Immunology of CELIAC Immunology of CELIAC DISEASEDISEASE

Ibrahim, IbrahimIbrahim, IbrahimMentor: Dr. Sanil ManavalanMentor: Dr. Sanil Manavalan

Columbia University, College of Physicians and Columbia University, College of Physicians and SurgeonsSurgeons

Celiac diseaseCeliac disease

•a digestive disease that damages the small a digestive disease that damages the small intestine and interferes with absorption of intestine and interferes with absorption of nutrients from foodnutrients from food•it is a genetic disorderit is a genetic disorder•some of the symptoms of celiac disease are some of the symptoms of celiac disease are diarrhea, weight loss, malnutrition, etc…diarrhea, weight loss, malnutrition, etc…•when people with celiac disease eat foods when people with celiac disease eat foods containing gluten, their immune system responds containing gluten, their immune system responds by damaging the small intestineby damaging the small intestine•Diagnosis is by intestinal biopsy histological Diagnosis is by intestinal biopsy histological grading and presence of serum auto- antibodies.grading and presence of serum auto- antibodies.•because the body's own immune system causes because the body's own immune system causes the damage, celiac disease is considered an the damage, celiac disease is considered an autoimmune disorderautoimmune disorder

AUTOIMMUNE DISEASE: condition in AUTOIMMUNE DISEASE: condition in which the immune system mistakenly which the immune system mistakenly attacks the body's own organs and attacks the body's own organs and tissuestissues

• Type 1 Diabetes – affects the pancreasType 1 Diabetes – affects the pancreas• Systemic Lupus erythmatotosus Systemic Lupus erythmatotosus

(Lupus)– affects the skin(Lupus)– affects the skin• Crohn’s Disease- affects the gutCrohn’s Disease- affects the gut• Multiple Sclerosis- affects the brainMultiple Sclerosis- affects the brain

Key Terms• Cytokines – proteins secreted by macrophages

and leukocytes that stimulate or inhibit the proliferation or function of immune cells. – Interleukin – cytokines that only affect

leukocytes.

• Lymphocyte – A type of leukocyte (white blood cell) of the immune system.– T-Regulatory Cell – slows down and stops

immune response– T-Helper Cell – has antigen-receptors, releases

cytokines– Natural Killer Cell – kills macrophage and bad

stuff– Cytotoxic T-Cell – kills cells that produce

foreign antigens such as cells infected by viruses– B-Cell – creates antibodies.

• Antigen – invading microbe• Antibody – protein that fights bacteria and viruses.

Lymphocytes

The Gut LumenThe Gut Lumen• VilliVilli- (singular: villus) are tiny, finger-- (singular: villus) are tiny, finger-

like structures that protrude from the like structures that protrude from the wall of the intestinewall of the intestine

--MicrovilliMicrovilli- hair-like structure on the - hair-like structure on the surface of absorptive and secretorysurface of absorptive and secretory epithelial cells epithelial cells

• Epithelial cells Epithelial cells - layer of cells which - layer of cells which line the GI tract line the GI tract

Diagram

Glaidin

Enterocytes

GUT LUMEN

Antibodiesanti-glaidin

anti-endomysialAnd tissue trasgliutaminase

Cytokines(Interferon-, TNF-IL-15 etc)

Lymphocytes(T cells , Natural Killer cells and B cells)

Plasma cells

T cell receptor

Helper T cell

HLA-DQ2/DQ8molecule

Antigen Presenting Cell

GUT LUMEN

LAMINA PROPRIA AND INTRA EPITHILIAL SPACE

Inflammation &Damaged

enterocytes

ObjectiveObjective• To find the cytokine levels in the serum of patients in To find the cytokine levels in the serum of patients in

the following groupsthe following groups– Healthy Patients (Control)Healthy Patients (Control)– Active Celiac Disease PatientsActive Celiac Disease Patients– Celiac Disease patients on Gluten-free dietsCeliac Disease patients on Gluten-free diets– Refractory Disease PatientsRefractory Disease Patients

• The following cytokines were checked in the serum of The following cytokines were checked in the serum of these patients;these patients;– IL-1IL-1ββ– IL-6IL-6– IL-8IL-8– IL-12 p70IL-12 p70– IFN-IFN-γγ– TNF-TNF-αα

MethodsMethods• Dilution of Standards

Standards were used to calibrate the flow cytometer. The Standards were diluted by 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:125, 1:250, and 1:500. The standards were just FACS buffer solutions in water.

• Calibration of CytometerTo do this, we had solutions of the cytokines we were testing. After this we calibrated the equipment so it recognizes which wavelengths of light are representative of specific cytokines.

• Excel SpreadsheetTo analyze the results, and present them to the scientific community, we used Excel spreadsheet to put the data into a comprehensible graph format.

Flow Cytometry

DEFINITION:

technique for counting, examining and sorting microscopic particles (such as cells) suspended in a stream of fluid

ROLE IN OUR EXPERIMENT:

To analyze concentrations of cytokines in the sera of patients using capture beads which detect the cytokines.

Hypothesis

Increased serum levels of inflammatory cytokines in patients with active and refractory celiac disease

Normal levels of cytokines in gluten –free diet

Results

0.0

20.0

40.0

60.0

80.0

100.0

120.0

pg/mL

IFN-γTH1/TH2

IL-1β IK IL-2TH1/TH2

IL-4TH1/TH2

IL-6 IK IL-8 IK IL-10 IK IL-12 p70IK

TNF-α IK

Cytokines

Cytokine Concentrations

Controls (n=9)

Gluten-Free Diet (n=20)

Active Celiac Disease (n=14)

Refractory Disease (n=11)

Results Cont.Cytokine Concentrations

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

Controls (n=9) Gluten-Free Diet (n=20) Active Celiac Disease (n=14) Refractory Disease (n=11)

Patients

pg/m

L

IFN-γ TH1/TH2

IL-1β IK

IL-2 TH1/TH2

IL-4 TH1/TH2

IL-6 IK

IL-8 IK

IL-10 IK

IL-12 p70 IK

TNF-α IK

Conclusions

Pro-inflammatory cytokines such as Interferon –gamma and IL-8 seemed to be increased in both patients with active disease and Celiac patients on a gluten-free diet. We are analyzing the data on an individual patient by patient basis to determine if there is a correlation with the biopsy grade for each patient

Acknowledgements

Dr. Sanil Manavalan - MentorDr. Jamal Naiyer – Co-mentorSindhu Kunduvaram – Undergraduate StudentDr. Sat Bhattacharya – Coordinator OF HCSMrs. Susan Farenholtz – Coordinator of Project SeedHarlem Children SocietyProject Seed

Cytokines Cytokine IL-1β

IL-6

IL-8IL-12 p70

IFN-γTNF-α

Principal cell Targets and biological effectsEndothelial cells: activation B cells: proliferation of antibody producing cellsActivator of neutrophils NK cells and T cells: IFN-gamma synthesis, increased cytolytic activityMacrophages: ActivationEndothelial cells: Activation