Quality not quantity for transglutaminase antibody 2: the performance of an endomysial and tissue transglutaminase test in screening coeliac disease remains stable over time

National Institute of Clinical Excellence (NICE) and European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidance for the diagnosis of coeliac disease has been published. However, there is some controversy regarding the advice on the use of stratifying levels of immunoglobulin (IgA) tissue transglutaminase antibody (TG2) test positivity in the absence of test standardization and the vagueness of the indication to test equivocal samples. Using repeat service audit, we demonstrate that a combination of TG2 followed by IgA endomysial antibodies (EMA) is the best strategy for all degrees of mucosal abnormality using our test combination. Reliance upon immunoassay titre is not as effective, and cannot be applied consistently across populations in the absence of assay standardization. Guidelines advocating the use of tests should involve experts in laboratory diagnostics and external quality assurance to ensure that errors of generalization do not occur and that test performance is achievable in routine diagnostic use.     

Assessment of the response to gluten-free diet in an Iraqi population with coeliac disease. A histological and serological follow-up study

Introduction

Coeliac disease (CD) is a common diagnosis among children and adults in Iraq; however, removal of gluten from the diet is essential for patients with CD. The aim of this study, the first such study in Iraq, was to assess the serological and histological recovery profiles of coeliac patients, in both children and adults groups after commencing a gluten-free diet (GFD) for at least 1 year ± 1 month.

Material and methods

The study group comprised 78 proved coeliac patients (46 children and 32 adults, median age: 15 years, range: 1–66 years) who all agreed to undergo endoscopy in addition to serological assessment before and after treatment. The duodenal biopsies were interpreted histologically according to modified Marsh criteria and the sera were tested for anti-gliadin antibody (AGA), endomysium antibody (EMA) and anti-tissue transglutaminase antibody (tTG).

Results

Complete histological remission was seen in 29 (63.1%) of 46 treated children CD patients, while only 5 (10.9%) showed Marsh IIIa changes compared with 11 (24%) before GFD. Similarly none of the 32 adults after GFD showed Marsh IIIb and Marsh IIIc compared with 46.9% and 28.1% before treatment respectively (p = 001). Meanwhile, there was strongly significant reduction in AGA, EMA, and tTG antibodies levels (p = 0.00001) following GFD.

Conclusions

Repeating the duodenal biopsy 1 year ±1 month after diagnosis and starting a GFD supports the routine measurement of using histological findings as a gold standard test to confirm recovery of Iraqi CD patients along with using known coeliac serology antibodies.     

Intestinal anti‐tissue transglutaminase antibodies in potential coeliac disease

Anti‐tissue transglutaminase 2 (anti‐TG2) antibodies are present in the serum of the great majority of untreated coeliac disease (CD) patients. They are produced and deposited in the small intestinal mucosa. Potential CD patients present serum anti‐TG2 antibodies higher than cut‐off, but a normal duodenal mucosa where mucosal deposits of anti‐TG2 are not always detectable. The aim of our work was to investigate the presence of anti‐TG2 intestinal antibodies in patients with potential CD, and identify the most sensitive test to detect them. Twelve active CD patients, 28 potential CD patients and 39 non‐CD controls were enrolled. Biopsy fragments from all patients were analysed by double immunofluorescence to detect mucosal deposits of anti‐TG2 antibodies. Fragments from the same subjects were also cultured for 24 h with medium in the presence or absence of gliadin peptides. Anti‐TG2 autoantibodies secreted into supernatants were measured by enzyme‐linked immunosorbent assay. All active CD, 68% of potential CD patients and 20% of non‐CD controls showed mucosal deposits of immunoglobulin (Ig)A anti‐TG2; at the same time 100, 96 and 8% of active CD, potential CD and non‐CD control patients secreted these antibodies in culture supernatants, respectively. Our data showed that, to detect intestinal anti‐TG2 antibodies, the measurement of antibodies secreted into culture supernatants has higher sensitivity and specificity (97·5 and 92·3%, respectively) than the detection of mucosal deposits (77·5 and 80·0%, respectively). The measurement of intestinal anti‐TG2 antibodies may prove useful in clinical practice to predict evolution towards mucosal atrophy in potential coeliac patients and identify patients with gluten sensitivity.     

Development of an immunocapture method for measuring IgA antibodies to tissue transglutaminase in the sera of patients with coeliac disease

One of the most reliable sero-diagnostic tests for coeliac disease (CD) is the measurement, by ELISA, of serum IgA antibodies to tissue transglutaminase (tTG) adsorbed to the wells of microtitre plates. In spite of its reliability, however, some discrepancies exist with the results obtained by the antiendomysium histological assay (EMA) and by biopsy the accepted gold standard. Among the reasons for these differences in titres between the ELISA and the last 2 mentioned assays are the conformational changes that proteins undergo on adsorption and the importance of conformational epitopes on tTG for diagnosing CD. To address this problem, a novel procedure was developed using guinea-pig tTG (gptTG) free in solution to interact with IgA antibodies in the sera of CD patients. Any immune complexes so formed are then captured by anti-tTG antibodies preadsorbed to the wells of microtitre plates. This immunocapture method was optimized for the amount of soluble gptTG needed to interact with all the IgA's anti-tTG present in fixed dilutions of serum samples, the amount of rabbit IgG anti-gptTG used to coat the wells of microtitre plates and the order of addition of the reaction components. Comparison of the IgA titres obtained by immunocapture with those by EMA and ELISA (adsorbed tTG) on 9 highly positive and 6 weakly positive sera from clinically characterized CD patients and 5 negative sera from non-CD control subjects revealed that the IgA titres by the immunocapture procedure were well correlated with those obtained by EMA, whereas the titres on ELISA showed discrepancies with both immunocapture and EMA.     

Anti-tissue transglutaminase, anti-endomysium and anti-R1-reticulin autoantibodies-the antibody trinity of coeliac disease.

Anti-tissue transglutaminase has been recently described as the predominant autoantigen in coeliac disease. We purified serum anti-tissue transglutaminase antibodies from three patients with coeliac disease by column chromatography and eluted tissue section-bound R1-anti-reticulin antibodies from sections of rat tissue for two of these. Lastly, we generated seven mouse MoAbs to guinea pig tissue transglutaminase. Each preparation was examined for anti-tissue transglutaminase, anti-endomysium, anti-R1 reticulin and anti-gliadin antibodies. Column-purified patient antibodies and 2/7 mouse MoAbs gave characteristic anti-endomysium/anti-R1 reticulin reactivity on rat, monkey and human tissue. All positive sera gave indistinguishable patterns of immunofluorescence on rat liver, kidney and stomach, monkey oesophagus, and human umbilical cord. Anti-R1-reticulin eluted from sections showed anti-tissue transglutaminase reactivity in 2/2 cases, but 0/2 showed anti-gliadin reactivity. In both, tissue section-eluted anti-R1 reticulin gave endomysial staining on monkey oesophagus. None of the mouse monoclonals, or any of the purified patient's anti-tissue transglutaminase or anti-R1-reticulin antibody showed any reactivity with gliadin. These data confirm tissue transglutaminase as the predominant autoantigen in coeliac disease and suggest that both anti-endomysium and anti-R1 reticulin reactivities seen in coeliac disease arise due to an immune response to tissue transglutaminase. Rigorous immunoabsorption was sufficient to abrogate reactivity in the tissue transglutaminase ELISA, but failed to completely absorb anti-endomysium and anti-reticulin activity. The possibility remains that some of the anti-endomysium and anti-reticulin activity was directed against antigens other than tissue transglutaminase.     

Serum cytokine pattern in young children with screening detected coeliac disease

Coeliac disease is an autoimmune disease characterized by inflammation localized to the small bowel, but less is known about systemic signs of inflammation. The aim was to measure cytokines of the T helper 1 (Th1) and T helper 2 (Th2) cell patterns in children with screening‐detected coeliac disease before and after treatment with a gluten‐free diet. Serum samples selected before and after the start of a gluten‐free diet from 26 3‐year‐old children diagnosed with biopsy‐proven coeliac disease and from 52 matched controls were assayed in an multiplex enzyme‐linked immunosorbent assay (ELISA) for the 10 cytokines: interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐8, IL‐10, IL‐12p70, IL‐13 and tumour necrosis factor (TNF)‐α. Among Th1 cytokines, IFN‐γ and IL‐12p70 were elevated significantly in children with coeliac disease compared to controls (P < 0·001 and P = 0·001, respectively). Similar findings were demonstrated for the Th2 cytokines IL‐5 (P < 0·001), IL‐10 (P = 0·001) and IL‐13 (P = 0·002). No difference in cytokine levels between the two groups was found for TNF‐α, IL‐1β, IL‐2, IL‐4 and IL‐8. After gluten‐free diet, levels of IL‐5, IL‐12 and IL‐10 decreased significantly (P < 0·001, P = 0·002 and P = 0·007) and IFN‐γ levels were reduced (P = 0·059). Young children with coeliac disease detected by screening demonstrate elevated levels of serum cytokines at time of diagnosis. A prolonged systemic inflammation may, in turn, contribute to long‐term complications known to be associated with untreated coeliac disease.     

Serum autoantibodies directed against transglutaminase‐2 have a low avidity compared with alloantibodies against gliadin in coeliac disease

Coeliac disease is characterized by intolerance to gliadin and related gluten components present in wheat, barley and rye. Coeliac disease patients harbour antibodies directed against alloantigens such as gliadin, but also against the autoantigen transglutaminase‐2 (TG2). The type and quality of antibody responses provides insight into the underlying immune activation processes. Therefore, in this study we have analysed the avidity of the antibody response directed against the autoantigen TG2 and compared this with antibody responses against the alloantigens gliadin and Escherichia coli. We observed that the immunoglobulin (Ig)A autoantibody response directed against TG2 is of low avidity compared with the IgA response against the alloantigens gliadin and E. coli in the same patients; the same was true for IgG, both in IgA‐deficient and in ‐sufficient coeliac patients. The observed avidities appear not to be related to disease stage, antibody levels, age or duration of exposure to gluten. In conclusion, in coeliac disease there is a clear difference in avidity of the antibody responses directed against the auto‐ and alloantigens, indicating different regulation or site of initiation of these responses.     

A clinicopathological approach to the diagnosis of coeliac disease

Coeliac disease is defined as a small bowel enteropathy due to immune mediated damage on exposure to gluten in the diet, occurring in those with a genetic predisposition to this condition. Previously considered rare, the prevalence of coeliac disease is increasing due to a genuine rise in incidence and also better detection. The diagnosis of coeliac disease involves many disciplines, presentation is varied and if the diagnosis is delayed there is a risk of poor quality of life and a small increase in malignancy. Serology is a first line test, followed by confirmatory small intestinal biopsy. This review discusses the clinicopathological approach to diagnosis, through serology and biopsy and discusses complications which occur in some individuals, namely refractory coeliac disease and dermatitis herpetiformis. The entity of non-coeliac gluten sensitivity is also entering the spectrum of coeliac diagnosis and may lead to an extension of the diagnostic parameters of coeliac disease.     

Immunological indicators of coeliac disease activity are not altered by long‐term oats challenge

Coeliac disease is a gluten‐sensitive enteropathy that develops in genetically susceptible individuals. The disease exhibits many features of an autoimmune disorder. These include the production of highly specific anti‐endomysial autoantibodies directed against the enzyme tissue transglutaminase. It is well accepted that wheat‐, barley‐ and rye‐based foods should be excluded in the gluten‐free diet. Although several studies report that oats ingestion is safe in this diet, the potential toxicity of oats remains controversial. In the current study, 46 coeliac patients ingested oats for 1 year and were investigated for a potential immunogenic or toxic effect. Stringent clinical monitoring of these patients was performed and none experienced adverse effects, despite ingestion of a mean of 286 g of oats each week. Routine histological analysis of intestinal biopsies showed improvement or no change in 95% of the samples examined. Furthermore, tissue transglutaminase expression in biopsy samples, determined quantitatively using the IN Cell Analyzer, was unchanged. Employing immunohistochemistry, oats ingestion was not associated with changes in intraepithelial lymphocyte numbers or with enterocyte proliferation as assessed by Ki‐67 staining. Finally, despite the potential for tissue transglutaminase to interact with oats, neither endomysial nor tissue transglutaminase antibodies were generated in any of the patients throughout the study. To conclude, this study reaffirms the lack of oats immunogenicity and toxicity to coeliac patients. It also suggests that the antigenic stimulus caused by wheat exposure differs fundamentally from that caused by oats.     

Anti-endomysial antibody of IgG1 isotype detection strongly increases the prevalence of coeliac disease in patients affected by type I diabetes mellitus

A strong association between type 1 insulin-dependent diabetes mellitus (IDDM1) and coeliac disease (CD) is well documented, but it is known that prevalence values are underestimated. Serum anti-endomysial antibodies (EMA), considered diagnostic for CD because of their high sensitivity and specificity, belong to the IgA class, but the existence of EMA of IgG1 isotype in the presence or absence of IgA deficiency was reported. In order to re-evaluate the occurrence of CD in IDDM1 patients we performed a screening in IDDM1 patients using EMA of both isotypes. Ninety-four adults affected by IDDM1 (unaffected by CD before enrolling) were enrolled and 83 blood donors as controls. All subjects were on a gluten-containing diet. Histology and biopsy culture were performed. EMA IgA and IgG1 in sera and culture supernatants were detected. Serum EMA were positive in 13 of 94 IDDM1 patients (13.8%). Six of 13 presented IgA-EMA, seven of 13 presented IgG1-EMA. No EMA were found in the control population. Total intestinal atrophy was found in all six patients with serum IgA-EMA and in five of seven with serum IgG1-EMA. Diagnosis of CD was confirmed by histology and organ culture in all 13 patients with serum EMA. The prevalence of CD in the patients affected by IDDM1 was 6.4% for IgA-EMA-positive and 7.4% for IgG1-EMA-positive patients. We confirmed the prevalence of CD in the IDDM1 population obtained with IgA-EMA screening only (6.4%). This prevalence value increases dramatically to 13.8% when IgG1-EMA are also used in the screening. We conclude that IgG1-EMA should also be sought whenever an IDDM1 patient undergoes screening for CD.     

Gliadin and tissue transglutaminase complexes in normal and coeliac duodenal mucosa

Tissue transglutaminase (tTG) seems to be the target self-antigen for endomysial antibodies in coeliac disease (CD) and to catalyse the critical deamidation of gliadin which strengthens its recognition by HLA-restricted gut-derived T cells. To date, it has not been demonstrated whether gliadin is cross-linked to tTG within the gut wall, a phenomenon known to occur in vitro. We therefore investigated the putative presence of tTG and gliadin complexes directly in duodenal mucosa. The immunoprecipitation and Western blotting experiments were performed on mucosal biopsies obtained from untreated, treated CD patients and biopsied controls, by using either anti-tTG or anti-gliadin antibodies, in both denaturating/reducing or nondenaturating/nonreducing conditions. A subset of experiments was performed by using anti-tTG antibodies purified by affinity chromatography from sera of untreated coeliac patients. The localization of tTG and gliadin was studied by immunofluorescence at confocal laser microscopy on seriate sections of diseased and normal duodenal mucosa by using the same antibodies of the coimmunoprecipitation section. The amounts of tTG and gliadin coimmunoprecipitated with anti-tTG monoclonal antibody in untreated CD mucosa were significantly increased compared to those of the other two groups. When performing the experiments in nondenaturating/nonreducing conditions, a high molecular weight band formed by both molecules, was evidenciated. Also the anti-tTG antibodies purified from patients' sera turned out to be able to coimmunoprecipitate the two molecules. The analysis by confocal microscopy showed that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD, and only in the lamina propria of the villi in normal mucosa. Our findings firstly demonstrated that gliadin was directly bound to tTG in duodenal mucosa of coeliacs and controls, and the ability of circulating tTG-autoantibodies to recognize and immunoprecipitate the tTG-gliadin complexes.     

Intestinal titres of anti‐tissue transglutaminase 2 antibodies correlate positively with mucosal damage degree and inversely with gluten‐free diet duration in coeliac disease

It has always been known that anti-tissue transglutaminase 2 (anti-TG2) antibodies are produced in the small intestine. Their serum titres correlate with mucosal damage degree and decrease on a gluten-free diet (GFD).We aimed to correlate intestinal anti-TG2 antibodies levels with degree of mucosal damage and GFD duration.Thirty-four active, 71 potential and 24 CD patients on GFD for at least 2 years were enrolled. Anti-TG2 deposits were detected in intestinal biopsies by double immunofluorescence. Biopsies were cultured for 24 h with medium, and with gliadin peptic tryptic digest (PTG) or A-gliadin peptide 31–43 (P31-43). Anti-TG2 antibodies secreted into supernatants were measured by enzyme-linked immunosorbent assay (ELISA). All active CD patients secreted high titres of anti-TG2 antibodies into culture medium that increased with the worsening of mucosal injury (Spearman''s r = 0·71; P < 0·0001). Seventy of 71 potential CD patients and 15 of 24 treated CD patients secreted low titres of anti-TG2 antibodies into supernatants, eight of nine negative treated patients being on GFD for more than 10 years. An inverse correlation between antibody titres and duration of GFD was found, (Spearman''s r = −0·52; P < 0·01). All active, 53 of 71 potential and six of 24 treated, CD patients showed anti-TG2 mucosal deposits. Five of six positive treated CD patients had been on GFD for fewer than 6 years and were also positive for secreted anti-TG2. In treated patients, PTG/P31-43 was not able to induce secretion of anti-TG2 antibodies into culture medium.Measurement of anti-TG2 antibodies in biopsy supernatants proved to be more sensitive than detection by immunofluorescence to reveal their intestinal production. Intestinal antiTG2 antibodies titres correlated positively with the degree of mucosal damage and inversely with the duration of GFD.     

Affinity maturation of immunoglobulin A anti-tissue transglutaminase autoantibodies during development of coeliac disease

Coeliac disease (CD) is an immune-mediated enteropathy triggered by ingestion of wheat gluten and related cereals in genetically predisposed individuals. Circulating immunoglobulin A (IgA) class autoantibodies against tissue transglutaminase (IgA-TGA) are highly specific and sensitive serological markers for CD, which is ultimately confirmed by duodenal biopsy. Although CD is considered a life-long disorder, transient or fluctuating IgA-TGA seropositivity has been observed in asymptomatic individuals on a gluten-containing diet. We set out to explore possible differences in the maturation of IgA-TGA avidity between individuals progressing to CD and subjects remaining healthy despite occasional expression of autoantibodies. We developed a time-resolved fluorometric IgA-TGA assay based on human recombinant tissue transglutaminase (tTG), and further modified the method to also measure urea-dependent avidity of the autoantibodies. We measured the autoantibody titres and avidities of sequential serum samples from 10 children developing CD and 10 children presenting transient or fluctuating autoantibodies. Both the initial titres at seroconversion and peak values of transient/fluctuating IgA-TGA were significantly lower than corresponding autoantibody titres in samples drawn from individuals with progressing CD (P = 0.004 and P = 0.0002, respectively). However, there were no statistically significant differences in the initial or peak avidity index values between the two groups of children. The avidity index values increased during the follow-up period (P = 0.013 for both groups) with no significant difference in the rate of avidity maturation between children with transient/fluctuating IgA-TGA and children developing CD. According to our results, high autoantibody titres have a higher predictive value than avidity maturation of TGA-IgA in screening for CD.     

Mucosal reactivity to cow's milk protein in coeliac disease

Patients with coeliac disease (CD) on a gluten-free diet may still have gastrointestinal symptoms. On clinical grounds cow's milk (CM) protein sensitivity may be suspected. Here, using rectal protein challenge, we investigated the local inflammatory reaction to gluten and CM protein in adult patients with CD in remission. Rectal challenges with wheat gluten and dried CM powder were performed in 20 patients with CD and 15 healthy controls. Fifteen hours after challenge the mucosal reaction was recorded by the mucosal patch technique with measurements of local release of neutrophil and eosinophil granule constituents; myeloperoxidase (MPO) and eosinophil cationic protein (ECP). We measured the mucosal production of nitric oxide (NO) simultaneously. Six of the patients who reacted to CM were also challenged with alpha-lactalbumin and casein. In 18 of 20 patients gluten challenge induced neutrophil activation defined as increased MPO release and increased NO synthesis. Ten of these 20 patients showed a similarly strong inflammatory reaction to CM challenge. Six of the CM sensitive patients were challenged with specific CM proteins: casein and alpha-lactalbumin. Casein, in contrast to alpha-lactalbumin, induced an inflammatory response similar to that produced by CM. A mucosal inflammatory response similar to that elicited by gluten was produced by CM protein in about 50% of the patients with coeliac disease. Casein, in particular, seems to be involved in this reaction.     

Glossodynia and coeliac disease

Coeliac disease is an immune-mediated chronic inflammatory disorder of the small bowel caused by irritant gluten and, possibly, other environmental cofactors, in genetically prone people. Coeliac disease is characterized by no (or elusive or varied) symptoms. Oral clinical settings include aphthous stomatitis and dental enamel defects. Association with other signs in the oral mucosa (such as, for example, soreness, a burning sensation, erythema or atrophy) is much less common and, often, not considered by clinicians. We report on a 72-year-old woman with a four months history of oral burning sensation as a single clinical manifestation of coeliac disease. Clinical presentation and symptomatology are discussed in relation to the differential diagnosis of oral glossodynia. This case history highlights the importance of considering coeliac disease in managing cases of idiopathic glossodynia.     

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.     

The prevalence of coeliac disease in infertility.

An increased incidence of reproductive problems, including infertility, miscarriage, low birth weight newborns, and shorter duration of breast-feeding, are known to exist in women with coeliac disease; some of these conditions are improved by a gluten-free diet. We have tried to ascertain the prevalence of coeliac disease in 99 couples who were being evaluated for infertility, compared with the known prevalence of silent disease in the population of Northern Sardinia, in which it is endemic. Of all women, four tested positive for at least two out of three markers: immunoglobulin A (IgA) antigliadin, immunoglobulin (IgG) antigliadin, and anti-endomysium antibodies, and underwent a jejunal biopsy; three had histological evidence of coeliac disease. One male partner was positive for two markers, and had a diagnostic jejunal biopsy. The prevalence of coeliac disease in infertile women seems higher (three out of 99, 3. 03%) in the study group than in the general population (17 out of 1607, 1.06%), and particularly in the subgroup with unexplained infertility (two out of 25, 8%, P < 0.03). Screening for coeliac disease should be part of the diagnostic work-up of infertile women, particularly when no apparent cause can be ascertained after standard evaluation.