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author:("maki, Markku")
2.  Elevated Serum anti-Saccharomyces Cerevisiae, anti-I2 and anti-OmpW Antibody Levels in Patients with Suspicion of Celiac Disease 
Journal of clinical immunology  2008;28(5):10.1007/s10875-008-9200-9.
Objectives
Expression of anti-Saccharomyces Cerevisiae antibodies (ASCA) identifies patients and individuals at risk for Crohn’s disease and has also been reported in 40–60% of Celiac disease (CD) cases, suggesting a role of host response to enteric microbiota in the development of inflammatory lesions. In this prospective study in patients with suspicion of CD, we evaluate the frequency and association of ASCA to serological responses for other host microbial targets formally associated with Crohn’s disease, including the P.fluorescens associated sequence I2 and a Bacteroides caccae TonB-linked outermembrane protein, OmpW.
Methods
Small bowel mucosal biopsies were taken from 242 patients with CD suspicion, their sera were tested for antibodies to tissue transglutaminase (tTG), ASCA, I2 and OmpW. 80 adult healthy blood donors were used as controls.
Results
The diagnosis of CD was confirmed on biopsy in 134 cases. The occurence of ASCA and I2 positivity was significantly higher in adult CD patients as compared to patients with non-CD disease. Anti-I2 levels in the sera were significantly higher in adult CD patients compared to non-CD disease or the controls and anti-OmpW levels in CD and non-CD patients when compared to controls. Positive seroreactivity to OmpW seemed to increase with the age. 90% of CD patients were seropositive for at least one microbial antigen tested.
Conclusions
This study demonstrates a mosaic of disease-related serological responses to microbial antigens in patients with CD. Immune responses to commensal enteric bacteria may play a role in the small intestine mucosal damage in CD.
doi:10.1007/s10875-008-9200-9
PMCID: PMC3818910  PMID: 18496744
tissue transglutaminase; I2; OmpW; ASCA; serology; Celiac disease
3.  Long-Term Consumption of Oats in Adult Celiac Disease Patients 
Nutrients  2013;5(11):4380-4389.
Many celiac disease patients tolerate oats, but limited data are available on its long-term consumption. This was evaluated in the present study, focusing on small-bowel mucosal histology and gastrointestinal symptoms in celiac adults maintaining a strict gluten-free diet with or without oats. Altogether 106 long-term treated celiac adults were enrolled for this cross-sectional follow-up study. Daily consumption of oats and fiber was assessed, and small-bowel mucosal morphology and densities of CD3+, αβ+ and γσ+ intraepithelial lymphocytes determined. Gastrointestinal symptoms were assessed by a validated Gastrointestinal Symptom Rating Scale questionnaire. Seventy (66%) out of the 106 treated celiac disease patients had consumed a median of 20 g of oats (range 1–100 g) per day for up to eight years; all consumed oat products bought from general stores. Daily intake and long-term consumption of oats did not result in small-bowel mucosal villous damage, inflammation, or gastrointestinal symptoms. Oat-consumers had a significantly higher daily intake of fiber than those who did not use oats. Two thirds of celiac disease patients preferred to use oats in their daily diet. Even long-term ingestion of oats had no harmful effects.
doi:10.3390/nu5114380
PMCID: PMC3847736  PMID: 24201240
celiac disease; gluten-free diet; morphology; oats; questionnaire; small-bowel
4.  Validation of Morphometric Analyses of Small-Intestinal Biopsy Readouts in Celiac Disease 
PLoS ONE  2013;8(10):e76163.
Background
Assessment of the gluten-induced small-intestinal mucosal injury remains the cornerstone of celiac disease diagnosis. Usually the injury is evaluated using grouped classifications (e.g. Marsh groups), but this is often too imprecise and ignores minor but significant changes in the mucosa. Consequently, there is a need for validated continuous variables in everyday practice and in academic and pharmacological research.
Methods
We studied the performance of our standard operating procedure (SOP) on 93 selected biopsy specimens from adult celiac disease patients and non-celiac disease controls. The specimens, which comprised different grades of gluten-induced mucosal injury, were evaluated by morphometric measurements. Specimens with tangential cutting resulting from poorly oriented biopsies were included. Two accredited evaluators performed the measurements in blinded fashion. The intraobserver and interobserver variations for villus height and crypt depth ratio (VH:CrD) and densities of intraepithelial lymphocytes (IELs) were analyzed by the Bland-Altman method and intraclass correlation.
Results
Unevaluable biopsies according to our SOP were correctly identified. The intraobserver analysis of VH:CrD showed a mean difference of 0.087 with limits of agreement from −0.398 to 0.224; the standard deviation (SD) was 0.159. The mean difference in interobserver analysis was 0.070, limits of agreement −0.516 to 0.375, and SD 0.227. The intraclass correlation coefficient in intraobserver variation was 0.983 and that in interobserver variation 0.978. CD3+ IEL density countings in the paraffin-embedded and frozen biopsies showed SDs of 17.1% and 16.5%; the intraclass correlation coefficients were 0.961 and 0.956, respectively.
Conclusions
Using our SOP, quantitative, reliable and reproducible morphometric results can be obtained on duodenal biopsy specimens with different grades of gluten-induced injury. Clinically significant changes were defined according to the error margins (2SD) of the analyses in VH:CrD as 0.4 and in CD3+-stained IELs as 30%.
doi:10.1371/journal.pone.0076163
PMCID: PMC3795762  PMID: 24146832
5.  Thioredoxin Is Involved in Endothelial Cell Extracellular Transglutaminase 2 Activation Mediated by Celiac Disease Patient IgA 
PLoS ONE  2013;8(10):e77277.
Purpose
To investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation.
Methods
TG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study.
Results
We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity.
Conclusions
Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.
doi:10.1371/journal.pone.0077277
PMCID: PMC3793942  PMID: 24130874
6.  Serological Responses to Microbial Antigens in Celiac Disease Patients During a Gluten-Free Diet 
Journal of clinical immunology  2008;29(2):190-195.
Background
Immunoglobulin A (IgA) autoantibodies to tissue transglutaminase (tTG) are commonly used for screening and diagnosing of celiac disease (CD). Seroreactivity for anti-Saccharomyces cerevisiae antibody (ASCA) and bacterial antigens have also been detected in CD patients. The aim of this study was to examine prospectively serologic responses to microbial targets in adult CD patients at the time of diagnosis and during a gluten-free diet (GFD). Further, we wanted to evaluate whether these serologic specificities could provide new tools for the follow-up of CD patients.
Methods
Data on 55 adult biopsy-proven CD patients were available for follow-up study. Upper gastrointestinal endoscopy was performed on all patients. Sera from patients were tested for antibodies to tTG and ASCA and additionally analyzed with IgA enzyme-linked immunosorbent assays to Pseudomonas fluorescens-associated sequence, I2, and to a Bacteroides caccae TonB-linked outer membrane protein, OmpW.
Results
At the time of diagnosis, 91% of CD cases were positive for tTG and 49% for ASCA; positive seroreactivity to I2 was found in 86% and to OmpW in 60% of CD patients at the time of diagnosis. The frequency of seropositivity and serum levels of these antibodies decreased during GFD. Moreover, we found that the decline in the serum levels was significant in all of these markers (p<0.005). Interestingly, we also found that serum levels of ASCA correlated with the grade of mucosal morphology (p=0.021), as the ASCA serum levels declined in accordance with mucosal healing.
Conclusions
Commensal enteric bacteria seem to play a role in the small intestinal mucosal damage in CD. This was proven by the serological responses to different microbial antigens shown in this study. Serum levels of ASCA, anti-I2, and anti-OmpW antibodies decreased significantly during GFD, indicating that these serologic markers are gluten dependent in CD patients. These specificities could provide new tools in the follow-up of CD patients.
doi:10.1007/s10875-008-9255-7
PMCID: PMC3770459  PMID: 18987962
Celiac disease; gluten-free diet; ASCA; I2; OmpW
7.  Fingertip rapid point-of-care test in adult case-finding in coeliac disease 
BMC Gastroenterology  2013;13:115.
Background
Coeliac disease (CD), due to its protean clinical manifestation, is still very under diagnosed in adults and delays in diagnosis may take years and even decades. Simple tools to find cases in primary care may help to identify patients for further diagnostic tests. We have evaluated the usefulness of an on site rapid fingertip whole blood point-of-care test (POCT) for such a purpose.
Methods
As CD is known to run within families, we tested 148 healthy relatives of 70 Romanian index cases with biopsy-proven CD (87% of all first-degree family members, median age 36 years) for the presence of circulating autoantibodies. In addition to performing the POCT (which measures blood erythrocyte self-TG2-autoantibody complexes) on site, blood was drawn for later evaluations of serum IgA-class endomysial antibodies (EMA). EMA-positive sera were further tested for transglutaminase 2 antibodies (TG2-IgA). All serological parameters were analyzed blindly in a centralized laboratory that had no knowledge of the on site POCT result. Endoscopic small intestinal biopsies was recommended for all POCT- or EMA-test positive subjects.
Results
In on site testing the POCT was positive in 12/148 first-degree relatives (8%) and all these subjects were also serum EMA-positive. A positive EMA test was found only in one other subject. All remaining 135 healthy first-degree relatives were negative for both POCT and EMA. Four subjects positive for both POCT and EMA were negative for TG2-IgA. Ten out of thirteen of the antibody-positive subjects agreed to undergo endoscopy. The POCT was found to be positive in 8/9 first-degree relatives having coeliac-type mucosal lesions of grade Marsh 2 (n = 3) or Marsh 3 (n = 6). The three POCT-positive subjects not agreeing to undergo endoscopy were also both EMA- and TG2-IgA-positive.
Conclusion
The fingertip whole blood rapid POCT might fulfill the unmet need for a simple and cheap case-finding biomarker for early detection and presumptive diagnosis of CD. Confirmatory studies are warranted in adult case-finding in specialized outpatient clinics and in primary care.
doi:10.1186/1471-230X-13-115
PMCID: PMC3717015  PMID: 23849178
Coeliac disease; Healthy relatives; Case-finding; Point-of-care test
8.  Celiac Disease–Specific TG2-Targeted Autoantibodies Inhibit Angiogenesis Ex Vivo and In Vivo in Mice by Interfering with Endothelial Cell Dynamics 
PLoS ONE  2013;8(6):e65887.
A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility.
doi:10.1371/journal.pone.0065887
PMCID: PMC3688809  PMID: 23824706
9.  Predictors of persistent symptoms and reduced quality of life in treated coeliac disease patients: a large cross-sectional study 
BMC Gastroenterology  2013;13:75.
Background
Evidence suggests that many coeliac disease patients suffer from persistent clinical symptoms and reduced health-related quality of life despite a strict gluten-free diet. We aimed to find predictors for these continuous health concerns in long-term treated adult coeliac patients.
Methods
In a nationwide study, 596 patients filled validated Gastrointestinal Symptom Rating Scale and Psychological General Well-Being questionnaires and were interviewed regarding demographic data, clinical presentation and treatment of coeliac disease, time and place of diagnosis and presence of coeliac disease-associated or other co-morbidities. Dietary adherence was assessed by a combination of self-reported adherence and serological tests. Odds ratios and 95% confidence intervals were calculated by binary logistic regression.
Results
Diagnosis at working age, long duration and severity of symptoms before diagnosis and presence of thyroidal disease, non-coeliac food intolerance or gastrointestinal co-morbidity increased the risk of persistent symptoms. Patients with extraintestinal presentation at diagnosis had fewer current symptoms than subjects with gastrointestinal manifestations. Impaired quality of life was seen in patients with long duration of symptoms before diagnosis and in those with psychiatric, neurologic or gastrointestinal co-morbidities. Patients with persistent symptoms were more likely to have reduced quality of life.
Conclusions
There were a variety of factors predisposing to increased symptoms and impaired quality of life in coeliac disease. Based on our results, early diagnosis of the condition and consideration of co-morbidities may help in resolving long-lasting health problems in coeliac disease.
doi:10.1186/1471-230X-13-75
PMCID: PMC3651340  PMID: 23631482
Coeliac disease; Symptoms; Quality of life; Gluten-free diet; Adults
10.  Type 1 Diabetes Is Associated With Enterovirus Infection in Gut Mucosa 
Diabetes  2012;61(3):687-691.
Enterovirus infections have been linked to type 1 diabetes in several studies. Enteroviruses also have tropism to pancreatic islets and can cause β-cell damage in experimental models. Viral persistence has been suspected to be an important pathogenetic factor. This study evaluates whether gut mucosa is a reservoir for enterovirus persistence in type 1 diabetic patients. Small-bowel mucosal biopsy samples from 39 type 1 diabetic patients, 41 control subjects, and 40 celiac disease patients were analyzed for the presence of enterovirus using in situ hybridization (ISH), RT-PCR, and immunohistochemistry. The presence of virus was compared with inflammatory markers such as infiltrating T cells, HLA-DR expression, and transglutaminase 2–targeted IgA deposits. Enterovirus RNA was found in diabetic patients more frequently than in control subjects and was associated with a clear inflammation response in the gut mucosa. Viral RNA was often detected in the absence of viral protein, suggesting defective replication of the virus. Patients remained virus positive in follow-up samples taken after 12 months’ observation. The results suggest that a large proportion of type 1 diabetic patients have prolonged/persistent enterovirus infection associated with an inflammation process in gut mucosa. This finding opens new opportunities for studying the viral etiology of type 1 diabetes.
doi:10.2337/db11-1157
PMCID: PMC3282798  PMID: 22315304
11.  Use of health care services and pharmaceutical agents in coeliac disease: a prospective nationwide study 
BMC Gastroenterology  2012;12:136.
Background
Approximately 1% of the population suffer from coeliac disease. However, the disease is heavily underdiagnosed. Unexplained symptoms may lead to incremented medical consultations and productivity losses. The aim here was to estimate the possible concealed burden of untreated coeliac disease and the effects of a gluten-free diet.
Methods
A nationwide cohort of 700 newly detected adult coeliac patients were prospectively evaluated. Health care service use and sickness absence from work during the year before diagnosis were compared with those in the general population; the data obtained from an earlier study. Additionally, the effect of one year on dietary treatment on the aforementioned parameters and on consumption of pharmaceutical agents was assessed.
Results
Untreated coeliac patients used primary health care services more frequently than the general population. On a gluten-free diet, visits to primary care decreased significantly from a mean 3.6 to 2.3. The consumption of medicines for dyspepsia (from 3.7 to 2.4 pills/month) and painkillers (6.8-5.5 pills/month) and the number of antibiotic courses (0.6-0.5 prescriptions/year) was reduced. There were no changes in hospitalizations, outpatient visits to secondary and tertiary care, use of other medical services, or sickness absence, but the consumption of nutritional supplements increased on treatment.
Conclusions
Coeliac disease was associated with excessive health care service use and consumption of drugs before diagnosis. Dietary treatment resulted in a diminished burden to the health care system and lower use of on-demand medicines and antibiotic treatment. The results support an augmented diagnostic approach to reduce underdiagnosis of coeliac disease.
Trial registration
ClinicalTrials.gov NCT01145287
doi:10.1186/1471-230X-12-136
PMCID: PMC3503835  PMID: 23016889
Coeliac disease; Gluten-free diet; Burden of illness; Health care service use; Sickness absence
12.  Multiple independent variants in 6q21-22 associated with susceptibility to celiac disease in the Dutch, Finnish and Hungarian populations 
Celiac disease is an inflammatory enteropathy caused by intolerance to gluten. Previous linkage studies in the Dutch, Finnish and Hungarian populations have revealed a locus on chromosome 6q21-22 conferring susceptibility to celiac disease. This locus has previously been implicated in susceptibility to other autoimmune diseases such as Crohn's disease and type 1 diabetes. We performed fine mapping on 446 independent individuals with celiac disease and 641 controls of Dutch origin, testing 872 tagging SNPs in a 22 Mb region of chromosome 6. The 12 most promising SNPs were followed up in 2071 individuals from 284 Finnish and 357 Hungarian celiac disease families to identify risk variants in this region. Multiple markers in the region were significantly associated with celiac disease in the Dutch material. Two SNPs, rs9391227 and rs4946111, were significantly associated with celiac disease in the Finnish population. The association to rs9391227 represents the strongest association signal found in the Finnish (P=0.003, OR 0.66) as well as the combined Dutch, Finnish and Hungarian populations (P=3.6 × 10−5, OR 0.76). The rs9391227 is situated downstream of the HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1 (HACE1) gene and is contained within a region of strong linkage disequilibrium enclosing HACE1. Two additional, independent, susceptibility variants in the 6q21-22 region were also found in a meta-analysis of the three populations. The 6q21-22 region was confirmed as a celiac disease susceptibility locus and harbors multiple independent associations, some of which may implicate ubiquitin-pathways in celiac disease susceptibility.
doi:10.1038/ejhg.2011.2
PMCID: PMC3110053  PMID: 21326284
Celiac; linkage; association; genome-wide; 6q21-22
13.  Endomysial antibodies predict celiac disease irrespective of the titers or clinical presentation 
AIM: To investigate the association between serum antibody levels and a subsequent celiac disease diagnosis in a large series of children and adults.
METHODS: Besides subjects with classical gastrointestinal presentation of celiac disease, the study cohort included a substantial number of individuals with extraintestinal symptoms and those found by screening in at-risk groups. Altogether 405 patients underwent clinical, serological and histological evaluations. After collection of data, the antibody values were further graded as low [endomysial (EmA) 1:5-200, transglutaminase 2 antibodies (TG2-ab) 5.0-30.0 U/L] and high (EmA 1: ≥ 500, TG2-ab ≥ 30.0 U/L), and the serological results were compared with the small intestinal mucosal histology and clinical presentation.
RESULTS: In total, 79% of the subjects with low and 94% of those with high serum EmA titers showed small-bowel mucosal villous atrophy. Furthermore, 96% of the 47 EmA positive subjects who had normal mucosal villi and remained on follow-up either subsequently developed mucosal atrophy while on a gluten-containing diet, or responded positively to a gluten-free diet.
CONCLUSION: Irrespective of the initial serum titers or clinical presentation, EmA positivity as such is a very strong predictor of a subsequent celiac disease diagnosis.
doi:10.3748/wjg.v18.i20.2511
PMCID: PMC3360449  PMID: 22654448
Celiac disease; Diagnosis; Endomysial antibodies; Transglutaminase 2 antibodies; Clinical presentations
14.  Clinical benefit of gluten-free diet in screen-detected older celiac disease patients 
BMC Gastroenterology  2011;11:136.
Background
The utility of serologic screening for celiac disease is still debatable. Evidence suggests that the disorder remains undetected even in the older population. It remains obscure whether screening makes good or harm in subjects with long-standing gluten ingestion. We evaluated whether older subjects benefit from active detection and subsequent gluten free dietary treatment of celiac disease.
Methods
Thirty-five biopsy-proven patients aged over 50 years had been detected by serologic mass screening. We examined the disease history, dietary compliance, symptoms, quality of life and bone mineral density at baseline and 1-2 years after the commencement of a gluten-free diet. Symptoms were evaluated by gastrointestinal symptom rating scale and quality of life by psychological general well-being questionnaires. Small bowel biopsy, serology, laboratory parameters assessing malabsorption, and bone mineral density were investigated.
Results
Dietary compliance was good. The patients had initially low mean serum ferritin values indicating subclinical iron deficiency, which was restored by a gluten-free diet. Vitamin B12, vitamin D and erythrocyte folic acid levels increased significantly on diet. Celiac patients had a history of low-energy fractures more often than the background population, and the diet had a beneficial effect on bone mineral density. Alleviation in gastrointestinal symptoms was observed, even though the patients reported no or only subtle symptoms at diagnosis. Quality of life remained unchanged. Of all the cases, two thirds would have been diagnosed even without screening if the family history, fractures or concomitant autoimmune diseases had been taken carefully into account.
Conclusions
Screen-detected patients benefited from a gluten-free diet. We encourage a high index of suspicion and active case-finding in celiac disease as an alternative to mass screening in older patients.
doi:10.1186/1471-230X-11-136
PMCID: PMC3377922  PMID: 22176557
15.  Small- bowel mucosal changes and antibody responses after low- and moderate-dose gluten challenge in celiac disease 
BMC Gastroenterology  2011;11:129.
Background
Due to the restrictive nature of a gluten-free diet, celiac patients are looking for alternative therapies. While drug-development programs include gluten challenges, knowledge regarding the duration of gluten challenge and gluten dosage is insufficient.
We challenged adult celiac patients with gluten with a view to assessing the amount needed to cause some small-bowel mucosal deterioration.
Methods
Twenty-five celiac disease adults were challenged with low (1-3 g) or moderate (3-5g) doses of gluten daily for 12 weeks. Symptoms, small-bowel morphology, densities of CD3+ intraepithelial lymphocytes (IELs) and celiac serology were determined.
Results
Both moderate and low amounts of gluten induced small-bowel morphological damage in 67% of celiac patients. Moderate gluten doses also triggered mucosal inflammation and more gastrointestinal symptoms leading to premature withdrawals in seven cases. In 22% of those who developed significant small- intestinal damage, symptoms remained absent. Celiac antibodies seroconverted in 43% of the patients.
Conclusions
Low amounts of gluten can also cause significant mucosal deterioration in the majority of the patients. As there are always some celiac disease patients who will not respond within these conditions, sample sizes must be sufficiently large to attain to statistical power in analysis.
doi:10.1186/1471-230X-11-129
PMCID: PMC3240817  PMID: 22115041
16.  Latent coeliac disease or coeliac disease beyond villous atrophy? 
Gut  2007;56(10):1339-1340.
New diagnostic criteria for coeliac disease are warranted
doi:10.1136/gut.2006.113084
PMCID: PMC2000254  PMID: 17872565
17.  Multiple common variants for celiac disease influencing immune gene expression 
Nature genetics  2010;42(4):295-302.
We performed a second-generation genome wide association study of 4,533 celiac disease cases and 10,750 controls. We genotyped 113 selected SNPs with PGWAS<10−4, and 18 SNPs from 14 known loci, in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome wide significance (Pcombined<5×10−8), most contain immune function genes (BACH2, CCR4, CD80, CIITA/SOCS1/CLEC16A, ICOSLG, ZMIZ1) with ETS1, RUNX3, THEMIS and TNFRSF14 playing key roles in thymic T cell selection. A further 13 regions had suggestive association evidence. In an expression quantitative trait meta-analysis of 1,469 whole blood samples, 20 of 38 (52.6%) tested loci had celiac risk variants correlated (P<0.0028, FDR 5%) with cis gene expression.
doi:10.1038/ng.543
PMCID: PMC2847618  PMID: 20190752
18.  DNA-Binding and -Bending Activities of SAP30L and SAP30 Are Mediated by a Zinc-Dependent Module and Monophosphoinositides ▿ †  
Molecular and Cellular Biology  2008;29(2):342-356.
Deacetylation of histones is carried out by a corepressor complex in which Sin3A is an essential scaffold protein. Two proteins in this complex, the Sin3A-associated proteins SAP30L and SAP30, have previously been suggested to function as linker molecules between various corepressors. In this report, we demonstrate new functions for human SAP30L and SAP30 by showing that they can associate directly with core histones as well as naked DNA. A zinc-coordinating structure is necessary for DNA binding, one consequence of which is bending of the DNA. We provide evidence that a sequence motif previously shown to be a nuclear localization signal is also a phosphatidylinositol (PI)-binding element and that binding of specific nuclear monophosphoinositides regulates DNA binding and chromatin association of SAP30L. PI binding also decreases the repression activity of SAP30L and affects its translocation from the nucleus to the cytoplasm. Our results suggest that SAP30L and SAP30 play active roles in recruitment of deacetylating enzymes to nucleosomes, and mediate key protein-protein and protein-DNA interactions involved in chromatin remodeling and transcription.
doi:10.1128/MCB.01213-08
PMCID: PMC2612513  PMID: 19015240
19.  Phylogenetic analysis of the SAP30 family of transcriptional regulators reveals functional divergence in the domain that binds the nuclear matrix 
Background
Deacetylation of histones plays a fundamental role in gene silencing, and this is mediated by a corepressor complex containing Sin3 as an essential scaffold protein. In this report we examine the evolution of two proteins in this complex, the Sin3-associated proteins SAP30L and SAP30, by using an archive of protein sequences from 62 species.
Results
Our analysis indicates that in tetrapods SAP30L is more similar than SAP30 to the ancestral protein, and the two copies in this group originated by gene duplication which occurred after the divergence of Actinopterygii and Sarcopterygii about 450 million years ago (Mya). The phylogenetic analysis and biochemical experiments suggest that SAP30 has diverged functionally from the ancestral SAP30L by accumulating mutations that have caused attenuation of one of the original functions, association with the nuclear matrix. This function is mediated by a nuclear matrix association sequence, which consists of a conserved motif in the C-terminus and the adjacent nucleolar localization signal (NoLS).
Conclusion
These results add further insight into the evolution and function of proteins of the SAP30 family, which share many characteristic with nuclear scaffolding proteins that are intimately involved in regulation of gene expression. Furthermore, SAP30L seems essential to eukaryotic biology, as it is found in animals, plants, fungi, as well as some taxa of unicellular eukaryotes.
doi:10.1186/1471-2148-9-149
PMCID: PMC2711940  PMID: 19566944
20.  Increasing prevalence and high incidence of celiac disease in elderly people: A population-based study 
BMC Gastroenterology  2009;9:49.
Background
Celiac disease may emerge at any age, but little is known of its appearance in elderly people. We evaluated the prevalence of the condition in individuals over 55 years of age, and determined the incidence of biopsy-proven celiac disease (CDb) and celiac disease including seropositive subjects for anti-tissue transglutaminase antibodies (CDb+s).
Methods
The study based on prevalence figures in 2815 randomly selected subjects who had undergone a clinical examination and serologic screening for celiac disease in 2002. A second screening in the same population was carried out in 2005, comprising now 2216 individuals. Positive tissue transglutaminase antibodies were confirmed with small bowel biopsy.
Results
Within three years the prevalence of CDb increased from 2.13 to 2.34%, and that of CDb+s from 2.45 to 2.70%. Five new cases were found among patients previously seronegative; two had minor abdominal symptoms and three were asymptomatic. The incidence of celiac disease in 2002–2005 was 0.23%, giving an annual incidence of 0.08% in this population.
Conclusion
The prevalence of celiac disease was high in elderly people, but the symptoms were subtle. Repeated screening detected five biopsy-proven cases in three years, indicating that the disorder may develop even in the elderly. Increased alertness to the disorder is therefore warranted.
doi:10.1186/1471-230X-9-49
PMCID: PMC2711095  PMID: 19558729
21.  IL23R in the Swedish, Finnish, Hungarian and Italian populations: association with IBD and psoriasis, and linkage to celiac disease 
BMC Medical Genetics  2009;10:8.
Background
Association of the interleukin-23 receptor (IL23R) with inflammatory bowel disease (IBD) has been confirmed in several populations. IL23R also associates with psoriasis, suggesting that the gene may be an important candidate for many chronic inflammatory diseases.
Methods
We studied association of single-nucleotide variants in IL23R with IBD in Swedish patients, in both Crohn's disease (CD) and ulcerative colitis (UC) subsets. The same genetic variants were also studied in Finnish patients with psoriasis or celiac disease, and in Hungarian and Italian patients with celiac disease.
Results
Association of IL23R with IBD was replicated in our Swedish patients, and linkage and association of the IL23R region with psoriasis was found in the Finnish population. The IL23R region was also linked to celiac disease in Finnish families, but no association of IL23R variants with celiac disease was found in the Finnish, Hungarian or Italian samples.
Conclusion
Our study is the first to demonstrate association of IL23R with CD and UC in Swedish patients with IBD. It is also the first study to report linkage and association of the IL23R region with psoriasis in the Finnish population. Importantly, this is the first report of linkage of the IL23R region to celiac disease, a chronic inflammatory condition in which IL23R has not been previously implicated.
doi:10.1186/1471-2350-10-8
PMCID: PMC2642807  PMID: 19175939
22.  Transcription factors GATA-4 and GATA-6 in normal and neoplastic human gastrointestinal mucosa 
Background
Human gastrointestinal mucosa regenerates vigorously throughout life, but the factors controlling cell fate in mature mucosa are poorly understood. GATA transcription factors direct cell proliferation and differentiation in many organs, and are implicated in tumorigenesis. GATA-4 and GATA-6 are considered crucial for the formation of murine gastrointestinal mucosa, but their role in human gastrointestinal tract remains unexplored. We studied in detail the expression patterns of these two GATA factors and a GATA-6 down-stream target, Indian hedgehog (Ihh), in normal human gastrointestinal mucosa. Since these factors are considered important for proliferation and differentiation, we also explored the possible alterations in their expression in gastrointestinal neoplasias. The expression of the carcinogenesis-related protein Indian hedgehog was also investigated in comparison to GATA factors.
Methods
Samples of normal and neoplastic gastrointestinal tract from children and adults were subjected to RNA in situ hybridization with 33P labelled probes and immunohistochemistry, using an avidin-biotin immunoperoxidase system. The pathological tissues examined included samples of chronic and atrophic gastritis as well as adenomas and adenocarcinomas of the colon and rectum.
Results
GATA-4 was abundant in the differentiated epithelial cells of the proximal parts of the gastrointestinal tract but was absent from the distal parts. In contrast, GATA-6 was expressed throughout the gastrointestinal epithelium, and in the distal gut its expression was most intense at the bottom of the crypts, i.e. cells with proliferative capacity. Both factors were also present in Barrett's esophagus and metaplasia of the stomach. GATA-6 expression was reduced in colon carcinoma. Ihh expression overlapped with that of GATA-6 especially in benign gastrointestinal neoplasias.
Conclusion
The results suggest differential but overlapping functions for GATA-4 and GATA-6 in the normal gastrointestinal mucosa. Furthermore, GATA-4, GATA-6 and Ihh expression is altered in premalignant dysplastic lesions and reduced in overt cancer.
doi:10.1186/1471-230X-8-9
PMCID: PMC2323380  PMID: 18405344
23.  Secretion of celiac disease autoantibodies after in vitro gliadin challenge is dependent on small-bowel mucosal transglutaminase 2-specific IgA deposits 
BMC Immunology  2008;9:6.
Background
In celiac disease gluten, the disease-inducing toxic component in wheat, induces the secretion of autoantibodies which are targeted against transglutaminase 2 (TG2). These autoantibodies are produced in the small-intestinal mucosa, where they can be found deposited extracellularly below the epithelial basement membrane and around mucosal blood vessels. In addition, during gluten consumption these autoantibodies can also be detected in patients' serum but disappear from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The toxicity of gluten and the secretion of the disease-specific autoantibodies have been widely studied in organ culture of small-intestinal biopsy samples, but results hitherto have been contradictory. Since the mucosal autoantibodies disappear slowly after a gluten-free diet, our aim was to establish whether autoantibody secretion to organ culture supernatants in treated celiac disease patient biopsies is related to the duration of the diet and further to the pre-existence of mucosal TG2-specific IgA deposits in the cultured biopsy samples.
Results
In the organ culture system conducted with biopsies derived from treated celiac disease patients, gliadin induced secretion of autoantibodies to culture supernatants, reduced epithelial cell height and increased the density of lamina proprial CD25+ cells. However, these changes could be demonstrated only in biopsies from short-term treated celiac disease patients, where the small-intestinal mucosal TG2-specific IgA autoantibody deposits were still present. Furthermore, in these biopsies autoantibody secretion could be stimulated fully only after a 48-hour gliadin challenge.
Conclusion
Our results show that studies focusing on the toxic effects of gliadin in the organ culture system should be carried out with biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal IgA deposits present. In addition to providing an explanation for the discrepancies in previous publications, the present study also enables further validation of the organ culture method.
doi:10.1186/1471-2172-9-6
PMCID: PMC2275217  PMID: 18312620
24.  Population screening for coeliac disease in primary care by district nurses using a rapid antibody test: diagnostic accuracy and feasibility study  
BMJ : British Medical Journal  2007;335(7632):1244-1247.
Objective To evaluate the feasibility and diagnostic accuracy of screening for coeliac disease by rapid detection of IgA antibodies to tissue transglutaminase performed in primary care.
Design District nurses screened 6 year old children using rapid antibody testing of finger prick blood. They also collected capillary blood samples for laboratory determination of IgA and IgG antibodies to endomysium and IgA antibodies to tissue transglutaminase. Children with positive rapid test results were directly sent for biopsy of the small intestine.
Setting Primary care in Jász-Nagykun-Szolnok county, Hungary.
Participants 2690 children (77% of 6 year olds living in the county) and 120 nurses.
Main outcome measures Positivity for antibodies to endomysium or transglutaminase in the laboratory and coeliac disease confirmed at biopsy.
Results 37 children (1.4%, 95% confidence interval 0.9% to 1.8%) had biopsy confirmed coeliac disease. Only five of these children had been diagnosed clinically before screening. Rapid testing had a 78.1% sensitivity (70.0% to 89.3%) and 100% specificity (88.4% to 100%) for a final diagnosis of coeliac disease by biopsy. Sensitivity was 65.1% (50.2% to 77.6%) and specificity was 100% (99.8% to 100%) compared with combined results of IgA and IgG laboratory tests. Trained laboratory workers detected 30 of the 31 newly diagnosed IgA competent patients with the rapid test kit used blindly. Median time to biopsy after a positive rapid test result was significantly shorter (20 days, range 4-148) than after a positive laboratory result (142 days, 70-256; P<0.001). Children with coeliac disease detected at screening were smaller and had worse health status than their peers but they improved on a gluten-free diet.
Conclusions A simple rapid antibody test enabled primary care nurses to detect patients with coeliac disease in the community who were not picked up in clinical care. Extra training is needed to improve sensitivity.
doi:10.1136/bmj.39405.472975.80
PMCID: PMC2137074  PMID: 18063612
25.  Gene expression in TGFbeta-induced epithelial cell differentiation in a three-dimensional intestinal epithelial cell differentiation model 
BMC Genomics  2006;7:279.
Background
The TGFβ1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFβ1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation.
Results
The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFβ1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes.
Conclusion
Our results reveal potential new signaling pathways and several new genes affected by TGFβ in epithelial cell differentiation. The differentiation induced by TGFβ1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells.
doi:10.1186/1471-2164-7-279
PMCID: PMC1635984  PMID: 17074098

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