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ORIGINAL ARTICLE
Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac
intestinal mucosa and intestinal cell lines
SANDRO DRAGO
1,2
, RAMZI EL ASMAR
1
, MARIAROSARIA DI PIERRO
1,2
,
MARIA GRAZIA CLEMENTE
1
, AMIT TRIPATHI
1
, ANNA SAPONE
1
,
MANJUSHA THAKAR
1
, GIUSEPPE IACONO
3
, ANTONIO CARROCCIO
3
,
CINZIA D’AGATE
4
, TARCISIO NOT
5
, LUCIA ZAMPINI
6
, CARLO CATASSI
1,6
&
ALESSIO FASANO
1
1
Mucosal Biology Research Center, Center for Celiac Research and Division of Pediatric Gastroenterology and Nutrition,
University of Maryland, School of Medicine, Baltimore, USA,
2
Bionat Italia S.r.l., Palermo, Italy,
3
Clinica Medica,
Policlinico Universita
`
di Palermo, Palermo, Italy,
4
Azienda Ospedaliero Universitaria Policlinico-Univerita’ degli Studi di
Catania, Cattedra di Gastroenterologia, Catania, Italy,
5
Clinica Pediatrica Universita’ di Trieste and IRCCS Burlo Garofolo
Trieste, Italy, and
6
Dipartimento Scienze Materno-Infantile, Universita’ Politecnico delle Marche, Ancona, Italy
Abstract
Objective. Little is known about the interaction of gliadin with intestinal epithelial cells and the mechanism(s) through
which gliadin crosses the intestinal epithelial barrier. We investigated whether gliadin has any immediate effect on zonulin
release and signaling. Material and methods. Both ex vivo human small intestines and intestinal cell monolayers were
exposed to gliadin, and zonulin release and changes in paracellular permeability were monitored in the presence and absence
of zonulin antagonism. Zonulin binding, cytoskeletal rearrangement, and zonula occludens-1 (ZO-1) redistribution were
evaluated by immunofluorescence microscopy. Tight junction occludin and ZO-1 gene expression was evaluated by real-
time polymerase chain reaction (PCR). Results. When exposed to gliadin, zonulin receptor-positive IEC6 and Caco2 cells
released zonulin in the cell medium with subsequent zonulin binding to the cell surface, rearrangement of the cell
cytoskeleton, loss of occludin-ZO1 protein
/protein interaction, and increased monolayer permeability. Pretreatment
with the zonulin antagonist FZI/0 blocked these changes without affecting zonulin release. When exposed to luminal gliadin,
intestinal biopsies from celiac patients in remission expressed a sustained luminal zonulin release and increase in intestinal
permeability that was blocked by FZI/0 pretreatment. Conversely, biopsies from non-celiac patients demonstrated a limited,
transient zonulin release which was paralleled by an increase in intestinal permeability that never reached the level of
permeability seen in celiac disease (CD) tissues. Chronic gliadin exposure caused down-regulation of both ZO-1 and
occludin gene expression. Conclusions. Based on our results, we concluded that gliadin activates zonulin signaling
irrespective of the genetic expression of autoimmunity, leading to increased intestinal permeability to macromolecules.
Key Words: Celiac disease, gliadin, gut permeability, tight junctions, zonulin
Introduction
Gliadin, the main fraction of wheat gluten respon-
sible for the intestinal damage typical of celiac disease
(CD), is the environmental factor that triggers this
disorder [1]. It is known that CD is the result of an
inappropriate T-cell-mediated immune response
against ingested gliadin [2]. CD is associated with
the HLA alleles DQA1*0501/DQB1*0201, and in
the continued presence of gliadin the disease is self-
perpetuating [3]. One of the autoimmune targets of
CD is tissue transglutaminase (TTG) [4]. The
deamidating activity of this enzyme generates gliadin
peptide fragments that bind to DQ2 and to DQ8 so
as to be recognized by disease-specific intestinal T
cells [5]. This process activates a cascade of events in
which cytokines and matrix metalloproteinases are
Correspondence: Alessio Fasano, MD, Mucosal Biology Research Center, University of Maryland School of Medicine, 20 Penn Street, HSF II Building,
Room 345, Baltimore, Md. 21201, USA. Tel:
/1 410 706 5501. Fax: /1 410 706 5508. E-mail: afasano@mbrc.umaryland.edu
Scandinavian Journal of Gastroenterology, 2006; 41: 408/419
(Received 22 March 2005; accepted 30 June 2005)
ISSN 0036-5521 print/ISSN 1502-7708 online # 2006 Taylor & Francis
DOI: 10.1080/00365520500235334
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Maryland on 12/15/10
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up-regulated and the intestinal mucosa is destroyed
[6,7]. CD is currently regarded as a paradigm of
autoimmune disease for which the main genetic
predisposition (HLA-DQ2/DQ8), the exo-
genous trigger (gluten), and one of the autoantigens
(TTG) are known. In recent years much has been
discovered about the genetic and immuno-
logic aspects of CD [2]. However, little is known
about the possible interactions of gliadin (and/or its
peptide derivatives) with intestinal epithelia and the
mechanism(s) through which it crosses the epithelial
barrier to reach the submucosa. Under physiological
circumstances, intestinal epithelia are almost im-
permeable to macromolecules such as gliadin [8].
Several studies reported that CD is a condition in
which paracellular permeability is enhanced and the
integrity of the tight junction (tj) system is compro-
mised [9,10]. The up-regulation of zonulin, a re-
cently described intestinal peptide involved in tj
regulation [11], seems to be responsible, at least in
part, for the increased gut permeability characteristic
of CD [12]. Following stimulation of normal rat
intestinal cells (IEC6) with gliadin, zonulin is re-
leased and induces a protein kinase C-mediated
polymerization of intracellular actin filaments, which
are directly connected to tj complex proteins, thereby
regulating epithelial permeability [13]. Furthermore,
the persistent presence of inflammatory mediators
such as tumor necrosis factor-a (TNF-a) and inter-
feron-g (INF-g) have been shown to increase the
permeability across the endothelial and epithelial
layers [14,15].
The aim of this study was to investigate the early
effects of gliadin on intestinal epithelial mucosa and
the structures that dictate mucosal tj competency.
Human intestinal specimens obtained from either
treated CD or non-CD subjects and intestinal
epithelial cell lines were used. Our results provide
evidence that gliadin activates the zonulin signaling,
resulting in immediate reduction of intestinal barrier
function and passage of gliadin into the subepithelial
compartment. This process is dependent on the
presence of the zonulin receptor but independent of
individual genetic predisposition, suggesting that
gliadin-induced, zonulin-mediated paracellular per-
meability could be required but is not sufficient to
develop the autoimmune process typical of CD.
Material and methods
Intestinal cell cultures
Both human (Caco2 and T84) and rat-derived
(IEC6) intestinal cells (passages 25
/40) were cul-
tured from frozen stocks. Dulbecco’s Modified Eagle
Medium (D-MEM; Gibco, Grand Island, NY, USA)
containing 4500 mg/l D-glucose and pyridoxine
hydrochloride was supplemented with 5% heat
inactivated (568C, 30 min) fetal bovine serum
(FBS), 0.1 U/ml bovine insulin, 4 mM L-glutamine,
100 U/ml penicillin and 100 mg/ml streptomycin for
IEC-6-cell propagation, or supplemented with 10%
heat inactivated FBS, 0.1 mM non-essential amino
acids, 1.0 mM sodium pyruvate, 2 mM L-glutamine,
2.5 mg/ml amphotericin B, 50 U/ml penicillin and
50 mg/ml streptomycin for Caco2 cell propagation.
A 1:1 mixture of DMEM and Ham’s F12 medium
(D-MEM/F-12; Life Technologies) containing
15 mM HEPES, 2.5 mM L-glutamine and 0.5 mM
sodium pyruvate was supplemented with 10% heat
inactivated FBS, 100 U/ml penicillin and 100 mg/ml
streptomycin for T84-cell propagation. Cells were
cultured in a 5% CO
2
atmosphere at 378C into
6-well tissue culture plates. For the immunofluores-
cence experiments, cells were grown to sub-conflu-
ence (non-polarized), while for the Caco2
monolayer, experimental cells were grown to con-
fluence, followed by additional 10 days of growth to
allow cell differentiation.
Subcellular fractionation
Cells were grown in 175 cm
3
culture flasks until
/90% confluence was reached. An enzyme-
free solution (Gibco, Grand Island, NY, USA)
was used to detach the cells. After centrifugation
for 5 min at 1000 rpm
/48C, the medium was
discharged, cells were resuspended in 0.25 M su-
crose-20 mM Tris-Cl solution and homogenized by
passing 15 times through a G-25 needle. The
homogenate was first centrifuged for 15 min at
1000g to separate nuclei, plasma membrane sheets,
and cell debris (pellet); the supernatant obtained was
then centrifuged for 15 min at 10,000g to separate
mitochondria, lysosomes, peroxisomes (pellet); the
remaining supernatant was finally ultracentrifuged
for 1 h at 100,000g to separate all vesicles from
endoplasmic reticulum and Golgi (pellet) from the
soluble cytosolic protein fraction (supernatant).
Gliadin and casein digests
Gliadin was digested as previously described [16,17]
with minor modifications. Briefly, 50 g gliadin
(crude-wheat-Sigma, St. Louis, Mo., USA) was
dissolved in 500 ml 0.2 N HCl for 2 h at 378C
with 1 g pepsin (Sigma). The resultant peptic digest
was further digested by addition of 1 g trypsin
(Sigma) after pH adjusted to 7.4 using NaOH 2M.
The solution was stirred vigorously at 378C for 4 h,
and then boiled (1008C) for 30 min, freeze-dried,
lyophilized in 10 mg batches, and stored at
/208C
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until used. Pepsin-trypsin digested gliadin (PT-
gliadin) was freshly prepared by suspending it in
PBS to a final concentration of 1 mg/ml. Pancreatic
digested (PD)-casein (BactoTryptone, Becton Dick-
enson, Sparks, Md., USA) was used as a negative
control in all the experiments performed.
Synthesis of the zonulin peptide inhibitor FZI/0
The zonulin synthetic peptide inhibitor FZI/0, which
is capable of competitively blocking the zonulin
receptor and the consequent zonulin-dependent
opening of the tj, was identified and prepared as
previously described in structure-function analysis
studies of the Zot/zonulin receptor binding motif
[18] and was obtained from the Biopolymer Labora-
tories, University of Maryland, Baltimore, USA.
Immunofluorescence microscopy on intestinal cell lines
Cells were washed in phosphate buffered saline
(PBS) and gently detached by exposure to 0.25%
trypsin, 1 mM EDTA solution (Gibco brl) for 2
/
3 min. The cells (2/10
4
cells/ml) were suspended in
medium and seeded onto 8 chamber slides (Nalge
Nunc International, Naperville, IL, USA). At a cell
confluence of 60
/70% PT-gliadin (1 mg/ml) was
added for 30 min of incubation at 378 Cin5%CO
2
atmosphere. Cells were then washed twice in PBS,
fixed in 3.7% paraformaldeyde in PBS (pH 7.4) for
15 min at room temperature. To analyze intracellular
F-actin, cells were permeabilized with 0.5% Tri-
tonX-100 in PBS (Sigma) for 10 min at room
temperature and incubated for 30 min with 0.3 mM
solution of FITC-phalloidin (SIGMA). To study
zonulin surface binding, cells were incubated with
zonulin-specific anti-Zonula occludens toxin (Zot)
^G antibodies (10 mg/ml) [18] for 1 h at room
temperature without permeabilization, washed 3
times in PBS and than incubated for 30 min at
room temperature with the anti-rabbit FITC-con-
jugated antiserum (SIGMA). After 3 additional
washes, the cover slips were mounted with gly-
cerol-PBS (1:1) at pH 8.0. The results were blindly
analyzed by two independent observers using a
fluorescence microscope (Zeiss) and images taken
with a digital camera (Nikon).
Immunofluorescence microscopy on intestinal tissues
These experiments were performed as previously
described [19]. Briefly, small-bowel biopsies
obtained by endoscopy were freshly embedded in
OCT compound and stored at
/808C. Cryosec-
tions of 5
/7 mm were incubated with zonulin-spe-
cific, polyclonal anti-Zot antibodies, extensively
washed, and incubated with fluorescein isothiocya-
nate (FITC)-conjugated goat anti-rabbit antibody
(Sigma) in the dark. After repeated washing, the
tissue sections were mounted on slides with mount-
ing medium and blindly examined under fluores-
cence microscopy.
Direct immunofluorescence and ZO1 localization
migration in intestinal cell monolayers
Intestinal cells were grown on 8-chamber slides
using the growth conditions outlined above and
incubated with either PT-gliadin or the negative
control PT-casein (final concentration 1 mg/ml) at
increasing time intervals. PBS-exposed monolayers
were used as additional controls. Cells were then
washed gently with PBS and permeated with metha-
nol at
/208C for 2 min. The monolayers were then
washed three times with PBS, and incubated with
primary antibodies (FITC-conjugated anti-ZO1
monoclonal antibody; Zymed Laboratories Inc.,
San Francisco, Calif., USA). After 60 min of
incubation, the slides were washed twice with PBS,
air-dried, and blindly analyzed by fluorescence
microscopy.
Caco2 monolayers experiments
Culture conditions. Caco2 cells (passage 25
/40) were
grown on filter clusters until confluence (average
10
/15 days post-seeding); 200 ml DMEM was
added to the mucosal (apical) side and 3 ml of the
same medium was added to the serosal (basolateral)
side. The system was incubated at 378Cinan
atmosphere of 95% air and 5% CO
2
. The culture
medium was changed every third day. The mono-
layer was washed with PBS twice and incubated with
DMEM supplemented as above but without anti-
biotics. Replicates of Caco2 monolayers were incu-
bated at increasing time intervals with either 1 mg/ml
PT-gliadin or 1 mg/ml PD-casein, used as control.
Both preparations were added to the mucosal
(apical) side of the Caco2 monolayers. Medium
samples for each compartment were obtained to
test for zonulin release and passage of lactulose.
Transepithelial electrical resistance (TEER)
measurements. The baseline TEER of Caco2 mono-
layers grown on filter clusters was measured using
a dual planar electrode (Endhom SNAP Evom G
WPI analyzer; World Precision Instruments) and
expressed in V.cm
2
. TEER values were measured for
each incubation time after the addition of PT-gliadin
and PD-casein and corrected for the baseline resis-
tance values.
410 S. Drago et al.
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Measurement of lactulose flux from the apical to
basolateral side of Caco2 monolayers. Lactulose, a
probe used to check paracellular permeability, was
added at 40 mM/ml final concentration to the apical
side of all monolayers at time 0. Samples were
collected from the basolateral side at increasing
time intervals and lactulose concentration measured
by high performance anion exchange chromato-
graphy (HPAEC). After deproteination with aceto-
nitrile 1:1 v/v, samples were centrifuged at 4000 rpm
for 10 min, the supernatant collected, filtered
through a 0.22 mm membrane (Millipore, Bedford,
Mass., USA), and diluted with water 1 to 10
(basolateral samples) or 1 to 100 (apical samples).
For HPAEC analysis we used an HPAEC-PAD
system (Pulsed Amperometric Detector) 4500 Dio-
nex, comprising a CarboPac PA-1 column (4
/
250 mm) (Dionex), in serial with a precolumn
CarboPac PA-1 Guard (3
/25 mm) (Dionex); a
pulsed amperometric detector (PAD II) connected
by a gold electrode.
Intestinal tissue from CD and non-CD patients
Samples of small-intestine mucosa were taken from
the second/third portion of the duodenum from
subjects undergoing upper gastrointestinal (GI)
endoscopy. Patients included: 1) subjects with active
CD at diagnosis; 2) subjects with CD on treatment
with a gluten-free diet for at least 2 years, and
3) non-CD GI controls with persistent dyspeptic
complaints. All patients had clinical indications for
the procedure and gave their informed consent to
undergo an additional biopsy for the purpose of this
study. The study protocol was approved by the
Ethics Committee of the University of Ancona.
The small-intestine biopsies were oriented under a
dissecting microscope (16
/) with the villi facing
upward and mounted onto the modified microsnap-
well system [20]. The study was then conducted
as described above for the Caco2 monolayers
experiments.
Zonulin determination by sandwich enzyme-linked
immunosorbent assay (ELISA)
Zonulin content from either cell cultures (both cell
culture media and subcellular protein fractions) or
intestinal segments mounted in the microsnapwell
system was quantified using a sandwich ELISA as
previously described [20].
Quantitative polymerase chain reaction with the
TaqMan procedure
To analyze the effect of both acute and prolonged
intestinal exposure to gliadin on tj structural ele-
ments, occludin and ZO-1 genes expression was
analyzed by real-time PCR [21]. Cytokine TNF-a
gene expression was also determined using the same
technology. Total tissue RNA was extracted
(QiAamp RNA Blood Mini Kit; QIAGEN Inc.,
Valencia, Calif., USA) from each intestinal frag-
ment (from both CD and non-CD subjects)
mounted in the microsnapwell system. In order to
eliminate any DNA traces, the RNA obtained was
treated with deoxyribonuclease I, amplication grade,
according to the manufacturer’s instructions (Invi-
trogen Carlsbad, Calif., USA) and the reverse
transcription of RNA to cDNA was performed using
GeneAmp RNA PCR kit components (Perkin Elmer
Applied Biosystems, Forster City, Calif., USA). The
cycling protocol consisted of 428C 15 min, 998C
5 min. To quantify the occludin and ZO-1 RNA
level, primers (ZO-1: Forward: 5?-TTAAGC-
CAGCCTCTCAACAGAAA, Reverse: 5?-GGT
TGA TGA TGC TGG GTT TGT. Occludin:
Forward: 5?- TAT AAA TCC ACG CCG GTT
CCT, Reverse: 5?- ACG AGG CTG CCT GAA
GTC AT) and probe (ZO-1: 6FAM- ATC TCC
AGT CCC TTA CCT TTC GCC TGA A-
TAMRA. Occludin: 6FAM- AAG TGG TTC
AGG AGC TTC CAT TAA CTT CGC-TAMRA)
was designed (PRIMER EXPRESS software Perkin
Elmer Biosystems) and used in a multiplex real-time
PCR assay. Primers and probe were obtained from
Oligo Synthesis Group from Applied Biosystems.
For each PCR reaction a primers pair and probe
(VIC Probe) amplifying the human GAPDH was
used as an internal positive control (IPC) (TaqMan
†
RNA Control Reagents; Perkin Elmer Applied
Biosystems). To analyze the TNF-a expression, the
cDNA was amplified by using Pre-Developed Taq-
Man Assay Reagents (20
/, Perkin Elmer Applied
Biosystems).
PCR reagents consisted of TaqMan Universal
PCR Master Mix (50 mM KCl, 10 mM Tris-HCl,
0.01 M EDTA, 60 nM Passive Reference, pH 8.3 at
room temperature, 3.5 mM MgCl2, 200 mMdATP,
200 mM dCTP, 200 mM dGTP, 400 mM dUTP,
0025 U/mL AmpliTaq Gold, 0.01 U/ml Ampera-
seUNG), forward primers 0.6 mM, reverse primers
0.6 mM TaqMan probes 0.2 mM, 5 ml of target
cDNA and DEPC water up to 50 ml final volume.
Cycle parameters were 508C for 10 min (1 cycle),
958C for 10 min (1 cycle), 958C for 30 s
/608C for
1 min (40 cycles). (ABI Prism 7700 Sequence
Detection System; Perkin Elmer Applied BioSys-
tems). The target cDNA in duplicate and plasmid
DNA (prRNA18S) as standard DNA were included
in a single 96-well plate.
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Construction of standard plasmid DNA
In order to quantify occludin, ZO-1 and TNFa
mRNA levels we constructed a recombinant plasmid
to produce a standard DNA molecule (prGAPDH).
Briefly, the cDNA encoding human GAPDH gene
was amplified by PCR from a cDNA template
derived from human intestine; the amplicon
obtained was analyzed using agarose gel electrophor-
esis (2%). Finally, the PCR product was cloned into
the PCR
†
2.1-TOPO
†
vector, as recommended by
the manufacturer (TOPO TA Cloning Kit; Invitro-
gen). Ten-fold dilutions from 10 ng/reaction to 1 fg/
reaction were used in TaqMan procedures as stan-
dard DNA.
Statistical analysis
All values are expressed as means9
/SE (standard
error). The analysis of differences was performed by
t-test for either paired or unpaired varieties. A p -
value B
/0.05 was considered statistically significant.
Results
Experiments on intestinal cell lines
Effect of PT-gliadin on zonulin release, actin
cytoskeleton, and surface binding in intestinal epithelial
cells. Human intestinal epithelial cells Caco2 and
T84 as well as rat small-intestine IEC6 cells ex-
posed to PT-gliadin showed zonulin release in the
culture medium, irrespective of the cell line chal-
lenged (Figure 1A). Conversely, only zonulin recep-
tor-positive IEC6 and Caco2 cells [22] showed
zonulin surface immunostaining, while no signal
was detectable in zonulin receptor-negative T84
cells (Figure 1B). Caco2 cell subfractionation re-
vealed that the endoplasmic reticulum/Golgi
fraction was the most enriched in zonulin content
(1.149
/0.03 ng/mg protein) as compared with the
plasma membrane (0.059
/0.07 ng/mg protein), cy-
toplasm (0.289
/0.08 ng/mg protein), and mito-
chondria (0.169
/0.14 ng/mg protein) fractions. We
have already reported that zonulin binding in IEC6
cells was associated with an early reorganization of
intracellular actin filaments characterized by a redis-
tribution of F-actin to the cell subcortical compart-
ment [13]. Here, we report similar results obtained
in Caco2 cells (Figure 2), whereas no significant
changes were observed in T84 cells (Figure 2) in
which the zonulin release (Figure 1A) was not
followed by its binding to the cell surface (Figure
1B). Two hours after gliadin withdrawal, the reorga-
nization of the actin cytoskeleton recovered, and the
cytoskeleton had returned to its basal state (data not
shown).
Direct immunofluorescence microscopy of ZO-1
localization on intestinal cell monolayers exposed to PT-
gliadin. ZO-1 is one of the best-characterized com-
ponents of the junctional complex and it localizes in
the cytoplasmic submembranous plaque underlying
intercellular tj. Its presence at the cell boundary is
considered a sign of tj competency [23]. Immuno-
fluorescence analysis of both Caco2 and IEC6 cells
was used to establish whether the zonulin release and
actin rearrangement following PT-gliadin exposure
could affect the distribution of ZO-1. Casein was
used as a food-derived protein negative control. Both
untreated Caco2 monolayers and PD-casein-
exposed Caco2 monolayers showed the typical
ZO1 localization at the cell periphery 90 min post-
incubation (Figure 3). Conversely, PT-gliadin-
exposed cells showed a fluorescent irregular pattern
at the edge of the cells due to a redistribution of the
Figure 1. Zonulin release and surface binding in intestinal
epithelial cell cultures following gliadin exposure. A. Zonulin
release was detected in the medium of PT-gliadin-exposed cells
(closed bars) but not in PD-casein-exposed cells (open bars). B.
Immunofluorescence analysis also revealed that this release was
associated with zonulin binding on the surface of zonulin receptor-
positive Caco2 and IEC6 cells (see arrows ) but not zonulin
receptor-negative T84 cells. Magnification:
/40; n/4/8;
*p B
/0.0001; **p B/0.003; ***p B/0.009.
412 S. Drago et al.
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ZO1 protein, (Figure 3). Reciprocal co-immunopre-
cipitation experiments showed a time-dependent
disassociation between ZO-1 and occludin (data
not shown) that, combined with the immunofluor-
escence results, is suggestive of tj disassembly [24].
Effect of PT-gliadin on Caco2 monolayers barrier
function. The addition of PT-gliadin to the mucosal
aspect of Caco2 monolayers induced a signi-
ficant decrease in TEER, while no changes were
detected in monolayers challenged with PD-casein
(Figure 4A). The loss of barrier function was
preceded by a significant luminal (but not basolat-
eral) secretion of zonulin from cells incubated with
PT-gliadin (Figure 4B). No detectable zonulin was
found in monolayers exposed to PD-casein (Figure
4B). To confirm that the zonulin-mediated TEER
decrease involved the opening of intercellular tj, the
mucosal-to-serosal transport of the paracellular
marker lactulose was also monitored. In monolayers
challenged with PT-gliadin, an increase in serosal
lactulose (0.0809
/0.03 mg/ml) was observed 90 min
after PT-gliadin exposure compared to untreated
monolayers (0.0259
/0.005, N /7, p B/0.01 com-
pared to PT-gliadin-treated monolayers). Pretreat-
ment with the competitive zonulin antagonist FZI/0
prevented the increased paracellular lactulose trans-
port (0.029
/0.02), further supporting the hypothesis
that the PT-gliadin-induced changes in TEER and
paracellular permeability are zonulin dependent.
Experiments on human intestinal tissues
Zonulin release and TEER changes in human intestinal
tissues of CD patients in remission and non-CD patients
exposed to PT-gliadin. In addition to the up-regulation
of zonulin in acute CD, we have previously reported
that gliadin activates the zonulin-signaling pathway
even when added to normal rabbit intestinal epithe-
lium [13]. Based on these results, we studied the
effect of PT-gliadin on the zonulin signaling ex vivo
by using human duodenal biopsies obtained during
routine diagnostic endoscopies from both CD
patients in remission and non-CD subjects. The
tissues were mounted in the microsnapwell system
[20] immediately after their collection and zonulin
release and TEER were monitored both at base-
line and following exposure to either PT-gliadin or
negative control. At baseline, the zonulin con-
centration in tissue culture media collected from
the luminal side of biopsies from CD patients
Figure 2. Effect of gliadin on intestinal epithelial cells cytoskele-
ton. Incubation of both Caco2 and IEC6 cells with PT-gliadin
leads to a reorganization of actin filaments characterized by
their redistribution to the cell subcortical compartment. F-actin
fluorescence pattern remains unchanged in T84 cells exposed to
PT-gliadin. Magnification:
/40.
Figure 3. Effects of PT-gliadin on the junctional complex protein ZO-1 localization in Caco2 cells. Human intestinal Caco2 cells were
exposed to PT-gliadin, fixed after 90 min post-incubation, and immunostained using anti-ZO1 antibodies. Control monolayers show the
typical ZO-1 localization at the periphery of the cells. Monolayers exposed to PT-gliadin show different degrees of ZO-1 redistribution, with
areas with reduced or loss of ZO-1 staining at the edge of the cell and areas in which no significant changes were detected. No changes in
ZO-1 localization were detected in monolayers exposed to PD-casein.
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(0.679/0.13 ng/mg protein) was greater than that
from non-CD controls (0.029
/0.01, p B/0.01). This
baseline zonulin up-regulation was paralleled by a
lower baseline TEER in tissues obtained from CD
patients in remission (409
/8.5 Vcm
2
) as com-
pared to tissues obtained from non-CD patients
(1339
/12.8, p B/0.001) (see below). Following PT-
gliadin exposure, two different responses were ob-
served. Tissue from CD patients showed a sustained
zonulin release into the mucosal (but not serosal)
medium that was higher than the baseline levels,
beginning as early as 5 min post-incubation
(Figure 5A) and persisting for ]
/1 h. Conversely,
biopsies from non-CD patients exhibited transient
increases in luminal but not serosal zonulin levels
that peaked at 15 min and returned to baseline
within 30 min (Figure 5A). The zonulin release in
both groups was temporally associated with the
observed changes in TEER (Figure 5B). While the
Figure 4. Transepithelial electrical resistance (TEER) and zonu-
lin secretion changes in Caco2 monolayers exposed to PT-gliadin.
A. Monolayers exposed to PT-gliadin (squares) show significant
TEER decrease (*p B
/0.01; **p B/0.005) starting 90 min post-
incubation compared to both media control monolayers (circles)
and PT-casein-exposed monolayers (triangles). B. Kinetics of
zonulin release from Caco2 monolayers exposed to media control
(circles), inoculated with PT-gliadin (squares), or casein (trian-
gles). PT-gliadin-exposed monolayers show a significantly higher
zonulin release starting 60 min post-incubation compared to
both control and PD-casein-challenged monolayers (*p B
/0.01;
**p B
/0.005); n /3/5 determinations.
*
**
**
**
0
20
40
60
80
100
120
140
160
0 5 15 30 60
Time (min)
TEER (Ω.cm
2
)
*
**
**
**
A
B
Figure 5. Zonulin release and transepithelial electrical resistance
(TEER) changes in intestinal biopsies obtained from celiac
disease (CD) patients in remission and non-CD patients
exposed to PT-gliadin. Intestinal duodenal biopsies were
mounted in microsnapwell chambers and polarized zonulin
release and changes in TEER monitored. A. Zonulin release.
Following exposure to PT-gliadin, tissues obtained from CD
patients showed a luminal (squares) but not serosal (diamonds)
zonulin release. Conversely, biopsies from non-CD patients
showed a transitory zonulin release in the mucosal (triangles)
but not serosal (circles) media that reached its peak at 15 min
post-PT-gliadin incubation and returned to baseline within 30
min. *p B
/0.005 compared to serosal zonulin n/17 CD patients
and n /5 non-CD controls. B. TEER changes. Tissues obtained
from both CD patients in remission and non-CD controls
showed a TEER decrement temporally associated with zonulin
release. In CD tissues, a significant drop in TEER compared
to baseline was observed starting 15 min post-PT-gliadin
incubation (squares). No significant changes were detected in
negative controls exposed tissues (diamonds). Conversely, in
tissues from non-CD patients, a significant decrease in TEER
was observed only after 60 min post-PT-gliadin incubation
(triangles). Again, no changes were detected in tissues exposed
to negative controls (circles). *p B
/0.05; **p B/0.001 compared
to respective negative controls; n /17 CD patients and n /5
non-CD controls.
414 S. Drago et al.
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TEER decrease in tissues obtained from CD patients
reached significance 15 min following PT-gliadin
exposure, the TEER changes in tissues from non-
CD subjects did not reach statistical significance
until 60 min after PT-gliadin incubation (Figure 5B).
The TEER of CD patient-derived tissues was always
lower than the TEER of non-CD tissues, irrespective
of gliadin exposure and time of incubation (Figure
5B). No significant changes were detected in either
group when tissues were exposed to the negative
control (Figure 5B).
Effect of the zonulin inhibitor FZI/0 on TEER changes.
To establish whether the PT-gliadin-induced zonulin
release and TEER changes were causally re-
lated, intestinal biopsies obtained from CD patients
in remission were pretreated with the zonu-
lin inhibitor FZI/0 prior to PT-gliadin exposure.
Tissue preincubation for 15 min with FZI/0 did not
affect zonulin release (Figure 6B) but did prevent the
drop in PT-gliadin-induced TEER (Figure 6A).
These results provide direct evidence that
the gliadin-induced TEER changes are zonulin
dependent.
Zonulin binding to human intestinal tissues following
exposure to PT-gliadin. To establish whether PT-
gliadin-induced zonulin release was followed by
zonulin binding to the intestinal epithelium (as
observed in intestinal cell lines, see above), in situ
immunofluorescence microscopy studies using zo-
nulin-specific anti-Zot antibodies were performed.
Tissues obtained from CD patients in remission and
exposed to PT-gliadin showed a homogeneous stain-
ing on the mucosal surface (Figure 7A), while no
zonulin binding was detected on tissues exposed to
negative controls (Figure 7B). Tissues from non-CD
patients exposed to PT-gliadin also showed mucosal
surface staining; however, the zonulin binding
appeared patchy (Figure 7C), suggesting less bind-
ing to its surface receptor.
Effect of both acute and chronic exposure to gliadin on
ZO1 and occludin gene expression. Occludin and ZO-1
are considered key elements on the tj complex and
their expression and localization dictate tj compe-
tency. To establish whether the TEER changes
induced by PT-gliadin were associated with modifi-
cation in tj occludin and ZO-1 expression, RNA
levels of the two proteins were quantified by real-
time PCR
21
following both acute (ex vivo) and
chronic (in vivo) exposure to gliadin.
Acute exposure. Human intestinal tissues mounted in
the microsnapwell assay (see above) were harvested
at increasing time intervals, mRNA extracted and
both ZO-1, and occludin quantitative expression
measured by real-time PCR. Tissues obtained from
CD patients in remission and exposed to PT-gliadin
showed a progressive decrement in occludin but not
ZO-1 gene expression up to 60 min post-incubation
(Table I). Tissue from non-CD patients and exposed
to PT-gliadin also showed a decrement in occludin
genes expression, but the reduction was less pro-
nounced as compared with that seen in CD patients
(Table I). No significant changes in TNF-a gene
expression were detected in both groups, further
supporting our previous observation that the TEER
Figure 6. Effect of the zonulin inhibitor FZI/0 on PT-gliadin
induced zonulin release and transepithelial electrical resistance
(TEER) changes in duodenal tissues from celiac disease (CD)
patients in remission. Duodenal tissues from CD patients in
remission were mounted in the microsnapwell system and exposed
to PT-gliadin, either alone or following 15 min preincubation with
FZI/0. A. The TEER decrement induced by PT-gliadin (squares)
was prevented by pretreatment with FZI/0 (10 mg/ml) (triangles).
PD-casein-treated tissues (circle) are shown as negative controls.
B. FZI/0 pretreatment (triangles) did not affect the zonulin release
by PT-gliadin (squares) (B). *p B
/0.02 compared with PT-gliadin;
n
/17.
Gliadin and intestinal mucosa biology 415
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changes following gliadin-induced zonulin release
are not mediated by cytokine production [20].
Chronic exposure. Intestinal tissues obtained from CD
patients (both during the acute phase of the disease
and in remission following a gluten-free diet) as
well as from non-CD patients were immediately
processed following their collection for mRNA
extraction in order to perform ZO-1 and occludin
quantitative gene expression by real-time PCR.
Patients during the acute phase of CD showed
significant lower expression of both genes compared
to non-CD controls (Figure 8). Conversely, CD in
remission showed ZO-1 and occludin gene expres-
sion levels similar to those observed in non-CD
controls (Figure 8).
Discussion
During the past decade, progress has been made in
elucidating the biochemical characteristics of the
multiple gluten epitopes and in understanding the
activation of the immune system by these immuno-
modulatory/cytotoxic peptides [25]. Alpha-gliadin,
which we tested in this study, is a well-characterized
gluten fraction containing several different peptides
that are capable of activating the immune response
that is seen in CD patients [2]. Despite these
advances in understanding the pathogenesis of CD,
little is known about how gliadin actually gains access
to the subepithelial compartment in order to interact
with the gut-associated lymphoid tissue (GALT) and
stimulate autoimmunity. The enterocyte brush-bor-
der membrane has carrier systems (PepT1 and
PepT2) that facilitate the transcellular transport of
di-/tripeptides, while transport of larger peptides is
mainly limited to the paracellular route owing to the
physical-chemical characteristics of the cellular phos-
pholipid membrane [26,27]. Two, not necessarily
mutually exclusive pathways have been proposed to
explain the passage of gluten peptides in celiac
disease: transcellular transport [28] and para-
cellular permeability[12,13]. While neither pathway
has been validated as the definitive locus of entry of
immunologically active gliadin fragments into the
intestinal submucosa, the following observations
suggest that the paracellular pathway is of particular
importance in the pathogenesis of CD. The associa-
tion of increased small intestinal permeability with
CD is well established, and clinical studies have
shown that patients with active CD demonstrate
elevated fractional sugar permeability due to in-
creased intestinal paracellular passage of larger
probes (disaccharides, such as cellobiose and lactu-
lose) [29]. Abnormal paracellular permeability pre-
cedes the development of pathology in an animal
model of gluten-sensitive enteropathy [30]. A high
proportion of 1st degree relatives of CD patients have
an increased urinary lactulose/mannitol ratio without
Figure 7. Zonulin surface binding on intestinal biopsies from celiac
disease (CD) patients in remission and non-CD patients following
PT-gliadin exposure. A. Intestinal biopsies from CD patients in
remission exposed to PT-gliadin showed a homogeneous zonulin
binding on the mucosal surface (arrows ) and an increase in staining
in the submucosa. B. No surface staining was observed in tissues
exposed to negative control. C. Tissues obtained from non-CD
patients and exposed to PT-gliadin also showed zonulin surface
staining (arrows ) but the binding pattern was patchy. Magnification:
/40.
416 S. Drago et al.
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developing the disease, suggesting that the increased
intestinal permeability and the gliadin-induced in-
testinal damage are two distinct phenomena [31].
Acute gliadin-challenge studies in celiac patients in
remission suggest that one of the immediate con-
sequences of gluten exposure is increased paracellu-
lar permeability, occurring within 36 h [32]. Taken
together, these observations suggest that increased
permeability is an early event and not just a conse-
quence of chronic intestinal inflammation and par-
enchymal injury, and that paracellular permeability
may represent the key route through which gliadin
initially gains access into the submucosa.
Zonulin, a pathway that modulates tj competency,
is involved in innate immunity [20] and is up-
regulated in both CD [12] and type 1 diabetes
[33,34]. In the BB/wor rat animal model of type 1
diabetes, increases in zonulin-mediated permeability
precede the onset of the autoimmune process by 3
/4
weeks and are prevented by pretreatment with
zonulin inhibitor [34]. Therefore, it is conceivable
that a similar temporal relationship between expo-
sure to environmental agents, loss of intestinal
barrier function, and onset of pathology occurs in
CD.
Our results suggest that PT-gliadin peptides in-
duce a series of early mucosal events including
zonulin release and binding to receptor-positive cells
(Figure 1A and B), cytoskeleton rearrangement
(Figure 2), ZO-1 displacement from the junctional
complex (Figure 3) and tj disassembly (Figures 4
and 5).
We have previously reported that the interaction of
enteric bacteria with intestinal mucosa induces
zonulin release as a host-driven innate immunity
response [20]. Given the complexity of the cell
signaling events and intracellular structures involved
in the zonulin system [35], it is not surprising that
this pathway may be up-regulated in response to
proximal bowel contamination. Intestinal barrier
dysfunction, triggered by bacterial infection, has
previously been hypothesized as a key factor in
several pathological conditions involving the GI tract
[36
/38]. Increased tj permeability seems to be a
common denominator in these diseases and may be
responsible for the repeated passage of luminal
antigens to the mucosal immune system. The
response to this submucosal exposure is dictated by
host genetic predisposition either to recognize or
misinterpret environmental antigens when these are
presented to the GI tract [39]. The results of this
Table I. Occludin, ZO-1, and TNF-a gene expression in both celiac and non-celiac human intestine after acute exposure to gliadin in
microsnapwells.
Occludin
*
ZO-1
*
TNF-a
?
Controls CD patients Controls CD patients Controls CD patients
0 min 811,6359
/9,456 824,6819/12,811 703,2939/12,594 722,1669/17,451 675,8479/13,547 577,7469/11,547
5 min 784,2309/15,689 772,8019/13,141 692,0399/6,939 755,2309/5,170 668,5919/9,004 573,4469/4,004
15 min 763,7549/1,633 723,8149/7,603 687,8649/9,697 781,3499/12,451 698,3459/10,251 605,5589/11,840
30 min 778,6549
/3,256 750,4049/4,638 675,8479/4,454 751,3499/10,451 628,2139/12,890 590,9749/11,001
60 min 766,5329/2,986 735,6359/12,580 690,5529/3,945 748,2919/16,451 648,6729/18,255 593,5889/8,256
^(60
/0 min) /45,1039/9,748 /89,0469/21,110 /12,7419/7,358 /26,1259/13,104 /27,1759/29,884 /15,8429/11,475
p (vs time 0) 0.05 0.01
**
NS NS NS NS
N copies/10
6
GAPDH.
**
p B/0.05 compared to controls; n /9.
Figure 8. ZO-1 and occludin genes expression following chronic
exposure to gliadin. Intestinal tissues obtained from celiac disease
(CD) patients (during the acute phase of the disease and also after
remission following a gluten-free diet) and from non-CD patients
were treated for mRNA extraction immediately following their
collection. Quantitative real-time polymerase chain reaction
(PCR) using specific primers for ZO-1 (closed bars) and occludin
(open bars) genes was performed and the results normalized by
the number of GAPDH gene copies. Expression of both ZO-1 and
occludin genes was suppressed in CD patients during the active
phase of the disease compared to non-CD controls. Expression of
ZO-1 and occludin genes reverted to normal values during the
disease remission following a gluten-free diet. *p B
/0.01 compared
to both controls and treated celiacs; n
/9 for each group.
Gliadin and intestinal mucosa biology 417
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Maryland on 12/15/10
For personal use only.
study suggest that a direct effect of gliadin on the
activation of the zonulin system can be proposed as a
driver for antigen access to the GALT via the
paracellular route.
Recent reports suggest that CD is one of the most
common chronic genetic disorders of humankind
[40,41]. This observation raises an interesting ques-
tion. Why is a disease with significant morbidity and
increased mortality not segregated by genetic evolu-
tion, and why does it remains so frequently ex-
pressed? One possible explanation is that gluten, a
protein introduced in large quantities in the human
diet only after the advent of agriculture, activates
mechanisms of innate immunity (e.g. zonulin signal-
ing) that are designed to protect against proximal
bowel contamination and are too important for
human survival to be eliminated. This hypothesis is
further supported by recent reports on the role of
early dietary introduction of gluten and the increased
risk of developing type 1 diabetes [42,43].
This also implies that gliadin should activate the
zonulin system irrespective of the genetic predisposi-
tion to autoimmunity. Our results obtained on
intestinal tissues from both CD and non-CD
patients (Figure 5) support this theory. Nevertheless,
zonulin is markedly up-regulated in subjects affected
by CD, even when treated with a gluten-free diet
(Figure 5A). This up-regulation is associated with
increased baseline gut permeability (Figure 5B), and
an increased amplitude and duration of gluten-
induced zonulin release when compared with non-
CD intestinal samples. Despite the presence of a
measurable zonulin response in both CD and non-
CD subjects, CD patients appear to reach a critical
threshold of intestinal permeability upon gliadin
exposure that is not reached in non-CD intestinal
mucosa (Figure 5). Pretreatment with the zonulin
inhibitor FZI/0 prevents the gliadin-induced TEER
decrement, confirming that zonulin is the mediator
of these gluten-induced changes and possibly paving
the way for treatment alternatives to a gluten-free
diet.
Acknowledgements
This study was partially supported by the National
Institute of Health, Grants DK-48373 and
DK-66630 (A.F.). Sandro Drago and Ramzi El
Asmar contributed equally to the execution of this
project.
References
[1] Trier JS. Diagnosis of celiac sprue. Gastroenterology 1998;/
115:/211/6.
[2] Schuppan D. Current concepts of celiac disease pathogen-
esis. Gastroenterology 2000;
/119:/234/42.
[3] Sollid LM, Thorsby E. HLA susceptibility genes in celiac
disease: genetic mapping and role in pathogenesis. Gastro-
enterology 1993;
/105:/910/22.
[4] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken
EO, et al. Identification of tissue transglutaminase as the
autoantigen of celiac disease. Nat Med 1997;
/3:/797/801.
[5] Molberg O, McAdam SN, Sollid LM. Role of tissue
transglutaminase in celiac disease. J Pediatr Gastroenterol
Nutr 2000;
/30:/232 /40.
[6] Molberg O, Mcadam SN, Korner R, Quarsten H, Kristian-
sen C, Madsen L, et al. Tissue transglutaminase selectively
modifies gliadin peptides that are recognized by gut-derived
T cells in coeliac disease. Nat Med 1998;
/4:/713/7.
[7] Pender SL, Tickle SP, Docherty AJ, Howie D, Wathen NC,
MacDonald TT. A major role for matrix metalloproteinases
in T cell injury in the gut. J Immunol 1997;
/158:/1582/90.
[8] Fasano A. Modulation of intestinal permeability: an innova-
tive method of oral drug delivery for the treatment of
inherited and acquired human diseases. Molec Genet Metab
1998;
/64:/12/8.
[9] Madara JL, Trier JS. Structural abnormalities of jejunal
epithelial cell membranes in celiac sprue. Lab Invest 1980;
/
43:/254 /61.
[10] Schulzke JD, Bentzel CJ, Schulzke I, Riecken EO, Fromm
M. Epithelial tight junction structure in the jejunum of
children with acute and treated celiac sprue. Pediatr Res
1998;
/43:/435/41.
[11] Wang W, Uzzau S, Goldblum SE, Fasano A. Human
zonulin, a potential modulator of intestinal tight junctions.
J Cell Sci 2000;
/113:/4435/40.
[12] Fasano A, Not T, Wang W, Uzzau S, Berti I, Tommasini A,
et al. Zonulin, a newly discovered modulator of intestinal
permeability, and its expression in coeliac disease. Lancet
2000;
/355:/1518/9.
[13] Clemente MG, De Virgiliis S, Kang JS, Macatagney R,
Musu MP, Di Pierro MR, et al. Early effects of gliadin on
enterocyte intracellular signalling involved in intestinal
barrier function. Gut 2003;
/52:/218/23.
[14] Leach L, Firth JA. Structure and permeability of human
placental microvasculature. Microsc Res Tech 1997;
/38:/137/
44.
[15] Madsen KL, Lewis SA, Tavernini MM, Hibbard J, Fedorak
RN. Interleukin 10 prevents cytokine-induced disruption of
T84 monolayer barrier integrity and limits chloride secre-
tion. Gastroenterology 1997;
/113:/151/9.
[16] De Ritis G, Occorsio P, Auricchio S, Gramenzi F, Morisi G,
Silano V. Toxicity of wheat flour proteins and protein-
derived peptides for in vitro developing intestine from rat
fetus. Pediatr Res 1979;
/13:/1255/61.
[17] Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM,
et al. Structural basis for gluten intolerance in celiac sprue.
Science 2002;
/297:/2275/9.
[18] Di Pierro M, Lu R, Uzzau S, Wang W, Margaretten K,
Pazzani C, et al. Zonula occludens toxin structure-function
analysis. Identification of the fragment biologically active on
tight junctions and of the zonulin receptor binding domain. J
Biol Chem 2001;
/276:/19160/5.
[19] Fasano A, Uzzau S, Fiore C, Margaretten K. The enter-
otoxic effect of zonula occludens toxin on rabbit small
intestine involves the paracellular pathway. Gastroenterology
1997;
/112:/839/46.
[20] El Asmar R, Panigrahi P, Bamford P, berti I, Not T, Coppa
GV, et al. Host-dependent zonulin secretion causes the
impairment of the small intestine barrier function after
bacterial exposure. Gastroenterology 2002;
/123:/1607/15.
[21] Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K.
Oligonucleotides with fluorescent dyes at opposite ends
418 S. Drago et al.
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Maryland on 12/15/10
For personal use only.
provide a quenched probe system useful for detecting PCR
product and nucleic acid hybridization. PCR Methods Appl
1995;
/4:/357/62.
[22] Uzzau S, Lu R, Wang W, Fiore C, Fasano A. 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.
[23] Mitic LL, Anderson JM. Molecular architecture of tight
junctions. Annu Rev Physiol 1998;/60:/121 /42.
[24] Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita
S. Direct binding of three tight junction-associated
MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini
of claudins. J Cell Biol 1999;
/147:/1351/63.
[25] Koning F. The molecular basis of celiac disease. J Mol Recog
2003;/16:/333/6.
[26] Daniel H. Molecular and integrative physiology of intestinal
peptide transport. Annu Rev Physiol 2004;
/66:/361 /84.
[27] Walker WA. Antigen handling by the small intestine. Clin
Gastroenterol 1986;/15:/1 /20.
[28] Matysiak-Budnik T, Candalh C, Dugave C, Namane A,
Cellier C, Cerf-Bensussan, et al. Alterations of the intestinal
transport and processing of gliadin peptides in celiac disease.
Gastroenterology 2003;
/125:/696/707.
[29] Catassi C, Pierani P, Natalini G, Gabrielli O, Coppa GV,
Giorgi PL. Clinical application of a simple HPLC method
for the sugar intestinal permeability test. J Pediatr Gastro-
enterol Nutr 1991;
/12:/209/12.
[30] Hall EJ, Batt RM. Abnormal permeability precedes the
development of a gluten sensitive enteropathy in Irish setter
dogs. Gut 1991;
/32:/749/53.
[31] van Elburg RM, Uil JJ, Mulder CJ, Heymans HS. Intestinal
permeability in patients with celiac disease and relatives of
patients with celiac disease. Gut 1993;
/34:/354/7.
[32] Di Cagno R, De Angelis M, Auricchio S, Greco L, Clarke C,
De Vincenzi M, 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.
[33] Sapone A, Watts T, Counts D, Zielke R, Bizzari, Fasano A.
Inhibition of the zonulin-dependent increased intestinal
permeability prevents the onset of type 1 diabetes in BB/
wor rats. Gastroenterology 2004;126A
/518.
[34] Watts T, Berti I, Sapone A, Gerarduzzi T, Not T, Zielke R, et
al. Role of the intestinal tight junction modulator zonulin in
the pathogenesis of type-I diabetes in BB diabetic prone rats.
Proc Natl Acad Sci USA 2005;
/102:/2916/21.
[35] Fasano A, Fiorentini C, Donelli G, Uzzau S, Kaper JB,
Margaretten K, et al. Zonula occludens toxin modulates
tight junctions through protein kinase C-dependent actin
reorganization, in vitro. J Clin Invest 1995;
/96:/710/20.
[36] Fleckenstein JM, Kopecko DJ. Breaching the mucosal
barrier by stealth: an emerging pathogenic mechanism for
enteroadherent bacterial pathogens. J Clin Invest 2001;
/107:/
27 /30.
[37] Wittig BM, Zeitz M. The gut as an organ of immunology. Int
J Colorectal Dis 2003;
/18:/181/7.
[38] Mowat AM. Anatomical basis of tolerance and immunity to
intestinal antigens. Nat Rev Immunol 2003;
/3:/331/41.
[39] Fasano A. Pathological and therapeutical implications of
macromolecules passage through the tight junction. In:
Cereijido M, Anderson J, editors. Tight functions. Boca
Raton: CRC Press; 2001. pp 697
/722.
[40] Fasano A, Berti I, Gerarduzzi T, Not T, Colletti RB, Drago
S, 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.
[41] Maki M, Mustalahti K, Kokkonen J, Kulmala P, Haapalahti
M, Karttunen TJ, et al. Prevalence of celiac disease among
children in Finland. N Engl J Med 2003;
/348:/2517 /24.
[42] Norris JM, Barriga K, Klingensmith G, Hoffman M,
Eisenbarth GS, Erlich HA, et al. Timing of initial cereal
exposure in infancy and risk of islet autoimmunity. JAMA
2003;
/290:/1713/20.
[43] Ziegler AG, Schmid S, Huber D, Hummel M, Bonifacio E.
Early infant feeding and risk of developing type 1 diabetes-
associated autoantibodies. JAMA 2003;
/290:/1721/8.
Gliadin and intestinal mucosa biology 419
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