Rapid Immunochromatography on Fresh Intestinal Biopsy Is Highly Accurate in Detecting Intestinal Antitransglutaminase Antibodies.

INTRODUCTION: Intestinal antitransglutaminase antibodies (I-anti-TG2) are a specific marker of celiac disease (CeD). The aim of this study was to evaluate the diagnostic accuracy of a novel application of an immunochromatographic assay referred to as Rapid_AntiTG2 to detect I-anti-TG2 on intestinal biopsy lysate. METHODS: Consecutive pediatric patients referred to a single center for elective upper endoscopy were enrolled. Biopsies were taken from duodenal bulb and distal duodenum. For each sampling site, 2 biopsies were analyzed for standard histology, 1 biopsy was cultured to perform the reference standard assay for I-anti-TG2 detection (endomysium [EMA] biopsy), and 1 biopsy was mechanically lysed to perform Rapid_AntiTG2. The primary outcome was the diagnostic accuracy of Rapid_AntiTG2 on biopsy lysate compared with that of the gold standard (serology + histopathology) for CeD diagnosis. The secondary outcome was the agreement of Rapid_AntiTG2 with EMA biopsy. RESULTS: One hundred forty-eight patients were included. Of them, 79 were those with CeD (64 classical CeD, 2 seronegative CeD, and 13 potential CeD) and 69 were controls. Rapid_AntiTG2 on biopsy lysate had very high diagnostic accuracy (sensitivity 100%, specificity 97%, LR+ 34.1, LR- 0.01) in separating patients with CeD from controls. Diagnostic accuracy was unchanged in patients with potential and seronegative CeD. Rapid_AntiTG2 on biopsy lysate had almost perfect agreement with the EMA biopsy reference test (99% agreement, Cohen K 0.97). DISCUSSION: I-anti-TG2 can be detected with an immunochromatographic assay after simple mechanical lysis of fresh intestinal biopsy with very high diagnostic accuracy. The test is quick and easy to perform and can be widely available in any endoscopy unit. Its implementation would allow a better understanding of the prognostic value of I-anti-TG2 and help clinicians in cases of suspected CeD that are difficult to classify.

Celiac Disease Frequency Is Increased in IgE-Mediated Food Allergy and Could Affect Allergy Severity and Resolution.

OBJECTIVES: An increased frequency of celiac disease (CeD) has been reported in severe Immunoglobulin E (IgE) -mediated food allergy (FA). This observation requires confirmation, and whether CeD affects FA severity and resolution is unknown. The study aims to estimate the prevalence of CeD in patients with FA and to investigate whether CeD affects FA severity and oral tolerance. METHODS: Consecutive patients with FA referred for allergen reintroduction, either to evaluate allergy resolution or to start oral immunotherapy (OIT), were evaluated for CeD and for FA severity. The primary outcome was the prevalence of CeD. Secondary outcomes were the frequency of severe FA and the level of clinical tolerance at study entry and at last follow-up in patients with isolated FA versus patients with FA + CeD. RESULTS: Two hundred twenty-eight patients were included. CeD was confirmed in 15 patients (6.6%) of whom, 8 patients had a previously established diagnosis of CeD and were on a gluten-free diet. Severe FA was observed in 12 patients with FA + CeD (80%) versus 88 patients with FA (42%) ( P = 0.006). At baseline, patients with FA + CeD had significantly higher median allergen-specific IgE levels [61.8 kU/L; interquartile range (IQR) 11.6-279.0] compared to patients with FA (20.3 kU/L; IQR 2.9-72.7) ( P < 0.001). Complete clinical tolerance was observed in 1 of 15 patients (7%) with FA + CeD versus 98 of 205 patients (48%) with FA ( P = 0.002). CONCLUSIONS: CeD is highly prevalent in patients with FA and could affect FA severity and response to OIT. CeD screening should be considered in patients with severe or persistent FA.

20-Year Follow-up Study of Celiac Patients Identified in a Mass School Screening: Compliance to Gluten-Free Diet and Autoimmunity.

OBJECTIVES: To investigate the compliance to the gluten-free diet in a cohort of adult celiac patients 20 years after the diagnosis, received in childhood through a mass screening. METHODS: This is an observational historic cohort follow-up study. It was carried out at the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy. Two matched cohorts of adult celiac patients, diagnosed in childhood through a mass screening or for symptoms were enrolled. Adherence to the gluten free-diet and development of autoimmune diseases were investigated through a questionnaire administrated in the course of a phone interview.The primary study outcome was the adherence to the gluten-free diet, measured through the Biagi questionnaire, in the two cohorts of celiac patients. RESULTS: We contacted 25 patients (mean age 28 years, 19 females) diagnosed with screening and 34 patients (mean age 25 years, 26 females) diagnosed in the same period for symptoms. After 20 years, in the cohort diagnosed with screening and in the cohort diagnosed for symptoms the adherence to the gluten-free diet was optimal in 14 (56%) and 26 (81%), improvable in 5 (20%) and 3 (9%), inadequate in 6 (24%) and 3 (9%), respectively. In the two cohorts, four patients (16%) and six patients (18%) developed other autoimmune diseases. CONCLUSIONS: Twenty years after the diagnosis, near half of the patients diagnosed in a mass screening, does not have an optimal adherence to the gluten-free diet and a remarkable proportion of them have developed another autoimmune disease.

Circulating PV-1 as a marker of celiac disease-associated liver injury.

Aim: To investigate the role of endothelial PV-1 in patients with untreated celiac disease (CD)-associated liver injury. Materials & methods: PV-1 and PV-1 mRNA were measured in intestinal biopsies from untreated CD patients with elevated or normal alanine transaminase levels, controls, patients with inflammatory bowel disease and patients with toxic liver injury. Circulating PV-1 levels were also evaluated. Results: Circulating PV-1 levels were significantly increased in the serum of patients with CD-associated liver injury and reverted to normal following a gluten-free diet. Mucosal PV-1 and PV-1 mRNA were no different in patients with CD-associated liver injury. Conclusion: Serum but not mucosal PV-1 represents a marker of gluten-dependent liver injury and response to a gluten-free diet in patients with untreated CD.

Diagnostic accuracy and applicability of intestinal auto-antibodies in the wide clinical spectrum of coeliac disease.

BACKGROUND: Intestinal coeliac auto-antibodies are the marker of coeliac disease (CD). Since the determination of these antibodies is still not widely available, we used immunoassays to identify the most suitable technology for revealing intestinal auto-antibodies in the wide clinical spectrum of CD. METHODS: Intestinal auto-antibodies have been prospectively investigated in CD suspected children using two immunoassays: intestinal-deposits of IgA anti-tissue transglutaminase antibodies (anti-tTG) and biopsy-culture IgA anti-endomysium (AEA). Intestinal IgM antibodies have been determined in IgA-deficient subjects. FINDINGS: Two-hundred and twenty-one suspected CD patients were enrolled. Intestinal antibodies were tested positive for both assays in classical CD patients (n = 178) with villous atrophy and positive serum-CD antibodies, potential CD patients (n = 16) with normal intestinal mucosa and positive serum-CD antibodies, and pre-potential CD patients (n = 14) with normal intestinal mucosa and negative serum-CD antibodies. In 13/221 with normal intestinal mucosa, negative CD-serum antibodies and negative intestinal antibodies CD has been excluded. All classical, 14/16 potential and 11/14 pre-potential CD patients on gluten-free diet (GFD) improved their symptoms. In 9/11 pre-potential patients intestinal antibodies disappeared on GFD. Both assays were negative in 69/71 control subjects. The two assays showed high diagnostic sensitivity (100%) and specificity (99%). INTERPRETATION: Intestinal CD-antibodies make prompt diagnosis in the wide clinical spectrum of CD reducing the delay in diagnosis and treatment, especially in pre-potential CD patients. The easy handling biopsy culture assay is an effective diagnostic tool which should be carried out by any gastroenterology unit to recognize all CD clinical manifestations. FUNDING: Interreg Central-Europe, IRCCS "Burlo Garofolo".

Ghrelin Derangements in Idiopathic Dilated Cardiomyopathy: Impact of Myocardial Disease Duration and Left Ventricular Ejection Fraction.

BACKGROUND: Ghrelin may exert positive effects on cardiac structure and function in heart failure (HF) patients. METHODS: We assessed ghrelin levels in 266 dilated cardiomyopathy (DCM) patients and in 200 age, gender and body mass index (BMI) matched controls. Further, we evaluated the expression of ghrelin and growth hormone secretagogue-receptor (GHSR) in the myocardium of 41 DCM patients and in 11 controls. RESULTS: DCM patients had significantly lower levels of total, acylated and unacylated ghrelin when compared to controls (p < 0.05 for all). In controls, we observed a negative correlation of ghrelin with age, male gender and BMI. These correlations were lost in the DCM group, except for male gender. Total ghrelin was higher in patients with more recent diagnosis when compared to patients with longer duration of the DCM (p = 0.033). Further, total ghrelin was higher in patients with lower left ventricular systolic function (<40% LVEF, vs. 40% ≤ LVEF < 49% vs. LVEF ≥ 50%: 480.8, vs. 429.7, vs. 329.5 pg/mL, respectively, p = 0.05). Ghrelin prepropeptide was expressed more in DCM patients than in controls (p = 0.0293) while GHSR was expressed less in DCM patients (p < 0.001). Furthermore, ghrelin showed an inverse correlation with its receptor (= -0.406, p = 0.009), and this receptor showed a significant inverse correlation with Interleukin-1 (= -0.422, p = 0.0103). CONCLUSION: DCM duration and severity are accompanied by alterations in the ghrelin-GHSR system.

Galectin 3 and Galectin 3 Binding Protein Improve the Risk Stratification after Myocardial Infarction.

BACKGROUND: Acute myocardial infarction (AMI) survivors are at risk of major adverse cardiac events and their risk stratification is a prerequisite to tailored therapeutic approaches. Biomarkers could be of great utility in this setting. METHODS: We sought to evaluate the utility of the combined assessment of Galectin 3 (Gal-3) and Galectin 3 binding protein (Gal-3bp) for post-AMI risk stratification in a large, consecutive population of AMI patients. The primary outcomes were: Recurrent angina/AMI and all-cause mortality at 12 months after the index event. RESULTS: In total, 469 patients were included. The median Gal-3bp was 9.1 µg/mL (IQR 5.8-13.5 µg/mL), while median Gal-3 was 9.8 ng/mL (IQR 7.8-12.8 ng/mL). During the 12 month follow-up, 34 patients died and 41 had angina pectoris/reinfarction. Gal-3 was associated with all-cause mortality, while Gal-3bp correlated with the risk of angina/myocardial infarction even when corrected for other significant covariates. The final multivariable model for mortality prediction included patients' age, left ventricular ejection fraction (LVEF), Gal-3, and renal function. The ROC curve estimated for this model has an area under the curve (AUC) of 0.84 (95%CI 0.78-0.9), which was similar to the area under the ROC curve obtained using the GRACE score 1-year mortality. CONCLUSIONS: The integrated assessment of Gal-3 and Gal-3bp could be helpful in risk stratification after AMI.

A Functional Idiotype/Anti-Idiotype Network Is Active in Genetically Gluten-Intolerant Individuals Negative for Both Celiac Disease-Related Intestinal Damage and Serum Autoantibodies.

An unbalance between Abs that recognize an autoantigen (idiotypes; IDs) and Igs that bind such Abs (anti-IDs) is considered a functional event in autoimmune disorders. We investigated the presence of an ID/anti-ID network in celiac disease (CD), a condition in which antitissue transglutaminase 2 (TG2) Abs are suspected to contribute to CD pathogenesis. To characterize the ID side, we reproduced by in vitro yeast display the intestine-resident Abs from CD and control patients. These TG2-specific IDs were used to identify potential anti-IDs in the serum. We observed elevated titers of anti-IDs in asymptomatic patients with predisposition to CD and demonstrated that anti-ID depletion from the serum restores a detectable humoral response against TG2. Our study provides an alternative approach to quantify CD-related autoantibodies in cases that would be defined "negative serology" with current diagnostic applications. Therefore, we suggest that developments of this technology could be designed for perspective routine tests.

Immunohistologic analysis of the duodenal bulb: a new method for celiac disease diagnosis in children.

BACKGROUND AND AIMS: Anti-tissue transglutaminase antibodies (anti-tTG) have simplified celiac disease (CD) diagnosis. However, in atypical forms of CD, intestinal biopsy sampling is still required. This prospective study investigates whether histologic analysis of the duodenal bulb combined with intestinal IgA anti-tTG deposit immunoassay makes CD diagnosis possible in at-risk children with low concentrations of serum anti-tTG. METHODS: Histologic and intestinal IgA anti-tTG deposit immunoassays were used. RESULTS: Two hundred forty-five symptomatic children positive for serum anti-tTG (>7 U/mL) were enrolled and divided into 3 groups: extensive duodenal atrophy (n = 209), with IgA anti-tTG deposits throughout the duodenum and high serum anti-tTG concentrations (157 ± 178 U/mL); bulb duodenal atrophy (n = 22), with widespread IgA anti-tTG deposits in 9 and in the bulb alone in 13 and low serum anti-tTG concentrations (13.9 ± 8.7 U/mL); and normal duodenum (n = 14), with widespread IgA anti-tTG deposits in 8 and in the bulb alone in 6 and low serum anti-tTG concentrations (10.6 ± 6.2 U/mL). All patients in the first 2 groups were diagnosed with CD and 8 from the third group. All improved after 1 year of gluten-free diet. Bulb duodenal analysis led to a 12% (30/245) increase in CD diagnosis. No CD-related lesions were observed in the 30 control subjects. CONCLUSIONS: In children at risk for CD, bulb duodenum biopsy sampling is essential to identify villous atrophy and detect IgA anti-tTG deposits even in absence of intestinal lesions. These mucosal autoantibodies could well represent a new standard for diagnosing CD.

Anti-transglutaminase 6 Antibody Development in Children With Celiac Disease Correlates With Duration of Gluten Exposure.

OBJECTIVES: Antibodies against transglutaminase 6 (anti-TG6) have been implicated in neurological manifestations in adult patients with genetic gluten intolerance, and it is unclear whether autoimmunity to TG6 develops following prolonged gluten exposure. We measured the anti-TG6 in children with celiac disease (CD) at the diagnosis time to establish a correlation between these autoantibodies and the duration of gluten exposure. We investigated a correlation between anti-TG6 and the presence of neurological disorders. METHODS: Anti-TG6 (IgA/IgG) were measured by ELISA in sera of children with biopsy-proven CD and of children experiencing gastrointestinal disorders. CD patients positive for anti-TG6 were retested after 2 years of gluten-free diet (GFD). RESULTS: We analyzed the sera of 274 CD children and of 121 controls. Anti-TG6 were detected in 68/274 (25%) CD patients and in 19/121 (16%) controls, with significant difference between the 2 groups (P = 0.04). None of the CD patients and of the controls testing positive for anti-TG6 were experiencing neurological disorders. Eleven of 18 (61%) CD patients with other autoimmune diseases were positive for anti-TG6. In CD patients, a significant correlation between the gluten exposure before the CD diagnosis and anti-TG6 concentration was found (P = 0.006 for IgA; P < 0.0001 for IgG). After GFD anti-TG6 concentrations were significantly reduced (P < 0.001). No significant correlation was observed between anti-TG6 and anti-TG2 serum concentrations. CONCLUSIONS: Anti-TG6 are more prevalent in children with untreated CD in the absence of overt neurological disorders. The synthesis of the anti-TG6 is related to a longer exposure to gluten before the CD diagnosis, and the autoimmunity against TG6 is gluten dependent and disappeared during GFD.

Intestinal Production of Anti-Tissue Transglutaminase 2 Antibodies in Patients with Diagnosis Other Than Celiac Disease.

It has been hypothesized that gluten-dependent production of anti-tissue-transglutaminase 2 (anti-TG2) antibodies may occur only at an intestinal level. We have investigated intestinal production of anti-TG2 antibodies in 136 patients with normal serum levels of anti-TG2 antibodies and normal duodenal mucosa. Intestinal deposits of anti-TG2 antibodies were evaluated by immunofluorescence and anti-TG2 antibodies released in organ culture supernatants measured by ELISA. Intestinal antibody libraries were obtained from 10 subjects. Immunohistochemistry for CD25⁺, CD3⁺, and TCR-γδ⁺ was assessed in subjects with positive (n = 32) and negative (n = 31) intestinal anti-TG2 antibodies. Globally 33/136 (24%) seronegative patients produced anti-TG2 autoantibodies at an intestinal level. Antibody libraries analysis confirmed the anti-TG2 antibodies mucosal production in all (n = 8) positive subjects. Lamina propria CD25⁺ cell count was significantly (p < 0.05) higher in patients with intestinal anti-TG2. Moreover, 13/32 (41%) of them showed high TCR-γδ⁺/CD3⁺ ratios. Intestinal anti-TG2 antibody production does not show absolute specificity for CD. It is seen more often in association with inflamed mucosa. Further investigations are necessary to prove the possible role of dietary gluten.

Cross-sectional study of coeliac autoimmunity in a population of Vietnamese children.

OBJECTIVE: The prevalence of coeliac disease (CD) in Vietnam is unknown. To fill this void, we assessed the prevalence of serological markers of CD autoimmunity in a population of children in Hanoi. SETTING: The outpatient blood drawing laboratory of the largest paediatric hospital in North Vietnam was used for the study, which was part of an international project of collaboration between Italy and Vietnam. PARTICIPANTS: Children having blood drawn for any reason were included. Exclusion criteria were age younger than 2 years, acquired or congenital immune deficiency and inadequate sample. A total of 1961 children (96%) were enrolled (838 females, 1123 males, median age 5.3 years). OUTCOMES: Primary outcome was the prevalence of positive autoimmunity to both IgA antitransglutaminase antibodies (anti-tTG) assessed with an ELISA test and antiendomysial antibodies (EMA). Secondary outcome was the prevalence of CD predisposing human leucocyte antigens (HLA) (HLA DQ2/8) in the positive children and in a random group of samples negative for IgA anti-tTG. RESULTS: The IgA anti-tTG test was positive in 21/1961 (1%; 95% CI 0.61% to 1.53%); however, EMA antibodies were negative in all. HLA DQ2/8 was present in 7/21 (33%; 95% CI 14.5% to 56.9%) of the anti-tTG-positive children and in 72/275 (26%; 95% CI 21% to 32%) of those who were negative. CONCLUSIONS: Coeliac autoimmunity is rare in Vietnam, although prevalence of HLA DQ2/8 is similar to that of other countries. We hypothesise that the scarce exposure to gluten could be responsible for these findings.

Serum anti-tissue transglutaminase antibodies detected during febrile illness may not be produced by the intestinal mucosa.

Anti-transglutaminase antibodies are the diagnostic marker of celiac disease, and are considered to be synthesized only by intestinal B-lymphocytes. During an infectious disease, these antibodies are transiently detected in serum. We show that these infection-triggered antibodies may not originate in the intestinal mucosa and are not an indication of celiac disease.

Cryptic genetic gluten intolerance revealed by intestinal antitransglutaminase antibodies and response to gluten-free diet.

BACKGROUND AND OBJECTIVE: Antitransglutaminase (anti-TG2) antibodies are synthesised in the intestine and their presence seems predictive of future coeliac disease (CD). This study investigates whether mucosal antibodies represent an early stage of gluten intolerance even in the absence of intestinal damage and serum anti-TG2 antibodies. METHODS: This study investigated 22 relatives of patients with CD genetically predisposed to gluten intolerance but negative for both serum anti-TG2 antibodies and intestinal abnormalities. Fifteen subjects were symptomatic and seven were asymptomatic. The presence of immunoglobulin A anti-TG2 antibodies in the intestine was studied by creating phage-antibody libraries against TG-2. The presence of intestinal anti-TG2 antibodies was compared with the serum concentration of the intestinal fatty acid-binding protein (I-FABP), a marker for early intestinal mucosal damage. The effects of a 12-month gluten-free diet on anti-TG2 antibody production and the subjects' clinical condition was monitored. Twelve subjects entered the study as controls. RESULTS: The intestinal mucosa appeared normal in 18/22; 4 had a slight increase in intraepithelial lymphocytes. Mucosal anti-TG2 antibodies were isolated in 15/22 subjects (68%); in particular symptomatic subjects were positive in 13/15 cases and asymptomatic subjects in 2/7 cases (p=0.01). No mucosal antibodies were selected from the controls' biopsies. There was significant correlation between the presence of intestinal anti-TG2 antibodies and positive concentrations of I-FABP (p=0.0008). After a gluten-free diet, 19/22 subjects underwent a second intestinal biopsy, which showed that anti-TG2 antibodies had disappeared in 12/15 (p=0.002), while I-FABP decreased significantly (p<0.0001). The diet resolved both extraintestinal and intestinal symptoms. CONCLUSIONS: A new form of genetic-dependent gluten intolerance has been described in which none of the usual diagnostic markers is present. Symptoms and intestinal anti-TG2 antibodies respond to a gluten free-diet. The detection of intestinal anti-TG2 antibodies by the phage-antibody libraries has an important diagnostic and therapeutic impact for the subjects with gluten-dependent intestinal or extraintestinal symptoms. Clinical trial number NCT00677495.

Cost-effective HLA typing with tagging SNPs predicts celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations.

Human leukocyte antigen (HLA) genes, located on chromosome 6p21.3, have a crucial role in susceptibility to various autoimmune and inflammatory diseases, such as celiac disease and type 1 diabetes. Certain HLA heterodimers, namely DQ2 (encoded by the DQA1*05 and DQB1*02 alleles) and DQ8 (DQA1*03 and DQB1*0302), are necessary for the development of celiac disease. Traditional genotyping of HLA genes is laborious, time-consuming, and expensive. A novel HLA-genotyping method, using six HLA-tagging single-nucleotide polymorphisms (SNPs) and suitable for high-throughput approaches, was described recently. Our aim was to validate this method in the Finnish, Hungarian, and Italian populations. The six previously reported HLA-tagging SNPs were genotyped in patients with celiac disease and in healthy individuals from Finland, Hungary, and two distinct regions of Italy. The potential of this method was evaluated in analyzing how well the tag SNP results correlate with the HLA genotypes previously determined using traditional HLA-typing methods. Using the tagging SNP method, it is possible to determine the celiac disease risk haplotypes accurately in Finnish, Hungarian, and Italian populations, with specificity and sensitivity ranging from 95% to 100%. In addition, it predicts homozygosity and heterozygosity for a risk haplotype, allowing studies on genotypic risk effects. The method is transferable between populations and therefore suited for large-scale research studies and screening of celiac disease among high-risk individuals or at the population level.

IL23R in the Swedish, Finnish, Hungarian and Italian populations: association with IBD and psoriasis, and linkage to celiac disease.

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.

Association study of the IL18RAP locus in three European populations with coeliac disease.

Coeliac disease is caused by dietary gluten, triggering a chronic inflammation of the small intestine in genetically predisposed individuals. Recently, a risk locus on chromosome 2q11-q12, harbouring interleukin 18 receptor accessory protein (IL18RAP) and three other genes, was suggested for coeliac disease. IL18 has been shown to play an important role in T helper type 1 activity in coeliac disease, making this locus a highly interesting candidate. In this study, two previously indicated risk variants at the IL18RAP locus (rs13015714 and rs917997) were tested for genetic association in 1638 cases with coeliac disease and 1385 control individuals from the Finnish, Hungarian and Italian populations. The protein expression level of IL18RAP was also compared between risk allele carriers and non-carriers by Western blotting. Furthermore, immunohistochemical analysis was performed to study IL18RAP protein expression in small intestinal biopsies of untreated and treated coeliac patients and controls. We confirmed genetic association and dose effects of variants at the 2q12.1 locus with coeliac disease in the Hungarian population. The GA haplotype of the markers rs13015714 and rs917997 showed the strongest association (P = 0.0001, odds ratio = 1.475, 95% confidence interval 1.21-1.80). Two putative isoforms of IL18RAP were detected and the ratios and total levels of these isoforms may contribute to the aetiology of coeliac disease. Our study supports IL18RAP as a novel predisposing gene for coeliac disease and highlights the need for further functional studies on this relatively unknown gene in coeliac disease pathogenesis.

Characterization of the anti-tissue transglutaminase antibody response in nonobese diabetic mice.

Type 1 diabetes mellitus is an autoimmune disorder characterized by destruction of insulin-producing pancreatic beta cells by T lymphocytes. In nonobese diabetic (NOD) mice, a role has been hypothesized for dietary gluten proteins in the onset of diabetes, and because gluten dependence is the major feature of celiac disease, together with production of Abs to the autoantigen tissue transglutaminase (tTG), we looked for the presence of anti-tTG Abs in the serum of NOD mice and, to establish their origin, analyzed the Ab repertoire of NOD mice using phage display Ab libraries. We found significant levels of serum anti-tTG Abs and were able to isolate single-chain Ab fragments to mouse tTG mainly from the Ab libraries made from intestinal lymphocytes and to a lesser extent from splenocytes. Data from NOD mice on a gluten-free diet suggest that the anti-tTG response is not gluten-dependent. The intestinal Ab response to tTG is a feature of NOD mice, but the underlying mechanisms remain obscure.

One-step cloning of anti tissue transglutaminase scFv from subjects with celiac disease.

Celiac disease is characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals with a prominent role being played by gliadins, specific antigenic determinants found in wheat gluten. Patients suffering from celiac disease have serum antibodies recognizing gliadin, as well as the endomysial autoantigen tissue transglutaminase. Phage display antibody libraries have revealed ectopic production of anti-transglutaminase antibodies by intestinal lymphocytes with a biased use of the VH5 antibody gene family. Here we report a study on the pairing of VH and VL families in the antibodies to transglutaminase. Our results led to the construction of small phage display antibody libraries based on the amplification of the two genes in the VH5 family from intestinal lymphocytes. This method can be used for the rapid characterization of the anti-transglutaminase response in a potentially large number of subjects including asymptomatic patients whose serum antibodies may be undetectable.