Tissue Transglutaminase, Endomysial Antibodies, and Celiac Disease David Lucas, M.D. CP Conference, January 18, 2008

Tissue Transglutaminase, Tissue Transglutaminase, Endomysial Antibodies, and Endomysial Antibodies, and

Celiac DiseaseCeliac Disease

David Lucas, M.D.David Lucas, M.D.

CP Conference, January 18, CP Conference, January 18, 20082008

Celiac DiseaseCeliac Disease Celiac disease is characterized by small Celiac disease is characterized by small

intestinal damage with loss of absorptive intestinal damage with loss of absorptive villi and hyperplasia of crypts typically villi and hyperplasia of crypts typically leading to malabsorptionleading to malabsorption

Associated with nutrient deficiencies and Associated with nutrient deficiencies and increased risk of malignancy (especially increased risk of malignancy (especially intestinal T-cell lymphoma) with chronic intestinal T-cell lymphoma) with chronic diseasedisease

Precipitated by ingestion of the protein Precipitated by ingestion of the protein gliadin (component of wheat gluten) and gliadin (component of wheat gluten) and resolves with withdrawal of proteinresolves with withdrawal of protein

Gliadin initiates mucosal damage via an Gliadin initiates mucosal damage via an immunological process in individuals with immunological process in individuals with a genetic predispositiona genetic predisposition

Celiac DiseaseCeliac Disease Mechanism responsible for damage is still Mechanism responsible for damage is still

under debateunder debate Small intestinal biopsy is “gold standard” Small intestinal biopsy is “gold standard”

and reveals flattened/blunted villiand reveals flattened/blunted villi IgA antibodies against gliadin and IgA antibodies against gliadin and

endomysium (a smooth muscle connective endomysium (a smooth muscle connective tissue) have been the sole serological test tissue) have been the sole serological test used for both diagnosis and therapy controlused for both diagnosis and therapy control

IgA antibodies to endomysium are very IgA antibodies to endomysium are very specific indicators of celiac disease, specific indicators of celiac disease, suggesting one or more target autoantigenssuggesting one or more target autoantigens

Tissue Transglutaminase Tissue Transglutaminase IdentificationIdentification

In 1997, tissue transglutaminase (tTG) as the In 1997, tissue transglutaminase (tTG) as the unknown endomysial autoantigen was unknown endomysial autoantigen was identifiedidentified

tTG – otherwise known as erythrocyte, tTG – otherwise known as erythrocyte, cellular, endothelial, cytoplasmic, Type II, or cellular, endothelial, cytoplasmic, Type II, or liver TGliver TG

Family of calcium-dependent enzymes that Family of calcium-dependent enzymes that catalyze the crosslinking of proteins resulting catalyze the crosslinking of proteins resulting in the formation of epsilon-lysine bondsin the formation of epsilon-lysine bonds

Normally localized within the cytoplasm and Normally localized within the cytoplasm and released during wound healing, where it released during wound healing, where it interacts with cell surfaces or certain interacts with cell surfaces or certain extracellular matrix moleculesextracellular matrix molecules


tTG can also crosslink with fibronectin, tTG can also crosslink with fibronectin, osteonectin, collagen II, V, and XI, osteonectin, collagen II, V, and XI, procollagen III, and nidogenprocollagen III, and nidogen

Thought to stabilize the provisional Thought to stabilize the provisional extracellular matrix in granulation extracellular matrix in granulation tissuetissue

Also enhanced during apoptosis leading Also enhanced during apoptosis leading to irreversible crosslinking of to irreversible crosslinking of intercellular proteinsintercellular proteins

Gliadin proved to be an excellent Gliadin proved to be an excellent substrate for tTGsubstrate for tTG


Indirect immunofluorescence Indirect immunofluorescence performed with high titer Celiac disease performed with high titer Celiac disease serum placed on monkey esophagus serum placed on monkey esophagus with or without tTG pretreatmentwith or without tTG pretreatment

Untreated serum revealed a Untreated serum revealed a characteristic endomysial characteristic endomysial immunofluorescence pattern consistent immunofluorescence pattern consistent with disease whereas the treated with disease whereas the treated serum pattern nearly completely serum pattern nearly completely abolished endomysial abolished endomysial immunofluorescence immunofluorescence


This demonstrated that tTG was the This demonstrated that tTG was the predominant, if not sole, endomysial predominant, if not sole, endomysial autoantigenautoantigen

An ELISA for the detection of IgA An ELISA for the detection of IgA anti-tTG was establishedanti-tTG was established

PathogenesisPathogenesis Fundamental disorder is sensitivity to Fundamental disorder is sensitivity to

gluten, which is the alcohol-soluble, water-gluten, which is the alcohol-soluble, water-insoluble protein component (gliadin) of insoluble protein component (gliadin) of wheat and closely related grains (oat, wheat and closely related grains (oat, barley, and rye)barley, and rye)

T-cell mediated chronic inflammatory T-cell mediated chronic inflammatory reaction with autoimmune componentreaction with autoimmune component

Most likely develops as a consequence of a Most likely develops as a consequence of a loss of tolerance to gluten loss of tolerance to gluten

Interplay between genetic predisposing Interplay between genetic predisposing factors, the host immune response, and factors, the host immune response, and environmental factors is central to disease environmental factors is central to disease pathogenesispathogenesis

PathogenesisPathogenesis When exposed to gluten, the small intestine When exposed to gluten, the small intestine

accumulates intraepithelial CD8+ T cells accumulates intraepithelial CD8+ T cells and large numbers of lamina propria CD4+ and large numbers of lamina propria CD4+ T cells, which are sensitized to gliadin T cells, which are sensitized to gliadin

Also major histocompatibility complex class Also major histocompatibility complex class II HLA-DQ2 or HLA-DQ8 haplotypeII HLA-DQ2 or HLA-DQ8 haplotype

Recognition of these peptides by CD4+ T Recognition of these peptides by CD4+ T cells leads to secretion of interferon γ, cells leads to secretion of interferon γ, which damages the intestinal wallwhich damages the intestinal wall

Epithelial cells secrete large amounts of IL-Epithelial cells secrete large amounts of IL-15 that activates CD8+ T cells and 15 that activates CD8+ T cells and increases the risk of lymphoma increases the risk of lymphoma development.development.

Thoughts regarding EMA and tTGThoughts regarding EMA and tTG

EMA is nearly 100% specific and is the EMA is nearly 100% specific and is the gold standard in serologic testing for celiac gold standard in serologic testing for celiac diseasedisease

The ease of the assay for anti-tTG (ELISA) The ease of the assay for anti-tTG (ELISA) as opposed to EMA (immunofluorescence) as opposed to EMA (immunofluorescence) has influenced many reference labshas influenced many reference labs

In celiac disease, there are EMA and anti-In celiac disease, there are EMA and anti-tTG negative patients, as well as lack of tTG negative patients, as well as lack of concordance between anti-tTG and EMA in concordance between anti-tTG and EMA in up to one-third of patientsup to one-third of patients

A celiac panel for celiac disease should A celiac panel for celiac disease should include both EMA and anti-tTG serologyinclude both EMA and anti-tTG serology

Thoughts regarding EMA and Thoughts regarding EMA and tTGtTG

IgA EMA using human umbilical cord had a IgA EMA using human umbilical cord had a sensitivity of 90% and a specificity of 99%sensitivity of 90% and a specificity of 99%

ELISA anti-tTG antibodies using guinea pig ELISA anti-tTG antibodies using guinea pig liver tTG has shown a sensitivity of 93% liver tTG has shown a sensitivity of 93% and specificity of 95%and specificity of 95%

The introduction of human recombinant The introduction of human recombinant tTG has shown to enhance the sensitivity tTG has shown to enhance the sensitivity of the anti-tTG testof the anti-tTG test

A combination of antibodies should be A combination of antibodies should be used – to include anti-tTG and EMA used – to include anti-tTG and EMA antibodies, with at least one being IgG antibodies, with at least one being IgG based (IgA deficient patients)based (IgA deficient patients)

Additional PointsAdditional Points

2.6% of patients with celiac disease 2.6% of patients with celiac disease have selective IgA deficiencyhave selective IgA deficiency– Should have both IgA and IgG base Should have both IgA and IgG base

serological screening tests and total IgA if serological screening tests and total IgA if the above is suspectedthe above is suspected

Two further subgroups:Two further subgroups:– Patients with only IgG-type antibodies to Patients with only IgG-type antibodies to

both tTG and EMA, but with normal IgAboth tTG and EMA, but with normal IgA– Patients with anti-tTG and anti-R1 reticulin Patients with anti-tTG and anti-R1 reticulin

antibodies, but lacking typical EMAantibodies, but lacking typical EMA

ReferencesReferences Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Dieterich W, Ehnis T, Bauer M, Donner P, Volta U,

Riecken EO, Schuppan D. Identification of tissue Riecken EO, Schuppan D. Identification of tissue transglutaminase as the autoantigen of celiac transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3:797–801disease. Nat Med 1997;3:797–801

Dickey W, Hughes DF, McMillan SA. Reliance on Dickey W, Hughes DF, McMillan SA. Reliance on serum endomysial antibody testing serum endomysial antibody testing underestimates the true prevalence of coeliac underestimates the true prevalence of coeliac disease by one fifth. Scand J Gastroenterol disease by one fifth. Scand J Gastroenterol 2000;35:181–1832000;35:181–183

Stern M. Comparative evaluation of serologic Stern M. Comparative evaluation of serologic tests for celiac disease: a European initiative tests for celiac disease: a European initiative toward standardization. J Pediatr Gastroenterol toward standardization. J Pediatr Gastroenterol Nutr 2000;31:513–519Nutr 2000;31:513–519

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Tissue Transglutaminase, Endomysial Antibodies, and Celiac Disease David Lucas, M.D. CP Conference, January 18, 2008