Celiac Disease: From Pathogenesis to Novel Therapies
Detlef Schuppan, Yvonne Junker, Donatella Barisani
To appear in:
Please cite this article as: Schuppan, D., Junker, Y., Barisani, D., Celiac Disease: From
Pathogenesis to Novel Therapies, Gastroenterology (2009), doi:
This is a PDF file of an unedited manuscript that has been accepted for publication. As a
service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
20 July 2009
2 September 2009
11 September 2009
Celiac Disease: From Pathogenesis to Novel Therapies
Detlef Schuppan1, Yvonne Junker1, Donatella Barisani1,2
1 Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, MA 02215, USA
2 Department of Experimental Medicine, University of Milano Bicocca, 20053 Monza, MI, Italy
Key words: Antibody, biopsy, autoantibody, barley, CCR9, CD4, CD8, celiac disease, celiac sprue,
cereal, clinical trial, Crohn’s disease, deamidated gliadin, decision tool, diagnosis, dendritic cell,
duodenal biopsy, EATL, endoscopy, gene, GWAS, gliadin, gluten, gut homing, histology, HLA-
DQ2, HLA-DQ8, IL-15, inflammatory bowel disease, inhibitor, interleukin, intestine, intestinal T
cell lymphoma, intraepithelial lymphocyte, lymphocyte homing, macrophage, Marsh classification,
MMP, mouse model, prolyl endopeptidase, NK cell, NKG2D, permeability, proteomics, regulatory
T cell, rye, sensitivity, serology, specificity, stem cell, T cell, therapy, tight junction, tissue
transglutaminase, transepithelial, ulcerative colitis, wheat
Word count: Abstract: 250; Text: 8720; References: 283
Detlef Schuppan, M.D., Ph.D.
Division of Gastroenterology and Hepatology
Beth Israel Deaconess Medical Center
Harvard Medical School
330 Brookline Ave
Boston, MA 02215
phone: 617-6672371, 617-6678377
Abbreviations used: BMT, bone marrow transplantation; cd, celiac disease; DC, dendritic cell;
GWAS, genome wide association study ; IBD, inflammatory bowel disease; IEL, intraepithelial
lymphocyte; IFN, interferon; IL, interleukin; MMP, matrix metalloproteinase; MSC, mesechymal
stem cell; PEP, prolyl endopeptidase; Rcd, refractory celiac disease; TG2, tissue transglutaminase;
TGF?, transforming growth factor beta; RA, rheumatoid arthritis
Acknowledgements: The authors’ work on CD is supported by grant 1R21DK073254-02 from the
National Institutes of Health and a grant from the German Ministery for Education and Research to
DS, a one year fellowship grant from the German Ministery for Education and Research to YJ, and
a Fulbright Research Scholar fellowship to DB.
Celiac disease (cd) has become one of the best understood human lymphocyte antigen-linked
disorders. While it shares many immunological features with inflammatory bowel disease, cd is
uniquely characterized by 1) a defined trigger (gluten proteins from wheat and related cereals), 2)
the necessary presence of HLA-DQ2 or -DQ8, and 3) the generation of circulating autoantibodies to
the enzyme tissue transglutaminase (TG2). TG2 deamidates certain gluten peptides increasing their
affinity to HLA-DQ2 or -DQ8. This generates a more vigorous CD4+ T helper 1 T cell activation
which can result in intestinal mucosal inflammation, malabsorption, and numerous secondary
symptoms and autoimmune diseases. Moreover, gluten elicits innate immune responses that act in
concert with the above adaptive immunity. Exclusion of gluten from the diet reverses many disease
manifestations, but is usually not or less efficient in patients with refractory cd or associated
autoimmune diseases. Based on the advanced understanding of cd pathogenesis, targeted nondietary
therapies have been devised, and some of these are already in phase 1 or 2 clinical trials. Examples
are 1) modified flours that have been depleted of immunogenic gluten epitopes, 2) degradation of
immunodominant gliadin peptides that resist intestinal proteases by exogenous endopeptidases, 3)
decrease of intestinal permeability by blockage of the epithelial ZOT receptor, 4) inhibition of
intestinal TG2 activity by transglutaminase inhibitors, 5) inhibition of gluten peptide presentation
by HLA-DQ2 antagonists, 6) Modulation or inhibition of proinflammatory cytokines, and 7)
induction of oral tolerance to gluten. These and other experimental therapies will be discussed
and other cancers of the gastrointestinal tract 23-26. If and to what extent silent or oligosymptomatic,
Celiac disease, gluten sensitivity and associated disorders
Celiac disease (cd) is a common inflammatory disease of the small intestine that is mainly triggered
and maintained by the storage proteins (gluten) of wheat, barley and rye in genetically predisposed
individuals. Patients display various degrees of intestinal inflammation, ranging from mere
intraepithelial lymphocytosis to severe subepithelial (lamina propria) mononuclear cell infiltration
resulting in total villous atrophy coupled with crypt hyperplasia. Accordingly, clinical symptoms
and laboratory indices range from completely asymptomatic to global malabsorption
Autoantibody screening and biopsy confirmation of cd reveals prevalences in the USA and in most
Western and middle Eastern countries between 1:70–1:200 1-5, 9-11. This appears to further increase
with age, since a recent study from Finland showed a prevalence of 1:47 in randomly selected
subjects above 52 years of age 12.
The majority (>80%) of screening-detected celiacs shows no, minor or non-diarrhea associated
clinical symptoms (clinically silent, oligosymptomatic or atypical cd, respectively).
Oligosymptomatic cd is associated with anemia, osteoporosis and an often compromised wellbeing
and quality of life 13, which overlaps with atypical cd that is characterized by extra-intestinal
symptoms, such as arthritis, infertility, hypertransaminasemia, and even liver failure 1-5, 10, 11.
Furthermore, gluten sensitivity without intestinal lesions but circulating celiac autoantibodies or
mere antibodies to gliadin - which lack specificity for classical cd 14 - has been linked to otherwise
unexplained neurological or psychiatric disorders like cerebellar ataxia, peripheral neuropathy,
schizophrenia, or autism 15-18. Since symptoms in patients may improve on a gluten free diet, this
has lead to the suggestion of nutritional gluten sensitivity that does not manifest itself as the
classical intestinal lesion but rather as extraintestinal, e.g., neurological disease 19-21. Its relation to
cd is discussed controversially.
Classical cd is frequently found in conjunction with (other) autoimmune diseases, such as type 1
diabetes, autoimmune thyroiditis, autoimmune hepatitis, dermatitis herpetiformis and autoimmune
alopecia 22. In addition, patients with long-standing undetected and untreated symptomatic cd are at
an increased risk to develop enteropathy associated T cell lymphoma, small bowel adenocarcinoma
screening-detected celiacs may develop overt cd, secondary autoimmune diseases or even
malignancy, when continuing on a gluten containing diet, remains to be shown.
merely deamidates these glutamines to negatively charged glutamic acid residues 59-61. Due to their
The currently only available treatment of cd is a life-long strict gluten-free diet which is difficult to
maintain and which can lead to social isolation, since modern diets are heavily based on products
that contain gluten.
Pathogenesis of celiac disease
Virtually all patients with cd share the heterodimeric human lymphocyte antigen class II genes
HLA-DQ2 (or -DQ8) as common genetic background. These class II molecules are expressed on
antigen presenting cells, mainly macrophages, dendritic cells (DC) and B cells. Gluten peptides are
presented by these cd-associated HLA class II molecules. This can lead to activation of gluten-
specific CD4+ T helper 1 (Th1) cells in the lamina propria that are central effector cells of the
intestinal inflammation resulting in crypt hyperplasia and villus atrophy 6, 27. However, HLA-DQ2
or -DQ8 are expressed in 30-35% of the populations where cd is prevalent, with only ~2-5% of gene
carriers devoloping cd. This implicates other genetic as well as environmental factors as
contributors to the manifestation of cd 28, 29. Recent genetic studies in large numbers of celiacs,
relatives and matched controls revealed additional risk factors, most of which are related to T cell
regulation and inflammation 30-33. Yet the overall genetic contribution of these polymorphisms
combined was estimated at only 3-4% as compared to 30-35% for HLA-DQ2 or -DQ8 33, 34. The 13
susceptibility loci that have been identified to date are summarized in Table 1 31, 33, 35-49.
Furthermore, early exposure of infants to dietary gluten 50, early infection with enteropathic viruses,
or a change of the bacterial flora 51-56 were shown to favour the evolution of clinically manifest cd
in childhood. These observations indicate that cd results from dysregulation of a usually suppressed
T cell response to gluten in a subset of carriers of HLA-DQ2 or -DQ8.
Almost all patients with cd develop immunoglobulin A autoantibodies to the enzyme tissue
transglutaminase (transglutaminase 2, TG2) 57, 58 which is expressed by many cell types and which
associates with the extracellular matrix (endomysium or reticulin fibers). TG2 targets certain
glutamine residues in some extra- and intracellular proteins, usually tethering them to a lysine
residue of a second protein which results in crosslinking of both proteins. Alternatively, TG2
high content in glutamine and neigbouring proline and hydrophobic amino acid residues, gluten
proteins, especially the alcohol soluble fraction (i.e., gliadins of wheat, secalins of barley and
hordeins of rye), but also the glutenins are preferred substrates for TG2 57, 62. When deamidated,
gluten in patients that carry HLA-DQ2 or -DQ8 81. ?2 gliadin peptide p31-43, which is distinct from
most of the resultant negatively charged gluten peptides bind more strongly to HLA-DQ2 (or -DQ8)
which leads to a more rigorous gluten specific CD4+ Th1 T cell activation. A large number (>50) of
distinct (deamidated) gluten peptides that can trigger such T cell responses has been identified, or
deduced from consensus sequences for TG2 63-72. These gluten peptides are usually fairly resistant
to digestion by gastrointestinal proteases which increases their survival and availability in the small
intestine 67, 69. A 33mer peptide from ?2 gliadin contains 6 partly overlapping HLA-DQ2 binding
amino acid sequences that can be deamidated by TG2 68. This peptide is considered a celiac
“superantigen” and used as model peptide in preclinical studies.
How the immunogenic gluten peptides reach the lamina propria from the intestinal lumen remains
controversial. There is evidence that they can traverse via a paracellular pathway through defective
tight junctions 73, but other studies showed that much of the transport occurs via epithelial
transcytosis, especially in the inflamed mucosa of patients with cd 74-77. How far an association of
gluten peptides with luminal anti-gluten IgA and retrotranscytosis from the apical to the basal pole
of the epithelium may contribute to transcytosis in vivo remains to be shown 77. A third but yet
unproven possibility is the sampling of gluten peptides by lamina propria DC. It was demonstrated
in mice but not in humans that these cells can project protrusions between intestinal epithelial cells
reaching the intestinal lumen 78. Similarly, sampling of gluten peptides by DC could occur
preferentially via specialized microfold (M) cells that are part of the follicle associated epithelium
of the mucosal associated lymphoid tissue 79.
Innate immunity to gluten
While the adaptive immune reponse to gluten is well established, proteins from wheat, rye or barley
– apparently in contrast to “nontoxic” cereal proteins derived from, e.g. corn or rice - can elicit an
innate immune response in professional antigen presenting cells (monocytes, macrophages and
dendritic cells) that activates predominantly intraepithelial lymphocytes (IEL) but also intestinal
epithelial cells 80-84. This innate immune response is an immediate reaction and is usually directed
against relatively uniform microbial antigens but also against yet ill-defined constituents of cereals
85, 86. In cd the innate immune response appears to favor the development of adaptive immunity to
peptides that elicit adaptive immunity, was shown to trigger innate immunity in intestinal epithelia
and intestinal organ cultures 81, 87. Other peptides reportedly stimulated rodent monocyte or
macrophage cytokine release 80, 82, 83. However, these peptides have not been generally confirmed
emerged as an additional driving force of innate immunity which often acts in concert with IL15 103
and none of the studies identified a receptor on a responsive cellular subset. Two recent studies that
rigorously ruled out contamination by lipopolysaccharide implicated MyD88, the major
downstream signal transducer of toll like receptor 4 (TLR4) on monocytes, macrophages and DC,
and TLR4 itself as primary receptor for innate responses to cereal proteins 84 [Junker Y et al.,
Gastroenterology 2009, 36 (Suppl 1):M2022].
The role of intraepithelial lymphocytes
Progress has been made in our understanding how IEL are activated by luminal ceral proteins. The
perforin/granzyme and/or Fas/FasL pathways are central to the observed cytotoxicity and apoptosis-
inducing activity of IEL on the intestinal epithelium in cd 88-90. Innate immune activation of IEL by
gluten induces expression of the non-classical class I molecule MICA on the intestinal epithelium
which serves as ligand for the heterodimeric NKG2D receptor on NK, ?? T cells and on subsets of
CD4+ and CD8+ T cells 91. Epithelial MICA and upregulated epithelial production of IL-15 leads to
activation of NKG2D on IEL 92. NKG2D also links innate and adaptive immunity, since it both
triggers antigen-specific CTL-mediated cytotoxicity and induces a direct cytolytic function
independent of TCR specificity in effector CD8 T cells 93. Similarly, the NKG2C receptor that is
activated by its epithelial ligand HLA-E is implicated in cd pathogenesis, stimulating IEL
proliferation and cytokine secretion in patients wth cd 94-96. IEL can also have an immunoregulatory
capacity through the secretion of TGF?1, as reported for a subset of CD8+TCR??+ IEL that
express the inhibitory NK receptor NKG2A. Interestingly, this subset of regulatory cells was
reduced in duodenal biopsies of active cd patients as compared to controls or patients on gluten free
The central role of IL-15 in the activation of innate and adaptive immunity in cd has been confirmed
by several authors 89, 98-101, coupled with an increased expression of IL15R and a lower threshold for
activation on IEL 99. Both intestinal epithelia and DC/macrophages are major sources of IL-15 89,
102. Apart from being a potent growth factor for IEL, IL-15 blocks Smad3-dependent transcription
via the activation of JNK and phosphorylation of c-jun, and thus counteracts the
immunosuppressive TGF? pathway 98. Recently, IL-21 which is produced by CD4+ Th1 T cells has
Figs. 1 and 2 summarize key concepts of cd pathogenesis.
Regulatory T cells
Cytokines and matrix remodeling in celiac disease
CD4+ regulatory T cells (Treg) can downregulate destructive T cell responses, either in
autoimmunity or infection. While their role in murine models of autoimmunity and inflammatory
bowel diseases (IBD) is well established 104, their relevance as suppressors of human T cell
mediated disease is just emerging. CD4+CD25+Foxp3+ Treg have been demonstrated in peripheral
blood mononuclear cells and in intestinal biopsies of patients with cd, but functional studies are
lacking 105, 106. In mouse models these cells are generated in the periphery (predominantly in
mesenteric lymph nodes, MLN), and to a lesser degree in the intestine (Peyer’s patches, PP, and the
lamina propria) from naïve CD4+ T cells in the presence of transforming TGF?1 and retinoic acid
107. This maturation occurs in the presence of (retinoic acid producing) DCs that present the target
antigen, followed by homing of the Treg mainly to the gut where they downregulate immune
responses. The process is dependent on the chemokine receptors CCR7 on DC (for homing to MLN
and PP), and on CCR9 and the integrin ?4?7 on the T cells for their homing to the intestinal lamina
propria via adherence to the ?4?7 ligand MadCAM on high endothelial venules 108-110. Interestingly,
when retinoic acid is substituted by IL-6 naïve T cells are converted to destructive Th17 cells 107, 111.
CD8+ Treg have only recently re-emerged as important suppressors of intestinal immune responses,
largely due to a better understanding of underlying mechanisms 112, 113. Like the CD8+ cytotoxic T
cells that are implicated in mucosal destruction, both in IBD and cd, the CD8+ Treg mainly reside
in the epithelial compartment of the intestine as IEL. In mouse models of IBD, the cytotoxic CD8+
IEL appear to initiate and maintain the destructive, CD4+ T cell mediated immune response within
the lamina propria, leading to a breach of the mucosal barrier, entry of luminal antigens and massive
stimulation of a CD4+ Th1 T cell response 114-116. These aggressive IEL express the T cell receptor
(TCR) ?? or ?? heterodimer in conjunction with the CD8?? heterodimer. In contrast, CD8+ Treg
express FoxP3 and the CD8?? homodimer. Generation of these Treg apparently occurs during
thymic selection in the presence of cognate antigen when CD8?? are deselected.
While some mechanisms need confirmation in the human system and particularly in cd, the
improved understanding of mucosal immunology opens the possibility for a causal treatment aimed
at inducing tolerance to ingested gluten (see below).
HLA class II-restricted gliadin-specific T cell clones express interferon-? (IFN?). An IFN? blocking
antibody can prevent histological damage to healthy mucosa in an intestinal organ culture system
exposed to supernatants of gliadin-specific T cell clones from celiac patients 117, while IL-10 from
from biopsies which shows monoclonality, and flow cytometry of duodenal T cells can diagnose
Rcd type 2 and EATL when the fraction of aberrant T cells exceeds 20% 130. In analogy to other
Treg suppresses Th1 cells and likely acts as a mildly counter-regulatory cytokine 118. Cytokines are
important driving forces of tissue remodeling which results in the villus atrophy and crypt
hyperplasia that are characteristic of cd. In human fetal intestinal explant cultures IFN? activates
macrophages which in turn secrete TNF? and proteolytic matrix metalloproteinases (MMPs), such
as MMP-12 and -13. In intestinal myofibroblasts both TNF?? and IFN? stimulate the expression of
proteolyic MMP-1 and -3. This composite MMP release and activation induces extracellular matrix
proteolysis, a precondition for the architectural remodeling observed in IBD and cd 119-121. As in
Crohn’s disease, these MMPs may represent a therapeutic target 122.
Refractory celiac disease and intestinal T cell lymphoma
Refractory celiac disease (Rcd) can develop in 5-10% of adults with long-standing (often
undetected) cd. Patients do not respond to or relapse while on a strictly gluten-free diet. The
diagnosis of Rcd can only be made when (inadvertent) gluten ingestion or other diseases that can
cause diarrhea and villus atrophy have been ruled out. Thus 82-90% of patients with ‘refractory’ cd
referred to two large tertiary care centers had proven gluten ingestion or an incorrect diagnosis 123,
Rcd is now classified into Rcd type 1 and 2 125-128. Rcd type 1 is responsive to corticosteroids and
other immunosuppressants and only rarely evolves into enteropathy associated T cell lymphoma
(EATL). In contrast, Rcd type 2 can be considered a premimalignant condition, and roughly 50% of
patients with Rcd type 2 develop EATL within 5 years of diagnosis 125-128. Patients with Rcd type 2
and EATL frequently have lost autoantibodies to TG2 and display clonal growth of (intrapithelial)
natural killer and cytotoxic T cells which primarily belong to the innate immune system 85, 129.
Normally, 70% of intraepithelial lymphocytes express the suppressor/cytotoxic phenotype CD8, and
only 5–10% the helper CD4 phenotype. In RCD type 2 and EATL immunohistochemistry reveals
infiltration of the intestinal epithelium by small lymphocytes that lack expression of CD8, CD4, and
T cell receptors, while they retain intracytoplasmic but not surface staining for the general T cell
marker CD3. Furthermore, PCR for T cell receptor gamma gene rearrangements can be performed
lymphomas treatment of EATL (and Rcd type 2) is based on cytotoxic agents like cladribine, but
results for EATL are disappointing 125. However, autologous and especially allogenic bone marrow
transplantation offer hope 131.
epitopes have become highly useful in 1) confirming the uniform HLA-DQ2 or -DQ8 dependency
Recent data indicate a relative risk of ~3 of patients with (untreated) cd to develop EATL 25, 132,
much lower than in previous studies. This is likely due to the much higher (5-13-fold) prevalence of
silent or atypical cd on which today’s studies are based when compared to earlier studies that used
classical cd 133. When patients are on a gluten free diet for five years or more, the risk of developing
lymphoma (and GI cancers) appears to approach that of the normal population 25, 132.
Malignant clones of originally intraepithelial lymphocytes may still depend on innate immune
triggers and their downstream signal transduction, such as IL-15 and IL-21 (see above) are
implicated in Rcd type 2 and EATL.
Preclinical models of celiac disease
These models have been highly useful to explore the pathogenesis of cd and assess the efficacy of
non-dietary therapies. They will be discussed in brief, since they have recently been reviewed in
In vitro models
Culture of intestinal biopsies from celiac patients has been used as plausible approximation to the in
vivo disease process. Exposure of the biopsies to a peptic-tryptic digest of gluten or gliadin
replicates some of the pathology that is found in vivo, such as intestinal epithelial apoptosis,
intraepithelial and lamina propria T cell activation, even villus atrophy, and secretion of
autoantibodies to endomysium (tTG) into the culture medium 135-137. However, interpretation of the
results is limited by the harsh conditions imposed by ex vivo organ culture, leading to hypoxic
damage especially of non-inflammatory cells, usually within 1-2 days. Nonetheless, it may yield
important information that can only be obtained in a multicellular context, such as the
proinflammatory role of IFN? 117 and IL-15 81, 100, or the anti-inflammatory activity of IL-10 90.
Gluten reactive T cell clones
Intestinal T cells from patients with active cd can mount a strong adaptive Th1 (IFN? dominated)
response to certain gluten epitopes, especially those generated by TG2-mediated deamidation. Ex
vivo expanded T cell lines and especially T cell clones against an increasing spectrum of these
of adaptive immunity in cd, 2) demonstrating the large number of distinct T cell epitopes within
wheat, barley and rye, including glutenins as well as gliadins, 3) allowing a comparison of the
potency of the epitopes to trigger T cell activation, and 4) elucidating epitope spreading from
infancy to adulthood 63-72. Furthermore, they permit the testing of novel therapies that are aimed at
autoantibodies to TG2 and only 2 animals developed an increase in IEL. Furthermore, backcrossing
inactivation of antigenic T cell epitopes in cereals or at inhibition of the DQ2 or DQ8 molecules on
the surface of antigen presenting cells (see below).
Animal models of celiac disease
The Irish setter can develop mucosal atrophy in response to wheat ingestion 138 but the pathogenesis
is unlike cd, since disease does not develop when the first gluten exposure occurs after an age of 8-9
months 139, villous atrophy is not linked to MHC class genes and no serum antibodies to gluten can
be detected 140.
Since all patients with cd bear the human leukocyte antigens HLA-DQ2 or DQ8, HLA-DQ2 or DQ8
transgenic mice should render suitable models that replicate cd pathogenesis. Several transgenic
mice have been developed that express human CD4 and DQ8 in the absence of their murine
counterparts that would interfere with human immunology 141, 142. After being immunized with
gliadin these mice’s T cells showed in vitro responses to gluten in a HLA-DQ8 and CD4 restricted
manner, while T cells from DQ6 CD4+ control mice did not develop a gliadin specific immune
response 141, 143, 144. However, apart from high levels of anti-gliadin IgG antibodies the mice did not
show any celiac pathology 141, 143, 144. The cytokine profile in these mice resembled that of a
regulatory phenotype, characterized by CD4+ CD25+ T cells and production of IL-10 and TGF?1
141, likely leading to tolerance to gliadin, whereas cd is driven by a Th1 response dominated by
IFN?. Furthermore, mice did not have circulating anti-TG2 or IgA anti-gliadin antibodies. The same
group crossed human HLA-DQ8 into non-obese diabetic (NOD) mice 145. Sensitization of these
mice with gluten did not cause intestinal pathology, but 15 out of 90 animals developed blistering
skin lesions resembling those of Dermatitis herpetiformis, a disorder that occurs in up to 10% of
patients with cd. However, affected mice did not develop IgA antibodies to gliadin, or antibodies to
Given the fact that more than >90% of all celiac patients possess HLA-DQ2 whereas only 5-10%
bear HLA-DQ8 146, in vivo studies in mice transgenic for human CD4 and HLA-DQ2 are attractive.
Yet, similar to the results in HLA-DQ8 transgenic mice, and even after co-immunization with
pertussis toxin, only 2 out of 14 gluten fed HLA-DR3-DQ2 transgenic mice developed IgA
the mice to a NOD background or generating mice transgenic for a gliadin specific T cell receptor
did not lead to intestinal or dermal pathology 147, 148.
assessment using these parameters in large numbers of patients in remission who receive either
Spontaneously occurring gluten sensitivity was detected in 3% of a rhesus macaque strain. Upon
oral gluten ingestion the affected monkeys developed small intestinal pathology reminiscent of cd,
combined with malabsorption and weight loss. Affected monkeys recovered after reinstitution of a
gluten-free diet 149, 150. Gluten-sensitive animals had circulating IgA and IgG antibodies to gliadin,
and 3 of 15 displayed mildly elvated IgG anti-tTG. A problem is the rare spontaneous occurrence of
the complete cd phenotype (0.6%) and the animal species (primates) which currently precludes
large-scale exploration of novel non-dietary therapies in this model.
Recently, a cd mouse model was established by transfer of presensitized effector/memory T cells
(CD4+CD45RBlowCD25-) from gliadin-immunized wildtype mice to T and B cell deficient (Rag1-
/-) or T cell deficient (nude) mice 151. Recipient mice gained less weight and suffered from severe
duodenitis upon challenge with oral gluten when compared to recipients on a gluten-free diet or to
recipients of control (ovalbumin)-presensitized T cells. This was accompanied by mucosal
histological features characteristic of cd (villous atrophy and crypt hyperplasia), and a Th1/Th17 T
cell polarization in the duodenum and the periphery. Reintroduction of a gluten-free diet led to
weight gain, improvement of histological duodenitis, and a decrease in duodenal proinflammatory
transcripts. Moreover, B cell-competent nude recipients of gliadin-presensitized effector/memory T
cells produced high levels of serum anti-gliadin IgA and IgG1/IgG2c only when challenged with
oral gluten. While further refinement towards an HLA-DQ2 or -DQ8 background is desirable, this
model should be useful for the study of the breach of oral tolerance in cd, and for preclinical testing
of novel non-dietary therapies for cd.
Diagnostic methods to assess therapy response
Duodenal histology showing intraepithelial lymphocytosis, crypt hyperplasia and various degrees of
villous atrophy, coupled with clinical signs and laboratory parameters of malabsorption can still be
considered the gold standard to assess the severity of cd. Histology should be performed on ?6
biopsies from all quadrants of the proximal small intestine and specimens have to be correctly
oriented 1-7. Rigorous testing of novel therapies for cd still requires a pre- and post-treatment
gluten alone or gluten with the novel therapy. However, changes of these parameters may occur
within a few days in some patients and within weeks or months in others. Furthermore, due to focal
disease there may be sampling error, even when several biopsies are taken. Staining for and
However, the sensitivity of the test was questioned in a larger dose escalation study of AT-1001 170.
semiquantification of immune activation markers in biopsies, such as IFN?, TNF?, TGF?, ???IL-2, IL-
6, IL-10, IL-15, both at the RNA and protein level 117, 152-154 may be useful but has not been
validated in clinical studies. The same applies to quantitative PCR for genes encoding these
cytokine markers and certain MMPs 121, 153, 155, 156. Although autoantibodies to TG2 or antibodies to
deamidated gliadin peptides are excellent tools to detect patients with untreated cd or diagnosed
patients with frequent gluten exposure, and antibody titers show some correlation with histological
or clinical severity 157-161, they are not sensitive enough for early detection of (minor) gluten
exposure. In addition, they lack sensitivity to detect therapeutic effects due to their long half-life (at
least 30 days) 162, 163. Immunohistological detection of IgA anti-TG2 deposits in intestinal biopsies
precedes the appearance of serum autoantibodies 164 (see also Fig.1), but they may persist despite
the lack of other histological abnormalities, making the test unsuitable for therapeutic studies.
Therefore, alternative and preferably noninvasive methods are urgently needed.
Fecal fat and sugar absorption tests
A 3-day fecal fat collection is an accurate, quantitative test for malabsorption, but most patients
with cd do not have steatorrhea. Equally, the sensitivity and specificity of oral sugar tests, such as
D-xylose absorption, is low, even in many patients with classical cd 165. Both parameters were
measured in patients in remission before and during a 21 day moderate gluten challenge (5-10g per
day) 166. Although tests were pathological in most patients after 15 days of gluten challenge,
roughly 50% had already pathological baseline results. The low sensitivity and specificity of these
tests was confirmed in the first clinical trial with oral prolyl endopeptidase to digest immunogenic
gluten epitopes 166.
The absorption of usually nonabsorbable vs. absorbable sugars has been used to reflect small
intestinal epithelial (tight junctional) leakiness as occurs in Crohn’s disease and cd. An early study
in 17 cd patients and 12 controls showed an excellent predictive value of the absorbed
lactulose/mannitol uptake for predicting villus atrophy 167, and the test was recommended as a good
screening tool for cd in 111 first degree relatives of celiacs 168. In a pilot study that assessed the
paracellular permeability inhibitor AT-1001, 70% of the 14 patients with cd in remission but none
of the 7 controls who were exposed to 3g of gluten showed an increased lactulose/mannitol ratio 169.
Clinical scores are an important adjunct to studies evaluating novel therapeutics. To date only
symptom scores that were derived from general health queries for other gastrointestinal diseases
desired and necessary 8, 9, 187. Such treatments should be of low risk, reasonable cost and lend
were evaluated in cd, lacking important disease specific characteristics 171, 172. A cd specific celiac
symptom index (CSI) was recently validated in 154 patients based on 16 items that correlated
highly with general health and adherence to the gluten free diet 173. The CSI will serve as important
adjunct tool for future clinical studies.
Follow up of T cell activation with HLA-DQ2 (DQ8) tetramers and interferon ? ELISpot
Based on the identification of several gluten epitopes reccognized by T cells from cd patients 63, 64,
174-178 gliadin peptide-DQ2 tetramers that activate human T cell clones were developed 179. When
used in flow cytometry these tetramers detected and quantified gluten-specific CD4 T cells in the
peripheral blood of patients with cd after a short-term bread challenge 180, making this technology
an attractive tool to detect early gluten responses, e.g., in clinical trials. However, tetramers cannot
assess T cell function, the appearance of tetramer positive T cells in peripheral blood is transient,
occuring only within 3-6 days after short-term gluten challenge, and tetramer staining of activated T
cells is quickly diminished due to antigen-induced down regulation of the T cell receptor 63. In
contrast, the IFN? ELISpot permits functional characterization, but is equally limited by the
transient nature of the peripheral T cell response 181-183.
Search for better serum markers via proteomics
Proteomic projects to detect novel serological markers of cd activity are attractive, but no data for
cd have been published to date. They are based on the serum proteome of cd patients in remission
who are challenged with gluten. The serum proteome of the patients before and after challenge can
be interrogated using several approaches that are based on depletion of abundant proteins, protein
fractionation, mass spectrometry (MS) and bioinformatics 184-186.
Novel therapies for celiac disease An effective therapy for patients with cd is adherence to a strict
gluten-free diet, which often restricts social activities, limits nutritional variety, is costly, and is
difficult to maintain in many countries. Furthermore, a sizable proportion of patients with high level
gluten sensitivity, possibly including patients with proven Rcd type 1, is affected by trace amounts
of gluten in foods that are declared gluten free. Therefore, alternative or adjunctive treatments are
moderate to high efficacy for the majority of patients. Their major realistic aim would be the
“neutralization” of low amounts of gluten, e.g., up to 3 g per day (as compared to the 13-15 g in a
normal Western diet), in order to protect patients from minor unintentional or unavoidable gluten
low molecular weight glutenin 192. Interestingly, the gluten digest from T. tauschii (DD genome)
which contains sequences of the 33mer T cell “superantigen” from ?-gliadin 68 that is encoded by
ingestion. In patients with Rcd effective therapies that are more costly and incur a higher risk are
acceptable, since these patients have few alternatives. The same would apply to a curative
(immunological) approach, even in patients with classical cd.
The following discusses therapeutic strategies that have been tested in in vitro or in vivo models of
cd, and approaches that may be promising in the near future. Therapies can be subdivided as to their
intraluminal, epithelial or sub-epithelial action (Fig.3, Table 2).
Intraluminal therapies are focused either on reducing gluten immunogenicity or on sequestering
gluten, in order to prevent its uptake across the intestinal epithelium.
Wheat variants and genetic modification
Wheat strains with lower immunogenicity, i.e., a decreased number of immunogenic T cell
epitopes, can either be selected from already existing varieties or created by genetic modification.
Ideally, this should lead to preservation of the desired baking properties. The hexaploid Triticum
aestivum is the most widely used wheat variety in the food industry. It was generated by
hybridization between tetraploid T. turgidum (genes AABB, “pasta wheat”) and the diploid T.
tauschii (genes DD). The tetraploid T. turgidum likely originated from the wild growing diploid T.
monococcum (AA genome) and T. speltoides (BB genome) 188. Using duodenal biopsies from cd
patients a peptic-tryptic digest of tetraploid wheat gluten showed decreased toxicity when compared
to a digest of hexaploid wheat 189. Similarly, two wheat varieties, one poor in alpha and beta
gliadins and the other in alpha, beta, gamma and omega gliadins, revealed decreased toxicity on
duodenal biopsies 190.
The availability of gluten-specific T cell clones from duodenal biopsies of cd patients and the
identification of key immunogenic T cell epitopes, including specific antibodies directed to some of
these epitopes 64, 67, 71, 178, 191 provided reproducible tools for the characterization of less toxic wheat
species. Thus 16 wheat varieties (diploid, tetraploid and hexaploid) displayed highly variant
abilities to trigger the activation of T cell clones which was independent of the ploidy of the
genomes but correlated with the presence of specific epitopes derived from ?-gliadin, ?-gliadin and
the D genome elicited the strongest T cell responses, whereas T cell responses to gluten derived
from the AA, and BB genome species that lack these sequences were dampened 193. These findings
Pretreatment of flours
were confirmed by an in silico approach which analyzed 230 ?-gliadin sequences derived from
ancestral haplotypes for the presence of T cell stimulatory epitopes that bind to HLA-DQ2/8, with
all major immunogenic peptides being present in the DD genotype, except for ?9 sequences in the
AA genotype 194. Similarly, gliadin derived from T. monococcum was unable to induce cellular
damage on intestinal T cell lines 195 or IFN? production and histological damage in duodenal
biopsies from cd patients 196.
The effect of genetic deletion of certain gliadin genes has been analyzed in deletion lines of T.
aestivum (cultivar Chinese Spring), which lack one locus containing gluten genes, by using in silico
analysis based on the known DNA sequences and by Western blotting with epitope specific
antibodies 197. Complete deletion of the ?-gliadin locus on chromosome 6 lead to a decrease in total
T-cell stimulatory epitopes, but also impaired the baking properties, whereas deletion of ?-gliadins,
?-gliadins, and LMW glutenins on chromosome 1 lowered the immunostimulatory capacity without
compromising baking properties. The authors concluded that deleted gliadins need to be replaced by
non-immunogenic gliadin variants or avenins (the largely non-immune stimulatory prolamins from
oats), since an altered ratio of gliadins to glutenins changes dough elasticity.
Using a similar approach wheat varieties characterized by a reduced function in the enzymes
involved in gliadin and low molecular weight glutenin synthesis can be identified. Such varieties
can also be generated by mutagenesis, employing radiation, azide treatment or site-directed
mutagenesis, but significant effort is needed for the screening of numerous recombinants. The
TILLING (targeting induced local lesions in genomes) approach allows to screen a large number of
mutagenesis-induced recombinants based on the known sequence and the use of endonucleases to
identify the presence of mutations 198-202, whereas ecoTILLING detects the presence of natural
variants. Here, the 5-methylcytosine deglycosylases of wheat represent an attractive target. These
enzymes have to demethylate the promoters of all gliadin and low molecular weight glutenin genes
prior to their transcription and translation at the beginning of endosperm development, while the
high molecular weight glutenin gene promoters are not DNA methylated and would thus not be
affected, theoretically preserving much of the baking quality.
Certain lactobacilli added to sourdough for fermentation are able to proteolyze the
proline/glutamine-rich gluten peptides and thus decrease immunotoxicity
containing 30% fermented wheat flour plus a mixture of oat, millet, and buckwheat permits to
acceptance of (truly genetically) modified cereal products may be low and their consumption may
produce bread with a texture comparable to that of regular wheat sourdough bread. A pilot study in
17 patients with cd suggested that this sourdough bread was well tolerated. However, patients were
only challenged for 2 days, much too short to draw final conclusions 206.
Similarly, intrinsic proteases produced during germination of wheat, when the amino acids from the
gluten storage proteins are being utilized for the growing plant, can degrade immunogenic T cell
epitopes. This opened the possibility that flour based on germinating wheat, barley or rye may be
used to create “nontoxic” cereal products for patients with cd 207. However, removal of all major
storage proteins is expected to go hand in hand with loss of baking quality. Nonetheless, the
germinating proteases are used for oral enzyme therapy (see below).
Another approach is to inactivate immunogenic gluten epitopes by exploiting the same substrate
specificity of TG2 that generates more potent immunostimulatory gluten peptides via deamidation
208. Thus, incubation of gliadin with TG2 and lysine-methylester lead to quantitative cross-link
formation between the TG2-target sequences in gliadin and the terminal amino group of lysine-
methylester. These lysine-modified gliadins lost their affinity to bind to HLA-DQ2 which in turn
abrogated IFN-? production by intestinal T cell lines derived from HLA-DQ2 positive cd patients.
Furthermore, treatment of whole wheat flour with a low-molecular-weight microbial
transglutaminase (mTG) derived from Streptomyces moboraensis equally abrogated the flour’s
stimulatory effect on gluten-reactive T-cell lines. Importantly, treatment with mTG improves loaf
volume and crumb texture of breads 209. This could make pretreatment of flour with mTG (and non-
toxic lysine-methylester) attractive for patients with cd. mTG is already applied by the food industry
all over the world to improve doughs or the texture of foods in general 209, 210. However, a note of
caution is necessary, since treatment of flour with mTG increased rather than decreased the
stimulation of gliadin-specific T cell lines 211, 212.
Future studies have to show how far the above modifications can lead to cereal products that are
largely devoid of immunogenic epitopes. Furthermore, the products must maintain the desired
consistency and baking properties, large-scale industrial production must be cost effective, and
issues such as the nutritional value of the products, the degree of removal of immunogenic epitopes
and the lack of e.g. de novo generated antigenic epitopes need to be addressed. Finally, the general
be mainly limited to the patients’ households.
Oral enzyme therapy
In general, proteins reaching the intestinal lumen are digested by gastric pepsin, pancreatic
proteases and further degraded by brush border enzymes to yield single amino acids, di- or
tripeptides which are transported across the epithelial layer. However, the large quantity of proline
residues 213, especially in immunodominant gliadin peptides like the 33mer, causes them to be
highly resistant to human digestive proteases 68, 214, 215. Hence, one strategy to prevent those peptides
from reaching the lamina propria has been to make use of prolyl endopeptidases (PEP) that are
expressed in various microorganisms such as Flavobacterium meningosepticum, Sphingomonas
capsulata and Myxococcus xanthus and that are able to cleave the immunodominant proline rich
regions 216-220. A pilot and safety study using PEP from Flavobacterium meningosepticum admixed
to a daily drink with 5g of gluten over 2 weeks could prevent fat malabsorption and symptoms in
some patients with previously diet controlled cd 166. However, neither the potency of the enzyme
nor the sensitivity of the readouts (stool fat and D-xylose absorption) were considered sufficient to
draw clear conclusions. The effectiveness of PEPs can be limited by restrictions on the length of
their substrates 221, 222, their activity maximum at near neutral pH and the long time necessary to
completely digest the gliadin peptides 223, 224. A reasonable approach is therefore the use of enzymes
with a broader activity spectrum and of combination enzyme therapy. Thus PEP from Aspergillus
niger is active at acidic pH and has a higher specific activity than PEP from Flavobacterium
meningosepticum to inactivate immunodominant gluten epitopes 218, 219. Furthermore, endoprotease
(EP)-B2, a glutamine-specific protease of germinating barley in combination with PEP from
Sphingomonas capsulata can efficiently break down whole wheat gluten in vitro and in a rat model
in vivo, largely abrogating its immunogenic potential, as assessed with several gluten specific T cell
lines 225, 226. Both enzymes are active and stable at acid pH and can therefore be administered as
lyophilized powders or simple capsules or tablets 226. Both Aspergillus niger PEP and the EP-
B2/PEP combination enzyme therapy are currently in phase 1 clinical studies (Table 1). As with
most therapies discussed here, oral enzyme therapy will probably not be able to sufficiently degrade
immunogenic epitopes of a normal daily gluten ingestion amounting to >13g, but rather eliminate
the detrimental effect of a few 100 mg to a few g of gluten in patients with high gluten sensitivity or
Rcd type 1.
Intraluminal binding of gluten peptides
This approach has been suggested in a study that used a copolymer of polyhydroxy methacrylate
and polystyrene sulfonate to bind gliadin in a fairly specific manner 227. The polymer blocked
gliadin digestion to smaller immunogenic peptides, and attenuated the gliadin-induced intestinal
lower urinary nitrate excretion when compared to the placebo controls [Kelly et al.,
permeability increase and T cell activation in CD4 HLA-DQ8 transgenic mice. However, it is
expected that many other nutrient proteins will interact with the polymer and limit its activity in
patients with cd.
Neutralizing gluten antibodies
Orally ingested immunoglobulin G is highly resistant to gastric acidity and roughly 50% of
neutralizing activity survives when reaching the terminal ileum 228. Cow’s milk antibodies are easy
and cheap to produce. Based on this rationale large scale production of gluten neutralizing
antibodies is attractive. Importantly, these antibodies can be considered a safe nutritional product,
similar to milk products, which would not be subject to strict regulatory approval. A clinical phase 1
trial in the US is expected.
Inhibition of intestinal permeability
An increase of intestinal permeability via opening of the epithelial tight junctions appears to be an
important contributor to the influx of gluten peptides into the subepithelial lamina propria where the
destructive adaptive T cell response to gluten is triggered and maintained. Vibrio cholerae secretes
the ZOT toxin that opens the intestinal epithelial tight junctions via the 66 kD ZOT receptor 229, 230.
In addition, the injured epithelium of cd patients releases a paracrine protein product (zonulin) that
acts similar to ZOT. An octapeptide (AT-1001) with homology to ZOT (or zonulin) was developed
that blocks the ZOT/zonulin receptor and thus protects tight junctional integrity. A pilot study using
AT-1001 in 14 cd patients in remission and 7 controls who were challenged with a single dose of
gluten prevented the decrease in intestinal permeability, and ameliorated PBMC IFN? production
and urinary secretion of nitric oxide (a marker of NO synthase activation and inflammation) 169.
AT-1001 is currently the best studied pharmacological agent to treat cd. Thus a phase 2 dose
escalation study (1, 4 and 8mg daily) was performed in 184 patients in remission who were
challenged with 3x0.9g of daily gluten over 42 days. Although the primary endpoint (a significant
decrease of the lactulose to mannitol ratio vs. the placebo group) was not reached, AT-1001 treated
patients had a significantly improved symptom score, a less pronounced autoantibody response, and
Gastroenterology 2009;136 (Suppl 1):M2048]. As with the above “glutenases”, the effect of this
approach alone will likely be limited. However, its combination with other treatments could be
This peptide prevented agglutination of K562 erythroleukemic cells induced by PT digested gliadin
or ?-gliadin p31-43 242, 244 and protected Caco2 intestinal epithelial cell from apoptosis induced by
gliadin 245. The inhibitory effect was also present when lymphocytes 246 or duodenal biopsies 248
Dampening of the adaptive immune response
The use of TG2 inhibitors has been hypothesized as a possible therapeutic approach, since
inhibiting gliadin peptide deamidation could reduce their binding to HLA-DQ2 and -DQ8, and thus
their T cell stimulatory capacity. Since the >7 known transglutaminases share a high degree of
sequence similarity, especially in their catalytic center, inhibitors did not display unique selectivity
for TG2. Inhibitors that target the TG crosslinking activity have been developed and mainly tested
in vitro 231-234. These are 1) competitive inhibitors (putrescine, spermidine, histamine, monodansyl
cadaverin, cadaverine, 5-pentylamine, fluoresceine, cystamine, and cysteamine) 235, 2) reversible
inhibitors (mainly guanosine triphopsphate analogues)
236, and 3) irreversible inhibitors
(iodoacetamide, 3-halo-4,5-dihydroisoxazoles, carbobenzyloxy-L-glutaminylglycine derivatives, 6-
diazo-5-oxo-norleucine, 2-[(2-oxopropyl)thio]imidazolium derivatives) 237, 238. Cystamine and the 2-
[(2-oxopropyl)thio]imidazolium inhibitors (L682777 or R283) have also been tested ex vivo in
cultures of small intestinal biopsies of cd patients, where they blunted T cell stimulatory activity of
gliadin peptides 239, 240. The approach of transglutaminase inhibition, though potential useful, is
risky, since 1) transglutaminases fulfill many important functions in tissue homeostasis and
inhibition of other transglutaminases is expected, 2) agents need to be designed and tested that are
taken up by the intestine and do not reach the systemic circulation, and 3) even a complete
inhibition of transglutaminase-mediated gluten deamidation will not eliminate all immunogenic
epitopes, especially in children 71. Of note, transglutaminase inhibitors based on 6-diazo-5-oxo-
norleucine were recently developed that display 70-225fold specificity for TG2 over TG1, TG3,
TG6, and factor XIII when tested in vitro (Pasternack R, Hils M, Zedira Company, Darmstadt,
Germany, personal communication), raising hopes for increased safety of this approach.
Gluten peptides that downregulate innate responses
An “innate inhibitory” decapeptide (sequence QQPQDAVQPF) was isolated by affinity
chromatography and gel filtration from durum wheat and tested in various in vitro systems 241-248.
from celiac patients were challenged with PT gliadin in vitro. The authors postulated that the
First, while it is still not well known how intact gliadin peptides reach the lamina propria, this is
decapeptide induced a switch from a Th1 to a Th2 T cell phenotype, since it downregulated IFN?
and upregulated IL-10 production of celiac patients’ intestinal T cells
modifications of “toxic” gliadin peptides, to obtain “antagonistic” peptides 181, 249-251. Modification
247. Others tested
of the proline residues P38, P39 and P42 of ?-gliadin p31-43 abrogated its pathogenicity as
evaluated by morphometric analysis on duodenal biopsies of cd patients, but their activity as
antagonists of the wild type peptide or total gluten was not studied 249. Therefore, while serving as
proof of principle, the application of single modified peptides is unlikely to yield therapeutic agents.
Adaptive immunity in cd is driven by presentation of gliadin peptides on HLA-DQ2 in the majority
of cd patients, followed by activation of CD4+ T cells that initiate and maintain the Th1
inflammatory response. Therefore, blocking DQ2 represents an attractive target to prevent immune
activation. Similar approaches have already been investigated in other autoimmune diseases like
multiple sclerosis, rheumatoid arthritis or type 1 diabetes, though without demonstrating clinical
benefit, mainly due to inefficient drug delivery 252-254. In view of the accessibility of the small
intestine via the oral route, drug delivery should be easier in cd.
Based on gliadin peptides that drive adaptive immunity in cd, several peptides with high affinity to
HLA-DQ2 were designed by amino acid substitution, dimerization or introduction of aldehyde
groups. Modification of ?2-gliadin p57–73 lead to partial agonists that significantly inhibited the
IFN? production by PBMC from cd patients in the presence of the stimulatory unmodified peptide
181. Furthermore, replacement of leucine L11 and L18 residues in the ?-gliadin 33mer
“superantigen” with sterically bulky groups retained high DQ2 affinity but decreased T cell
recognition 251. Similar results were obtained employing azidoproline containing gluten peptides,
that per se were not able to activate T cells, although they could block the effect of a stimulatory
?9-gliadin peptide only when used at high concentrations 250. However, most of the modified
peptides still showed agonistic effects when tested on gliadin specific T cell lines. Moreover,
binding affinity for most of the peptides was not high enough to efficiently block access of
stimulatory gliadin peptides to DQ2 250, 251, 255, 256. Furthermore, this approach poses other problems.
even less clear for the modified peptides that may have to compete with the (luminal) gliadin
peptides to reach their target cells. Second, side effects like immunosuppression or hypersensitivity
responses are potential safety concerns. Therefore, this ambitious approach will require significant
HLA-DQ8 transgenic mice after parenteral sensitization to the peptide, resulting in a diminished
work to develop a highly specific, high affinity, nontoxic and non immunogenic compound prior to
testing in humans.
Immune modulation and induction of tolerance to gluten
The perhaps most attractive and causal treatment would be the restoration of tolerance to ingested
gluten. That this is feasible is exemplified by the observation that 1) only 1 out of 30 carriers of the
major predisposition for cd, i.e., HLA-DQ2 or –DQ8, will develop cd in their lifetime, and 2) 20%
of 61 subjects whose cd was diagnosed in childhood and who remained on a gluten free diet for
several years did not develop cd despite having resumed a normal gluten containing diet in
adolescence for an average of 10 years 257. Induction of tolerance has been attempted by intranasal
administration of gliadin peptides in gliadin sensitized Balb/c or transgenic DQ8 mice, resulting in a
decreased T cell proliferative response to gliadin and a decrease in the production of inflammatory
Another strategy used three select immunogenic 16mer peptides derived from ?-gliadin, ?-gliadin
and hordein that account for 60% of the overall gluten T cell response to immunize gliadin-specific-
TCR/DQ2 transgenic mice via subcutaneous injections. This “gluten vaccination” suppressed CD4+
T cell proliferation, IL-2 and IFN? production, and increased the expression of Treg markers by
splenic CD4+ cells in response to a gluten challenge 261. A clinical study is on the way in Australia.
A simple, safe and cost-effective method would be to downregulate the proinflammatory
(microbial) milieu of the small intestine in patients with cd. Thus addition of Bifidobacterium
strains suppressed the proinflammatory effect of fecal extracts on peripheral blood mononuclear
cells from patients with active cd 262. Clinical studies have not yet been performed. Another group
from Australia has initiated a phase 1 clinical trial in celiacs using non-infectious larvae from the
hookworm Necator Americanus. It is hoped that similar to trichiuris suis therapy of inflammatory
bowel disease this treatment will skew the proinflammatory Th1 T cell response to a less aggressive
Th2 or a suppressive Treg response 263-265.
Another approach ulilized probiotic Lactococcus lactis that were engineered to secrete an
immunogenic DQ8 restricted deamidated gliadin peptide. These bacteria were then administered to
delayed type hypersensitivity response, a diminished T cell response to the peptide and an increase
in Foxp3 positive Treg in the mesenteric lymph nodes 266.
Therapies targeted at immune cells
a phase 1 safety trial (ChemoCentryx). A phase 2 clinical trial has been planned, but the inhibitors
are currently on hold for cd. Similarly, the ?4?7 integrin blocking antibody LDP-02 is used in a
Most of these targeted therapies are currently used or evaluated in autoimmune diseases like
rheumatoid arthritis and/or in inflammatory bowel disease. While they are not justified to treat
classical cd, due to side effects and costs in view of a usually effective gluten free diet, they hold
promise in the treatment of Rcd and EATL. For most of these therapies there exist case reports on
their clinical utility at best (Table 1). The following discusses some of the targets and treatments
that show promise for (refractory) cd and EATL.
CCR9 and integrin ?4?7 antagonists
Chemokine and chemokine receptors play an important role in the selective recruitment of
leukocytes from the circulation to target tissues. Effector/memory T cells that home to the small
intestine, i.e., the intestinal segment affected by cd, express both chemokine receptor 9 (CCR9) and
integrin ?4?7. CCR9 mediates small intestinal homing via binding to chemokine ligand 25
(CCL25) that is secreted by the intestinal epithelium, and integrin ?4? mediates attachment to the
mucosal vascular addressin MadCam-1 on intestinal high venular endothelium 267-269. Thus
increased CCR9 expression is found in Crohn’s disease, both in intestinal and peripheral
lymphocytes 268. In cd discordant results have been obtained: augmented CCR9 expression was
detected in peripheral lymphocytes 268, whereas CCR9 protein levels were reduced in IEL and
lamina propria lymphocytes of duodenal biopsies, and the decreased CCR9 expression was
associated with activated PBMC 270. Blockage of CCR9/CCL25 improved histological damage in
early phases of a mouse model of spontaneous ileitis 271, supporting the role of CCR9 as possible
therapeutic target. Thus CCX282-B, a CCR9 inhibitor, ameliorated the severity of ileitis in a TNF-
?-driven model of chronic ileitis 272, and a phase 2 clinical trial in patients with moderate to severe
Crohn’s disease demonstrated a reduction of the Crohn’s disease activity index in 61% of the
patients versus 47% for placebo [Keshav S et al., Gastroenterology 2007;132:A157; Keshav S et al.,
Gastroenterology 2009;136 (Suppl 1)]. A study to evaluate the effect of CCX282-B compared to
placebo on the villous height/crypt depth ratio of small intestinal biopsies taken from subjects with
celiac disease before and after gluten exposure has been planned [Hamilton G et al.
Gastroenterology 2008;134 (Suppl 1):A493]. CCX025, a second oral CCR9 inhibitor is currently in
phase 2 clinical trial for Crohn’s disease (NCT00655135), but no trial in cd has been initiated yet.
The overall benefit of blocking lymphocyte homing to the small intestine in cd is not clear, since
beneficial immunosuppressive Treg are equally inhibited.
devised to treat cd. With further advances in the development of preclinical models and better
The central role of IL-15 in the pathogenesis of (refractory) cd has been highlighted above. IL-15-
blocking antibodies have been tested in patients with rheumatoid arthritis 273. Furthermore, an
inhibitor of the downstream Jak3 signal transducer is currently tested in phase 2 clinical trials for
rheumatoid arthritis, transplant rejection, psoriasis and inflammatory bowel disease 274. Much hope
has been invested in anti-IL-15 therapy, especially for Rcd type 2 and EATL in which the expansion
of malignant lymphocytes appears to be driven by IL-15, but industry has so far been reluctant to
support a clinical trial.
Bone marrow transplantation
Autologous bone marrow transplantation (BMT) has been used to induce remission in patients with
EATL 131. While remissions have been achieved, patients have relapsed due to residual cells that
reside in the transplanted autologous bone marrow. Therefore, heterologous BMT using cells from
unaffected donors is more promising, but also more risky. No studies using heterologous BMT have
been reported yet.
Mesenchymal stem cell therapy
A novel modality is the infusion of mesenchymal stem cells (MSC) 275. MSC differentiate in vitro
and in vivo into multiple mesodermal tissues including bone, cartilage, adipose tissue, tendon,
ligament or even muscle 276. These cells can be produced in large quantities ex vivo from human
donors. Importantly, they have low immunogenicity due to the lack of HLA class I or II, and of
costimulatory molecules 277. MSC can therefore be infused safely into allogeneic recipients. MSC
preferentially home to sites of organ damage where they suppress lymphocyte proliferation 278-280.
Clinical studies (Prochymal, Osiris) are ongoing in numerous inflammatory and degenerative
diseases showing benefit in severe (intestinal) graft vs. host and therapy resistant Crohn’s disease
276. It is conceivable that MSC infusion can dampen or even abrogate the immune response to gluten
in patients with cd, and perhaps in patients with Rcd or EATL. A clinical trial is planned.
Due to advanced understanding of its pathogenesis numerous therapeutic strategies have been
noninvasive activity markers clinical validation of many of these therapies is anticipated in the next
years. Of particular interest are 1) immune based treatments that induce oral tolerance to gluten and
are thus curative, and 2) combination therapies that increase efficacy while at the same time having
reduced side effects. The advances in cd will also spawn therapeutic developments for other
immune mediated disorders such as inflammatory bowel disease, or autoimmune disease of other
organs for which cd can serve as a well defined model disease.
Table 1: Non-HLA loci of celiac disease susceptibility.
GWAS, genome wide association study; SNP, single nucleotide polymorphism.
Table 2: Novel therapies for celiac disease.
MCP-1 which can potentiate the adaptive immune response to gluten. APC, antigen presenting cell;
Fig.1: Pathogenesis of celiac disease
Gluten peptides that are highly resistant to intestinal proteases reach the lamina propria, either via
epithelial transcytosis or an increased epithelial tight junctional permeability. Cross-linking and
particularly deamidation of gluten peptides by TG2 creates potent immunostimulatory epitopes
which are presented via HLA-DQ2 or -DQ8 on antigen presenting cells. Subsequently, CD4+ T cells
are activated, secreting mainly Th1 cytokines like IFN? which induce the release and activation of
matrix metalloproteinases by myofibroblasts, finally resulting in mucosal remodelling and villus
atrophy. Additionally, Th2 cytokines are produced driving the production of (auto-) antibodies to
gluten and TG2. Other cytokines like IL-18, IFN? or IL-21 seem to play a role in polarizing and
maintaining the Th1 response. Furthermore, IL-15 links the adaptive immune system to innate
immune responses (see Fig. 2). The scheme is simplified. It does not show that T cells circulate to
mesenteric lymph nodes where they encounter and are primed by APC (mainly dendritic cells) and
from where they home back to the lamina propria, a process that is driven by the lymphocyte
homing receptors chemokine receptor CCR9 and integrin ?4?7.
Fig. 2: Innate immune responses in celiac disease
Upon stimulation with gliadin peptide p31-49 (and other peptides) epithelial cells and macrophages
& dendritic cells secrete IL-15 that in turn upregulates both the NKG2D receptor on intraepithelial
lymphocytes (IEL) and its epithelial ligand MICA. The thus stimulated cytotoxic lymphocytes
(CTL) induce increased epithelial apoptosis and permeability. Furthermore, the NKG2C receptor on
a subset of NK-like IEL is stimulated by its epithelial ligand HLA-E on epithelial cells resulting in
their proliferation and cytotoxicity, whereas stimulation of ??+ CD8+ IELs bearing the NKG2A
receptor via HLA-E induces TGF? secretion and therefore a regulatory phenotype. Gliadin (cereal)
peptides can also directly elicit innate immune responses in macrophages and dendritic cells via
pattern recognition receptors like TLR4 or other MyD88 dependent pathways. This drives
maturation of these cells and secretion of inflammatory cytokines like IL-1?, IL-8, TNF?, and
pDC, plasmacytoid dendritic cell; TG2, tissue transglutaminase 2; IELs, intraepithelial lymphocyte;
MMP, matrix metalloproteinase.
Fig. 3: Novel therapeutic approaches
Use of ancestral and/or modified wheat strains with lower immunogenecity. Intraluminal therapies
that either bind or degrade ingested gluten peptides in the intestine (glutenases, gluten binders,
neutralizing antibodies). Blocking the ZOT receptor with the octapeptide AT1001 to decrease
intestinal permeability is another option. Furthermore, since the deamidation of gluten peptides by
TG2 and the subsequent presentation by HLA-DQ2/8 initiates the adaptive immune responses, TG2
inhibitors and DQ2 blocking peptides seem to be an attractive possibility to prevent inflammation.
Another promising alternative especially for patients with refractory celiac disease is directly
targeting the immune cells either by lymphocyte blocking (anti-IL-15, anti-CCR9, anti-?4?7) or
Smedby KE, Akerman M, Hildebrand H, et al. Malignant lymphomas in coeliac disease:
evidence of increased risks for lymphoma types other than enteropathy-type T cell
lymphoma. Gut 2005;54:54-9.
Viljamaa M, Kaukinen K, Pukkala E, et al. Malignancies and mortality in patients with
coeliac disease and dermatitis herpetiformis: 30-year population-based study. Dig Liver Dis
1. Abdulkarim AS, Murray JA. Celiac Disease. Curr Treat Options Gastroenterol 2002;5:27-
Ciclitira PJ, King AL, Fraser JS. AGA technical review on Celiac Sprue. American
Gastroenterological Association. Gastroenterology 2001;120:1526-40.
Farrell RJ, Kelly CP. Celiac sprue. N Engl J Med 2002;346:180-8.
Green PH, Cellier C. Celiac disease. N Engl J Med 2007;357:1731-43.
Green PH, Jabri B. Coeliac disease. Lancet 2003;362:383-91.
Schuppan D. Current concepts of celiac disease pathogenesis. Gastroenterology
Trier JS. Celiac sprue. N Engl J Med 1991;325:1709-19.
Sollid LM, Lundin KE. Diagnosis and treatment of celiac disease. Mucosal Immunol
Di Sabatino A, Corazza GR. Coeliac disease. Lancet 2009;373:1480-93.
Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in at-risk and not-at-risk
groups in the United States: a large multicenter study. Arch Intern Med 2003;163:286-92.
Maki M, Mustalahti K, Kokkonen J, et al. Prevalence of Celiac disease among children in
Finland. N Engl J Med 2003;348:2517-24.
Vilppula A, Kaukinen K, Luostarinen L, et al. Increasing prevalence and high incidence of
celiac disease in elderly people: A population-based study. BMC Gastroenterol 2009;9:49.
Green PH. The many faces of celiac disease: clinical presentation of celiac disease in the
adult population. Gastroenterology 2005;128:S74-8.
Uibo O, Uibo R, Kleimola V, et al. Serum IgA anti-gliadin antibodies in an adult population
sample. High prevalence without celiac disease. Dig Dis Sci 1993;38:2034-7.
Cascella NG, Kryszak D, Bhatti B, et al. Prevalence of Celiac Disease and Gluten
Sensitivity in the United States Clinical Antipsychotic Trials of Intervention Effectiveness
Study Population. Schizophr Bull 2009.
Catassi C, Ratsch IM, Fabiani E, et al. Coeliac disease in the year 2000: exploring the
iceberg. Lancet 1994;343:200-3.
Fasano A, Catassi C. Current approaches to diagnosis and treatment of celiac disease: an
evolving spectrum. Gastroenterology 2001;120:636-51.
Genuis SJ, Bouchard TP. Celiac Disease Presenting as Autism. J Child Neurol 2009.
Ford RP. The gluten syndrome: a neurological disease. Med Hypotheses 2009;73:438-40.
Grossman G. Neurological complications of coeliac disease: what is the evidence? Pract
Verdu EF, Armstrong D, Murray JA. Between celiac disease and irritable bowel syndrome:
the "no man's land" of gluten sensitivity. Am J Gastroenterol 2009;104:1587-94.
Ventura A, Magazzu G, Greco L. Duration of exposure to gluten and risk for autoimmune
disorders in patients with celiac disease. SIGEP Study Group for Autoimmune Disorders in
Celiac Disease. Gastroenterology 1999;117:297-303.
families. Am J Hum Genet 2002;70:51-9.
Monsuur AJ, de Bakker PI, Alizadeh BZ, et al. Myosin IXB variant increases the risk of
celiac disease and points toward a primary intestinal barrier defect. Nat Genet
Naluai AT, Nilsson S, Gudjonsdottir AH, et al. Genome-wide linkage analysis of
Scandinavian affected sib-pairs supports presence of susceptibility loci for celiac disease on
chromosomes 5 and 11. Eur J Hum Genet 2001;9:938-44.
25. Gao Y, Kristinsson SY, Goldin LR, et al. Increased risk for non-Hodgkin lymphoma in
individuals with celiac disease and a potential familial association. Gastroenterology
Goldacre MJ, Wotton CJ, Yeates D, et al. Cancer in patients with ulcerative colitis, Crohn's
disease and coeliac disease: record linkage study. Eur J Gastroenterol Hepatol 2008;20:297-
Sollid LM. Coeliac disease: dissecting a complex inflammatory disorder. Nat Rev Immunol
Wolters VM, Wijmenga C. Genetic background of celiac disease and its clinical
implications. Am J Gastroenterol 2008;103:190-5.
Macdonald TT, Monteleone G. Immunity, inflammation, and allergy in the gut. Science
Dubois PC, van Heel DA. Translational mini-review series on the immunogenetics of gut
disease: immunogenetics of coeliac disease. Clin Exp Immunol 2008;153:162-73.
Hunt KA, Zhernakova A, Turner G, et al. Newly identified genetic risk variants for celiac
disease related to the immune response. Nat Genet 2008;40:395-402.
Romanos J, van Diemen CC, Nolte IM, et al. Analysis of HLA and non-HLA alleles can
identify individuals at high risk for celiac disease. Gastroenterology 2009.
van Heel DA, Franke L, Hunt KA, et al. A genome-wide association study for celiac disease
identifies risk variants in the region harboring IL2 and IL21. Nat Genet 2007;39:827-9.
Petronzelli F, Bonamico M, Ferrante P, et al. Genetic contribution of the HLA region to the
familial clustering of coeliac disease. Ann Hum Genet 1997;61:307-17.
Adamovic S, Amundsen SS, Lie BA, et al. Association study of IL2/IL21 and FcgRIIa:
significant association with the IL2/IL21 region in Scandinavian coeliac disease families.
Genes Immun 2008;9:364-7.
Babron MC, Nilsson S, Adamovic S, et al. Meta and pooled analysis of European coeliac
disease data. Eur J Hum Genet 2003;11:828-34.
Dema B, Martinez A, Fernandez-Arquero M, et al. Association of IL18RAP and CCR3 with
celiac disease in the Spanish population. J Med Genet 2009.
Djilali-Saiah I, Schmitz J, Harfouch-Hammoud E, et al. CTLA-4 gene polymorphism is
associated with predisposition to coeliac disease. Gut 1998;43:187-9.
Garner CP, Murray JA, Ding YC, et al. Replication of Celiac Disease UK Genome-Wide
Association Study Results in a US Population. Hum Mol Genet 2009.
Greco L, Corazza G, Babron MC, et al. Genome search in celiac disease. Am J Hum Genet
Haimila K, Einarsdottir E, de Kauwe A, et al. The shared CTLA4-ICOS risk locus in celiac
disease, IgA deficiency and common variable immunodeficiency. Genes Immun
Koskinen LL, Einarsdottir E, Dukes E, et al. Association study of the IL18RAP locus in
three European populations with coeliac disease. Hum Mol Genet 2009;18:1148-55.
Liu J, Juo SH, Holopainen P, et al. Genomewide linkage analysis of celiac disease in Finnish
epitope. Nat Med 2000;6:337-42.
Arentz-Hansen H, McAdam SN, Molberg O, et al. Celiac lesion T cells recognize epitopes
that cluster in regions of gliadins rich in proline residues. Gastroenterology 2002;123:803-9.
Fleckenstein B, Molberg O, Qiao SW, et al. Gliadin T cell epitope selection by tissue
transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the
transamidation versus deamidation process. J Biol Chem 2002;277:34109-16.
46. Naluai AT, Nilsson S, Samuelsson L, et al. The CTLA4/CD28 gene region on chromosome
2q33 confers susceptibility to celiac disease in a way possibly distinct from that of type 1
diabetes and other chronic inflammatory disorders. Tissue Antigens 2000;56:350-5.
Romanos J, Barisani D, Trynka G, et al. Six new coeliac disease loci replicated in an Italian
population confirm association with coeliac disease. J Med Genet 2009;46:60-3.
van Belzen MJ, Mulder CJ, Zhernakova A, et al. CTLA4 +49 A/G and CT60
polymorphisms in Dutch coeliac disease patients. Eur J Hum Genet 2004;12:782-5.
Woolley N, Holopainen P, Ollikainen V, et al. A new locus for coeliac disease mapped to
chromosome 15 in a population isolate. Hum Genet 2002;111:40-5.
Norris JM, Barriga K, Hoffenberg EJ, et al. Risk of celiac disease autoimmunity and timing
of gluten introduction in the diet of infants at increased risk of disease. Jama 2005;293:2343-
Collado MC, Calabuig M, Sanz Y. Differences between the fecal microbiota of coeliac
infants and healthy controls. Curr Issues Intest Microbiol 2007;8:9-14.
Collado MC, Donat E, Ribes-Koninckx C, et al. Imbalances in faecal and duodenal
Bifidobacterium species composition in active and non-active coeliac disease. BMC
Collado MC, Donat E, Ribes-Koninckx C, et al. Specific duodenal and faecal bacterial
groups associated with paediatric coeliac disease. J Clin Pathol 2009;62:264-9.
Pavone P, Nicolini E, Taibi R, et al. Rotavirus and celiac disease. Am J Gastroenterol
Stene LC, Honeyman MC, Hoffenberg EJ, et al. Rotavirus infection frequency and risk of
celiac disease autoimmunity in early childhood: a longitudinal study. Am J Gastroenterol
Zanoni G, Navone R, Lunardi C, et al. In celiac disease, a subset of autoantibodies against
transglutaminase binds toll-like receptor 4 and induces activation of monocytes. PLoS Med
Dieterich W, Ehnis T, Bauer M, et al. Identification of tissue transglutaminase as the
autoantigen of celiac disease. Nat Med 1997;3:797-801.
Elli L, Bergamini CM, Bardella MT, et al. Transglutaminases in inflammation and fibrosis
of the gastrointestinal tract and the liver. Dig Liver Dis 2009.
Piacentini M, Rodolfo C, Farrace MG, et al. "Tissue" transglutaminase in animal
development. Int J Dev Biol 2000;44:655-62.
Aeschlimann D, Thomazy V. Protein crosslinking in assembly and remodelling of
extracellular matrices: the role of transglutaminases. Connect Tissue Res 2000;41:1-27.
Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic
functions. Nat Rev Mol Cell Biol 2003;4:140-56.
Schuppan D, Dieterich W, Ehnis T, et al. Identification of the autoantigen of celiac disease.
Ann N Y Acad Sci 1998;859:121-6.
Anderson RP, Degano P, Godkin AJ, et al. In vivo antigen challenge in celiac disease
identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell
Thomas KE, Sapone A, Fasano A, et al. Gliadin stimulation of murine macrophage
inflammatory gene expression and intestinal permeability are MyD88-dependent: role of the
innate immune response in Celiac disease. J Immunol 2006;176:2512-21.
Meresse B, Cerf-Bensussan N. Innate T cell responses in human gut. Semin Immunol
Palmer E. The generation of T cell tolerance. Swiss Med Wkly 2007;137 Suppl 155:99S-
66. Molberg O, McAdam SN, Korner R, et al. Tissue transglutaminase selectively modifies
gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med
Qiao SW BE, Molberg O, Xia J, Fleckenstein B, Khosla C, Sollid LM. Antigen presentation
to celiac lesion-derived T cells of a 33-mer gliadin peptide naturally formed by
gastrointestinal digestion. J Immunol 2004;173:1757-1762.
Shan L, Molberg O, Parrot I, et al. Structural basis for gluten intolerance in celiac sprue.
Vader LW, de Ru A, van der Wal Y, et al. Specificity of tissue transglutaminase explains
cereal toxicity in celiac disease. J Exp Med 2002;195:643-9.
Vader LW, Stepniak DT, Bunnik EM, et al. Characterization of cereal toxicity for celiac
disease patients based on protein homology in grains. Gastroenterology 2003;125:1105-13.
Vader W, Kooy Y, Van Veelen P, et al. The gluten response in children with celiac disease
is directed toward multiple gliadin and glutenin peptides. Gastroenterology 2002;122:1729-
van de Wal Y, Kooy Y, van Veelen P, et al. Selective deamidation by tissue
transglutaminase strongly enhances gliadin-specific T cell reactivity. J Immunol
Clemente MG, De Virgiliis S, Kang JS, et al. Early effects of gliadin on enterocyte
intracellular signalling involved in intestinal barrier function. Gut 2003;52:218-23.
Schumann M, Richter JF, Wedell I, et al. Mechanisms of epithelial translocation of the
alpha(2)-gliadin-33mer in coeliac sprue. Gut 2008;57:747-54.
Zimmer KP, Poremba C, Weber P, et al. Translocation of gliadin into HLA-DR antigen
containing lysosomes in coeliac disease enterocytes. Gut 1995;36:703-9.
Matysiak-Budnik T, Candalh C, Dugave C, et al. Alterations of the intestinal transport and
processing of gliadin peptides in celiac disease. Gastroenterology 2003;125:696-707.
Matysiak-Budnik T, Moura IC, Arcos-Fajardo M, et al. Secretory IgA mediates
retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease. J Exp
Niess JH, Brand S, Gu X, et al. CX3CR1-mediated dendritic cell access to the intestinal
lumen and bacterial clearance. Science 2005;307:254-8.
Man AL, Prieto-Garcia ME, Nicoletti C. Improving M cell mediated transport across
mucosal barriers: do certain bacteria hold the keys? Immunology 2004;113:15-22.
Cinova J, Palova-Jelinkova L, Smythies LE, et al. Gliadin peptides activate blood
monocytes from patients with celiac disease. J Clin Immunol 2007;27:201-9.
Maiuri L, Ciacci C, Ricciardelli I, et al. Association between innate response to gliadin and
activation of pathogenic T cells in coeliac disease. Lancet 2003;362:30-7.
Palova-Jelinkova L, Rozkova D, Pecharova B, et al. Gliadin fragments induce phenotypic
and functional maturation of human dendritic cells. J Immunol 2005;175:7038-45.
Tuckova L, Novotna J, Novak P, et al. Activation of macrophages by gliadin fragments:
isolation and characterization of active peptide. J Leukoc Biol 2002;71:625-31.
Meresse B, Verdier J, Cerf-Bensussan N. The cytokine interleukin 21: a new player in
coeliac disease? Gut 2008;57:879-81.
Izcue A, Coombes JL, Powrie F. Regulatory lymphocytes and intestinal inflammation. Annu
Rev Immunol 2009;27:313-38.
Frisullo G, Nociti V, Iorio R, et al. Increased CD4+CD25+Foxp3+ T cells in peripheral
blood of celiac disease patients: correlation with dietary treatment. Hum Immunol
87. Londei M, Ciacci C, Ricciardelli I, et al. Gliadin as a stimulator of innate responses in celiac
disease. Mol Immunol 2005;42:913-8.
Ciccocioppo R, Di Sabatino A, Parroni R, et al. Cytolytic mechanisms of intraepithelial
lymphocytes in coeliac disease (CoD). Clin Exp Immunol 2000;120:235-40.
Di Sabatino A, Ciccocioppo R, Cupelli F, et al. Epithelium derived interleukin 15 regulates
intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac
disease. Gut 2006;55:469-77.
Salvati VM, Mazzarella G, Gianfrani C, et al. Recombinant human interleukin 10 suppresses
gliadin dependent T cell activation in ex vivo cultured coeliac intestinal mucosa. Gut
Burgess SJ, Maasho K, Masilamani M, et al. The NKG2D receptor: immunobiology and
clinical implications. Immunol Res 2008;40:18-34.
Hue S, Mention JJ, Monteiro RC, et al. A direct role for NKG2D/MICA interaction in
villous atrophy during celiac disease. Immunity 2004;21:367-77.
Meresse B, Chen Z, Ciszewski C, et al. Coordinated induction by IL15 of a TCR-
independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells
in celiac disease. Immunity 2004;21:357-66.
Terrazzano G, Sica M, Gianfrani C, et al. Gliadin regulates the NK-dendritic cell cross-talk
by HLA-E surface stabilization. J Immunol 2007;179:372-81.
Jabri B, de Serre NP, Cellier C, et al. Selective expansion of intraepithelial lymphocytes
expressing the HLA-E-specific natural killer receptor CD94 in celiac disease.
Meresse B, Curran SA, Ciszewski C, et al. Reprogramming of CTLs into natural killer-like
cells in celiac disease. J Exp Med 2006;203:1343-55.
Bhagat G, Naiyer AJ, Shah JG, et al. Small intestinal CD8+TCRgammadelta+NKG2A+
intraepithelial lymphocytes have attributes of regulatory cells in patients with celiac disease.
J Clin Invest 2008;118:281-93.
Benahmed M, Meresse B, Arnulf B, et al. Inhibition of TGF-beta signaling by IL-15: a new
role for IL-15 in the loss of immune homeostasis in celiac disease. Gastroenterology
Bernardo D, Garrote JA, Allegretti Y, et al. Higher constitutive IL15R alpha expression and
lower IL-15 response threshold in coeliac disease patients. Clin Exp Immunol 2008;154:64-
Maiuri L, Ciacci C, Auricchio S, et al. Interleukin 15 mediates epithelial changes in celiac
disease. Gastroenterology 2000;119:996-1006.
Mention JJ, Ben Ahmed M, Begue B, et al. Interleukin 15: a key to disrupted intraepithelial
lymphocyte homeostasis and lymphomagenesis in celiac disease. Gastroenterology
Maiuri L, Ciacci C, Vacca L, et al. IL-15 drives the specific migration of CD94+ and TCR-
gammadelta+ intraepithelial lymphocytes in organ cultures of treated celiac patients. Am J
Leffler DA, Dennis M, Hyett B, et al. Etiologies and predictors of diagnosis in
nonresponsive celiac disease. Clin Gastroenterol Hepatol 2007;5:445-50.
Al-Toma A, Verbeek WH, Hadithi M, et al. Survival in refractory coeliac disease and
enteropathy-associated T-cell lymphoma: retrospective evaluation of single-centre
experience. Gut 2007;56:1373-8.
Cellier C, Delabesse E, Helmer C, et al. Refractory sprue, coeliac disease, and enteropathy-
associated T-cell lymphoma. French Coeliac Disease Study Group. Lancet 2000;356:203-8.
106. Gianfrani C, Levings MK, Sartirana C, et al. Gliadin-specific type 1 regulatory T cells from
the intestinal mucosa of treated celiac patients inhibit pathogenic T cells. J Immunol
Mucida D, Park Y, Cheroutre H. From the diet to the nucleus: vitamin A and TGF-beta join
efforts at the mucosal interface of the intestine. Semin Immunol 2009;21:14-21.
Iwata M, Eshima Y, Kagechika H. Retinoic acids exert direct effects on T cells to suppress
Th1 development and enhance Th2 development via retinoic acid receptors. Int Immunol
Mora JR, Iwata M, Eksteen B, et al. Generation of gut-homing IgA-secreting B cells by
intestinal dendritic cells. Science 2006;314:1157-60.
Papadakis KA PJ, Nelson V, Cheng L, Binder SW, Ponath PD, Andrew DP, Targan SR. The
role of thymus expressed chemokine and its receptor CCR9 on lymphocytes in the regional
specialization of the mucosal immune system. J Immunol 2000;165:5069-5076.
Mucida D, Park Y, Kim G, et al. Reciprocal TH17 and regulatory T cell differentiation
mediated by retinoic acid. Science 2007;317:256-60.
Smith TR, Kumar V. Revival of CD8+ Treg-mediated suppression. Trends Immunol
van Wijk F, Cheroutre H. Intestinal T cells: facing the mucosal immune dilemma with
synergy and diversity. Semin Immunol 2009;21:130-8.
Cheroutre H. In IBD eight can come before four. Gastroenterology 2006;131:667-70.
Nancey S, Holvoet S, Graber I, et al. CD8+ cytotoxic T cells induce relapsing colitis in
normal mice. Gastroenterology 2006;131:485-96.
Westendorf AM, Fleissner D, Deppenmeier S, et al. Autoimmune-mediated intestinal
inflammation-impact and regulation of antigen-specific CD8+ T cells. Gastroenterology
Przemioslo RT, Lundin KE, Sollid LM, et al. Histological changes in small bowel mucosa
induced by gliadin sensitive T lymphocytes can be blocked by anti-interferon gamma
antibody. Gut 1995;36:874-9.
Forsberg G, Hernell O, Melgar S, et al. Paradoxical coexpression of proinflammatory and
down-regulatory cytokines in intestinal T cells in childhood celiac disease. Gastroenterology
Pender SL, Tickle SP, Docherty AJ, et al. A major role for matrix metalloproteinases in T
cell injury in the gut. J Immunol 1997;158:1582-90.
Daum S, Bauer U, Foss HD, et al. Increased expression of mRNA for matrix
metalloproteinases-1 and -3 and tissue inhibitor of metalloproteinases-1 in intestinal biopsy
specimens from patients with coeliac disease. Gut 1999;44:17-25.
Ciccocioppo R, Di Sabatino A, Bauer M, et al. Matrix metalloproteinase pattern in celiac
duodenal mucosa. Lab Invest 2005;85:397-407.
Schuppan D, Freitag T. Fistulising Crohn's disease: MMPs gone awry. Gut 2004;53:622-4.
Abdulkarim AS, Burgart LJ, See J, et al. Etiology of nonresponsive celiac disease: results of
a systematic approach. Am J Gastroenterol 2002;97:2016-21.
model of gluten sensitivity. Immunol Lett 2008;119:78-83.
Senger S, Maurano F, Mazzeo MF, et al. Identification of immunodominant epitopes of
alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization. J Immunol
Marietta E, Black K, Camilleri M, et al. A new model for dermatitis herpetiformis that uses
HLA-DQ8 transgenic NOD mice. J Clin Invest 2004;114:1090-7.
127. Malamut G, Afchain P, Verkarre V, et al. Presentation and long-term follow-up of refractory
celiac disease: comparison of type I with type II. Gastroenterology 2009;136:81-90.
Rubio-Tapia A, Kyle RA, Kaplan EL, et al. Increased prevalence and mortality in
undiagnosed celiac disease. Gastroenterology 2009;137:88-93.
Jabri B, Sollid LM. Mechanisms of disease: immunopathogenesis of celiac disease. Nat Clin
Pract Gastroenterol Hepatol 2006;3:516-25.
Verbeek WH, Goerres MS, von Blomberg BM, et al. Flow cytometric determination of
aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more
accurately than T-cell clonality analysis in Refractory Celiac Disease. Clin Immunol
Al-toma A, Visser OJ, van Roessel HM, et al. Autologous hematopoietic stem cell
transplantation in refractory celiac disease with aberrant T cells. Blood 2007;109:2243-9.
Catassi C, Bearzi I, Holmes GK. Association of celiac disease and intestinal lymphomas and
other cancers. Gastroenterology 2005;128:S79-86.
Holmes GK, Prior P, Lane MR, et al. Malignancy in coeliac disease--effect of a gluten free
diet. Gut 1989;30:333-8.
Marietta E, Schuppan D, Murray JA. In vitro and in vivo models of celiac disease. Exp Opin
Drug Discov 2009;in press.
de Ritis G, Auricchio S, Jones HW, et al. In vitro (organ culture) studies of the toxicity of
specific A-gliadin peptides in celiac disease. Gastroenterology 1988;94:41-9.
Falchuk ZM, Gebhard RL, Sessoms C, et al. An in vitro model of gluten-sensitive
enteropathy. Effect of gliadin on intestinal epithelial cells of patients with gluten-sensitive
enteropathy in organ culture. J Clin Invest 1974;53:487-500.
Picarelli A, Maiuri L, Frate A, et al. Production of antiendomysial antibodies after in-vitro
gliadin challenge of small intestine biopsy samples from patients with coeliac disease.
Batt RM, McLean L, Carter MW. Sequential morphologic and biochemical studies of
naturally occurring wheat-sensitive enteropathy in Irish setter dogs. Dig Dis Sci
Hall EJ, Batt RM. Dietary modulation of gluten sensitivity in a naturally occurring
enteropathy of Irish setter dogs. Gut 1992;33:198-205.
Polvi A, Garden OA, Houlston RS, et al. Genetic susceptibility to gluten sensitive
enteropathy in Irish setter dogs is not linked to the major histocompatibility complex. Tissue
Black KE, Murray JA, David CS. HLA-DQ determines the response to exogenous wheat
proteins: a model of gluten sensitivity in transgenic knockout mice. J Immunol
Cheng S, Smart M, Hanson J, et al. Characterization of HLA DR2 and DQ8 transgenic
mouse with a new engineered mouse class II deletion, which lacks all endogenous class II
genes. J Autoimmun 2003;21:195-9.
D'Arienzo R, Maurano F, Luongo D, et al. Adjuvant effect of Lactobacillus casei in a mouse
transglutaminase autoantibodies is correlated with the clinical presentation of coeliac
disease. Scand J Immunol 2005;61:207-12.
Koleba T, Ensom MH. Pharmacokinetics of intravenous immunoglobulin: a systematic
review. Pharmacotherapy 2006;26:813-27.
Tursi A, Brandimarte G, Giorgetti GM. Lack of usefulness of anti-transglutaminase
antibodies in assessing histologic recovery after gluten-free diet in celiac disease. J Clin
146. Karell K, Louka AS, Moodie SJ, et al. HLA types in celiac disease patients not carrying the
DQA1*05-DQB1*02 (DQ2) heterodimer: results from the European Genetics Cluster on
Celiac Disease. Hum Immunol 2003;64:469-77.
Chen D, Ueda R, Harding F, et al. Characterization of HLA DR3/DQ2 transgenic mice: a
potential humanized animal model for autoimmune disease studies. Eur J Immunol
de Kauwe AL, Chen Z, Anderson RP, et al. Resistance to celiac disease in humanized HLA-
DR3-DQ2-transgenic mice expressing specific anti-gliadin CD4+ T cells. J Immunol
Sestak K, Merritt CK, Borda J, et al. Infectious agent and immune response characteristics
of chronic enterocolitis in captive rhesus macaques. Infect Immun 2003;71:4079-86.
Bethune MT, Borda JT, Ribka E, et al. A non-human primate model for gluten sensitivity.
PLoS ONE 2008;3:e1614.
Freitag T, Rietdijk S, Junker Y, et al. Gliadin-primed CD4+CD45RBlowCD25-
effector/memory T cells drive gluten-dependent small intestinal damage after adoptive
transfer into lymphopenic mice. Gut 2009.
Kontakou M, Przemioslo RT, Sturgess RP, et al. Expression of tumour necrosis factor-alpha,
interleukin-6, and interleukin-2 mRNA in the jejunum of patients with coeliac disease.
Scand J Gastroenterol 1995;30:456-63.
Nilsen EM, Jahnsen FL, Lundin KE, et al. Gluten induces an intestinal cytokine response
strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology
Westerholm-Ormio M, Garioch J, Ketola I, et al. Inflammatory cytokines in small intestinal
mucosa of patients with potential coeliac disease. Clin Exp Immunol 2002;128:94-101.
Castellanos-Rubio A, Santin I, Irastorza I, et al. TH17 (and TH1) signatures of intestinal
biopsies of CD patients in response to gliadin. Autoimmunity 2009;42:69-73.
Salvati VM, MacDonald TT, Bajaj-Elliott M, et al. Interleukin 18 and associated markers of
T helper cell type 1 activity in coeliac disease. Gut 2002;50:186-90.
Barker CC, Mitton C, Jevon G, et al. Can tissue transglutaminase antibody titers replace
small-bowel biopsy to diagnose celiac disease in select pediatric populations? Pediatrics
Hopper AD, Hadjivassiliou M, Hurlstone DP, et al. What is the role of serologic testing in
celiac disease? A prospective, biopsy-confirmed study with economic analysis. Clin
Gastroenterol Hepatol 2008;6:314-20.
Kotze LM, Utiyama SR, Nisihara RM, et al. IgA class anti-endomysial and anti-tissue
transglutaminase antibodies in relation to duodenal mucosa changes in coeliac disease.
Tursi A, Brandimarte G, Giorgetti GM. Prevalence of antitissue transglutaminase antibodies
in different degrees of intestinal damage in celiac disease. J Clin Gastroenterol 2003;36:219-
Schilling J, Spiekerkoetter U, Wohlrab U, et al. Immunoglobulin isotype profile of tissue
Anderson RP vHD, Tye-Din JA, Jewell DP, Hill AVS. Antagonists and non-toxic variants
of the dominant wheat gliadin T cell epitope in coeliac disease. Gut 2006;55:485-491.
Andersson EC, Hansen BE, Jacobsen H, et al. Definition of MHC and T cell receptor
contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and
characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice.
Proc Natl Acad Sci U S A 1998;95:7574-9.
164. Korponay-Szabo IR, Dahlbom I, Laurila K, et al. Elevation of IgG antibodies against tissue
transglutaminase as a diagnostic tool for coeliac disease in selective IgA deficiency. Gut
Farrell RJ, Kelly CP. Diagnosis of celiac sprue. Am J Gastroenterol 2001;96:3237-46.
Pyle GG, Paaso B, Anderson BE, et al. Low-dose gluten challenge in celiac sprue:
malabsorptive and antibody responses. Clin Gastroenterol Hepatol 2005;3:679-86.
Juby LD, Rothwell J, Axon AT. Lactulose/mannitol test: an ideal screen for celiac disease.
Vogelsang H, Wyatt J, Penner E, et al. Screening for celiac disease in first-degree relatives
of patients with celiac disease by lactulose/mannitol test. Am J Gastroenterol 1995;90:1838-
Paterson BM, Lammers KM, Arrieta MC, et al. The safety, tolerance, pharmacokinetic and
pharmacodynamic effects of single doses of AT-1001 in coeliac disease subjects: a proof of
concept study. Aliment Pharmacol Ther 2007;26:757-66.
Kelly CP, Green PH, Murray JA, et al. Intestinal Permeability of Larazotide Acetate in
Celiac Disease: Results of a
Phase IIB 6-Week Gluten-Challenge Clinical Trial. Gastroenterology 2009;136 (Suppl 1):M2048.
171. Johnston SD, Watson RG, Middleton D, et al. Genetic, morphometric and
immunohistochemical markers of latent coeliac disease. Eur J Gastroenterol Hepatol
172. Mustalahti K, Lohiniemi S, Collin P, et al. Gluten-free diet and quality of life in patients
with screen-detected celiac disease. Eff Clin Pract 2002;5:105-13.
173. Leffler DA, Dennis M, Edwards George J, et al. A Validated Disease Specific Symptom
Index for Adults with Celiac Disease. Clin Gastroenterol Hepatol 2009.
174. Arentz-Hansen H, Korner R, Molberg O, et al. The intestinal T cell response to alpha-
gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue
transglutaminase. J Exp Med 2000;191:603-12.
175. Sjostrom H, Lundin KE, Molberg O, et al. Identification of a gliadin T-cell epitope in
coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition.
Scand J Immunol 1998;48:111-5.
176. Stepniak D, Wiesner M, de Ru AH, et al. Large-scale characterization of natural ligands
explains the unique gluten-binding properties of HLA-DQ2. J Immunol 2008;180:3268-78.
177. van de Wal Y, Kooy YM, van Veelen P, et al. Glutenin is involved in the gluten-driven
mucosal T cell response. Eur J Immunol 1999;29:3133-9.
178. van de Wal Y, Kooy YM, van Veelen PA, et al. Small intestinal T cells of celiac disease
patients recognize a natural pepsin fragment of gliadin. Proc Natl Acad Sci U S A
179. Quarsten H, McAdam SN, Jensen T, et al. Staining of celiac disease-relevant T cells by
peptide-DQ2 multimers. J Immunol 2001;167:4861-8.
180. Raki M, Fallang LE, Brottveit M, et al. Tetramer visualization of gut-homing gluten-specific
T cells in the peripheral blood of celiac disease patients. Proc Natl Acad Sci U S A
van den Broeck HC, van Herpen TW, Schuit C, et al. Removing celiac disease-related
gluten proteins from bread wheat while retaining technological properties: a study with
Chinese Spring deletion lines. BMC Plant Biol 2009;9:41.
Comai L, Young K, Till BJ, et al. Efficient discovery of DNA polymorphisms in natural
populations by Ecotilling. Plant J 2004;37:778-86.
Greene EA, Codomo CA, Taylor NE, et al. Spectrum of chemically induced mutations from
a large-scale reverse-genetic screen in Arabidopsis. Genetics 2003;164:731-40.
183. Hansson T, Dannaeus A, Klareskog L. Cytokine-producing cells in peripheral blood of
children with coeliac disease secrete cytokines with a type 1 profile. Clin Exp Immunol
Hoffman SA, Joo WA, Echan LA, et al. Higher dimensional (Hi-D) separation strategies
dramatically improve the potential for cancer biomarker detection in serum and plasma. J
Chromatogr B Analyt Technol Biomed Life Sci 2007;849:43-52.
Issaq HJ, Veenstra TD. The role of electrophoresis in disease biomarker discovery.
Patterson SD, Aebersold RH. Proteomics: the first decade and beyond. Nat Genet 2003;33
Sollid LM, Khosla C. Future therapeutic options for celiac disease. Nat Clin Pract
Gastroenterol Hepatol 2005;2:140-7.
Feldman M, . The origin of cultivated wheat. In: Bonjean AP AW, eds., ed. The world wheat
book. London: Intercept, 2001:1-56.
Auricchio S DG, De Vincenzi M, Occorsio P, Silano V. . Effects of gliadin-derived peptides
from bread and durum wheats on small-intestine cultures from rat fetus and celiac children.
Pediatr Res 1982;16:1004–1010.
Frisoni M CG, Lafiandra D, De Ambrogio E, Filipponi C, Bonvicini F, Borasio E, Porceddu
E, Gasbarrini G. . Wheat deficient in gliadins: promising tool for treatment of coeliac
disease. Gut 1995;36:375-378.
Molberg O, Kett K, Scott H, et al. Gliadin specific, HLA DQ2-restricted T cells are
commonly found in small intestinal biopsies from coeliac disease patients, but not from
controls. Scand J Immunol 1997;46:103-8.
Spaenij-Dekking L, Kooy-Winkelaar Y, van Veelen P, et al. Natural variation in toxicity of
wheat: potential for selection of nontoxic varieties for celiac disease patients.
Molberg O UA, Jensen T, Flaete NS, Fleckenstein B, Arentz-Hansen H, Raki M, Lundin
KE, Sollid LM. Mapping of gluten T-cell epitopes in the bread wheat ancestors: implications
for celiac disease. Gastroenterology 2005;128:393-401.
van Herpen TWJM GS, van der Schoot J, Mitreva M, Salentijn E, Vorst O, Schenk MF, van
Veelen PA, Koning F, van Soest LJM, Vosman B, Bosch D, Hamer RJ, Gilissen LJWJ,
Smulders MJM. Alpha-gliadin genes from the A, B, and D genomes of wheat contain
different sets of celiac disease epitopes. BMC Genomics 2006;7:1 doi:10.1186/1471-2164-
Vincentini O MF, Gazza L, Silano M, Dessi M, De Vincenzi M, Pogna NE. Environmental
factors of celiac disease: cytotoxicity of hulled wheat species Triticum monococcum, T.
turgidum ssp. dicoccum and T. aestivum ssp. spelta. J Gastroenterol Hepatol 2007;22:1816-
Pizzuti D BA, D'Odorico A, D'Incà R, Chiarelli S, Curioni A, Martines D. Lack of intestinal
mucosal toxicity of Triticum monococcum in celiac disease patients. Scand J Gastroenterol
Technol Biomed Life Sci 2007;855:236-41.
Marti T, Molberg O, Li Q, et al. Prolyl endopeptidase-mediated destruction of T cell
epitopes in whole gluten: chemical and immunological characterization. J Pharmacol Exp
Cornell HJ, Macrae FA, Melny J, et al. Enzyme therapy for management of coeliac disease.
Scand J Gastroenterol 2005;40:1304-12.
200. McCallum CM, Comai L, Greene EA, et al. Targeting induced local lesions IN genomes
(TILLING) for plant functional genomics. Plant Physiol 2000;123:439-42.
Till BJ, Reynolds SH, Greene EA, et al. Large-scale discovery of induced point mutations
with high-throughput TILLING. Genome Res 2003;13:524-30.
Barkley NA, Wang ML. Application of TILLING and EcoTILLING as Reverse Genetic
Approaches to Elucidate the Function of Genes in Plants and Animals. Curr Genomics
Di Cagno R, De Angelis M, Lavermicocca P, et al. Proteolysis by sourdough lactic acid
bacteria: effects on wheat flour protein fractions and gliadin peptides involved in human
cereal intolerance. Appl Environ Microbiol 2002;68:623-33.
Rizzello CG, De Angelis M, Di Cagno R, et al. Highly efficient gluten degradation by
lactobacilli and fungal proteases during food processing: new perspectives for celiac disease.
Appl Environ Microbiol 2007;73:4499-507.
De Angelis M, Rizzello CG, Fasano A, et al. VSL#3 probiotic preparation has the capacity
to hydrolyze gliadin polypeptides responsible for Celiac Sprue. Biochim Biophys Acta
Di Cagno R, De Angelis M, Auricchio S, et al. Sourdough bread made from wheat and
nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients.
Appl Environ Microbiol 2004;70:1088-96.
Kiyosaki T, Matsumoto I, Asakura T, et al. Gliadain, a gibberellin-inducible cysteine
proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically
digests gliadin and is regulated by intrinsic cystatins. Febs J 2007;274:1908-17.
Gianfrani C, Siciliano RA, Facchiano AM, et al. Transamidation of wheat flour inhibits the
response to gliadin of intestinal T cells in celiac disease. Gastroenterology 2007;133:780-9.
Yokoyama K, Nio N, Kikuchi Y. Properties and applications of microbial transglutaminase.
Appl Microbiol Biotechnol 2004;64:447-54.
Pasternack R, Dorsch S, Otterbach JT, et al. Bacterial pro-transglutaminase from
Streptoverticillium mobaraense--purification, characterisation and sequence of the zymogen.
Eur J Biochem 1998;257:570-6.
Cabrera-Chavez F, Rouzaud-Sandez O, Sotelo-Cruz N, et al. Transglutaminase treatment of
wheat and maize prolamins of bread increases the serum IgA reactivity of celiac disease
patients. J Agric Food Chem 2008;56:1387-91.
Cabrera-Chavez F, Rouzaud-Sandez O, Sotelo-Cruz N, et al. Bovine milk caseins and
transglutaminase-treated cereal prolamins are differentially recognized by IgA of celiac
disease patients according to their age. J Agric Food Chem 2009;57:3754-9.
Wieser H. Relation between gliadin structure and coeliac toxicity. Acta Paediatr Suppl
Hausch F, Shan L, Santiago NA, et al. Intestinal digestive resistance of immunodominant
gliadin peptides. Am J Physiol Gastrointest Liver Physiol 2002;283:G996-G1003.
Mamone G, Ferranti P, Rossi M, et al. Identification of a peptide from alpha-gliadin
resistant to digestive enzymes: implications for celiac disease. J Chromatogr B Analyt
the pathogenesis of type I diabetes in BB diabetic-prone rats. Proc Natl Acad Sci U S A
Jeitner TM, Delikatny EJ, Ahlqvist J, et al. Mechanism for the inhibition of transglutaminase
2 by cystamine. Biochem Pharmacol 2005;69:961-70.
Pardin C, Roy I, Lubell WD, et al. Reversible and competitive cinnamoyl triazole inhibitors
of tissue transglutaminase. Chem Biol Drug Des 2008;72:189-96.
218. Stepniak D, Spaenij-Dekking L, Mitea C, et al. Highly efficient gluten degradation with a
newly identified prolyl endoprotease: implications for celiac disease. Am J Physiol
Gastrointest Liver Physiol 2006;291:G621-9.
Mitea C, Havenaar R, Drijfhout JW, et al. Efficient degradation of gluten by a prolyl
endoprotease in a gastrointestinal model: implications for coeliac disease. Gut 2008;57:25-
Pyle GG, Paaso B, Anderson BE, et al. Effect of pretreatment of food gluten with prolyl
endopeptidase on gluten-induced malabsorption in celiac sprue. Clin Gastroenterol Hepatol
Fulop V, Szeltner Z, Polgar L. Catalysis of serine oligopeptidases is controlled by a gating
filter mechanism. EMBO Rep 2000;1:277-81.
Shan L, Marti T, Sollid LM, et al. Comparative biochemical analysis of three bacterial
prolyl endopeptidases: implications for coeliac sprue. Biochem J 2004;383:311-8.
Cerf-Bensussan N, Matysiak-Budnik T, Cellier C, et al. Oral proteases: a new approach to
managing coeliac disease. Gut 2007;56:157-60.
Matysiak-Budnik T, Candalh C, Cellier C, et al. Limited efficiency of prolyl-endopeptidase
in the detoxification of gliadin peptides in celiac disease. Gastroenterology 2005;129:786-
Gass J, Vora H, Bethune MT, et al. Effect of barley endoprotease EP-B2 on gluten digestion
in the intact rat. J Pharmacol Exp Ther 2006;318:1178-86.
Gass J, Bethune MT, Siegel M, et al. Combination enzyme therapy for gastric digestion of
dietary gluten in patients with celiac sprue. Gastroenterology 2007;133:472-80.
Pinier M, Verdu EF, Nasser-Eddine M, et al. Polymeric binders suppress gliadin-induced
toxicity in the intestinal epithelium. Gastroenterology 2009;136:288-98.
Warny M, Fatimi A, Bostwick EF, et al. Bovine immunoglobulin concentrate-clostridium
difficile retains C difficile toxin neutralising activity after passage through the human
stomach and small intestine. Gut 1999;44:212-7.
Lu R, Wang W, Uzzau S, et al. Affinity purification and partial characterization of the
zonulin/zonula occludens toxin (Zot) receptor from human brain. J Neurochem
Uzzau S, Lu R, Wang W, et al. Purification and preliminary characterization of the zonula
occludens toxin receptor from human (CaCo2) and murine (IEC6) intestinal cell lines.
FEMS Microbiol Lett 2001;194:1-5.
Choi K, Siegel M, Piper JL, et al. Chemistry and biology of dihydroisoxazole derivatives:
selective inhibitors of human transglutaminase 2. Chem Biol 2005;12:469-75.
Lai TS, Slaughter TF, Peoples KA, et al. Regulation of human tissue transglutaminase
function by magnesium-nucleotide complexes. Identification of distinct binding sites for
Mg-GTP and Mg-ATP. J Biol Chem 1998;273:1776-81.
Siegel M, Khosla C. Transglutaminase 2 inhibitors and their therapeutic role in disease
states. Pharmacol Ther 2007;115:232-45.
Watts T, Berti I, Sapone A, et al. Role of the intestinal tight junction modulator zonulin in
Bolin DR, Swain AL, Sarabu R, et al. Peptide and peptide mimetic inhibitors of antigen
presentation by HLA-DR class II MHC molecules. Design, structure-activity relationships,
and X-ray crystal structures. J Med Chem 2000;43:2135-48.
Falcioni F, Ito K, Vidovic D, et al. Peptidomimetic compounds that inhibit antigen
presentation by autoimmune disease-associated class II major histocompatibility molecules.
Nat Biotechnol 1999;17:562-7.
237. de Macedo P, Marrano C, Keillor JW. Synthesis of dipeptide-bound epoxides and
alpha,beta-unsaturated amides as potential irreversible transglutaminase inhibitors. Bioorg
Med Chem 2002;10:355-60.
Hausch F, Halttunen T, Maki M, et al. Design, synthesis, and evaluation of gluten peptide
analogs as selective inhibitors of human tissue transglutaminase. Chem Biol 2003;10:225-
Molberg O, McAdam S, Lundin KE, et al. T cells from celiac disease lesions recognize
gliadin epitopes deamidated in situ by endogenous tissue transglutaminase. Eur J Immunol
Maiuri L, Ciacci C, Ricciardelli I, et al. Unexpected role of surface transglutaminase type II
in celiac disease. Gastroenterology 2005;129:1400-13.
De Vincenzi M DM, Giovannini C, Maialetti F, Mancini E. . Agglutinating activity of wheat
gliadin peptide fractions in coeliac disease. Toxicology 1995;96:29-35.
De Vincenzi M GG, Silano V. . A small peptide from durum wheat gliadin prevents cell
agglutination induced by prolamin-peptides toxic in coeliac disease. Toxicology
De Vincenzi M LR, Giovannini C, Pogna NE, Saponaro C, Galterio G, Gasbarrini G. . In
vitro toxicity testing of alcohol-soluble proteins from diploid wheat Triticum monococcum
in celiac disease. Biochem Toxicol 1996;11:313-318.
De Vincenzi M SA, Luchetti R, Silano M, Gasbarrini G, Silano V. . Structural specificities
and significance for coeliac disease of wheat gliadin peptides able to agglutinate or to
prevent agglutination of K562(S) cells. Toxicology 1998;127:97-106.
Giovannini C SM, Straface E, Scazzocchio B, Silano M, De Vincenzi M. . Induction of
apoptosis in caco-2 cells by wheat gliadin peptides. Toxicology 2000;145:63-71.
Silano M DBR, Trecca A, Arrabito G, Leonardi F, De Vincenzi M. A Decapeptide from
Durum Wheat Prevents Celiac Peripheral Blood Lymphocytes from Activation by Gliadin
Peptides. Pediatr Res 2007;61:67-71.
Silano M DBR, Maialetti F, De Vincenzi A, Calcaterra R, Trecca A, De Vincenzi M. A 10-
residue peptide from durum wheat promotes a shift from a Th1-type response toward a Th2-
type response in celiac disease. Am J Clin Nutr 2008;87:415-423.
Silano M LF, Trecca A, Mancini E, Di Benedetto R, De Vincenzi M. Prevention by a
decapeptide from durum wheat of in vitro gliadin peptide-induced apoptosis in small-bowel
mucosa from coeliac patients. Scand J Gastroenterol 2007;42:786-787.
Biagi F, Ellis HJ, Parnell ND, et al. A non-toxic analogue of a coeliac-activating gliadin
peptide: a basis for immunomodulation? Aliment Pharmacol Ther 1999;13:945-50.
Kapoerchan VV, Wiesner M, Overhand M, et al. Design of azidoproline containing gluten
peptides to suppress CD4+ T-cell responses associated with celiac disease. Bioorg Med
Xia J, Siegel M, Bergseng E, et al. Inhibition of HLA-DQ2-mediated antigen presentation
by analogues of a high affinity 33-residue peptide from alpha2-gliadin. J Am Chem Soc
Zabel BA, Agace WW, Campbell JJ, et al. Human G protein-coupled receptor GPR-9-6/CC
chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal
lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated
chemotaxis. J Exp Med 1999;190:1241-56.
254. Ishioka GY, Adorini L, Guery JC, et al. Failure to demonstrate long-lived MHC saturation
both in vitro and in vivo. Implications for therapeutic potential of MHC-blocking peptides. J
Siegel M, Xia J, Khosla C. Structure-based design of alpha-amido aldehyde containing
gluten peptide analogues as modulators of HLA-DQ2 and transglutaminase 2. Bioorg Med
Xia J, Bergseng E, Fleckenstein B, et al. Cyclic and dimeric gluten peptide analogues
inhibiting DQ2-mediated antigen presentation in celiac disease. Bioorg Med Chem
Matysiak-Budnik T, Malamut G, de Serre NP, et al. Long-term follow-up of 61 coeliac
patients diagnosed in childhood: evolution toward latency is possible on a normal diet. Gut
Maurano F, Siciliano RA, De Giulio B, et al. Intranasal administration of one alpha gliadin
can downregulate the immune response to whole gliadin in mice. Scand J Immunol
Rossi M, Maurano F, Caputo N, et al. Intravenous or intranasal administration of gliadin is
able to down-regulate the specific immune response in mice. Scand J Immunol 1999;50:177-
Senger S, Luongo D, Maurano F, et al. Intranasal administration of a recombinant alpha-
gliadin down-regulates the immune response to wheat gliadin in DQ8 transgenic mice.
Immunol Lett 2003;88:127-34.
Keech CL, Dromey J, Chen Z, et al. Immune Tolerance Induced by Peptide Immunotherapy
in an HLA-DQ2-Dependent Mouse Model of Gluten Immunity. Gastroenterology
Medina M, De Palma G, Ribes-Koninckx C, et al. Bifidobacterium strains suppress in vitro
the pro-inflammatory milieu triggered by the large intestinal microbiota of coeliac patients. J
Inflamm (Lond) 2008;5:19.
Elliott DE, Summers RW, Weinstock JV. Helminths as governors of immune-mediated
inflammation. Int J Parasitol 2007;37:457-64.
Summers RW, Elliott DE, Urban JF, Jr., et al. Trichuris suis therapy in Crohn's disease. Gut
Summers RW, Elliott DE, Urban JF, Jr., et al. Trichuris suis therapy for active ulcerative
colitis: a randomized controlled trial. Gastroenterology 2005;128:825-32.
Huibregtse IL, Marietta EV, Rashtak S, et al. Induction of antigen-specific tolerance by oral
administration of lactococcus lactis delivered immunodominant DQ8-restricted gliadin
peptide in sensitized nonobese diabetic abdegrees Dq8 transgenic mice. J Immunol
Berlin C, Berg EL, Briskin MJ, et al. Alpha 4 beta 7 integrin mediates lymphocyte binding
to the mucosal vascular addressin MAdCAM-1. Cell 1993;74:185-95.
Papadakis KA, Prehn J, Moreno ST, et al. CCR9-positive lymphocytes and thymus-
expressed chemokine distinguish small bowel from colonic Crohn's disease.
270. Olaussen RW KM, Lundin KEA, Jahnsen J, Brandtzaeg P, Farstad IN. Reduced chemokine
receptor 9 on intraepithelial lymphocytes in celiac disease suggests persistent epithelial
activation. Gastroenterology 2007;132:2371-2382.
Rivera–Nieves J HJ, Bamias G, Ivashkina N, Ley K, Oppermann M, Cominelli F. Antibody
Blockade of CCL25/CCR9 Ameliorates Early but not Late Chronic Murine Ileitis.
Wei Z EL, Baumgart T, Rubas W, Hor SY, Wright JJK, Howard M, Schall T, Keshav S. Cc
chemokine receptor 9 (ccr9) antagonist ameliorates experimental ileitis and colitis (abstr).
Gastroenterology 2005;128:A204 –A205.
Baslund B, Tvede N, Danneskiold-Samsoe B, et al. Targeting interleukin-15 in patients with
rheumatoid arthritis: a proof-of-concept study. Arthritis Rheum 2005;52:2686-92.
West K. CP-690550, a JAK3 inhibitor as an immunosuppressant for the treatment of
rheumatoid arthritis, transplant rejection, psoriasis and other immune-mediated disorders.
Curr Opin Investig Drugs 2009;10:491-504.
Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human
mesenchymal stem cells. Science 1999;284:143-7.
Garcia-Castro J, Trigueros C, Madrenas J, et al. Mesenchymal stem cells and their use as
cell replacement therapy and disease modelling tool. J Cell Mol Med 2008;12:2552-65.
Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by
human marrow stromal cells: implications in transplantation. Transplantation 2003;75:389-
Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune
cell responses. Blood 2005;105:1815-22.
Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-
lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood
Francois S, Bensidhoum M, Mouiseddine M, et al. Local irradiation not only induces
homing of human mesenchymal stem cells at exposed sites but promotes their widespread
engraftment to multiple organs: a study of their quantitative distribution after irradiation
damage. Stem Cells 2006;24:1020-9.
281. Costantino G, della Torre A, Lo Presti MA, et al. Treatment of life-threatening type I
refractory coeliac disease with long-term infliximab. Dig Liver Dis 2008;40(1):74-7.
282. Gillett HR, Arnott ID, McIntyre M, et al. Successful infliximab treatment for steroid-
refractory celiac disease: a case report. Gastroenterology 2002 Mar;122(3):800-5.
283. Vivas S, Ruiz de Morales JM, Ramos F, et al. Alemtuzumab for refractory celiac disease in
a patient at risk for enteropathy-associated T-cell lymphoma. N Engl J Med. 2006 Jun
Loci identified Type of Study employed
Origin of the cohort(s) Candidate genes (function) Reference
Sweden and Norway
Ireland, Italy, USA,
36, 40, 43, 45
CELIAC 3 2q33 Candidate gene approach CTLA4 (T cell response) 38, 41, 48
CELIAC 4 19p13.1
CELIAC 5 15q11-q13
CELIAC 6 4q27
Myosin IXB (Rho-family GTPase)
RGS1 (B cell activation)
31, 33, 35, 39,
CELIAC 7 1q31 GWAS (SNPs) UK, Netherland,
Ireland, Italy, USA
31, 33, 39,47
31,33, 42 CELIAC 8 2q11-q12 GWAS (SNPs) IL18RAP
CELIAC 9 3p21 GWAS (SNPs)
31, 33, 37
CELIAC 10 3q25-q26 GWAS (SNPs) UK, Netherland,
Ireland, Italy, USA
Ireland, Italy, USA
Ireland, Italy, USA
31, 33, 39, 47
31, 33, 39,47
31, 33, 47
CELIAC 11 3q28 GWAS (SNPs) LPP (zinc binding protein)
CELIAC 12 6q25.3 GWAS (SNPs) TAGAP (T cell activation)
CELIAC 13 12q24 GWAS (SNPs)
SH2B3 (TLR intracellular adaptor,
T cell activation)
31, 33, 39, 47
(Ancient) wheat variants with low
Genetic modified wheat variants with lower
Pretreatment with lactobacilli
Transamidation of gliadin
Prolyl endopeptidases (PEP) from
a) Aspergillus niger
b) Sphingomonas capsulate in combination with
(EP)-B2 from germinating barley
Intraluminal gliadin binding by polymers
Gluten neutralizing cow’s milk antibodies
State of development
tested biopsies and gliadin reactive T cell lines
Ingested gliadin peptides
Clinical trial on 17 patients
Preclinical, tested on gliadin reactive T cell lines
Phase I clinical trial (NCT00810654)
Phase I clinical trial (NCT00626184)
Epithelial tight junctions
Dampening of the adaptive immune response
Phase IIb clinical trial (NCT00889473)
Preclinical, tested ex vivo on biopsies
tested on biopsies and gliadin reactive T cell lines
Preclinical, tested on gliadin reactive T cell lines
Abstract (see text)
ZOT receptor antagonist AT1001
“inhibitory” innate gluten peptides
250, 251, 255, 256
Gluten “vaccination” (Nexvax2)
Phase II clinical trial (NCT00671138)
Phase I-II clinical trial (NCT00879749)
Phase II clinical trial planned (NCT00540657)
Phase II clinical trial for Crohn’s disease
Phase II clinical trial for RA (NCT00433875)
Phase II clinical trial for RA, transplant rejection
Clinical trial on patients with EATL
Phase II clinical trial for Crohn’s disease
Abstract (see text)
Biologicals (systemic T cell or cytokine blockers)
Small intestine homing T cells
Gut homing T cells
CCR9 antagonists (Ccx282-B, CCX025)
Anti-integrin ?4?7 (LDP-02)
Anti-IL-15 (AMG 714),
Clonal intestinal T cells
Autologous bone marrow transplantation
Mesenchymal stem cell transplantation
Mucosal destruction in Rcd
Case reports in celiac disease
Phase II clinical trial for Crohn’s disease
Case reports in celiac disease
ACCEPTED MANUSCRIPT Download full-text