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Original Articles: Gastroenterology

Increased Expression of Serum- and Glucocorticoid-regulated Kinase-1 in the Duodenal Mucosa of Children With Coeliac Disease

Szebeni, Beáta*; Vannay, Ádám; Sziksz, Erna; Prókai, Ágnes; Cseh, Áron; Veres, Gábor; Dezsőfi, Antal; Győrffy, Hajnalka; Szabó, IR Korponay§; Arató, András

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Journal of Pediatric Gastroenterology and Nutrition: February 2010 - Volume 50 - Issue 2 - p 147-153
doi: 10.1097/MPG.0b013e3181b47608
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Abstract

Apoptosis is central to the maintenance of the epithelial functions of the gut because it is involved in the normal enterocyte turnover. Under physiological conditions apoptotic cells are restricted to the tips of the villous in the small bowel and are replaced by an equal number of proliferating immature crypt cells.

In contrast, in immunomediated disorders such as coeliac disease (CD) or inflammatory bowel disease, an increased number of enterocytes undergo premature apoptosis all along the crypt-villous axis. The combination of markedly increased apoptosis and altered turnover rates results in architectural changes in the mucosa, characterised by villous atrophy and crypt hyperplasia (1).

Serum- and glucocorticoid-regulated kinases (SGKs) belong to a new family of serine/threonine kinases that are regulated at both transcriptional and posttranslational levels by external stimuli (2). The mRNA encoding SGK1, the best-studied member of the SGK family, is rapidly induced in response to a variety of stimuli including growth factors (3), steroid and peptide hormones (4), cytokines (5–7), excessive glucose concentrations (8), heat shock, ultraviolet radiation, and oxidative stress (9). To become functional, SGK1 requires phosphorylation of 2 regulatory sites, Ser-422 by an unknown kinase referred to tentatively as 3-phosphoinositide-dependent kinase-2 or “hydrophobic motif” kinase and Thr-256 by phosphoinositide-dependent kinase-1, which act in a phosphatidylinositol 3-kinase–dependent manner (10).

Functionally, active SGK1 plays a crucial role in promoting cell survival as well as activates several ion channels and carriers, regulates different enzymes and transcription factors, and participates in the regulation of transport, hormone release, and neuroexcitability (11). The antiapoptotic effect of SGK1 is attributed in part to the phosphorylation of forkhead transcription factors such as FKHRL1 (FOXO3a) leading to disruption of FKHRL1-dependent transcription, cell cycle arrest, and apoptosis (2). Moreover, SGK1 enhances the activity of nuclear factor (NF)-κB by association with and activation of IκB kinase β, which also inhibits apoptosis (12). The role of SGK1 in the cell survival response was sparsely investigated, but it was strongly correlated with the occurrence of cell death (13,14).

Although it is known that SGK1 expression is increased in the inflamed ileal mucosa of patients with Crohn disease (5), the significance of SGK1 in CD is elusive so far. Accordingly, our aim was to examine the expression and localisation of SGK1 in duodenal biopsy samples taken from children with untreated CD, children on a gluten-free diet (treated CD), and control patients.

PATIENTS AND METHODS

Patients

Duodenal biopsy samples from 16 children with untreated CD [6 boys, 10 girls, median age 6.7 years, range 3.7–13.9 years] and 9 children with treated CD [4 boys, 5 girls, median age 6.7 years, range 4.9–12.7 years] were collected. Biopsy samples of 7 children with untreated CD were taken only at the time of diagnosis, before the introduction of a gluten-free diet. In addition, the untreated CD group included 9 other children from whom duodenal biopsies were obtained before (untreated CD) and 1.5 years (range 1.1–2.5 years) after exclusion of gluten from the diet (treated CD) (Table 1). The diagnosis of CD was based on the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition criteria (15). All of the patients untreated for CD had anti-endomysium IgA positivity and subtotal villous atrophy of the intestinal mucosa (Fig. 1A). In case of treated patients for CD full clinical remission was observed in the diet and no serum anti-endomysium antibodies were detected (Fig. 1C). The control group consisted of 10 children (4 boys and 6 girls) with a median age of 8 years (range 1.7–13 years), who were investigated for either growth retardation or chronic diarrhea and an upper gastrointestinal endoscopy was part of their diagnostic procedure. The intestinal mucosa was normal in all of them (Fig. 1E) and no significant age- or sex-related differences were observed among children with untreated CD, treated CD, and controls (P = NS).

TABLE 1
TABLE 1:
Clinical characteristics of the study populations
FIGURE 1
FIGURE 1:
A, Subtotal villous atrophy (Marsh IIIB) in active (untreated) CD. Haematoxylin and eosin, original magnification × 200. B, Marked increase in the number of IELs (brown cells, arrowhead) in active (untreated) CD. CD3+ staining, original magnification × 600. C, No villous atrophy after gluten-free diet (treated CD). Haematoxylin and eosin, original magnification × 200. D, Raised IELs (brown cells, arrowhead) count with normal villous architecture after gluten-free diet (treated CD). CD3+ staining, original magnification × 600. E, Normal villous architecture in controls. Haematoxylin and eosin, original magnification × 200. F, Few IELs (brown cells, arrowhead) in controls. CD3+ staining, original magnification × 600. CD = coeliac disease; IELs = intraepithelial lymphocytes.

Biopsy samples were immediately frozen and stored at −80°C until further analysis. Written informed consent was obtained from the parents of each participant before the procedure, and the study was approved by the Semmelweis University Regional and Institutional Committee of Science and Research Ethics (TUKEB: 73/2003).

Methods

Histology

Duodenal biopsy specimens were routinely fixed in 10% formalin, embedded in paraffin wax, and 3-μm sections were stained with haematoxylin and eosin (Ventana Medical Systems Inc, Tucson, AZ). The number of intraepithelial lymphocytes (IELs) in every 100 epithelial cells was counted on the haematoxylin and eosin sections using anti-human CD3 antibody (Novocastra Lab Ltd, Newcastle upon Tyne, UK) diluted at 1:100. Randomly selected areas of biopsy samples were examined in a blinded fashion by the same qualified pathologist. The value for the number of IELs in every 100 epithelial cells was taken as the average of the 3 counts.

RNA Isolation and Real-Time RT-PCR

Total RNA was isolated from the duodenal biopsy samples by RNeasy Total RNA Isolations Kit (Qiagen GmbH, Hilden, Germany), according to the instructions of the manufacturer. The quality and quantity of the RNA were photometrically confirmed. One μg of total RNA was reverse transcripted using SuperScript II RNase H (Gibco/BRL, Eggenstein, Germany) to generate first-strand cDNA.

SGK1-mRNA expression was determined by SYBR Green real-time polymerase chain reaction (PCR) on a LightCycler system (Roche Diagnostics, Mannheim, Germany). Specific primer pairs for SGK1 were selected by Primer3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) (NCBI, gene accession number NM_005627). The SGK1-PCR was performed in glass capillaries, in a final volume of 20 μL containing 3 mmol/L MgCl2, 0.2 μmol/L of each primer, 2 μL of the LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics), and 1 μL of cDNA. The condition of PCR was as follows: 1 cycle at 95°C for 8 minutes, followed by 50 cycles at 95°C for 3 seconds, 58°C for 5 seconds, and 72°C for 28 seconds, with a single fluorescence detection point at the end of the extension.

To test the complementary DNAs for representation and full-length genes, reverse transcriptase (RT)-PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed. The mRNA expression of GAPDH was determined by real-time PCR quantification using fluorescence resonance energy transfer hybridisation probes on a LightCycler system (Roche Diagnostics). The GAPDH PCR was performed in a final volume of 20 μL containing 2 mmol/L MgCl2, 0.17 nmol/L of each hybridisation probe, 0.5 nmol/L of each primer, 2 μL of the LightCycler FastStart DNA Master HybProbe (Roche Diagnostics), and 1 μL of cDNA. The PCR reaction was carried out using the following conditions for GAPDH: 1 cycle at 95°C for 8 minutes, followed by 50 cycles at 95°C for 4 seconds, 55°C for 8 seconds, and 72°C for 22 seconds, with a single fluorescence detection point at the end of the annealing segment. The sequences of the specific primer pairs and probes (Tib Molbiol, Berlin, Germany) for SGK1 and GAPDH are presented in Table 2.

TABLE 2
TABLE 2:
Nucleotide sequence of specific primer pairs and probes applied for the real-time detection of SGK1 and GAPDH

After each LightCycler run, PCR products were separated by electrophoresis on 2.5% agarose gels and visualised by staining with ethidium bromide. To control the length of the generated PCR products, a 100-bp DNA ladder (Invitrogen Corp, Carlsbad, CA) was used.

Quantification was performed with second derivative method by monitoring the cycle number at which the fluorescent signal could be distinguished from the background (crossing point). Serially diluted cDNA samples were used as external standards. Results were analysed by using LightCycler software version 3.5.3. Expression level of SGK1 was normalised to that of GAPDH for the same biopsy sample.

Protein Isolation and Western Blotting

Duodenal biopsy specimens were solubilised in a sample buffer containing 10 μg/mL leupeptin, 10 μg/mL aprotinin, 1% Triton X-100, 0.1 mol/L Tris-HCl (pH 8.0), 1 mmol/L ethylene glycol-bis (2-aminoethylether),N,N,N′,N′-tetraacetic acid, 5 mmol/L NaF, 1 mmol/L phenylmethylsulphonylfluoride, and 10 mmol/L Na-orthovanadate (each substance was purchased from Sigma Chemical Co, St Louis, MO). The lysed samples were centrifuged (10,000g, 5 minutes, 4°C) to pellet nuclei and large cellular fragments. Protein concentration of the supernatants was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA).

A total of 20 μg of protein sample was separated for the determination of SGK1 and phosphorylated (P)-SGK1, respectively, by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis at 120 V and 40 mA for 90 minutes (Penguin Dual-Gel Water Cooled Systems, Owl, NH). Prestained protein mixture (BenchMark, Gibco/BRL) was used as marker of molecular mass. Separated proteins were then transferred to nitrocellulose membrane (Hybond ECL, AP Biotech, Buckinghamshire, UK) in transfer buffer containing 25 mmol/L Tris, 170 mmol/L glycine, and 20% methanol at 70 V, 220 mA for 120 minutes (MiniTank electroblotter, Owl, NH).

Nonspecific binding sites were blocked for 1 hour at room temperature (RT) in a blot solution containing 5% nonfat dry milk and phosphate-buffered saline (PBS). Blots were then incubated for 60 minutes at RT with rabbit polyclonal antibodies raised against amino acids 399-412 (C-GKSPDSVLVTASVK) of human SGK1 (rabbit polyclonal IgG, #07-315, Millipore, San Diego, CA) or against the phosphorylation site of Ser 422 of human P-SGK1 (rabbit polyclonal IgG, ab55281, Abcam, Cambridge, UK) both diluted to 1:500.

Blots were then washed and incubated for 30 minutes at RT with peroxidase conjugated secondary goat anti-rabbit IgG antibody (12-348, Millipore) diluted to 1:2000 (SGK1) or 1:1000 (P-SGK1). Immunoreactive bands were visualised using the enhanced chemiluminescence Western blotting detection protocol (AP Biotech). Computerised densitometry of the specific bands was analysed with Gel-Pro Analyser 3.1 software (Media Cybernetics, Silver Spring, MD). The values were normalised to an internal standard and expressed as relative optical density.

Immunofluorescent Staining

Duodenal biopsy samples were immediately snap-frozen for immunofluorescent analysis. They were embedded in Shandon cryomatrix (ThermoElectron Co, Waltham, MA), cut to 5-μm slides with a cryostat and stored at −80°C until further processing. Slides were incubated for 60 minutes at RT with mouse monoclonal IgG2b for SGK1 (sc-28338, Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal IgG for P-SGK1 (same antibody as in Western blot, ab55281, Abcam) diluted to 1:100. After repeated washing, slides were incubated with Alexa Fluor 488 F (ab')2 fragment of goat anti-mouse IgG (A-11017, Invitrogen, Carlsbad, CA) and Alexa Fluor 568 F (ab')2 fragment of goat anti-rabbit IgG (A-11036, Invitrogen) diluted to 1:100 for 30 minutes at RT. DNA was stained with Hoechst 33342 (Sigma-Aldrich Company Ltd, Gillingham, UK) for 10 minutes at RT, diluted to 1:1000. Finally, slides were rinsed in PBS and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA). Appropriate controls were performed omitting the primary antibodies to ensure their specificity and to avoid autofluorescence.

To visualise the stained tissues on the sections, a Zeiss LSM 510 Meta confocal laser scanning microscope (Carl Zeiss, Jena, Germany) was used and equipped with an inverted Axiovert 200M microscope, 20x Plan Apochromat (numerical aperture 0.80), and 63x Plan Apochromat oil immersion differential interference contrast (DIC) objectives (numerical aperture 1.4).

The SGK1 (green) was excited with a 488 Argon laser 4 and fluorescence was detected with a BP 505-570 filter. The 543 HeNe laser was used for the P-SGK1 (red) and fluorescence was detected with a BP 560-615 filter. We applied a 405-430 diode laser for excitation of Hoechst. The emitted light was collected with BP 420-480 BP. DIC was prepared by HBO-100 mercury lamp.

Statistical Analysis

Data were analysed using Statistica 6.0 software (StatSoft Inc, Tulsa, OK). After testing the normality with Shapiro-Wilk test, nonparametric Mann-Whitney U test was used to determine the levels of difference among all of the groups for IEL counts and for SGK1-mRNA expression as well as SGK1 and P-SGK1 protein levels in the duodenal biopsy samples. P values less than or equal to 0.05 were considered statistically significant.

RESULTS

Histology

The average IEL count was significantly higher in untreated CD (median 38, range 27–70) as well as in treated CD (median 18, range 14–27) than in controls (median 12, range 2–20) (P < 0.0001 and P < 0.01, respectively). However, this value was significantly lower in treated CD than in untreated CD (P = 0.0001) (Table 1, Fig. 1).

SGK1-mRNA Expression

Figure 2 shows the mRNA expression of SGK1 using real-time RT-PCR as detected in the duodenal mucosa of representative samples from children with untreated CD, children with treated CD, and controls.

FIGURE 2
FIGURE 2:
SGK1-mRNA expression in the duodenal mucosa of representative samples from children with untreated CD, children with treated CD, and controls. mRNA expression of SGK1 was determined by SYBR Green–based real-time RT-PCR. Results were analysed by using LightCycler software version 3.5.3. The type of standard curve analysis used was second derivative maximum withr = −0.98, error = 0.233 at SGK1 and r = −1.00 error = 0.101 at GAPDH. Data are expressed as mean ± SD. Analysis of significance was performed by Mann-Whitney U test. *P < 0.001 vs control, **P = 0.01 vs treated CD, ¥P < 0.05 vs control. CD = coeliac disease; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; RT-PCR = reverse transcription-polymerase chain reaction; SGK1 = serum- and glucocorticoid-regulated kinase-1.

SGK1-mRNA expression was significantly increased in the duodenal mucosa of children with untreated CD as well as children with treated CD compared with controls (P < 0.001 and P < 0.05, respectively). In the duodenal mucosa of children with treated CD, the SGK1-mRNA level was decreased in comparison to the children with untreated CD (P = 0.01).

SGK1 and P-SGK1 Protein Levels

Western blot analysis of duodenal biopsy specimens of representative samples from children with untreated CD, children with treated CD, and controls using anti-SGK1 and anti-P-SGK1 rabbit anti-human polyclonal antibodies revealed distinct bands at 50 kDa (SGK1) and approximately 60 kDa (P-SGK1) (Fig. 3).

FIGURE 3
FIGURE 3:
SGK1 and P-SGK1 protein levels in the duodenal mucosa of representative samples from children with untreated CD, children with treated CD, and controls. Top, Western blot analysis of the duodenal biopsy lysates with anti-SGK1, anti-P-SGK1 rabbit polyclonal antibodies reveal 1 distinct band at molecular weight of 50 kDa (SGK1) and 60 kDa (P-SGK1) (A). Bottom, data for protein levels of SGK1 (B) and P-SGK1 (C) were obtained by computerised analysis of the Western blots. Data are expressed as mean ± SD. Analysis of significance was performed by Mann-WhitneyU test. *P < 0.001 vs control, ¥P < 0.05 vs control #P < 0.01 vs treated CD, $P < 0.001 vs control. CD = coeliac disease; P-SGK1 = phosphorylated serum- and glucocorticoid-regulated kinase-1; SGK1 = serum- and glucocorticoid-regulated kinase-1.

SGK1 protein levels in the duodenal mucosa of children with untreated CD were 13 times higher than in controls (P < 0.001). In the duodenal mucosa of children with treated CD, SGK1 protein levels were 3 times lower than in untreated CD (P < 0.01).

We found about 7-fold elevation of the P-SGK1 (activated form) protein levels in the duodenal mucosa of children with untreated CD compared with controls (P < 0.001). In the duodenal mucosa of children with treated CD, P-SGK1 protein levels were 2-fold lower than in children with untreated CD (P < 0.01).

SGK1 and P-SGK1 Immunofluorescent Staining

Figure 4A–C show the cellular distribution of SGK1 and P-SGK1 proteins in the duodenal villi of children with untreated CD, children with treated CD, and controls. As shown in Figure 4A, heavy staining of both SGK1 and P-SGK1 was found in untreated CD, in the whole width of villous enterocytes: on their basal membrane, in their intracellular space, and in the apical part of their cytoplasm. At the same time fluorescent signal was also seen in the immune cells of the lamina propria. After the gluten-free diet (in treated CD) the distribution of SGK1 and P-SGK1 was similar to untreated cases—enterocytes and lamina propria immune cells—but both proteins got decreased (Fig. 4B) compared with untreated CD samples and still increased compared with the controls. In controls, weak SGK1 and weak P-SGK1 immunopositivity was observed in villous enterocytes, whereas no staining was detected in the immune cells of the lamina propria (Fig. 4C).

FIGURE 4
FIGURE 4:
Localisation of SGK1 and P-SGK1 in the duodenal mucosa of representative samples from children with untreated CD (A), children with treated CD (B), and controls (C). Frozen duodenal villous sections were fixed and double-labeled immunofluorescent staining was performed using anti-SGK1 (green) and anti-P-SGK1 (red) antibodies. Arrowheads indicate SGK1 and P-SGK1 staining of enterocytes and LPC. Heavy staining of both SGK1 and P-SGK1 was found in villous enterocytes of untreated patients with CD. SGK1 and P-SGK1 positive inflammatory cells also appeared in the lamina propria (A). Immunofluorescent staining revealed decreased SGK1 and P-SGK1-positivity in villous enterocytes and lamina propria immune cells of treated patients with CD compared with patients untreated for CD (B). Weak SGK1 and even weaker P-SGK1 immunopositivity was observed in villous enterocytes of controls, whereas no fluorescent signal was detected in the immune cells of the lamina propria (C). DIC pictures show longitudinal sections of duodenal villi. Confocal images were taken on a Zeiss Axiovert LSM510 with the plan apochromat 63x/1.40 Oil DIC. DIC = differential interference contrast; E = enterocytes; P-SGK1 = phosphorylated serum- and glucocorticoid-regulated kinase-1; LPC = lamina propria immune cells; SGK1 = serum- and glucocorticoid-regulated kinase-1.

DISCUSSION

In this study we demonstrated that the expression of SGK1, which has antiapoptotic effect, was elevated in duodenal enterocytes in CD.

Increased enterocyte apoptosis is responsible for villous atrophy in CD (16). Wheat gliadin and other cereal prolamins have been reported to be involved in the increased apoptosis of enterocytes of the small intestine in CD (1). Moss et al (16) revealed that enterocyte apoptosis fell to normal because of a gluten-free diet, before histological improvement. Giovanni et al (1) found that digested peptides from wheat gliadins induced enterocyte apoptosis by the CD95/Fas apoptotic pathway and this gliadin digest-induced apoptosis could be blocked by Fas cascade–blocking agents.

Although the role of SGK1 in cell survival/apoptosis is well defined, its role in different human diseases is sparsely investigated. Schoenebeck et al (13) showed that transcription of SGK1 was increased in several animal models of Parkinson disease and in a transgenic model of amyotrophic lateral sclerosis. They found that the upregulation of SGK1 strongly correlated with the occurrence of cell death. Aoyama et al (14) revealed that SGK1 was dynamically regulated during acute biomechanical stress in the heart and inhibited cardiomyocyte apoptosis.

In our study we aimed to depict the alteration of the amount and localisation of SGK1 and its activated form (P-SGK1) in duodenal biopsy samples taken from children with untreated CD, as well as children with treated CD, and controls.

We found increased SGK1 and P-SGK1 expression in the duodenal mucosa of children with untreated CD compared with controls. SGK1 and P-SGK1 immunopositivity detected in villous enterocytes and in the immune cells of the lamina propria was also stronger in the duodenal villi of children with untreated CD than in controls.

Apoptosis of enterocytes in CD can be induced by infiltrating both cytotoxic mucosal T lymphocytes and IELs via the perforin-granzyme mechanism or the Fas-Fas ligand pathway, respectively (17,18).

Ehrmann et al (19) found that the effect of perforin-granzyme can be modulated by the Bcl-2 family. SGK1 can stimulate NF-κB activity, which leads to transcriptional activation of genes whose products block apoptosis, including members of the Bcl-2 family (12). Maiuri et al (20) showed that NF-κB was activated in the small intestinal mucosa of patients with coeliac disease. Activated SGK1 is also capable of promoting cell survival by phosphorylating the forkhead transcription factor FKHRL1, leading to the repression of genes involved in apoptosis, such as Fas ligand (2,9).

In the duodenal mucosa of children with treated CD, where normal duodenal villous structure was observed, SGK1 and P-SGK1 expression were lower than in children with untreated CD but higher than in controls. SGK1 and P-SGK1 staining intensity was also weaker in villous enterocytes as well as in lamina propria immune cells of children with treated CD compared with untreated CD but stronger than in controls.

Selby et al (21) revealed that activation of cytotoxic mucosal T lymphocytes and ongoing proliferation of IELs may take place even in patients treated with a gluten-free diet and with no evidence for ingestion of trace amounts of gluten. In our study we also found raised IELs count in treated CD compared with controls. Thus, it is possible that the elevated SGK1 and P-SGK1 expression may inhibit the IELs and cytotoxic mucosal T lymphocytes-mediated enterocyte apoptosis in treated patients with CD as well.

To the best of our knowledge no previous study examined the potential role of SGK1 in the pathophysiology of CD; however, 1 previous study investigated the role of SGK1 in inflammatory bowel disease (5). Waldegger et al (5) analysed SGK1-mRNA expression and localisation in small intestinal mucosa of patients with Crohn disease and healthy people by in situ hybridisation. In congruence with our data they found that in normal ileum, SGK1-mRNA was selectively localised to the villous enterocytes. At the same time, in Crohn disease, abundant SGK1-positive inflammatory cells appeared in the lamina propria beside enterocytes. Moreover, they suggested that SGK1 expression in normal and inflamed intestinal mucosa should have been regulated by transforming growth factor (TGF)-β1.

It is known that TGF-β1 per se is a potent mediator of tissue repair; it is required for the differentiation of the intestinal epithelium and for mucosal wound healing (22). Hansson et al (23) revealed that TGF-β1 expression was also increased in the lamina propria of children with coeliac along with villous atrophy, so it may be one of the possible explanations for increased SGK1 expression observed in our study; however, Lionetti et al (24) showed lower TGF-β epithelial cell expression in the coeliac mucosa. Intracellular cytokine staining of mucosal denritic cells from patients with coeliac show higher interleukin (IL)-6 expression (25) and IL-6 stimulation can also increase SGK1-mRNA expression and protein level (6).

In summary, our results of increased SGK1 and P-SGK1 expression in untreated CD suggest the contribution of this kinase to the enterocyte survival against apoptosis in CD. Moreover, their decreased expression in treated CD may confirm the efficiency of the gluten-free diet, besides the normal histological finding.

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Keywords:

children; coeliac disease; duodenal biopsies; expression; serum-and glucocorticoid-regulated kinase-1

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