL may be diluted up to 2.5 liters). Label as Working Wash Buffer. Store both the

concentrate and the Working Wash Buffer in the refrigerator. The diluted buffer should be used

within 14 days.”

Reconstitution of Hu-IL-13 Standard: Test #1

Next, the Hu IL-13 Standard to 10,000 pg/mL was reconstituted using Standard Diluent

Buffer. After that, 0.015 mL of the reconstituted Hu IL-13 Standard was added to a tube with

2.985 mL of Standard Diluent Buffer and was labeled “50 pg/mL Hu IL-13 Mix.” For more

exact information on how to create the Standard Diluent Buffer, this is the information from the

thermofisher.com protocol page for the IL-13 Human ELISA Kit, Ultrasensitive:

1. “Reconstitute standard to 10,000 pg/mL with Standard Diluent Buffer. Refer to standard

vial label for instructions. Swirl or mix gently and allow to sit for 10 minutes to ensure

complete reconstitution. Use standard within 1 hour of reconstitution.

2. Add 0.015 mL of the reconstituted standard to a tube containing 2.985 mL Standard

Diluent Buffer. Label as 50 pg/mL Hu IL-13. Mix.

3. Add 0.300 mL of Standard Diluent Buffer to each of 6 tubes labeled 25, 12.5, 6.2, 3.1,

1.6, and 0.78 pg/mL Hu IL-13.

4. Make serial dilutions of the standard as described in the following dilution table. Mix

thoroughly between steps.”

Serial Dilution of Standards Test #1:

Hobson 36

Then the instructions in table 3.1 were followed to make the serial dilutions of the

standards.

Table 3.1: This is a table from thermofisher.com for the IL-13 ELISA Kit, Ultrasensitive protocol page.

Secondary Antibody Test #1

This information on how to construct the Secondary Antibody can be found from the

thermofisher protocol guide for the IL-13 Human ELISA Kit, Ultrasensitive (on

thermofisher.com): “The Streptavidin-HRP (100X) is in 50% glycerol. This solution is viscous. To

ensure accurate dilution, allow Streptavidin-HRP (100X) to reach room temperature. Gently

mix. Pipette Streptavidin-HRP (100X) slowly. Remove excess concentrate solution from pipette

tip by gently wiping with clean absorbent paper. 1.) Dilute 10 μL of this 100X concentrated

solution with 1 mL of Streptavidin-HRP Diluent for each 8-well strip used in the assay. Label as

Streptavidin-HRP Working Solution. For Example:”

Hobson 37

Well Plate Division Test #1 Table 4.1: This is a table of the possible ways of how to create the Secondary Antibody, depending on the number of wells you have in your well plate. This picture is from the thermofisher protocol guide for the IL-13 Human ELISA Kit, Ultrasensitive (which can be found on thermofisher.com).

Well Plate Standards Test #1

In rows G and H of the well plate the Standards were added. The 50 pg/mL dilution was

placed in G1, G2, and H1. In the H2 well, none of the 50 pg/mL dilution was used. The 25

pg/mL dilution was placed in G3, G4, and H3. In the H4 well, none of the 25 pg/mL dilution was

used. The 6.2 pg/mL dilution was placed in G5, G6, and H5. In the H6 well, none of the 6.2

pg/mL dilution was used. The 3.1 pg/mL dilution was placed in G7, G8, and H7. In the H8 well,

none of the 3.1 pg/mL dilution was used. The 0.78 pg/mL dilution was placed in G9, G10, and

H9. In the H10 well, none of the 0.78 pg/mL dilution was used. The 0 pg/mL dilution was

placed in G11, G12, and H11. In the H12 well, none of the 0 pg/mL dilution was used.

Well Plate Division test #1

Hobson 38

For rows A and B the control saliva samples were used. For rows C and D the saliva

samples that were exposed to wheat source material were used. For rows E and F the saliva

samples that were exposed to dairy source material were used. Columns 1-6 contained the

saliva samples from the EoE patient’s sister. Columns 7-12 contained the saliva samples from

the EoE patient.

Table 5.1: This is a visual representation of the ELISA well plate for test #1

Aspiration and Washing Directions Test #1

Here is some more information on how to complete the aspirating and washing portion of

the procedure. This is information from the thermofisher protocol guide for the IL-13 Human

ELISA Kit, Ultrasensitive (can be found on thermofisher.com): “Incomplete washing will

adversely affect the test outcome. All washing must be performed with Wash Buffer

Concentrate (25X) provided. Washing can be performed manually as follows: completely

aspirate the liquid from all wells by gently lowering an aspiration tip (aspiration device) into the

Hobson 39

bottom of each well. Take care not to scratch the inside of the well. After aspiration, fill the

wells with at least 0.4 mL of diluted wash solution. Let soak for 15 to 30 seconds, then aspirate

the liquid. Repeat as directed under ASSAY METHOD. After the washing procedure, the plate is

inverted and tapped dry on absorbent tissue. Alternatively, the wash solution may be put into

a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, completely filling all

wells. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. If

using an automated washer, the operating instructions for washing equipment should be

carefully followed.”

Appendix C: Test #2 Well Plate Design Test #2 Table 6.1: This is a table showing the placement and concentration of the standards for the allergy testing. The samples were ordered in a different way depicted in the figure below

Hobson 40

Figure 3.1: This is a picture of the well plate design used for the second ELISA test.

A1-H1 and A2-H2 are all of the standards. A1 and A2 are the 200 pg/mL standards. B1

and B2 are the 100 pg/mL standards. C1 and C2 are the 50 pg/mL standards. D1 and D2 are

the 25 pg/mL standards. E1 and E2 are the 12.5 pg/mL standards. F1 and F2 are the 6.25

pg/mL standards. G1 and G2 are the 3.13 pg/mL standards. H1 and H2 are the blank wells. A3-

5, B3-5, and C3-5 are the control samples from the other person. D3-5, E3-5, F3-5 are the saliva

samples from the other person that were exposed to dairy. G3-5, H3-5, and A6-8 are the saliva

samples from the other person that were exposed to wheat. B6-8, C6-8, D6-8 are the control

samples from the EoE patient. E6-8, F6-8, G6-8 are the saliva samples from the EoE patient that

were exposed to dairy. H6-8, A9-11, B9-11 are the saliva samples from the EoE patient that

were exposed to wheat. C9-11, D9-11, E9-11 are the control samples from the EoE patient’s

Hobson 41

sister. F9-11, G9-11, H9-11 are the saliva samples from the EoE patient’s sister that were

exposed to dairy. A12-H12 are the saliva samples from the EoE patient’s sister that were

exposed to wheat.

Aspiration and Washing Directions Test #2: This is information on how to complete the aspirating and washing can be found from the

thermofisher protocol guide for the IL-13 Human ELISA Kit, Ultrasensitive (on

thermofisher.com):” Remove adhesive film and empty wells. Wash the microwell strips 3 times

with approximately 400 μl Wash Buffer per well with thorough aspiration of microwell contents

between washes. Allow the Wash Buffer to sit in the wells for about 10 – 15 seconds before

aspiration. Take care not to scratch the surface of the microwells. After the last wash, tap

microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the

microwell strips immediately after washing or place upside down on a wet absorbent paper for

no longer than 15 minutes. Do not allow wells to dry.”

Appendix D: Raw Data Test #1 Table 6.1: This is the raw data from the ELISA test A01 0.041 A02 0.045 A03 0.376 A04 0.383 A05 0.365 A06 0.350 A07 0.402 A08 0.403 A09 0.447

Hobson 42

A10 0.423 A11 0.424 A12 0.034 B01 0.043 B02 0.044 B03 0.391 B04 0.405 B05 0.392 B06 0.368 B07 0.421 B08 0.445 B09 0.469 B10 0.431 B11 0.435 B12 0.040 C01 0.042 C02 0.046 C03 0.286 C04 0.285 C05 0.290 C06 0.276 C07 0.598 C08 0.567 C09 0.575 C10 0.592 C11 0.613 C12 0.042 D01 0.052 D02 0.057 D03 0.312 D04 0.314 D05 0.323

Hobson 43

D06 0.300 D07 0.583 D08 0.612 D09 0.491 D10 0.607 D11 0.640 D12 0.044 E01 0.041 E02 0.051 E03 0.094 E04 0.102 E05 0.102 E06 0.106 E07 0.080 E08 0.087 E09 0.083 E10 0.076 E11 0.082 E12 0.039 F01 0.041 F02 0.046 F03 0.104 F04 0.104 F05 0.119 F06 0.113 F07 0.107 F08 0.084 F09 0.075 F10 0.080 F11 0.086 F12 0.039 G01 0.069

Hobson 44

G02 0.067 G03 0.844 G04 0.826 G05 0.269 G06 0.274 G07 0.204 G08 0.189 G09 0.129 G10 0.124 G11 0.124 G12 0.038 H01 0.059 H02 0.042 H03 0.822 H04 0.087 H05 0.273 H06 0.098 H07 0.211 H08 0.092 H09 0.145 H10 0.095 H11 0.121 H12 0.038

Hobson 45

Table 6.2: This is the data formatted more clearly for the absorbance of the standards. Numbers in red were not used in the stats analysis due to human error during the experiment.

Table 6.3: This is the data formatted more clearly for the absorbance of the control, dairy, and wheat samples for the EoE patient’s sister’s saliva. Numbers in red were not used in the stats analysis due to human error during the experiment.

Hobson 46

Table 6.4: This is the data formatted more clearly for the absorbance of the control, dairy, and wheat samples for the EoE patient’s saliva. Numbers in red were not used in the stats analysis due to human error during the experiment.

Table 6.5: This is the data formatted more clearly for the absorbance of the standards. These numbers do not include the numbers in red from table 6.2. This was the data used in the stats analysis.

Table 6.6: This is the data formatted more clearly for the absorbance of the control, dairy, and wheat samples for the EoE patient’s sister’s saliva. These numbers do not include the numbers in red from table 6.3. This was the data used in the stats analysis.

Hobson 47

Table 6.7: This is the data formatted more clearly for the absorbance of the control, dairy, and wheat samples for the EoE patient’s saliva. These numbers do not include the numbers in red from table 6.4. This was the data used in the stats analysis.

Fixed Data Test #1

The fixed data includes the data from the raw data, but all values are subtracted from

the blank well with 0 pg/mL from the standards. But, due to human error, the blank well with

the 0 pg/mL was compromised so the blank well from the 0.78 pg/mL well was used instead.

Table 7.1: This is the data for the standard absorbance subtracted from the blank well of the 0.78 pg/mL mixture. These values were used in the data analysis.

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Table 7.2: This is the fixed data (taking into account the subtraction from the blank well of the 0.78 pg/mL standard absorbance) for the absorbance of the control, dairy, and wheat samples for the EoE patient’s sister’s saliva. This was the data used in the stats analysis.

Table 7.3: This is the fixed data (taking into account the subtraction from the blank well of the 0.78 pg/mL standard absorbance) for the absorbance of the control, dairy, and wheat samples for the EoE patient’s saliva. This was the data used in the stats analysis.

Appendix E: Raw Data Test #2 Table 8.1: This is the raw data from the ELISA test A01 3.037 A02 2.938 A03 0.073 A04 0.070 A05 0.071 A06 0.091 A07 0.070

Hobson 49

A08 0.089 A09 0.081 A10 0.105 A11 0.081 A12 0.082 B01 2.206 B02 2.052 B03 0.084 B04 0.082 B05 0.087 B06 0.096 B07 0.077 B08 0.079 B09 0.062 B10 0.087 B11 0.074 B12 0.099 C01 1.199 C02 1.215 C03 0.092 C04 0.088 C05 0.100 C06 0.098 C07 0.086 C08 0.087 C09 0.077 C10 0.084 C11 0.080 C12 0.101 D01 0.617 D02 0.617 D03 0.103 D04 0.152 D05 0.123 D06 0.096 D07 0.088 D08 0.087 D09 0.103 D10 0.101 D11 0.100 D12 0.053 E01 0.359 E02 0.357 E03 0.097 E04 0.136 E05 0.107 E06 0.115 E07 0.106

Hobson 50

E08 0.108 E09 0.112 E10 0.106 E11 0.088 E12 0.040 F01 0.242 F02 0.249 F03 0.095 F04 0.130 F05 0.111 F06 0.106 F07 0.095 F08 0.109 F09 0.114 F10 0.090 F11 0.091 F12 0.038 G01 0.189 G02 0.177 G03 0.098 G04 0.146 G05 0.100 G06 0.095 G07 0.106 G08 0.096 G09 0.113 G10 0.092 G11 0.054 G12 0.038 H01 0.106 H02 0.139 H03 0.106 H04 0.128 H05 0.099 H06 0.131 H07 0.091 H08 0.081 H09 0.088 H10 0.101 H11 0.039 H12 0.038 Table 8.2: This is the data formatted more clearly for the absorbance of the standards. Numbers in red were not used in the stats analysis due to human error during the experiment.

Hobson 51

Table 8.3: This is the data formatted more clearly for the absorbance of the control, dairy, and wheat samples for the other person’s saliva.

Table 8.4: This is the data formatted more clearly for the absorbance of the control, dairy, and wheat samples for the EoE patient’s saliva.

Table 8.5: This is the data formatted more clearly for the absorbance of the control, dairy, and wheat samples for the EoE patient’s sister’s saliva. Numbers in red were not used in the stats analysis due to human error during the experiment.

Hobson 52

Table 8.6: This is the data formatted more clearly for the absorbance of the control, dairy, and wheat samples for the EoE patient’s sister’s saliva. These numbers do not include the numbers in red from table 8.5. This was the data used in the stats analysis.

Fixed Data Test #2

The fixed data includes the data from the raw data, but all values are subtracted from

the blank well with 0 pg/mL from the standards.

Table 9.1: This is the data for the standard absorbance subtracted from the blank well of the 0 pg/mL mixture. These values were used in the data analysis.

Hobson 53

Table 9.2: This is the fixed data (taking into account the subtraction from the blank well of the 0 pg/mL standard absorbance) for the absorbance of the control, dairy, and wheat samples for the other person’s saliva. This was the data used in the stats analysis.

Table 9.3: This is the fixed data (taking into account the subtraction from the blank well of the 0 pg/mL standard absorbance) for the absorbance of the control, dairy, and wheat samples for the EoE patient’s saliva. This was the data used in the stats analysis.

Table 9.4: This is the fixed data (taking into account the subtraction from the blank well of the 0 pg/mL standard absorbance) for the absorbance of the control, dairy, and wheat samples for the EoE patient’s sister’s saliva. This was the data used in the stats analysis.

Hobson 54

Appendix F: Analysis Test #1 Raw Data The values used in these stats tests are from appendix D’s raw data for test #1

Figure 4.1: This is the graph of the absorbance levels of the salivary IL-13 cytokines from the EoE patient’s sister. The grey points represent the control group that just contained the saliva from the EoE patient’s sister. The orange points represent the saliva from the EoE patient’s sister that was exposed to wheat. The blue points represent the saliva from the EoE patient’s sister that was exposed to dairy. Table 10.1: These are the values for the average control, dairy, and wheat groups from the EoE patient’s sister

Table 10.2: These are the values for the differences between the wheat and control groups and the dairy and control groups for the EoE patient’s sister’s saliva.

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

1 2 3 4 5 6 7 8

Abso

rban

ce (n

m)

Sample Number


Control (M) Wheat (M) Dairy (M)

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Figure 4.2: This is the graph of the absorbance levels of the salivary IL-13 cytokines from the EoE patient. The grey points represent the control group that just contained the saliva from the EoE patient. The orange points represent the saliva from the EoE patient that was exposed to wheat. The blue points represent the saliva from the EoE patient that was exposed to dairy. Table 10.3: These are the values for the average control, dairy, and wheat groups from the EoE patient.

Table 10.4: These are the values for the differences between the wheat and control groups and the dairy and control groups for the EoE patient.

Table 10.5: These are the p-values for the control group and the wheat group for the EoE patient’s sister and the dairy group and control group for the EoE patient’s sister.

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

1 2 3 4 5 6 7 8 9 10

Abso

rban

ce (n

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Sample Number

Data from EoE Patient (TEST #1)

Control (B) Wheat (B) Dairy (B)

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Table 10.6: These are the p-values for the wheat group from the EoE patient’s sister and the wheat group from the EoE patient, the control group from the EoE patient’s sister and the control group from the EoE patient, and the dairy group from the EoE patient’s sister and the dairy group from the EoE patient.

Table 10.7: These are the p-values for the control group and the wheat group for the EoE patient’ and the dairy group and control group for the EoE patient.

Analysis Test #1 Fixed Data This is the analysis for the fixed data from appendix D Test #1 Table 11.1: These are the values for the average control, dairy, and wheat groups from the EoE patient’s sister.

Table 11.2: These are the values for the differences between the wheat and control groups and the dairy and control groups for the EoE patient’s sister’s saliva.

Table 11.3: These are the values for the average control, dairy, and wheat groups from the EoE patient.


Hobson 57


Table 11.6: These are the p-values for the control group and the wheat group for the EoE patient’ and the dairy group and control group for the EoE patient.


Analysis Test #2 Raw Data The values used in these stats tests are from appendix E’s raw data for test #2

Hobson 58

Figure 5.1: This is the graph of the absorbance levels of the salivary IL-13 cytokines from the other person. The grey points represent the control group that just contained the saliva from the other person. The orange points represent the saliva from the other person that was exposed to wheat. The blue points represent the saliva from the other person that was exposed to dairy. Table 12.1: These are the values for the average control, dairy, and wheat groups from the other person.

Table 12.2: These are the values for the differences between the wheat and control groups and the dairy and control groups for the other person.

0.0000.0200.0400.0600.0800.1000.1200.1400.160

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Sample Number

Data from the Other Person

Control O Dairy O Wheat O

Hobson 59

Figure 5.2: This is the graph of the absorbance levels of the salivary IL-13 cytokines from the EoE patient. The grey points represent the control group that just contained the saliva from the EoE patient. The orange points represent the saliva from the EoE patient that was exposed to wheat. The blue points represent the saliva from the EoE patient that was exposed to dairy. Table 12.3: These are the values for the average control, dairy, and wheat groups from the EoE patient.


0.0000.0200.040

0.0600.080

0.1000.120

0.140

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Sample Number

Data from EoE Patient

Control B Dairy B Wheat B

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Figure 5.3: This is the graph of the absorbance levels of the salivary IL-13 cytokines from the EoE patient’s sister. The grey points represent the control group that just contained the saliva from the EoE patient’s sister. The orange points represent the saliva from the EoE patient’s sister that was exposed to wheat. The blue points represent the saliva from the EoE patient’s sister that was exposed to dairy. Table 12.5: These are the values for the average control, dairy, and wheat groups from the EoE patient’s sister.

Table 12.6: These are the values for the differences between the wheat and control groups and the dairy and control groups for the EoE patient’s sister.

Table 12.7: These are the p-values for the control group and the wheat group for the other person and the dairy group and control group for the other person.

Table 12.8: These are the p-values for the control group and the wheat group for the EoE patient and the dairy group and control group for the EoE patient.

0.000

0.020

0.040

0.060

0.080

0.100

0.120

1 2 3 4 5 6 7 8 9

Abso

rban

ce (n

m)

Sample Number

Data from EoE Patient's Sister

Control (M) Dairy M Wheat M

Hobson 61


Table 12.10: These are the p-values for the wheat group from the other person and the wheat group from the EoE patient and the wheat group from the other person and the wheat group from the EoE patient’s sister.

Table 12.11: These are the p-values for the control group from the other person and the control group from the EoE patient and the control group from the other person and the control group from the EoE patient’s sister.

Table 12.12: These are the p-values for the dairy group from the other person and the dairy group from the EoE patient and the dairy group from the other person and the dairy group from the EoE patient’s sister.


Analysis Test #2 Fixed Data

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This is the analysis for the fixed data from appendix E’s Test #2 Table 13.1: These are the values for the average control, dairy, and wheat groups from the other person.

Table 13.2: These are the values for the differences between the wheat and control groups and the dairy and control groups for the other person.

Table 13.3: These are the values for the average control, dairy, and wheat groups from the EoE patient.


Table 13.5: These are the values for the average control, dairy, and wheat groups from the EoE patient’s sister.

Table 13.6: These are the values for the differences between the wheat and control groups and the dairy and control groups for the EoE patient’s sister.

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Table 13.7: These are the p-values for the control group and the wheat group for the other person and the dairy group and control group for the other person.

Table 13.8: These are the p-values for the control group and the wheat group for the EoE patient and the dairy group and control group for the EoE patient.


Table 13.10: These are the p-values for the wheat group from the other person and the wheat group from the EoE patient and the wheat group from the other person and the wheat group from the EoE patient’s sister.

Table 13.11: These are the p-values for the control group from the other person and the control group from the EoE patient and the control group from the other person and the control group from the EoE patient’s sister.

Table 13.12: These are the p-values for the dairy group from the other person and the dairy group from the EoE patient and the dairy group from the other person and the dairy group from the EoE patient’s sister.


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Appendix G: Notes

Source Title Do We Know What Causes Eosinophilic Esophagitis? A Mechanistic Update

Source citation Runge, T., & Dellon, E. (2015). Do we know what causes eosinophilic esophagitis? A mechanistic update. Current Gastroenterology Reports, 17(9), 1-10. doi:10.1007/s11894-015-0458-9

Source found by Searched Summon using “what is eosinophilic esophagitis”

Source type Journal Article

Keywords Eosinophilic Esophagitis, Pathogenesis, Etiology, Allergy, Inflammation

Summary This is some preliminary research on EoE

Reason for interest The article seemed like a good basic one to start out with. Knowing all of the causes of a certain disease is good information to know when doing a project. This article was also fairly recent, which is very important to me.

Notes Abstract: • “chronic immune/antigen-mediated disease” • diagnosed by “esophageal dysfunction” and “esophageal

eosinophilic infiltrate • ^ meaning esophagus not working correctly, and infiltration of

eosinophils in esophagus • “Th2-predominant inflammatory process triggered by

allergens” • “Proinflammatory cytokines and chemokines” gather

eosinophils into the esophagus epithelium • ôverflow of eosinophils in esophagus causes inflammation that

leads to chocking episodes • “single nucleotide polymorphisms have been identified and

associated with EoE” • ^single nucleotide differs from normal genetic code

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• Scientists don’t think that genetic factors are the only factors that contribute to EoE

• “Rapid epidemiologic changes in the incidence and prevalence” lead scientists to believe that environmental factors are a cause of EoE

Introduction:

• many symptoms “failure to thrive, abdominal or epigastric pain difficulty feeding, heartburn, chest pain, dysphagia, or food impactions”

• biopsy’s typically find “brisk mucosal infiltration of eosinophils” • In order to diagnose EoE, 15 eosinophils per high-power field • “Must rule out competing causes such as gastroesophageal

reflux disease (GERD), proton pump inhibitor-responsive esophageal eosinophilia (PPI-REE), drugs, infection, autoimmune conditions, and primarily hypereosinophilic syndromes to diagnose EoE”

• Males are affected 3 to 4 times more than females, also disease more commonly found in Caucasians.

• .5-1 case per 1000 individuals • Unknown what causes EoE – discoveries in understanding

pathogenesis of disease – they think it involves “an immune/allergen-mediated Th2 response”

• “Most studies show that 50–80% of patients with EoE, regardless of age, have comorbid allergic diseases including food allergies, allergic rhinitis or sinusitis, atopic dermatitis, and asthma”

• “Aeroallergens have been shown to play a role in EoE as well, either trigging it directly or correlating with disease activity.65, 66, 67There have also been a number of studies showing seasonal variation of EoE diagnosis, with more cases diagnosed in summer or fall months, again suggesting a link with environmental allergies.”

Questions/Things to look up further

1. What is Th2? 2. Look up more about proinflammatory cytokines and chemokins and

their role in EoE 3. The mucosal infiltration of eosinophils found during biopsy

Hobson 66

Source Title Food Allergy Testing in Eosinophilic Esophagitis: What the Gastroenterologist Needs to Know

Source citation Aceves, S. S. (2014). Food allergy testing in eosinophilic esophagitis: What the gastroenterologist needs to know. Clinical Gastroenterology and Hepatology : The Official Clinical Practice Journal of the American Gastroenterological Association, 12(8), 1216-1223. doi:10.1016/j.cgh.2013.09.007

Source found by Searched Summon using “allergy testing to eosinophilic esophagitis”


Keywords Eosinophilic Esophagitis, Elimination Diet, Allergy Test, Remodeling, Eosinophil

Summary EoE Introductory Information

Reason for interest

My project idea is based on the inaccuracy of current allergy tests to detect the allergens that cause EoE for the specific patient.

Notes • “The triggering antigen in EoE is often a food that initiates a cascade of Th2-associated interleukins such as interleukin-5 and interleukin-13 and chemokines such as eotaxin-3 as well as esophageal eosinophilia and mastocytosis.”

• Food elimination diets that are followed by single food introduction with repeat biopsy

• ^lengthy, but efficient, can be scary for patients, especially • “the ideal allergy test for identifying food antigens in EoE remains

to be elucidated” • doctors use food skin prick tests combined with atopy patch testing • others have not had successful results with those tests “Currently, a

positive test on food allergy evaluation suggests a food trigger for EoE but does not substitute for biopsy-based tissue evaluation after food removal and reintroduction”

Introduction:

Hobson 67

• EoE has a similar pathogenies to asthma and eczema, “in which an antigen induces a cascade of Th2 interleukins (ILs) and chemokines in addition to inflammatory cell infiltration”

• Eliminating specific food groups is “highly effective in controlling EoE-associated symptoms, endoscopic abnormalities, and eosinophilia”

• “In children and adults, the empiric elimination diet resolves EoE in more than 60% of subjects”

• Because this method is so successful, it is direct proof that food allergens contribute to EoE

Questions/Things to Research Further

1. Look up the specific interleukins, chemokines, esophageal eosinophilia, and mstocytosis

Source Title Eosinophilic Esophagitis From an Allergy Perspective: How to Optimally Pursue Allergy Testing & Dietary Modification in the Adult Population

Source citation McGowan, E., & Platts-Mills, T. (2016). Eosinophilic esophagitis from an allergy perspective: How to optimally pursue allergy testing & dietary modification in the adult population. Current Gastroenterology Reports, 18(11), 1-10. doi:10.1007/s11894-016-0531-z

Source found by Searched Summon using “accuracy of allergy testing eosinophilic esophagitis”


Keywords Eosinophilic esophagitis, six-food elimination diet, skin prick testing, food specific IgE

Summary Current allergy testing methods = not very reliable, dietary restrictions are more effective, but take much longer to complete and are very invasive. Not an optimal treatment method for EoE, due to fact 100% confident in the cause/physiopathology of EoE

Reason for interest To know more about the specific allergy testing methods and how they relate to EoE.

Hobson 68

Notes Abstract:

• EoE – clinicopathologic disease, symptoms: “esophageal dysfunction and eosinophil-predominant inflammation”

• Adult treatments: “swallowed steroids, elimination diets, and periodic esophageal dilations”

• Adults: elimination diets include – “elemental diets, allergy testing-directed diets, and empiric elimination diets”

Introduction:

• Characterized as a “chronic, immune/antigen-mediated esophageal disease”

• Affects 10-57 per 100,000 people • Both children and adults • ^similar histopathology, but adults more frequently display symptoms • studies suggest “that pediatric and adult EoE were driven by food and

aeroallergen exposure” • ^most initial studies performed in children, more studies show that this

is the same in adults • Adult treatment options: “proton pump inhibitor, swallowed steroids,

elimination diets, periodic esophageal dilations” • ^later will focus on benefits and limitations • dietary restriction can eliminate cause for inflammation (inflammation

lead to long term remission for patients with food triggers The Role of Allergy in Eosinophilic Esophagitis:

• considered allergic disease • many EoE patients also have “asthma, rhinitis, IgE-mediated food

allergy, urticarial, and/or atopic dermatitis” • other studies have seen seasonal variation • ^possible role of aeroallergen exposure that could contribute to the

disease • most patients have an “immunoglobulin that cross-links receptors on

mast cells and basophils and is intricately involved in the manifestations of allergic disease.”

• inflammation from “eosinophils, mast cells, and Th2 cytokines, such as IL-4, IL-5, and IL-13.”

• Strong correlation to “exposure to foreign antigens, most of which are allergens.”

Dietary Therapy in Eosinophilic Esophagitis:

Elemental Diet:

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• 1995 Kelly et al. tested elemental diet for children with esophageal eosinophilia for six weeks and receives a 90% histologic improvement (w/ <= 15 eosinpohils/high-powered field [hpf])

• (from knowledge, not article) an elemental diet is one where one eats liquid nutrients in place of normal food meals.

• ^^ “accepted, albeit cumbersome, means of treatment for pediatric EoE”

• adults: 92% improvement rate, 17/18 patients, but acceptance of diet very low, 33% dropped out and many lost a lot of weight

• not good for long term use for adults: expensive, not all covered by insurance, unpalatable, high lifestyle modification

Allergy Testing-Directed Diets:

• “Skin prick testing (SPT), atopy patch testing (APT), serum food-specific IgE testing (sIgE), and component-resolved diagnostics (CRD)”

Overview of Allergy Testing:

• used to access IgE-mediated (immediate) and non-IgE cell-mediated (delayed) response

• ÎgE = through SPT or quantifying sIgE • “presence of IgE to an allergen is referred ot as ‘allergic sensitization.’” • SPT – measures presence and function of IgE antibodies that bind to

mast cells • ^performed by using food extracts or fresh food • sIgE – presence and quantity of sIgE in bloodstream • IgE bound to receptors on mast cells and basophils – only fraction of all

IgE located in circulation • Both of these tests assess immediate reactions – hives, angioedema,

anaphylaxis • ^”false-positive tests are common with both of these testing

modalities, so they should be used only in combination with a careful clinical history, preferable under the care of a specialist.”

• Recent new testing method – presence of IgE antibodies known as CRD • âllergen extracts mixture of proteins, which are the target for IgE

antibodies known as components • ^CRD uses purified native/recombinant allergens to access the proteins

– used to diagnose IgE-mediated peanut alleregy and effectiveness has also been studied in EoE

• use APT testing to assess cell-mediated (delayed) reactions • ^”involve fresh foods in aluminum Finn chambers and placing them on

a patient’s back for 48 h.” • êxamine back at 48/72 hour for erythema and vesicles • because reactions occur within days, not within hours, many doctors

have looked at using this technique for EoE allergy diagnosing Skin Prick Testing and Atopy Patch Testing-Based Diets:

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• Spergel et al. – study 26 children went through SPT and APT testing and then went on either testing-directed diets or elemental formula diets for 6ish weeks

• 24 – 75% symptom improvement, >50% biopsy improvement with a decrease in the number of eosinophils/hpf

• 2012 study = 53% success rate for diet restriction method • ^”found low specificity and low negative predictive value (NPV) for SPT

and APT to milk” • ^means that low rate for proving that don’t have the disease • ^when empiric milk elimination added to dietary restriction success rate

= 77% • adults: test with subjects sensitivity to rye, grass, wheat … only one

adult showed improvement in symptoms • Molina-Infante et al. 22 adults w/ EoE SPT, APT, and prick-prick testing

to 26 different foods – 17/22 followed up with allergy testing-directed diets for 6 weeks and 5/17 (29%) showed improvement <=15e/hpf

• Wold et al. adult EoE – 32% elimination diet based on SPT and clinical history achieved <=15e/hpf

• SPT/APT diets have limited utility for gaining remission/successful results (<=15e/hpf) in adults

Serum-Specific IgE Diets:

• Sensitized (or responded) to a larger number of foods with sIgE than normal SPT

• “early studies suggested that elimination diets based on sIgE were effective in achieveing disease remission in adults with EoE.”

• 2014 Rodriguez-Sánchez et al. assessed sIgE-directed diets. Positive sIgE results = targeted elimination diet, negative results = six-food elimination diet

• ^73.1% of adults = remission w/ sIgE-directed diet/52.9% SFED diet (fewer endoscopies and fewer elimination foods)

• ^^sIgE testing = high accuracy in detecting cow’s milk as an allergic trigger

• ^^^results haven’t been replicated – “suggest that in those with food sensitization identified by sIgE, a targeted elimination diet may be a viable treatment option

Component-Resolved Diagnostic Diets:

• allergenic-molecules are cross-reactive between foods and aeroallergens

• some studies imply that food sensitizations in adults with EoE may be caused by aeroallergen cross-reactivity

• CRD-guided diets in adults with EoE, but ended prematurely due to low success rate (7%)

• Low correlation between CRD testing and sIgE testing possible due to “assay inhibition by IgG4 blocking antibodies – limiting design of elimination diet

Hobson 71

• Studies done with on particular method of CRD – wonder if other methodologies could have an impact on results

Limitations of Allergy Testing-Directed Diets:

• “low efficacy of allergy testing-directed diets” • “These testing modalities may be limited in EoE because they are based

on the presence of IgE, which now appears to have little role in the pathophysiology of EoE”

• îdea was reasoned in a recent study that used omalizumab (a monoclonal antibody blocking IgE - “found to be ineffective in improving clinical symptoms or histology in both children and adults with EoE”

• recent studies found that “IgG4 levels are higher in esophageal homogenates in patients with EoE compared to controls”

• înfiltrates of IgG4 producing plasma cells can be found in lamina propria of the esophagus in EoE patients

• could work in tandem together as B cells producing IgG4 can switch to produce IgE, but the cells that produce IgE cannot switch to produce IgG4

• so….EoE may not be an IgE-mediated food hypersensitivity…but overall pathophysiology remains unclear

Empiric Elimination Diets:

• because there is little success with allergy-testing based elimination diets, many use the technique of using empiric elimination diets – “foods most commonly associated with childhood food allergy and EoE are avoided without reliance on allergy testing”

• preliminary endoscopy and biopsy -> strict elimination diet for 6 weeks … repeated process then histologic remission is assessed

• îf remission = reintroduction of foods for 2-6 weeks with repeated endoscopies…if # of eosinophils increases, cause of inflammation

Six-Food Elimination Diet:

• Kagalwalla et al. – 6 most common foods associated with “IgE-mediated food allergy and EoE (milk, soy, egg, wheat, peanut/tree nuts, and seafood)” -> 35 children 26/35 = histological and clinical remission (<=10eosinophils/hpf)

• 2012 – Gonsalves et al. – 50 adults w/ EoE SFED for 6 weeks -> 70% histological remission (<=10e/hpf) 94% improvement

• ^20 “food reintroduction, with confirmatory histologic assessment, and wheat and milk were identified as the most common triggers”

• Lucendo et al. 67 adults w/ EoE with SFED – also eliminated rice, corn, and all legumes -> 73% improvements in symptoms and histology (<=15e/hpf)

• ^wheat and milk most common food triggers

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• ^âllergy testing (SPT and sIgE) also performed but was not found to be predictive of the food triggers

• ^^^2 years later, 30% still in remission • 3 more studies, lower success rate SFED -> 52-56% in adults • ^differences than others: Rodriguez-Sanchez et al. only prescribed to

adults with negative sIgE testing -> selection bias. Other two studies no consultation with dietician -> influenced compliance with elimination diet

Four-Food Elimination Diet:

• SFED – milk, wheat, egg, and soy/legumes = most common triggers • Molina-Infante et al. efficiency of these four restrictions – 52 patients

w/ EoE avoided these foods for 6 weeks – 28/52 (54%) histological remission (<=15e/hpf)

• Non-responders = 19 underwent SFED and 31% histologic remission • Not as effective, but shortens the treatment time, minimizing #

endoscopes Milk Elimination Diet:

• Milk = most frequent food trigger in EoE with children and adults • Kagalwalla et al. 17 children eliminated cow’s milk 11/17 (65%) =

histological remission (<=15e/hpf) • Similar study = compared empiric cow’s milk elimination diet to

swallowed fluticasone – histologic improvement 64% children • ^study = all patients PPI (protein pump inhibitor • ^^confirmed cow’s milk EoE patients can tolerate cow’s milk-based

hydrolyzed formula and baked milk … interesting • recent study histologic improvement with milk elimination have lower

levels of IgE than those who don’t respond to milk Limitations of Empiric Elimination Diets:

• initial studies of empiric elimination diets (mostly SFED) have “higher efficiency rates in achieving histologic remission than allergy testing-directed diets”

• recent studies not very successful, long period to figure out with reintroduction endoscopies, etc.

Our Approach to Implementing Allergy Testing and Dietary Modification in Adult Eosinophilic Esophagitis:

• patients w/ EoE – swallowed steroids/periodic endoscopic dilations or dietary modification as an alternative

• thorough dietary history, measure sIgE with foods most associated with EoE milk, wheat, egg, soy/legumes, peanut, tree nuts, fish, and shellfish) and others that are consumed in patients diet

• if positive sIgE to foods, target elimination diet for 6 weeks w/ registered dietitians opinion

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• if negative sIgEor >=8 positive tests, recommend SFED for 6 weeks • âfter 6 weeks repeated endoscopies w/ biopsy’s … if clinical and

histologic remission (>= 15 e/hpf) then foods introduced every 2 weeks with endoscopies every 4 weeks to identify triggers

• if haven’t experienced histologic remission, inflammation could be a result of “a.) dietary non-compliance, b.) cross-contamination of allergens in consumed foods, c.) consumption of a trigger food that has not been identified, or d.) persistent exposure to potential aeroallergen triggers.”

• After conversation w/ nutritionist about ^– positives go on SFED while negatives eliminate other allergenic foods or elemental formula

• Other options are also discussed – i.e. steroids/dilations Conclusions:

• Recent studies = dietary modifications • Diets based on SPT/APT more effective in children than adults • sIgE-directed and empiric food elimination diets are promising • dietary able to find cause of allergenic information -> long term

remission • êliminates long term usage of PPIs and corticosteroids • ^^limited success with allergy testing, repeat endoscopies, difficulty of

successfully sticking to dietary restrictions • future: improve IgE use and maybe IgG4 to identify food triggers and

noninvasive techniques to easily watch progress of the disease

Questions/Things to Research Further

• immunoglobulin that cross-links receptors, mast cells, basophils • Th2 cytokines • Erythema • Vesicles

Source Title Eosinophilic esophagitis: A clinicopathological review Source citation

Philpott, H., Nandurkar, S., Thien, F., Gibson, P. R., & Royce, S. G. (2015). Eosinophilic esophagitis: A clinicopathological review. Pharmacology & Therapeutics, 146, 12-22. doi:10.1016/j.pharmthera.2014.09.001

Source found by Searched Summon using “eosinophilic esophagitis AND inflammatory infiltrate”

Hobson 74


Keywords Eosinophil; Esophagitis; Dysphagia; Allergy; Remodelling Diet

Summary Types of cells involved in cell infiltrate

Reason for interest

This article seemed like it had good information about how the esophagus becomes inflamed in EoE.

Notes Abstract: • Antigen-driven • “food and/or aeroallergens induce a chronic inflammatory infiltrate

in the esophagus, resulting in pathological hyperplasia of the epithelia and muscular layers, and fibrosis of the lamina propria (referred to collectively as remodeling) and the symptoms of dysphagia and food impaction”

• àallergens (antigens – produce immune reponse/stimulate production of antibodies) lead to enlargement of epithelia/muscle layers + thickening of the lamina propria (tissue below epithelium)

• other similar atopic diseases like asthma and atopic dermatitis • ^similar characteristics: Th2 cytokine milieu, inflammatory infiltrate

of eosinophils, mast cells, and lymphocytes • à share similar over production of those cells/types of cells • expression of eosinophil chemokine eotaxin 3

Introduction:

• symptoms: dysphagia/food impaction • à difficulty swallowing / food getting stuck • “(1) acute narrowing of the esophageal lumen by inflammation and

oedema, (2) fixed narrowing and limited distensibility of the lumen by remodelling and (3) dynamic and variable narrowing caused by muscular contraction or spasm”

• à1.) esophageal lumen – tube structure, oedema – water fluid collecting/pooling in tissues 2.) distensibility – stretching of the tube 3.) contracting of muscles

Hobson 75

• normal^ “Esophageal barrier function is maintained by an orderly

arrangement of epithelial cells maintained by gap junction proteins. The muscularis is striated (upper 1/3 of the esophagus) and smooth (lower 2/3) of the esophagus. Antigen presentation may occur by dendritic cells or possibly epithelial cells.”

• à epithelial layer, lamina propria muscularis mucosae in normal esophagus, organized arrangement of cells

• EoE “Epithelial barrier integrity is disrupted, allowing greater contact between antigens and dendritic cells. The epithelial layer is thickened and disorderly, and an inflammatory infiltrate rich in eosinophils extends throughout all layers, and may contribute to dysmotility. Angiogenesis is present; the esophagus is friable and bleeds easily at endoscopy. The lamina propria is thickened and fibrotic. The clinical sequelae of the pathological changes are dysphagia and food bolus obstruction due to luminal narrowing, limited distensibility and disturbance of peristalsis.”

• à barrier is disrupted, this allows the antigens to have more acess/contact with the skin layers in the esophagus. Infiltrate occurs every layer à food impaction/food dysphagia because of luminal narrowing

Hobson 76

• “involve cells (e.g., eosinophils, mast cells, epithelial cells and fibroblasts) cytokines (e.g., interleukins IL-4,5 and 13, and the chemokine eotaxin 3) and adhesion molecules (e.g., integrins and vascular cell adhesion protein 1 (VCAM-1)”

• à specific types of cells, cytokines (signaling other cells), adhesion molecules (puts together)

• don’t know exactly how everything is involved/don’t know how the process, signaling, or sequence of events occur to get final result.

• Asthma/atopic dermatitis help to figure out pathogenesis of EoE • Prominently white males Antigen presentation:

• “direct contact with the esophageal mucosa leads to antigen presentation and a localized inflammatory infiltration”

• à contact directly with the layer leads to infiltration • possible exposure to small bowel (many lymphoid follicles)à

eosinophils in esophagus • possible = distant contact aeroallergens with contact to nasals

àesophageal infiltration of eosinophils • direct exposure of ingested food to esophagus à eosinophilic

infiltration … how inflamed? • êsophageal mucosa has about 30 layers of epithelium.

Underneath = separating food antigens from lamina propria where mast cells/eosinophils located. Secretion of mucus from submucosal glands lower esophagus – airway epithelium has ciliated epithelial cells containing goblet cells that produce mucus to potentially trap the antigen

• ^ BUT… physical contact with mast inflammatory cells residing in lamina propria = limited

• other possible ways that could cause the interaction of antigens with inflammatory cells … possible esophageal epithelium/eosinophils to act as antigen presenting cells (APC)

Inflammatory cell infiltration Eosinophils:

• esophagus normally doesn’t have eosinophils inside of it • “Eosinophils are derived from myeloid precursors in the bone

marrow and mature in response to IL-5, subsequently circulating in the blood for up to 20 h and residing in the tissues for between 2–14 days.”

Hobson 77

• à produced bone marrow, blood stream, inhabit themselves in tissue

• Patients w/ untreated EoE have a large number of eosinophils • Cytokines/chemokins à eosinophils to be located in esophagus • “Cytokines such as IL-5, IL-9 and IL-13 and the chemokines, eotaxins

1, 2, and 3, are of central importance, as determined by elevated circulating levels and mRNA expression of esophageal tissue in human patients with EoE”

• “Studies in vitro of human lung and endobronchiolar tissues suggest a role for vascular adhesion mediated by VCAM-1 and P-selectin on the endothelial surface along with P-selectin glycoprotein and very late protein-4 on eosinophils in facilitating eosinophil attachment and migration into tissues, a process governed by integrins”

• àblood vessel adhesion by those proteins on the inside skin of blood vessels, and those other proteins on the eosinophils --> attachment/migration to the tissues (sensed by intergrins – sense if adhesion occurs)

• in esophagus may be in intraepithelial spaces. There they’re able to form microabscesses when clustered in lamina propria/muscle layers

• ^hard to determine, not enough research done • Eosinophils release mediators from secondary granules such as

“major basic protein (MBP), eosinophil peroxidase, eosinophil catonic protein and eosinophil-derived neurotoxin the release of which causes local tissue and damage and esophageal dysmotility, and may secondarily activate mast cells”

• à Eosinophils release specific mediators causing tissue damage to the area and dysmotility. This could also active mast cells (creating worse infiltration and inflammation)

• Possible that the specific mediator, MBP (Makor basic protein) acts on esophageal epithelial cells à fibroblast growth factor-9 à epithelial hyperplasia

• Eosinophils produce transforming growth factor-B(beta symbol) • àactivation of myofibroblasts which stimulates the production of

fibrotic tissue in the lamina propria • TGF-B(beta symbol) possible contributes to smooth muscle

contraction, hyperplasia, and hypertrophy • àcontractions, enlargement tissue, and enlargement tissue to to

increase cell size • Eosinophils = attracted/activated by cytokines/interleukins secreted

by other cells • ^capable of cytokine secretion themselves

Hobson 78

• “importance of IL-9 production by eosinophils in patients with EoE and of the ability of this cytokine to attract mast cells were demonstrated”

• eosinophils contribute to pathological process of tissue remodeling, epithelial hyperplasia, subepithelial fibrosis, and muscular hypertrophy, and hyperplasia

Mast cells:

• small # found in normal esophagus, only located in lamina propria • increased # in EoE – found in connective

tissue/intraepithelial/muscle layers • derived from CD34+ progenitors in bone marrow – mature in

tissues, don’t circulate in blood • associated type 1 hypersensitivity reaction – antigen contact with

specific IgE bound to mast cells à activation, degranulation, release of range of mediators (histamine, eicosandoids, cytokines)

• àmast cells help carry out type 1 hypersensitivity reaction (B cells simulated to produce IgE antibody) – the antigen comes in contact with the IgE bound to the mast cell --> mediators are released to help combat effects of antigen (histamine,cytokines,eicosandoids)

• mast cells bearing IgE found in studies for EoE • “Mast cells may modulate remodeling via the production of TGF-β,

which in turn governs connective tissue production and also possibly smooth muscle contractility”

• à mast cells could aid in the remodeling process of esophaguses of EoE patients. They produce TGF-b which regulated the connective tissue production/possible the smooth muscle contractility as well

• mast cells, not eosinophils, could cause smooth muscle spasm EoE patients could experience

• role of mast cells = worth further research B-lyphocytes and IgE antibodies:

• B cells = inside esophageal mucosa of EoE patients • à B cell = lymphocyte responsible for producing antibodies located

in the inner lining of the esophagus • “switching to IgE antibody production by B cells occurs in response

to TH2 cytokines” • à TH2 cytokine (cell signaling molecules) cause B cells to produce

IgE antibodies • dietary removal of allergen reverses eosinophilic

infiltration/remodeling à My sentence “Notably, dietary

Hobson 79

restrictions decrease antigen exposure and reverse the cell infiltration”

• “Whilst EoE is considered an antigen-driven disease, the significance of IgE antibodies in the pathogenesis is debatable.”

• à “removing IgE from the circulation with the monoclonal antibody, omalizumab, may greatly decrease IgE levels” àsucces in some EoE patients

• à “The failure of this medication to deliver universally positive results also supports the suggestion that a TH2-mediated cytokine production, rather that B cell antibody production is of greater importance in the pathogenesis of EoE.”

• à possible that IgE isn’t a cause – varying positive/negative results with allergy testing. Methods to reduce IgE levels have had limited results in EoE patients. Due to the unclear results, scientists believe that TH2 cytokine production will be more beneficial to study than B cell production of IgE to the pathology of EoE

T lymphocytes:

• increased # of T cells (T lymphocyte) in EoE patients (CD8+, CD4+, CD3+)

• “The assertion that EoE is a TH2-mediated disease is upheld by the finding that the cytokines, IL-4, IL-5 and IL-13, are produced in greater quantities by monocytes following antigen exposures in the peripheral blood of patients with EoE compared to healthy controls, and that mRNA for these cytokines is overexpressed in esophageal biopsies.”

• à TH2 cytokines (produced by T lymphocytes) have an increased production after antigen exposure in EoE patients.

• “TH-1 cytokines, TNF-α and IFN-γ are also increased following antigen exposure of monocytes and are found in increased quantities in esophageal mucosal biopsies”

• à other structures produced by T lymphocytes are increased as well in EoE patients

• “The cytokines IL-4, IL-5, IL-13 and possibly IL-9 are produced by TH2 and TH-9 cells, and drive the eosinophilic and mastocytic infiltrates characteristic of EoE”

• à It has been proposed that these cytokines could “drive the eosinophilic and mastocytic infiltrates characteristic of EoE”

• “It has been proposed that the expression of eotaxin-3 by the esophageal epithelium, VEGF by the endothelium and integrins by the interstitium attracts these cells to the esophagus” à degranulation tissue damage

Hobson 80

• Furthermore, growth factors such as TFG-β and FGF-9 (fibroblast growth factor 9) are released by eosinophils and mast cells that activate quiescent fibroblasts to myofibroblasts, and drive hyperplasia of epithelium and smooth muscle that complete the cycle of remodeling

• à Many other cell signalers that assist in the process of the inflammation and cell infiltrate of the esophagus are produced by these T lymphocytes which is why these are central figuring out the pathogenesis of EoE

• T helper cells = central to pathogenesis of EoE à esophageal remodeling

Basophils and the TSLP basophil axis:

• “basophils and the epithelial cytokine thymic stromal lymphopoietin (TSLP) are integral to the development of EoE, and that disease development can occur in the absence of IgE and IL-5.”

• àA new experiment done in mice supports the hypothesis that basophils and TSLPs (thymic stromal lymphopoietin) could cause EoE.

NOT USING INFORMATION BELOW Remodeling: Epithelial cells

• lined by squamous partially keratinised epithelium • EoE develop epithelial hyperplasia à possible MBP/TGF-B(beta)

from eosinophils • “Eotaxin 3 is produced by esophageal epithelium in response to IL-

13, which in turn attracts eosinophils expressing the CCR-3 (eotaxin 3) receptor, promoting the remodelling described” = possible

• “mast cells may produce IL-9 in turn causing increased IL-13 production by TH2 cells, thus completing the loop” = other possible

• Exotaxin 3 = important for pathogenesis • “Furthermore, a single nucleotide polymorphism in the gene

encoding eotaxin 3 has been demonstrated in some patients with EoE”

• inherited/acquired defects in esophageal epithelial barrier function à possible lead to EoE

Hobson 81

Esophageal muscle: • “The esophageal muscle layer is predominantly striated in the

cervical esophagus (the upper third), a mixture of striated and smooth muscle in the middle third and smooth muscle alone in the lower third. The muscle layers themselves are oriented in a circular (inner) and longitudinal (outer) fashion”

• mast cells/eosinophils infiltrate the muscular layer • “it is possible that both structural alterations (myocyte hypertrophy

and hyperplasia along with inflammatory infiltration) and dynamic changes (muscular contraction) contribute to the clinical syndrome of dysphagia and food bolus obstruction.”

• Mediators from mast cells (histamine) àpossible muscle contraction/hyperplasia

• “it is yet to be determined, for example, whether the refractory dysphagia typical of some patients with EoE represents ongoing inflammation in the muscle layer or muscular dysmotility, or simply subepithelial fibrosis.”

Epithelial mesenchymal transition:

• “Epithelial mesenchymal transition (EMT) refers to the process whereby epithelial cells may lose their typical histological and immunohistochemical appearance, and functional properties to instead acquire the structure and function of mesenchymal cells”

• ^motility/depolarized cytoskeletal arrangements

• “The same process of EMT, and the resultant fibrosis and smooth muscle hyperplasia observed in asthma may occur in EoE”

Production of extracellular matrix(EMC) the process of subepithelial fibrosis:

• “Subepithelial fibrosis is characterised by an increase in the thickness and density of collagen bundles, and an increase in fibroblast density”

• “The production of the ECM by myofibroblasts that have been activated by TGFβ secreted by a range of cells (e.g., eosinophils, mast cells, epithelial cells) leads to SMAD-dependent signalling, upregulating fibrogenic genes such as collagen, alpha-SMA and periostin”

• “The reversal of the fibrous remodelling appears to correlate with the disappearance of eosinophils”

Hobson 82

• possibly involved in the fibrosis remodeling of EoE Angiogenesis:

• “Angiogenesis, the formation and development of new blood vessels, is a feature of eosinophilic esophagitis”

• “the angiogenic factor vascular endothelial growth factor alpha (VEGF-A), angiogenin and IL-8 have been implicated in promoting this pathological process, and it is notable that eosinophil-depleted mice have decreased angiogenesis “

Microbata:

• Microbiota of the esophagus and possible effects on barrier function may be important in the pathogenesis of EoE


VOCABULARY: • Antigen – toxin à immune response (stimulate production of

antibodies) • Pathological à cause/effects of disease • Hyperplasia à enlargement of organ/tissue • Fibrosis à thickening / scaring of connective tissue • Lamina propria à connective tissue below the epithelium • Dysphagia à difficulty swallowing • Impaction à collision/crash/smash • TH2 cytokines à cell signaling molecules that communicate

immune responses and cause cells to move to inflammation cites, produce inflammatory responses

• Eosinophils à white blood cells to combat toxins • Mast cells à white blood cells • Lymphocytes à come from leukocytes (white blood cells), occur

especially in lymphatic system • Chemokines à type of cytokines • Lumen à tube like structure • Oedema à excess water fluid in connective tissue • Distensibility à stretching • Angiogenesis à formation of new blood cells • Fibroblasts à connective tissue cells • Adhesion molecules à proteins on cell surface – binding other cells

with the extracellular matrix • Esophageal mucosa à inner lining of the esophagus • Lymphoid follicles à in lymph nodes – try to fight antigens • Airway epithelium à skin layer respiratory track • Ciliated epithelial cells à hairs help move particles

Hobson 83

• Interposed à inserted • Goblet cells à secrete mucus • Endobronchiolar tissues à lung tissue • Vascular à vessels (carry blood) • Endothelial à epithelium interior of surface blood vessels • Integrins à attaching cell cytoskeletons w/ extracellular matrix +

sensing whether adhesion occurred • Microabcesses à very small abscess – collection of neutrophils • Intraepithelial à within layers of cells form skin for esophagus • Mediators à help bring about changes • Granules à vesicles immune system • Dysmotility à strength of esophagus muscle doesn’t work as

should • Myofibroblasts à cell between fibroblast + smooth muscle cell • Fibrotic tissue à excess connective tissue • Hypertrophy à enlargement organ/tissue – increases cell size • Progenitors à cells divide not forever (pick path) • Type 1 hyperplasia à B cells stimulate _ produce IgE antibody • Degranulation à releases molecules from vesicles • Modulate à regulate/adjust • B cells à lymphocyte (responsible for producing antibodies) • Esophageal mucosa à inner lining of the esophagus • T lymphocytes – produce cytokines – a form of small leukocyte

(white blood cell) w/ a single round nucleus, occurring especially in the lymphatic system

Source Title Biology in Focus – 35.1 Source citation Urry, L. A. (2015). Campbell biology in

focus (2. revised Ed. ed.). Boston [u.a.]: Pearson.

Source found by Ms. Curran

Source type Book

Keywords Biology

Hobson 84

Summary General overview of the immune system

Reason for interest Background information on the immune system

Notes • Pathogen – a bacterium, fungus,

virus, or other disease-causing agent • Want to inhabit in animal • ^ nutrients, protection for growth and

development and reproduction, transportation to new environments

• adaptations have arised for animals to protect against invadors

• immune cells à affect and destroy pathogens

• immune cells release defense molecules into body’s fluids

• immune system – defense system to avoid or limit infections

• first lines = help prevent pathogens from gaining entrance

• if breaches defense and enters body, immune system must detect foreign object

• immune cells à receptor molecules that bind to molecules from foreign cells or viruses and activate defense response

• ^binding of immune receptors to foreign molecules = molecular recognition

• all animals = innate immunity – barrier defenses

• ^molecular recognition relies on small set of receptors that bind to molecules or structures that are absent from animal bodies but common to a group of viruses, bacteria, or other microbes

• this binding activates defense

Hobson 85

• adaptive immunity – only in vertebrates

• molecular recognition relies on vast number of receptors – each recognizes only specific part of specific pathogen – very specific

• aka acquired immune response • develop slowly after innate immune

response • enhanced by exposure to pathogen

35.1:

• In innate immunity, recognition and response rely on traits common to groups of pathogens

Innate immunity of invertebrates:

• Rely on exoskeleton as first line of defense

• Lysozyme (an enzyme that breaks down bacterial cell walls, acts as a chemical barrier against pathogens ingested with food

• Immune cells – hemocytes – travel throughout body in the hemolymph (insect circulatory fluid)

• Phagocytosis – hemocytes ingest and break down bacteria and other foreign objects

• Others release chemicals that kill pathogens and help entrap invaders

• Defense molecule – antimicrobial peptides – circulate thoughout body and inactivate/kill fungi and bacteria by disrupting their plasma membranes

• Immune cells of insects bind to molecules in outer layers of fungi/bacteria

• Fungal cell walls = polysaccharides, bacteria cell walls = polymers, “identity tags” pathogen recognition

Hobson 86

Innate Immunity of Vertebrates:

• Mostly about mammals Barrier Defenses:

• Block entry of pathogens (most) • Skin/mucus membranes in digestive,

respiratory, urinary, reproductive systems

• Produce mucus (fluid that traps microbes and other particles)

• Airway – ciliated epithelial cells sweep mucus/microbes upward – prevent infection of lungs

• Body secretions = hostile to many microbes

• Lysozymes in tears, saliva, mucus destroy cell walls bacteria as enter openings

• Acidic environment of stomach • Oil/sweat glands from skin (acid ph)

Cellular Innate Defenses:

• Pathogens engulfed by phagocytic cells detect invader using receptors

• Similar to Toll (key activator of innate immunity in insects)

• Toll-like receptor (TLR) finds to fragments of molecules characteristic of a set of pathogens

• Invertibrates – detection of invasion triggers phagocytosis/destruction

• Types of phagocytic cells in mammals are neutrophils (circulate in the blood and attracted by signals from infected tissue) and macrophages (larger phagocytic cells)

• Dendritic cells – found mainly in tissues, stimulate adaptive immunity against pathogens they encounter/engulf

• Eosinophils – often found beneath mucous membranes, important in

Hobson 87

defending against multicellular invaders, upon engagement with pathogen à release destructive enzymes

• Natural killer cells – circulate and detect abnormal array of surface protein characteristics of virus-infected/cancerous cells

• Release chemicals that lead to cell death (stopping spread virus/cancer)

• Many innate defences involve lymphatic system – some macrophages reside in lymph nodes and engulf pathogens that have entered the lymph from interstitial fluid

• Dendritic cells reside outside of lymphatic system but migrate to lymph notes after interaction with pathogens – interact with other immune cells to stimulate adaptive immunity

Antimicrobial Peptides and Proteins • Pathogen recognition triggers

production/release of peptides/proteins to attack pathogens

• Some damage broad groups of pathogens by disrupting membrane integrity / others (interferons/complement proteins)

• Interferons – proteins that provide innate defense by interfering with viral infections

• Virus infected cells secrete interferons à uninfected cells to produce substances that inhibit viral reproduction – limit cell to cell spread, controlling infections

• Some wbc secrete different interferon activate microphages (enhancing phyagocytic ability)

Hobson 88

• Complement system – infection fighting, consists of proteins in blood plasma

• ^ circulate in inactive state – activated by substances on surface of microbes à biochemical reaction àlysis (burning) of invading cells, and inflammatory response

Inflammatory Response:

• inflammatory response – changes brought about by signaling molecules released upon injury or infection

• signaling molecule = histamine – stored in granules (vescicles) of mast cells (found in connective tissue)

• histamine triggers nearby blood vessels to dilate and become more permeable

• microphages and neutrophils discharge cytokines (signaling molecules that in an immune response promote blood flow to the site of injury/infection

• încrease in local blood supply causes redness and increased skin temperature – blood-engorged capillaries leak fluid into neighboring tissues à swelling

• activated complement proteins promote further release of histamine (more phagocytic cells to enter tissues), enhanced blood flow (antimicrobial peptides) àaccumulation of pus (fluid rich in white blood cells, dead pathogens, cell debris from damaged tissue)

• minor – local, severe à systematic response (throughout body) some cells in infected area secrete moleculs à release of more neutrophils from bone marrow

• chronic (ongoing) inflammation

Hobson 89

Evasion of Innate Immunity by Pathogens:

• adaptations in pathogens able to avoid destruction f=by phagocytic cells

• other bacteria can be engulfed by host cell resisting breakdown by lysosomes


Nothing in particular. This section of the immune chapter cleared up many questions.

Source Title Biology in Focus – 35.2 Source citation Urry, L. A. (2015). Campbell biology in

focus (2. revised Ed. ed.). Boston [u.a.]: Pearson.

Source found by Ms. Curran

Source type Book

Keywords biology

Summary General overview of the immune system

Reason for interest Background information on the immune system

Notes 35.2: In adaptive immunity, receptors provide pathogen-specific recognition

• vertebrates

Hobson 90

• relies on T and B cells - types of white blood cells called lymphocytes

• originate from stem cells in bone marrow

• some migrate from bone marrow to thymus (organ in thoracic cavity above heart)

• ^mature into T cells • remain in bone marrow develop as B

cells • any substance that elicits a response

from a B cell or T cells in an antigen • recognition occurs when a T/B cell

binds to an antigen, such as a bacterial or viral protein, via a protein called an antigen receptor

• ^specific binds to one part of one molecule from specific pathogen

• all antigen receptors made by B/T cells = identical

• antigens – foreign, large molecules, proteins/polysaccharides

• part of antigen that binds to antigen receptor = epitope (antigenic determinant)

Antigen Recognition by B Cells and Antibodies:

• antigen receptor = y shaped molecule for polypeptide chains: two identical heavy chains/two identical light chains

• light and heavy chains each have C region (amino acid sequences vary little among receptors on different B cells

• tip = V region (amino acid sequences vary extensively from on B cell to another

• heavy chain V region/light chain V region à asymmetric binding site for an antigen

Hobson 91

• binding of B cell antigen to an antigen à formation of cells that secrete a soluble form of the receptor à secreted protein = antibody or immunoglobulin

• ^same Y shaped organization as B cell antigen receptors (but are secreted rather than membrane bound)

• antibodies specifically that defend against pathogens

• The antigen-binding site of a membrane-bound receptor or antibody has a unique shape that provides a lock and key fit for a particular epitope

• Antibodies can bind to antigens on pathogens or free in body fluids

Antigen Recognition by T Cells:

• Antigen receptor = polypeptide chains - alpha chain and beta chain

• Base of T cell receptor = transmembrane region that anchors the molecule in the cell’s plasma membrane

• Outer tip V region form a single antigen binding site

• Remainder = C region • Antigen receptors of T cells bind to

fragments of antigens that are on the surface of host cells

• Host protein that displays the antigen = major histocompatibility complex (MHC) molecule

• Recognition of protein antigens by T cells pathogen infects/is taken by a host cell – inside enzymes cleave the antigen into smaller peptides

• Each peptide (antigen fragment) binds to MHC molecule and bound antigen fragment to the cell surface à antigen presentation (display of

Hobson 92

the antigen fragment in an exposed groove of the MHC protein

• If cell displaying an antigen fragment encounters a T cell with the right specificity, the antigen receptor on the T cell can bind to both the antigen fragment and the MHC molecule

B cell and T cell development:

• Immense diversity of lymphocytes and receptors à immune system to detect pathogens never before encountered

• Adaptive immunity normally has self-tolerance (lack of reactivity against animals’ own molecules and cells)

• Cell proliferation triggered by activation increases B/T cells specific for an antigen

• Stronger/more rapid response to an antigen encountered previously due to a feature known as immunological memory

Generation of B Cell and T Cell diversity:

• Combining element à many different receptors

• Receptor light chain – variable segment, joining segment, and a constant segment

• Many combinations … heavy chain formation even greater number of combinations

Origin of Self Tolerance:

• Some immature lymphocytes produce receptors specific for epitopes on the organism’s own molecules

• If these self-reactive lymphocytes weren’t eliminated/inactivated couldn’t distinguish self from nonself

Hobson 93

• Instead – as mature antigen receptors tested for self-reactivity

Proliferation of B Cells and T Cells:

• An antigen is presented to a steady stream of lymphocytes in the lymph nodes until a match is made

• Match à changes in cell number and activity for the lymphocyte to which an antigen is bound

• Binding of antigen receptor to epitope initiates events that activate lymphocyte àB/T cell division – clone

• Some cells from the clone àeffector cells (short-lived cells that take effect immediately against the antigen and nay pathogens producing that antigen)

• Effector forms of B cells = plasma cells (secrete antibodies)

• Effector forms of T cells = T helper cells/cytotoxic T cells

• Remaining = memory cells (long-lived cells that can give rise to effector cells if the same antigen is encountered later in the animal’s life

• Process = clonal selection – encounter with an antigen selects which lymphocyte will divide to produce a clonal population of thousands of cells specific for a particular epitope

Immunological Memory:

• Long term protection that a prior infection provides against many diseases

• Before exposure – antigen alters speed, strength, and duration of the immune response

• Production of effector cells from a clone of lymphocytes during first

Hobson 94

exposure to an antigen = basis for primary immune response

• 10-17 days after initial exposure • B/T cells give rise to effector forms • If exposed again to same antigen –

response is faster (2-7) = secondary immune response

• Relies on T and B cell memory following initial exposure

• If an antigen is encountered again, memory cells specific for that antigen enable the rapid formation of clones of thousands of effector cells also specific for that antigen àenhanced immune defense

Questions/Things to look up further Nothing in particular. This section of the immune chapter cleared up many questions.

Source Title Eosinophilic Esophagitis Source citation Eosinophilic esophagitis | AAAAI. Retrieved

from http://www.aaaai.org/conditions-and-treatments/related-conditions/eosinophilic-esophagitis

Source found by Looking up “what makes EoE different than normal allergies” on Google

Source type Web Page

Keywords What is EoE?

Summary Background information on EoE

Reason for interest More background information on EoE

Notes • Chronic allergic/immune

Hobson 95

• Normally no eosinophils in the esophagus but EoE – found there

• Infants/toddlers – refuse food/not growing (sign)

• School-aged – abdominal pain, trouble swallowing, vomiting (sign)

• Teenagers/adults – difficulty swallowing solid foods

• Can get to the point – food impaction • Acid reflux /GERD (gastroesophageal

reflux disease)à EoE • Only way diagnose – biopsy of

esophagus • Biopsy = tissue samples

taken/analyzed • Majority EoE – atopic (family history

of allergies/asthma + symptoms of one of more allergic disorder – asthma, allergic rhinitis, atopic dermatitis (eczema) and food allergy)

• Allergy testing – information any allergic aspects of EoE can be tested properly, plan dietary therapy/reintroduction of foods to diet

• Environmental allergies • Food = main cause EoE • Relationship between food allergy

and EoE is complex • Many food allergies – easily

diagnosed by history of severe allergic reaction – i.e. hives after ingestion

• Difficult to establish role of foods since delayed reaction, can develop over days – hard to pinpoint specific food

• Several test (mostly w/ dairy, egg, soy, and wheat – main triggers)

• Cannot be easily proven by conventional allergy tests (skin, patch, blood)

Hobson 96

• Bc food allergies = delayed, caused by immune mechanisms other than classical IgE-mediated food allergy

• Prick Testing: introduced small amount of allergen into skin by making small puncture with a prick device that has a drop of allergen

• Skin prick à what not allergic to – allergy patients have allergic antibody = Immunoglobulin E (IgE)

• When test done = swelling/redness (15 min)

• Limited use in identifying • Blood Testing: serum specific immune

assay – can be helpful, both tests = limitations, - few (not many, more research) prick tests more helpful

• Food patch tests: some evidence more useful in children

• Small amoun of a fresh food in small aluminum chamber called a Finn chamber – taped on back for 48 hrs – removed and read at 72 hrs

• If inflamed = positive delayed reaction to the food

• All three tests have false positives – may suggest allergic to food but actually not

• Possible to have false negatives – negative but food is actually an allergen

• Treatment = food testing directed diets

• Empiric Elimination – difficult to follow – eliminate diary, egg, wheat, soy, peanut, tree nuts, fish/shellfish – introduce one at a time

• Elemental diets – all sources of protein removed – get nutrition from amino acid formula + simple sugar/oils – feeding tube might be needed (taste of formula – many don’t like)

Hobson 97

• Usually for people who don’t respond to other treatment forms

• No medications approved by FDA but some have shown to reduce eosinophils in esophagus / improve symptoms

• Corticosteroids – control inflammation – small doses – at first higher doses to control inflammation - but higher doses linked with greater risk of side effects – once inflammation controlled – go to smaller doses

• Proton pump inhibitors (help control amount of acid produced) help treat – some respond well w/ decrease of inflammation/eosinophils, can also improve symptoms w/o making inflammation better

Questions/Things to look up further Nothing

Source Title Allergic Reactions Source citation Allergic reactions TTR | AAAAI. Retrieved

from https://www.aaaai.org/conditions-and-treatments/library/at-a-glance/allergic-reactions

Source found by Looking up “what are normal allergies” on Google


Keywords Allergies

Summary Allergic reaction types and what happens when they occur

Reason for interest

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To distinguish the difference between normal allergic reactions and EoE reactions

Notes • An allergic reaction is actually a result

of a chain reaction that begins in your genes and is expressed by your immune system

• Immune system – controls how your body defends itself – once recognize allergen – overreacts by producing antibodies called Immunoglobulin E (IgE)

• Antibodies travel to cells that release chemicals à reaction

• Symptoms in nose, lungs, throat, sinuses, ears, lining of stomach, or on skin

• Each IgE has “radar” for each type of allergen – some people only allergic to cat dander – only have IgE antibodies specific to cat dander

• Some have multiple allergens bc have many types of IgE antibodies

• Allergic rhinitis may be seasonal or year-round

• Seasonal allergic rhinitis – “hay fever” spring/summer/fall

• Sneezing, stuffy, runny nose, itching nose eyes roof of mouth

• Year round – indoor – dust, mites, molds, pets

• Urticaria – hives – itchy, red bumps clumps large or small – triggered by certain food allergens / medications

• Allergic conjunctivitis (eye allergy) – eyes react to allergens – reddening, itching, swelling à atopic dermatitis, or eczema à allergen exposed to skin – itching, reddening, flaking, peeling

• Asthma – chronic lung disease – coughing, chest tightness, shortness

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of breath, wheezing – airways become narrow

• 78% also have allergic rhinitis • inhaling allergens may cause

increased swelling of airway lining + further narrowing of air passages

• if food allergy = immune system overreacts to particular protein found in that food – most common – cow’s milk, eggs, peanuts, wheat, soy, fish, shellfish, tree nuts

• rhinosinusitis – sinusitis – nasal congestion, facial pressure, cough, thick nasal discharge – swelling of sinuses

Severe Allergic Reaction

• anaphylaxis – life threatening – foods, insect stings, medications, latex

• feeling of warmth, flushing, a red, itchy rash, feelings of light-headedness, shortness of breath, throat tightness, anxiety, pain/cramps/vomiting/diarrhea

• severe – drop in blood pressure àloss of consciousness/shock

• immediate treatment, injection of epinephrine – can be fatal if not treated properly

Questions/Things to look up further Types of allergy testing

Source Title What are Cytokines Source citation What are cytokines? Retrieved

from https://www.news-medical.net/health/What-are-Cytokines.aspx

Source found by Searching “What are Cytokines” on Google


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Keywords Cytokines

Summary Function of cytokines and what they are

Reason for interest Cytokines are a main part of the cell infiltration for EoE

Notes • cyto = cell, kinos = movement • cell signaling molecules that aid cell to

cell communication in immune responses and stimulate the movement of cells towards sites of inflammation, infection, and trauma

• forms = peptide, protein, glycoprotein • large family of molecules • types of cytokines/examples:

interleukin/interferon – regulate immune system’s response to inflammation/infection


Nothing in particular concerning cytokines

Source Title Allergy skin test Source citation Allergy skin tests. Retrieved

from http://www.mayoclinic.org/tests-procedures/allergy-tests/basics/definition/prc-20014505

Source found by Searching “how do skin prick tests work” on Google


Keywords Skin prick test, patch test, allergies

Summary

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Overview of skin prick tests and patch tests

Reason for interest Possible ideas for my project

Notes • nurse administers – doctor interprets • 20-40 min – skin prick • SKIN PRICK: identify many allergens • Children – upper back, adults –

forearm • Uses needles (lancets) barely

penetrate surface • Wont bleed, mild discomfort • Draws small marks on skin and applies

a drop of allergen extract next to each mark

• Then uses a lancet to prick the extracts into the skins surface (new lancet for each allergen)

• To see if reacting normally, two more substances are scratched into skin’s surface:

• Histamine – causes skin response, if you don’t react to histamine – skin test may not reveal an allergy even if you have one

• Glycerin or saline – most people don’t cause any reaction – if do react, may have sensitive skin – test results need to be interpreted carefully – bc false positives

• If allergic – raised, red, itchy bump (wheal) – measure bump size and then clean skin with alcohol to remove marks

• PATCH TEST: to see whether particular substance is causing allergic skin irritation – can detect delayed allergic reactions – can take several days to develop

• Allergens are applied to patches which are placed on skin. May be

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exposed ot 20-30 extracts of substances that can cause dermatitis (skin red swollen)

• Wear in on arm/back for 48 hrs – avoid bathing sweating

• Irritated skin indicates allergy

Questions/Things to look up further More information about allergy testing

Source Title Food Patch Testing Source citation Food Patch Testing. Retrieved from

http://www.massgeneral.org/children/services/food-

patch-testing.aspx

Source found by Searching “how do patch tests work” on Google

Source type Webpage

Keywords Patch testing, allergies

Summary How patch testing works and how the test is administered

Reason for interest Possible ideas for my project

Notes • Food patch testing used to detect food

allergies • Skin prick/blood tests – immediate

reactions àhives/anaphylaxis • Patch tests – delayed-type reactions

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• Food patch test panels on back – prepared food extracts placed on shallow aluminum disks which are taped to the skin

• Remain for 48 hrs, can’t get wet or shift

• After 48 – removal and evaluation by nurse

• After 72 – physician for evaluation and interpretation of patch testing results

Questions/Things to look up further More information on allergy testing

Source Title Skin Prick Tests Source citation Skin Prick Tests. Retrieved from

https://www.foodallergy.org/life-food-

allergies/food-allergy-101/diagnosis-testing/skin-

prick-tests

Source found by Searching “how do skin prick tests work” on Google


Keywords Skin prick tests, allergies

Summary How skin prick tests work, accuracy of skin prick testing

Reason for interest More information on skin prick tests

Notes • Food allergy symptoms between food allergen and an antibody (IgE)

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• Skin prick test to measure presence of IgE antibodies for suspected food

• Drop of solution containing food allergen on forearm or back (fruits/vegetables fresh food may be used instead of solution)

• Prick skin to allow tiny amount to enter below surface (fingernail scratch)

• Positive results – wheal (raised bump surrounded by small circle of itchy red skin

Accuracy of Skin Prick Tests:

• Seldom produce “false negatives” – indicate not allergic when actually are

• Negative results mostly mean not allergic to food

• Positive tests not always accurate • 50-60% of all SPTs yield “false

positive” results à shows positive when not actually allergic to the food being tested

• occur for two reasons: • àwhen eat digestive gradually breaks

down food proteins into very small pieces allergenic proteins so small IgE antibodies unable to detect them – food is actually safe to eat

• SPTs and blood tests can’t mimic the digestive process

• Food proteins – bigger when interact w/ skin or blood – easier for IgE antibodies to recognize allergens and attack

• Why show false positives, more sensitive than actually are

• àfood “family” share similar proteins, if peanuts – results may show a positive response to other members of legume family

• cross-reactivity

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• test = positive recognizes similar protein in peanuts and other legume – test hasn’t detected real culprit (the other different protein that is found in only peanuts)


More info on allergy testing

Source Title Blood tests Source citation Blood tests. Retrieved from

https://www.foodallergy.org/life-food-

allergies/food-allergy-101/diagnosis-

testing/blood-tests

Source found by Searching “How do blood tests work” on Google

Source type Webpage

Keywords Blood tests, allergy tests

Summary Information on how blood testing works and is administered

Reason for interest Help gain ideas for my project

Notes • measure presence of IgE antibodies to specific foods (immunoglobulin E – antibody triggers food allergy symptoms)

• were called RASTs, but don’t any more

• both blood tests and skin tests detect for food-specific IgE

• skin tests – results immediate

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• blood tests – at least several days • blood test is not affected by

antihistamines and can be performed for people with extensive rashes that prevent using skin test

• results not very helpful for predicting severity of allergy

• gives information about chance there is an allergy

• 50/60% blood tests/skin tests – false positives

• rare false negatives • helpful in combination with history –

if have had several reactions to eating peanuts and tests say allergic to peanuts – most likely have peanut allergy

Questions/Things to look up further More information specific for new ways to allergy test for EoE

Source Title Alternate Food Allergy Tests to Avoid Source citation Alternate Food Allergy Tests to Avoid

http://www.kidswithfoodallergies.org/page/unproven-

methods-food-allergy-tests.aspx

Source found by Searching “New allergy testing methods” on Google

Source type Webpage

Keywords Allergy testing methods

Summary Summary of what a patch test is

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Reason for interest Inaccuracy of patch testing

Notes Patch Testing:

• used mainly for the diagnosis of contact dermatitis and delayed onset allergic reactions

• the test has a very limited role in evaluating the impact of foods in eczema and eosinophilic esophagitis

• these tests have not been standardized or validated for foods

• the results are often quite variable • low predictive value

Questions/Things to look up further Allergy testing methods EoE

Source Title Eosinophilic Esophagitis Related Cytokines in Saliva: Characterization and Methodological Considerations

Source citation Eosinophilic Esophagitis Related Cytokines in

Saliva: Characterization and Methodological

Considerations. Retrieved from

http://www.gastrojournal.org/article/S0016-

5085(14)60211-0/pdf

Source found by Searching “Eosinophilic Esophagitis saliva allergy testing”

Source type Webpage

Keywords Saliva, cytokines, allergies, EoE

Summary Cytokines are able to be tested with active saliva using an oral swab (OS)

Reason for interest

Hobson 108

Noninvasive way to test for EoE, idea for allergy testing method

Notes • saliva – biomarker, easy, noninvasive • technique to measure salivary

cytokines • early development for use for salivary

concentrations of EoE relevant cytokines

• two common saliva methods: 1 ml of saliva

• à passive droop (PD) • à active saliva using an oral swab

(OS) • transported 4 degrees Celsius, within

2 hr centrifuged at 3000 rpm for 15 min

• using magnetic high sensitivity human multiplex assays

• measured interleukins (IL) 4, 5, 13 on the Luminex 200 platform and

• eotaxin 3 (Eo3) and thymic stromal lymphopoietin (TSLP) by ELISA techniques

• Wilcoxon rank sum test used to detect potential differences between concentrations of cytokines in PD vs OS samples

• All participants able to provide OS while 2 children unable to provide PD

• All EoE relevant cytokines could be quantified in OS whereas Eo3 and TSLP were undetectable in 3 PD samples

• Median concentrations of all cytokines were similar between PD and OS

• Conclusion: EoE relevant cytokines can be characterized in saliva in children

• Suggest OS should be used in saliva collection and cytokine analyses

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Questions/Things to look up further • What is a PD test • What is a OS test • Email author about how to test

for cytokines and how to care for the cells

• What is a Luminex 200 platform • What types of cytokines should I

be looking for

Source Title Eosinophilic Esophagitis Source citation Dellon, E. S. (2013). Eosinophilic

esophagitis. Gastroenterology Clinics of North America, 42(1), 133-153. doi:10.1016/j.gtc.2012.11.008

Source found by Searching “Eosinophilic Esophagitis saliva cytokines”



Summary Types of cytokines

Reason for interest More in-depth about cytokines

Notes • Eosinophils, mast cells, basophils, and T cells that produce TH2 cytokines (interleukin IL 4 and IL-13)

• Association between a single nuclear polymorphism (SNP) in the gene encoding thymic stromal lymphopoietin (TSLP) and risk for EoE

• Polymorphism in eotaxin-3 – a potent eosinophil chemoattractant that signals through CCR3, a chemokine

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receptor expressed by activated eosinophil and mast cells

• Although IgE-mediated food allergies are common in EoE, one small pilot study of 2 patients found that anti-IgE therapy had no effect on EoE

• In addition, 5% to 10% of patients treated with oral immunotherapy for desensitization to IgE-meditated food allergy develop EoE, indicating a non-IgE mechanism

• Skin prick testing or specific sera IgE has not proven successful in definitive identification of causative food in EoE

• Cytokines and chemokins = essential role in inflammation

• Resulting in increased TH2 response, eosinophil survival, and fibrotic changes

• TSLP – type of cytokine – regulator of TH2 type allergic inflammation

• Secreted by cells of nonhematopoetic lineage, such as epithelial cells, fibroblasts, and smooth muscle cells, in response to atopic cytokines and environmental allergens

• TSLP is increased in the esophageal biopsy specimens of EoE patients compared with those without EoE and is overexpressed within epithelial barriers

• Polymorphisms in TSLP are lined to EoE, and deletion of TSLP in murine model of EoE completely eliminated EoE

• Expression of TSLP may be induced by tissue injury or stimulation of the esophageal epithelium, starting the TH2 cascade

• Eotaxin-3 (CCL-26) – potent eosinophilic chemoattractant that signals through CCR3, a chemokine

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receptor expressed by activated eosinophils and mast cells

• Plays a key role in the migration of eosinophils to the esophageal tissue

Questions/Things to look up further More on cytokines

Source Title Promising Modalities to Identify and Monitor Eosinophilic Esophagitis

Source citation Bae, J. (2014). Narrative reviews. Epidemiology and Health, 36, e2014018. doi:10.4178/epih/e2014018

Source found by Searching “Eosinophilic Esophagitis saliva cytokines”



Summary Info on relation of saliva to cytokines

Reason for interest Saliva and cytokines – specific to project

Notes Saliva: • Markers of EoE could be reflected in

oronasopharyngeal secretions (i.e. saliva)

• Saliva is rich in molecular and microbial analytes

• Salivary IL 4 and IL 5 can be increased significantly in children with EoE after controlling for their atopic comorbidities such as allergc rhinitis and asthma, and exposure to medications

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• MicroRNAs, known to act as diagnostic and prognostic markers of systemic diseases, can be expressed similarity in saliva as in serum

• Pilot studies have indicated massive dysregulation of salivary microRNAome in EoE when compared with healthy controls, potential for noninvasive biomarkers

Questions/Things to look up further More on cytokines

Source Title Sandwich ELISA Source citation

Sandwich ELIZA Retrieved from https://www.genwaybio.com/services/sandwich-elisa

Source found by

Searching “Sandwich ELISA techniques”

Source type Webpage


Summary Sandwich ELIZA explanation

Reason for interest

Possible way to produce my project

Notes • Sandwich Enzyme-Linked ImmunoSorbent Assay (ELIZA) à sensitive and robust method which measures the antigen concentration in an unknown sample

• The antigen of interest is quantified between two layers of antibodies: the capture and detection of antibody

• The antibodies must bind to non-overlapping epitopes on the antigen

Hobson 113


More info on sandwich ELIZA

Source Title A striking local esophageal cytokine expression profile in eosinophilic esophagitis

Source citation Blanchard, C., Stucke, E. M., Rodriguez-Jimenez, B., Burwinkel, K., Collins, M. H., Ahrens, A., . . . Rothenberg, M. E. (2011). A striking local esophageal cytokine expression profile in eosinophilic esophagitis. The Journal of Allergy and Clinical Immunology, 127(1), 7. doi:10.1016/j.jaci.2010.10.039

Source found by Searching “cytokines and EoE”


Keywords cytokines, allergies, EoE

Summary Relevant cytokines to EoE

Reason for interest Information for specific cytokine I want to work with

Hobson 114

Notes • Relevant cytokines to EoE

• A human inflammatory cytokine and receptor PCR array containing 84 genes followed by PCR validation and multiplex arrays were used to quantify cytokine mRNA in esophageal biopsies and blood samples

• Conclusion: Evidence is presented that IL13 and IL5 associate with eosinophil and eotaxin-3 levels, indicating the key role of adaptive TH2 immunity in regulating eotaxin-3-driven esophageal eosinophilia in the absence of a consistent systematic change in cytokines


More info on cytokines

Source Title T-helper 2 Cytokines, Transforming Growth Factor B(beta)1, and Eosinophil Products Induce Fibrogenesis and Alter Muscle Motility in Patients with Eosinophilic Esophagitis

Source citation Rieder, F., Nonevski, I., Ma, J., Ouyang, Z., West, G., Protheroe, C., . . . Fiocchi, C. (2014). T-helper 2 cytokines, transforming growth factor β1, and eosinophil products induce fibrogenesis and alter muscle motility in patients with eosinophilic esophagitis. Gastroenterology, 146(5), 1277.e9. doi:10.1053/j.gastro.2014.01.051

Source found by Searching “how cytokines affect EoE”



Hobson 115

Summary Specific cytokines and more information on cytokines

Reason for interest More info on cytokines in general

Notes • EoE associated with Th2-immune response, with increased expression of interleukin(IL)-4, IL-5, IL-13 or eotaxins

• IL-5 and IL-13 recruit and activate eosinophils, leading to secretion of transforming growth factor (TGF)-B(beta)1

• IL-4 and IL-13 activate fibroblasts to secrete enhanced amounts of extracellular matrix (ECM) and IL-13 activated epithelial cells to secrete eotaxins, powerful chemoattractants

• TGF-B(beta)1 is the main driver of fibrosis across all organs by inducing excessive ECM secretion by mesenchymal cells

• While different cell types are implicated in EoE pathogenesis, the eosinophil is still believed to play a central role

• Increased number and activation state in the mucosa, its ability to secrete virtually all mediators found in EoE, and its release of potent biologically active molecules, such as major basic proteins (MBP)

• Secretion of cytokines and growth factors associated with inflammation, eosinophilia, and fibrosis in mucosal biopsies obtained from EoE and control subjects. IL-5, IL-6, IL-13, eotaxin-1, and TGF-B(beta)1 were actively secreted in the undernatants and their levels were significantly increased in EoE compared to controls

Hobson 116

• Discussion: We found an elevated secretion of IL-5, IL-6, IL-13, eotaxin-1, and TFG-B(beta)1 in EoE compared to control samples, with a close correlation of their levels with eosinophil numbers, eosinophil degranulation, and the presence of microabscesses

• This implicated the combines presence, distribution and activation of eosinophils in the expression of those cytokines

Questions/Things to look up further More information cytokines

Source Title Understanding eosinophilic esophagitis: the cellular and molecular mechanisms of an emerging disease

Source citation Mulder, D. J., & Justinich, C. J. (2011). Understanding eosinophilic esophagitis: The cellular and molecular mechanisms of an emerging disease. Mucosal Immunology, 4(2), 139-147. doi:10.1038/mi.2010.88

Source found by Searching “cytokines and EoE”



Summary Specific information on important cytokines

Reason for interest Specific cytokine for my project

Notes • In general, eosinophil recruitment from blood to tissue in humans in

Hobson 117

influenced by a host of proinflammatory and chemoattractant cytokines, secreted by an organ in response to IL-4 and IL-13

• Chemoattractant cytokines and chemokines draw eosinophils into tissue via integrin-mediated migration

• Activation of eosinophils can result from interaction with cytokines (including IL-3, IL-5, and granulocyte macrophage-colony-stimulating factor), lipid and inflammatory mediators, and from immunogenic antigen exposure, although this may not occur directly

• The primary function of the eosinophil may be to act as an endeffector cell in antiparasite and atopic disease

• In EoE, CD3+, CD4+, and CD8+ T cells are increased in the esophageal mucosa and can be decreased with corticosteroid treatment

• Peripheral blood monocuclear cells (which are approx.. 75% T cells) have been isolated from EoE patients and stimulated with phytohemagglutinin, which caused an increased IL-13 production

IL-4: • Thought to be responsible for

initiating the TH2 response through differenciation of naïve T helper cells int o TH2 type cells

• The initial source of IL-4 in atopic disease remains unclear but it is produced by T helper cells during inflammatory response

IL-5: • Has a major role in many eosinophil-

related disorders by acting on the

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bone marrow as an eosinophil differentiation factor and activator

• In EoE, mRNA and protein expression of IL-5 is significantly increased, but plasma IL-5 does not correlate with esophageal eosinophil number

IL-13: • Appears to activate the local tissue

inflammatory response in TH2 associated diseases

• In the esophagus, IL-13 is increased at the mRNA level in EoE patient biopsies

• IL-13 decreases esophageal epithelial cell differentiation, a process that may be crititcal for maintaining the barrier function of the esophageal mucosa

• Acts through STAT6 on esophageal epithelial cells to upregulate eotaxin-1, eotaxin-2, and eotaxin-3 production

Cytokine: • A remarkable similarity exists

between gene expression profiles of mucosal biopsies from patients with EoE and primary esophageal epithelial cells treated with IL-13

• STAT6-dependent eotaxin-3 expression (and protein secretion) is increased in primary esophageal epithelial cell cultures treated with IL-13 and in esophageal epithelial cell cultures treated with IL-13 and in esophageal squamous carcinoma cell lines in an IL-13 dose-dependent manner

Questions/Things to look up further ELIZA and cytokines

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Source Title Approaches to determine expression of inflammatory cytokines

Source citation Amsen, D., de Visser, K. E., & Town, T. (2009). Approaches to determine expression of inflammatory cytokines. Methods in Molecular Biology (Clifton, N.J.), 511, 107-142. doi:10.1007/978-1-59745-447-6_5

Source found by Searching “sandwich ELIZA techniques”


Keywords cytokines, allergies, EoE, ELIZA

Summary VERY useful for project. Procedure on how to use cytokines and sandwich ELIZA technique

Reason for interest Specifics on how to do my project

Notes ELIZA basic principle: • ELISA assays allow quantitative

measurement of antigens in biological samples

• Wide variety of assay principles can be used

• Sandwich ELISA – an assay suitable to quantify cytokine levels in samples

• A capture antibody with specificity for the cytokine of interest is immobilized on microtiter wells

• Biological samples and standard samples (containing a known concentration of recombinant cytokine) are then allowed to react with the immobilized capture antibody

• Unbound protein is removed through extensive washing, after which a

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second specific antibody, conjugated to an enzyme such as horseradish peroxide (HRP) or alkaline phosphate (AP), is added to form a sandwich with the captured cytokine

• Sometimes this second antibody is conjugated to biotin, and detection occurs by subsequent addition of streptavidin linked to HRP or AP.

• A chromogenic substrate is added, which is chemically converted by the enzyme coupled to the detection antibody, resulting in a color change.

• The intensity of the color is proportional to the amount of cytokine bound to the capture antibody

• Optical density of reaction is measured with a spectrophotometer and compared with the optical density of the known standard samples to determine protein concentrations

3.2.2. à sample preparation … ? ELIZA protocols 3.2.3

• Refer to article for steps ELIZA Data Analysis 3.2.4

• Refer to article for steps

Questions/Things to look up further Materials to buy – cytokine antibodies

Documents

ALTERNATE ALLERGY TESTING FOR EOSINOPHILIC …users.wpi.edu/~sahobson/Pdfsused/STEMThesisFinalCopy.pdfHobson 6 Abstract Eosinophilic Esophagitis (EoE) is a chronic, antigen-driven