Intestinal anti-transglutaminase 2 immunoglobulin A deposits in children at risk for coeliac disease (CD): data from the PreventCD study.

In coeliac disease (CD), anti-tissue transglutaminase 2 immunoglobulin (Ig)A antibodies (anti-TG2) are produced and deposited in the intestine. PreventCD (www.preventcd.com) is a European multi-centre study, which investigates the influence of infant nutrition and that of genetic, immunological and other environmental factors on the risk of developing CD. The aim of the current study was to evaluate the appearance of intestinal anti-TG2 deposits in very early intestinal biopsies from at-risk infants and their predictive value for villous atrophy. Sixty-five small bowel biopsies, performed in 62 children, were investigated for the presence of intestinal anti-TG2 extracellular IgA deposits by using double immunofluorescence. The biopsies were performed in the presence of elevated serum levels of CD-associated antibodies and/or symptoms suggesting disease. Deposits of anti-TG2 IgA were present in 53 of 53 CD patients and three of three potential CD patients. In potential CD patients, mucosal deposits showed a patchy distribution characterized by some areas completely negative, whereas active CD patients had uniformly present and evident mucosal deposits. Only one of six patients without CD (negative for serum anti-TG2 and with normal mucosa) had intestinal deposits with a patchy distribution and a weak staining. Two of the 53 CD patients received a definitive diagnosis of CD after a second or third biopsy; mucosal deposits of anti-TG2 IgA were evaluated in all samples. Before developing villous atrophy, both patients had anti-TG2 deposits in normal mucosal architecture, antibodies in one patient being absent in serum. We demonstrated that in CD the intestinal deposits of anti-TG2 are a constant presence and appear very early in the natural history of disease.

Coeliac disease diagnosis: ESPGHAN 1990 criteria or need for a change? Results of a questionnaire.

BACKGROUND AND OBJECTIVES: A revision of criteria for diagnosing coeliac disease (CD) is being conducted by The European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN). In parallel, we have performed a survey aimed to evaluate present practices for CD among paediatric gastroenterologists and to learn their views on the need for modification of present criteria for CD diagnosis. PATIENTS AND METHODS: Questionnaires were distributed to experienced paediatric gastroenterologists (ESPGHAN members) via the Internet. RESULTS: Overall, 95 valid questionnaires were available for analysis, pertaining to 28 different countries, with the majority of responders treating patients with CD for >15 years. Only about 12% of the responders comply with present criteria, noncompliance being related mainly to the challenge policy. Approximately 90% request a revision and modification of the present criteria. Forty-four percent want to omit the small bowel biopsy in symptomatic children with positive anti-tissue transglutaminase immunoglobulin (Ig) A or endomysial IgA antibodies, especially if they are DQ2/DQ8 positive. For silent cases detected by screening with convincingly positive anti-tissue transglutaminase IgA or EMA IgA, about 30% consider that no small bowel biopsy should be required in selected cases. Adding human leukocyte antigen typing in the diagnostic workup was asked for by 42% of the responders. As for gluten challenge, a new policy is advocated restricting its obligation to cases whenever the diagnosis is doubtful or unclear. CONCLUSIONS: Based on these opinions, revision of the ESPGHAN criteria for diagnosing CD is urgently needed.

European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease.

OBJECTIVE: Diagnostic criteria for coeliac disease (CD) from the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) were published in 1990. Since then, the autoantigen in CD, tissue transglutaminase, has been identified; the perception of CD has changed from that of a rather uncommon enteropathy to a common multiorgan disease strongly dependent on the haplotypes human leukocyte antigen (HLA)-DQ2 and HLA-DQ8; and CD-specific antibody tests have improved. METHODS: A panel of 17 experts defined CD and developed new diagnostic criteria based on the Delphi process. Two groups of patients were defined with different diagnostic approaches to diagnose CD: children with symptoms suggestive of CD (group 1) and asymptomatic children at increased risk for CD (group 2). The 2004 National Institutes of Health/Agency for Healthcare Research and Quality report and a systematic literature search on antibody tests for CD in paediatric patients covering the years 2004 to 2009 was the basis for the evidence-based recommendations on CD-specific antibody testing. RESULTS: In group 1, the diagnosis of CD is based on symptoms, positive serology, and histology that is consistent with CD. If immunoglobulin A anti-tissue transglutaminase type 2 antibody titers are high (>10 times the upper limit of normal), then the option is to diagnose CD without duodenal biopsies by applying a strict protocol with further laboratory tests. In group 2, the diagnosis of CD is based on positive serology and histology. HLA-DQ2 and HLA-DQ8 testing is valuable because CD is unlikely if both haplotypes are negative. CONCLUSIONS: The aim of the new guidelines was to achieve a high diagnostic accuracy and to reduce the burden for patients and their families. The performance of these guidelines in clinical practice should be evaluated prospectively.

Genome-wide analysis of extended pedigrees confirms IL2-IL21 linkage and shows additional regions of interest potentially influencing coeliac disease risk.

Coeliac disease is a chronic inflammatory condition of the small intestine, triggered by dietary exposure to gluten in genetically susceptible individuals. Risk alleles at HLA-DQA1 and HLA-DQB1 are necessary for disease development, but are alone not sufficient for disease onset. We aimed to identify novel loci underlying susceptibility to coeliac disease through the use of extended Finnish and Hungarian families with multiple affected individuals. An initial whole-genome linkage approach yielded several loci that were followed up further using the Immunochip custom array. Loci with a parametric logarithm of odds (LOD) score of >1.3 were identified at 4q, 6p [human leukocyte antigen (HLA) region], 6q, 7p, 17p, 17q and at 22p. The 4q and 6q loci have been identified previously in coeliac disease risk, whereas follow-up analyses indicate that the 17p and 22p loci may be novel risk loci for coeliac disease. These loci harbour previously described risk variants for other autoimmune diseases, but their segregation patterns do not explain the linkage to coeliac disease. We followed up the linkage to the 4q region, containing the previously described interleukin (IL)2 and IL21 genes. The risk variants at 4q in the studied pedigrees are most likely distinct from previously described risk variants, indicating that the observed linkage may be due to rare high-risk variants of still unknown nature. The importance of this locus to coeliac disease risk was further shown by the finding that serum levels of IL21 were elevated in both untreated and treated coeliac patients compared to controls.

Epithelial transport and deamidation of gliadin peptides: a role for coeliac disease patient immunoglobulin A.

In coeliac disease, the intake of dietary gluten induces small-bowel mucosal damage and the production of immunoglobulin (Ig)A class autoantibodies against transglutaminase 2 (TG2). We examined the effect of coeliac patient IgA on the apical-to-basal passage of gluten-derived gliadin peptides p31-43 and p57-68 in intestinal epithelial cells. We demonstrate that coeliac IgA enhances the passage of gliadin peptides, which could be abolished by inhibition of TG2 enzymatic activity. Moreover, we also found that both the apical and the basal cell culture media containing the immunogenic gliadin peptides were able to induce the proliferation of deamidation-dependent coeliac patient-derived T cells even in the absence of exogenous TG2. Our results suggest that coeliac patient IgA could play a role in the transepithelial passage of gliadin peptides, a process during which they might be deamidated.

Fine mapping of the CELIAC2 locus on chromosome 5q31-q33 in the Finnish and Hungarian populations.

Celiac disease is a chronic inflammation of the small intestine, arising in genetically predisposed individuals as a result of ingestion of dietary gluten. The only confirmed and functionally characterised genetic risk factors for celiac disease are the DQ2 or DQ8 heterodimers at the major histocompatibility complex (MHC) class II locus (CELIAC1). These genes are necessary but alone not sufficient for disease onset. Genome-wide linkage scans have suggested chromosome 5q31-q33 (CELIAC2) as an important risk locus for celiac disease. This region has also been associated to other inflammatory disorders, although as yet, no clear gene associations have been found. In the current study, 11 celiac disease candidate loci were screened for genetic linkage in the Hungarian population. As the CELIAC2 locus showed the strongest evidence for linkage, this locus was selected for follow-up. Seventeen candidate genes were selected from the CELIAC2 locus, and genotyped using 48 haplotype tagging single nucleotide polymorphisms (SNPs) in large Finnish and Hungarian family materials. A subset of these, 40 tagging SNPs in 15 genes, were genotyped in an independent set of Finnish and Hungarian cases and controls. We confirmed linkage of this region with celiac disease and report strong linkage in both the Finnish and Hungarian populations. The association analysis showed modest associations throughout the whole region. These association findings were not replicated in the case-control datasets. Our study strongly supports the role of the CELIAC2 locus in celiac disease, but it also highlights the need for a more powerful study design in the region, to locate the true disease risk variants.

Linkage and association study of FcgammaR polymorphisms in celiac disease.

The Fcgamma receptor cluster on chromosome 1q23 contains a number of genes that may affect susceptibility to celiac disease, but previous studies have yielded contradictory results. We studied the FcgammaRIIa*A519G (rs1801274) and FcgammaRIIIa*A559C (rs396991) single nucleotide polymorphisms in celiac disease families from Hungary and Finland and in celiac disease case-control materials from Hungary and Italy. Neither the Hungarian nor the Italian case-control material or a meta-analysis of the combined case-control material showed significant single-marker or haplotype association. In addition, neither linkage nor family-based association tests showed evidence for association in the Finnish or Hungarian family material. This study thus does not support a previous publication showing FcgammaR association with celiac disease.

The shared CTLA4-ICOS risk locus in celiac disease, IgA deficiency and common variable immunodeficiency.

IgA deficiency (IgAD) and common variable immunodeficiency (CVID) often co-occur in families, associating with chronic inflammatory diseases such as celiac disease (CD). ICOS (inducible co-stimulator) and CTLA4 (cytotoxic T-lymphocyte-associated protein-4) may be important in both disorders, as ICOS is necessary for Ig class-switching and CTLA4 negatively regulates T-cell activation. Linkage and association of CD with CTLA4-ICOS is well documented, we thus aimed to further pinpoint CD susceptibility by haplotype-tagging analysis. We genotyped 663 CD families from Finland and Hungary, 575 additional CD patients from Finland, Hungary and Italy; 275 Swedish and Finnish IgAD individuals and 87 CVID individuals for 14-18 genetic markers in CTLA4-ICOS. Association was found between CTLA4-ICOS and both IgAD (P=0.0015) and CVID (P=0.0064). We confirmed linkage of CTLA4-ICOS with CD (LOD 2.38, P=0.0005) and found association of CTLA4-ICOS with CD (P=0.0009). Meta-analysis of the IgAD, CVID and CD materials revealed intergenic association (P=0.0005). Disease-associated markers were associated with lower ICOS and higher CTLA4 expression, indicating that the risk haplotypes contain functional variants. In summary, we identified a novel shared risk locus for IgAD, CVID and CD, the first report of association between CTLA4-ICOS and IgAD. Association between CD and CTLA4-ICOS was also confirmed in a large European data set.

Coeliac disease-specific autoantibodies targeted against transglutaminase 2 disturb angiogenesis.

Coeliac disease is characterized by immunoglobulin-A (IgA)-class autoantibodies targeted against transglutaminase 2 (TG2), a multi-functional protein also with a role in angiogenesis. These antibodies are present in patient serum but are also found bound to TG2 below the epithelial basement membrane and around capillaries in the small intestinal mucosa. Based on these facts and the information that the mucosal vasculature of coeliac patients on a gluten-containing diet is disorganized, we studied whether the coeliac disease-specific autoantibodies targeted against TG2 would disturb angiogenesis. The effects of coeliac disease-specific autoantibodies on in vitro angiogenesis were studied in angiogenic cell cultures. The binding of the antibodies to cells, endothelial sprouting, migration of both endothelial and vascular mesenchymal cells, the integrity of the actin cytoskeleton in both cell types and the differentiation of vascular mesenchymal cells were recorded. In vitro, IgA derived from coeliac disease patients on a gluten-containing diet binds to surface TG2 on endothelial and vascular mesenchymal cells and this binding can be inhibited by the removal of TG2. In addition, coeliac disease-specific autoantibodies targeting TG2 disturb several steps of angiogenesis: endothelial sprouting and the migration of both endothelial and vascular mesenchymal cells. Furthermore, the autoantibodies cause disorganization of the actin cytoskeleton in both capillary cell types that account most probably for the defective cellular migration. We conclude that coeliac disease-specific autoantibodies recognizing TG2 inhibit angiogenesis in vitro. This disturbance of the angiogenic process could lead in vivo to the disruption of the mucosal vasculature seen in coeliac disease patients on a gluten-containing diet.

Myosin IXB gene region and gluten intolerance: linkage to coeliac disease and a putative dermatitis herpetiformis association.

BACKGROUND: Coeliac disease is caused by dietary gluten, which triggers chronic inflammation of the small intestine in genetically predisposed individuals. In one quarter of the patients the disease manifests in the skin as dermatitis herpetiformis. Recently, a novel candidate gene, myosin IXB on chromosome 19p13, was shown to be associated with coeliac disease in the Dutch and Spanish populations. The same gene has previously been associated with inflammatory bowel disease, systemic lupus erythematosus and rheumatoid arthritis risk, making myosin IXB a potential shared risk factor in these inflammatory disorders. METHODS: In this study, previously reported myosin IXB variants were tested for genetic linkage and association with coeliac disease in 495 Hungarian and Finnish families and in an additional 270 patients and controls. RESULTS AND CONCLUSION: The results show significant linkage (logarithm of odds (LOD) 3.76, p = 0.00002) to 19p13 which supports the presence of a genuine risk factor for coeliac disease in this locus. Myosin IXB variants were not associated with coeliac disease in this study; however, weak evidence of association with dermatitis herpetiformis was found. The association could not explain the strong linkage seen in both phenotypes, indicating that the role of other neighbouring genes in the region cannot be excluded. Therefore, more detailed genetic and functional studies are required to characterise the role of the myosin IXB gene in both coeliac disease and dermatitis herpetiformis.

Increased mucosal expression of Toll-like receptor (TLR)2 and TLR4 in coeliac disease.

OBJECTIVES: The dysregulation of adaptive immunity is extensively investigated in celiac disease (CD). Recent data also suggest, however, the implication of innate immunity in CD. Toll-like receptors (TLRs) play a central role in the initiation or maintenance of innate immune responses. The aim of this study was to characterise the expression of TLR2, TLR3, and TLR4 in duodenal biopsy samples taken from children with CD and from controls. PATIENTS AND METHODS: Duodenal biopsy specimens were collected from 16 children with untreated CD, 9 children with treated CD, and 10 controls. The mRNA expression of TLR2, TLR3, and TLR4 was determined by semiquantitative reverse transcription-polymerase chain reaction. Protein levels of TLRs were determined by Western blot. RESULTS: We found higher TLR2 and TLR4 mRNA expression and protein levels in the duodenal mucosa of children with treated CD and untreated CD compared with controls. TLR2 and TLR4 mRNA expression and protein levels were even higher in the duodenal mucosa of children with treated CD than in untreated CD. TLR3 mRNA expression was increased in the duodenal mucosa of children with treated CD compared with untreated CD and controls. We were able to detect TLR3 protein only in the biopsy specimens of treated patients with CD. CONCLUSIONS: The alteration of TLR2 and TLR4 expression in the duodenal mucosa of patients with CD supports the potential implication of innate immune system in the pathomechanism of this disease.

Diagnostic significance of HLA-DQ typing in patients with previous coeliac disease diagnosis based on histology alone.

BACKGROUND: Coeliac disease is strongly associated with human leukocyte antigen (HLA)-DQ2 or DQ8 genotypes. The diagnosis is based on demonstrating crypt-hyperplastic villous atrophy, endomysial or transglutaminase antibodies and correlation of disease activity with gluten intake. AIM: To evaluate the clinical utility of HLA-DQ typing, when coeliac disease diagnosis had previously been established solely by histology. METHODS: HLA-DQ alleles, endomysial and transglutaminase antibodies were investigated and histology slides reviewed in 70 patients diagnosed 2-25 years earlier by small-intestinal biopsy but without measuring endomysial or transglutaminase antibodies. Patients without DQ2 or DQ8 or without unequivocal villous atrophy were followed-up on free diet by using serology and biopsies. RESULTS: All 40 endomysial/transglutaminase antibodies positive patients carried DQ2 or DQ8, and 39 of them had severe villous atrophy. Only 56% of patients without endomysial or transglutaminase antibodies positivity had DQ2 or DQ8 (P < 0.001). Seropositivity and relapse developed in 4 of 11 DQ2 positive but in none of 15 DQ2 and DQ8 negative patients on long-term gluten exposure. CONCLUSIONS: Coeliac disease diagnosis based solely on histology is not always reliable. HLA-DQ typing is important in identifying DQ2 and DQ8 negative subjects who need revision of their diagnosis, but it does not have additive diagnostic value if endomysial positivity is already known.

Immunoglobulin A autoantibodies against transglutaminase 2 in the small intestinal mucosa predict forthcoming coeliac disease.

BACKGROUND: Reliable markers of early developing coeliac diseases are needed. Coeliac autoantibodies in the serum or Marsh I inflammation may be indicators of subsequent coeliac disease. AIM: To investigate whether determination of intestinal transglutaminase 2-targeted autoantibody deposits would detect early developing coeliac disease better than previous methods. METHODS: The study investigated patients previously excluded for coeliac disease: 25 had positive serum coeliac autoantibodies (endomysial), 25 antibody-negative had Marsh I, and 25 antibody-negative had Marsh 0 finding. Seven (median) years after baseline investigation, new coeliac cases were recorded, and small bowel biopsy was offered to the rest of the patients. Serum and intestinal coeliac autoantibodies and intraepithelial lymphocytes were assessed as indicators of developing coeliac disease. RESULTS: Seventeen patients had developed coeliac disease: 13 in the autoantibody-positive group, three in the Marsh I group and one in the Marsh 0 group. At baseline, intestinal coeliac autoantibody deposits had a sensitivity and specificity of 93% and 93% in detecting subsequent coeliac disease, CD3+ 59% and 57%, gammadelta+ 76% and 60%, and villous tip intraepithelial lymphocytes 88% and 71%, respectively. CONCLUSIONS: Endomysial antibodies with normal histology indicates early developing coeliac disease. Transglutaminase 2-targeted intestinal autoantibody deposits proved the best predictor of subsequent coeliac disease.

Self transglutaminase-based rapid coeliac disease antibody detection by a lateral flow method.

BACKGROUND: The conventional coeliac disease antibody tests require patient's sera, and are laborious and time-consuming. AIM: To evaluate a newly developed rapid whole blood test in coeliac disease antibody detection, and its suitability for office use. METHODS: Endogenous tissue transglutaminase found in red blood cells in a whole blood fingertip or venous sample is liberated upon haemolysis and complexes with tissue transglutaminase antibodies, if present. The complexes, captured by a lateral flow system, are visualized within 5 min. Stored samples from 121 untreated, 106 treated coeliac disease patients and 107 controls were evaluated and compared with serum endomysium and tissue transglutaminase antibody tests and histology; 150 patients were prospectively tested on site in the doctor's office. RESULTS: The rapid test showed sensitivity (96.7%) comparable with the serum endomysium and tissue transglutaminase antibody tests from stored samples; specificity was slightly lower (93.5%). When tested on site the results were concordant in 96.7% of cases compared with endomysium and tissue transglutaminase antibody results. The test recognized the disappearance of tissue transglutaminase antibodies on a gluten-free diet. CONCLUSIONS: The self tissue transglutaminase-based rapid test can be easily carried out from a fingertip blood sample on site in the physician's office for both coeliac disease case finding and dietary monitoring purposes.

Endomysial antibody-negative coeliac disease: clinical characteristics and intestinal autoantibody deposits.

BACKGROUND: Some patients with untreated coeliac disease are negative for serum endomysial autoantibodies (EmA) targeted against transglutaminase 2 (TG2). AIMS: To evaluate the clinical and histological features of EmA-negative coeliac disease, and to examine whether EmA-equivalent autoantibodies against TG2 can be seen in the small-bowel mucosa when absent in serum. PATIENTS: Serum EmA was studied in 177 biopsy-proved specimens from adult patients with coeliac disease. 20 patients with intestinal diseases served as non-coeliac controls; three had autoimmune enteropathy with villous atrophy. METHODS: Clinical manifestations, small-bowel mucosal morphology, intraepithelial inflammation and TG2-specific extracellular immunoglobulin A (IgA) deposits were investigated in both serum EmA-negative and EmA-positive patients. RESULTS: 22 patients with IgA-competent coeliac disease were negative for serum EmA. Three of these had small-bowel lymphoma. Patients with EmA-negative coeliac disease were older, had abdominal symptoms more often, and the density of gammadelta+ intraepithelial lymphocytes in their intestinal mucosa was lower than in EmA-positive patients; otherwise the histology was similar. All serum EmA-negative patients with coeliac disease, but none of the disease controls, had gluten-dependent mucosal IgA deposits alongside TG2 in the small-bowel mucosal specimens. In vivo deposited IgA was shown to be TG2-specific by its ability to bind recombinant TG2. CONCLUSIONS: Negative serum EmA might be associated with advanced coeliac disease. TG2-targeted autoantibodies were deposited in the small-bowel mucosa even when absent in serum. This finding can be used in the diagnosis of seronegative coeliac disease when the histology is equivocal. It may also be helpful in the differential diagnosis between autoimmune enteropathy and coeliac disease.

Autoantibody targeting of brain and intestinal transglutaminase in gluten ataxia.

OBJECTIVE: To investigate the presence of autoantibody deposition against type 2 tissue transglutaminase (TG2; a reliable marker of the whole spectrum of gluten sensitivity) in the jejunal tissue and brain of patients with gluten ataxia and in control subjects. METHODS: The authors evaluated jejunal biopsy samples from nine patients with gluten ataxia and seven patients with other causes of ataxia for the presence of TG2-related immunoglobulin deposits using double-color immunofluorescence. Autopsy brain tissue from one patient with gluten ataxia and one neurologically intact brain were also studied. RESULTS: IgA deposition on jejunal TG2 was found in the jejunal tissue of all patients with gluten ataxia and in none of the controls. The intestinal IgA deposition pattern was similar to that seen in patients with overt and latent celiac disease and in those with dermatitis herpetiformis. Widespread IgA deposition around vessels was found in the brain of the patient with gluten ataxia but not the control brain. The deposition was most pronounced in the cerebellum, pons, and medulla. CONCLUSIONS: Anti-tissue transglutaminase IgA antibodies are present in the gut and brain of patients with gluten ataxia with or without an enteropathy in a similar fashion to patients with celiac disease, latent celiac disease, and dermatitis herpetiformis but not in ataxia control subjects. This finding strengthens the contention that gluten ataxia is immune mediated and belongs to the same spectrum of gluten sensitivity as celiac disease and dermatitis herpetiformis.

Coeliac disease case finding and diet monitoring by point-of-care testing.

BACKGROUND: Immunoglobulin A class transglutaminase autoantibodies are highly predictive markers of active coeliac disease, a disorder difficult to recognize solely on clinical grounds. AIMS: To develop and evaluate a simple rapid test for point-of-care detection of coeliac autoantibodies. METHODS: The novel whole blood test utilizes the patient's endogenous transglutaminase in red blood cells for detection of transglutaminase-specific immunoglobulin A antibodies present in the blood sample, with normal plasma immunoglobulin A detection as positive test control. We evaluated 284 patients under suspicion of coeliac disease and undergoing jejunal biopsy, and 263 coeliac patients on a gluten-free diet, 383 being tested prospectively in a point-of-care setting. Results were compared with histology, conventional serum autoantibody results and dietary adherence. RESULTS: The rapid test showed 97% sensitivity and 97% specificity for untreated coeliac disease, and identified all immunoglobulin A-deficient samples. Point-of-care testing found new coeliac cases as efficiently as antibody tests in laboratory. Coeliac autoantibodies were detected onsite in 21% of treated patients, while endomysial and transglutaminase antibodies were positive in 20% and 19%, respectively. The positivity rate correlated with dietary lapses and decreased on intensified dietary advice given upon positive point-of-care test results. CONCLUSIONS: Point-of-care testing was accurate in finding new coeliac cases and helped to identify and decrease dietary non-compliance.

In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies.

BACKGROUND: IgA class serum autoantibodies against type 2 (tissue) transglutaminase (TG2) bind to both intestinal and extraintestinal normal tissue sections in vitro, eliciting endomysial, reticulin, and jejunal antibody reactions. It is not known whether similar binding also occurs in coeliac patients in vivo, and may thereby contribute to disease manifestations. AIMS: To investigate intestinal and extraintestinal coeliac tissues for the presence of in vivo bound TG2 specific IgA and its relation to small intestinal mucosal atrophy. PATIENTS: We investigated jejunal samples with normal villous morphology from 10 patients with developing coeliac disease who subsequently progressed to a flat lesion, from 11 patients with dermatitis herpetiformis, and from 12 non-coeliac controls. Six extrajejunal biopsy samples (liver, lymph node, muscle, appendix), obtained based on independent clinical indications from patients with active coeliac disease, were also studied. METHODS: Double colour immunofluorescent studies for in situ IgA, TG2, and laminin were performed. IgA was eluted from tissue sections and tested for TG2 specificity by enzyme linked immunosorbent assay and indirect immunofluorescence. RESULTS: IgA (in one IgA deficient case IgG) deposition on extracellularly located TG2 was detected in jejunal and extrajejunal specimens of all coeliac patients, and also in seven of 11 dermatitis herpetiformis patients, of whom two had no circulating endomysial antibodies. IgA eluted from extraintestinal coeliac tissues was targeted against TG2. CONCLUSIONS: Coeliac IgA targets jejunal TG2 early in disease development even when endomysial antibodies are not present in the circulation. Extraintestinal target sites of coeliac IgA further indicate that humoral immunity may have a pathogenetic role.

Elevation of IgG antibodies against tissue transglutaminase as a diagnostic tool for coeliac disease in selective IgA deficiency.

BACKGROUND: IgA serum autoantibodies against tissue transglutaminase (tTG) have an established diagnostic value in coeliac disease, and high efficacy tests are widely available for their detection. However, serological evaluation of IgA deficient subjects is still difficult. AIMS: To evaluate the diagnostic potential of IgG class anti-tTG autoantibodies measured quantitatively using an enzyme linked immunosorbent assay (ELISA) compared with immunofluorescent detection of coeliac autoantibodies. PATIENTS: We tested serum samples from 325 IgA deficient subjects, including 78 patients with coeliac disease, 73 disease controls, and 174 blood donors. METHODS: IgG antibodies against human recombinant tTG were measured with an ELISA. IgG antiendomysium antibodies (EMA) were assayed by indirect immunofluorescence on human jejunum and appendix sections. RESULTS: The IgG anti-tTG ELISA had a sensitivity of 98.7% and a specificity of 98.6%, and the correlation with IgG EMA titres was high (r(s)=0.91). One coeliac patient, initially negative in all autoantibody tests, displayed both IgG anti-tTG antibodies and IgG EMA during later gluten exposure. IgG anti-tTG antibodies and EMA titres showed significant decreases (p<0.001) in treated patients. The frequency of IgG anti-tTG autoantibody positivity was 9.8% among IgA deficient blood donors and 11 of the 12 positive subjects with known HLA-DQ haplotypes carried DQ2 or DQ8 alleles. CONCLUSIONS: IgG anti-tTG and IgG EMA autoantibody tests are highly efficient in detecting coeliac disease in IgA deficient patients. The high prevalence of coeliac antibodies among symptom free IgA deficient blood donors who also carry coeliac-type HLA-DQ genes indicates that all IgA deficient persons should be evaluated for coeliac disease.

Missing endomysial and reticulin binding of coeliac antibodies in transglutaminase 2 knockout tissues.

BACKGROUND: Autoantibodies against transglutaminase 2 (TG2) are thought to be responsible for the endomysial (EMA), reticulin (ARA), and jejunal antibody (JEA) tissue binding of serum samples from coeliac patients but the exclusive role of TG2 in these staining patterns has not yet been established. AIMS: To evaluate whether antigens other than TG2 contribute to EMA/ARA/JEA reactions. PATIENTS: Serum samples from 61 EMA/ARA/JEA positive untreated patients with coeliac disease, 40 dermatitis herpetiformis patients, and 34 EMA/ARA/JEA negative non-coeliac controls were tested. METHODS: TG2 knockout (TG2-/-) and wild-type mouse oesophagus, jejunum, liver, and kidney sections, and TG2-/- sections coated with human recombinant TG2 were used as substrates in single and double immunofluorescent studies for patient IgA binding and tissue localisation of TG2, fibronectin, actin, and calreticulin. RESULTS: None of the patient serum samples elicited EMA, ARA, or JEA binding in TG2-/- morphologically normal tissues. In contrast, 96 of 101 gluten sensitive patient samples (95%) reacted with wild-type mouse tissues and all 101 reacted in EMA/ARA/JEA patterns with TG2-/- mouse tissues coated with human TG2. Serum IgA binding to TG2-/- smooth muscle cells was observed in low titres in 31.1%, 27.5%, and 20.5%, and to TG2-/- epithelium in 26.3%, 5.0%, and 8.8% of coeliac, dermatitis herpetiformis, and control samples, respectively. These positivities partly colocalised with actin and calreticulin but not with TG2 or fibronectin. CONCLUSIONS: EMA/ARA/JEA antibody binding patterns are exclusively TG2 dependent both in coeliac and dermatitis herpetiformis patients. Actin antibodies are responsible for some positivities which are not part of the EMA/ARA/JEA reactions.