Antonio Picarelli

Production of antiendomysial antibodies after in-vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease

BACKGROUND Diagnosis of many immune-mediated diseases is helped by detection of antibodies directed against the pathogenetic (self or foreign) antigen. In coeliac disease (CD), we have a situation in which antiendomysial antibodies (EMA), which are not specific for the pathogenetic antigen, reach a specificity and sensitivity of detection of CD approaching 100%, whereas detection of antibodies against gliadin (AGA), the pathogenetic antigen, is far less specific and sensitive in diagnosis. No direct evidence of a relation between gluten/gliadin challenge and EMA production exists. We tried to establish whether the small intestine of CD patients is the site of EMA production and whether gliadin challenge could induce their release. METHODS Small intestine biopsy samples from treated (23) and untreated (16) CD patients and controls (18) were cultured in vitro for 24-48 h in the presence of gliadin, another alimentary antigen, or medium. EMA and AGA were detected in the supernatants of these organ culture biopsy samples by ELISA and immunofluorescence technique, respectively. FINDINGS No EMA were found in the culture supernatants of biopsy samples of 18 controls, whereas they were detected in the culture supernatants of all 16 untreated CD patients irrespective of gliadin challenge. Conversely, EMA were not detected in supernatants of biopsy samples cultured in medium only from 23 treated CD patients, but were detected in 17 of the 23 biopsy samples challenged with gliadin. INTERPRETATION Our results suggest that a more complex pathogenetic mechanism than normally accepted is involved in CD. Furthermore, our findings raise the possibility that EMA, or the antigen recognised by them, are involved directly in the pathogenesis of CD.

In vitro Activities of A-Gliadin-Related Synthetic Peptides Damaging Effect on the Atrophic Coeliac Mucosa and Activation of Mucosal Immune Response in the Treated Coeliac Mucosa

BACKGROUND Gliadin amino acid sequence(s) responsible for toxicity in susceptible individuals have not been fully elucidated. Previous in vitro studies have suggested the presence of active sequences in the NH(2)-terminal part of the A-gliadin molecule. In this paper the in vitro activity of A-gliadin synthetic peptides 31-55, 31-43, and 44-55 has been investigated. METHODS Organ culture of jejunal mucosa from untreated and treated coeliac patients was used. In the first system enterocyte height was used as a measure of peptide toxicity; in the second system evidence of activated mucosal cell-mediated immune response was sought. RESULTS Peptides 31-55 and 31-43 were active on untreated coeliac mucosa at a concentration of 0.5 mg/ml and peptide 44-55 only at a concentration of 3 mg/ml. In in vitro-cultured treated coeliac mucosa peptides 31-55 and 31-43 at 1 mg/ml and peptide 44-55 at 3 mg/ml were able to induce enhanced epithelial expression of HLA-DR and 4F2 molecules and the appearance of CD25 positive cells. CONCLUSIONS Our results suggest that 31-43 and 44-55 A-gliadin peptides are both active, even if to different extents. In vitro systems remain essential tools to screen material to be subsequently tested in vivo.

Radioimmunoassay to detect antitransglutaminase autoantibodies is the most sensitive and specific screening method for celiac disease

OBJECTIVE:The aim of this study was to establish the most sensitive and specific screening method for celiac disease. We tested three methods based on different principles, which all detect autoantibodies against the same antigen (tissue transglutaminase).METHODS:Sixty-two celiac children at the first biopsy (group 1), 78 celiac children on a gluten-free diet (group 2), 14 celiac children on a gluten-challenge (group 3), and 56 controls with a normal duodenal mucosa (group 4) were studied. The methods used were: 1) radioimmunoprecipitation assay using recombinant tissue transglutaminase (RIA); 2) commercial enzyme immunoassay using guinea pig tissue transglutaminase (ELISA); and 3) indirect immunofluorescence method for detection of antiendomysium antibodies (IF-EMA).RESULTS:RIA antitransglutaminase autoantibodies were detected in 100% of group 1, 43.6% of group 2, 100% of group 3, and none of the control subjects. ELISA antitransglutaminase autoantibodies were detected in 90.3% of group 1, 9% of group 2, 78.6% of group 3, and in none of the control subjects. IF-EMA were detected in 95.2% of group 1, 11.5% of group 2, 92.3% of group 3, and 1.8% of the controls.CONCLUSIONS:Our results demonstrate a very high sensitivity and specificity of the RIA method to detect antitransglutaminase autoantibodies in comparison to ELISA and IF-EMA assays. We can explain this finding with the use of human recombinant antigen and the increased capacity of the RIA method to detect low titers of autoantibodies. If our data are confirmed by studies on larger series, tissue transglutaminase RIA could be proposed as the best screening method for celiac patients.

Anti-tissue transglutaminase antibodies in inflammatory bowel disease: new evidence

Abstract Anti-tissue transglutaminase, previously held to be identical to anti-endomysial antibodies in celiac sprue, has been reported in inflammatory bowel disease patients. To investigate these data further, we evaluated serum and intestinal anti-tissue transglutaminase in inflammatory bowel disease patients, with respect to the Crohn’s disease activity index and the integrated disease activity index. Study population comprised: 49 patients with Crohn’s disease and 29 patients with ulcerative colitis; 45 patients with celiac sprue and 85 autoimmune patients as disease controls; and 58 volunteers as healthy controls. Immunoglobulin A (IgA) anti-recombinant human tissue transglutaminase and anti-endomysial antibody detection in sera and fecal supernatants were performed. Adsorption of positive sera with recombinant human tissue transglutaminase were also performed. Marked increased anti-tissue transglutaminase concentrations were found in celiac sprue, while low-positive values were also found in Crohn’s disease and ulcerative colitis. Anti-endomysial antibodies were detectable only in celiac sprue. Antigen adsorption resulted in a significant reduction of the anti-tissue transglutaminase either in celiac sprue or inflammatory bowel disease sera. A significant correlation between anti-tissue transglutaminase and Crohn’s disease activity index or integrated disease activity index scores was found. Anti-tissue transglutaminase was also detectable in fecal supernatants from inflammatory bowel disease patients. Data highlight that both circulating and intestinal anti-tissue transglutaminases are detectable in inflammatory bowel disease, and that they are related to disease activity. These features underline that, in addition to anti-tissue transglutaminase, an anti-endomysial antibody test is necessary in the diagnostic work-up of celiac sprue, especially in patients with known inflammatory bowel disease.

Serologic and genetic markers of celiac disease: A sequential study in the screening of first degree relatives

Objectives: The prevalence of celiac disease (CD) among the relatives and the complications of an undiagnosed CD prompted us to identify a useful disease screening strategy. Methods: We studied 441 first degree relatives of 208 CD patients by immunoglobulin (Ig)A antiendomysium antibodies (EMA) and radioimmunoprecipitation assay (RIA) IgA antitransglutaminase autoantibodies (TGAA). Of these, 364 were typed for human leukocyte antigen-DRB1, -DQA1, and -DQB1 genes by the polymerase chain reaction sequence specific primers method. It was suggested to the autoantibody-positive subjects that they should undergo intestinal biopsy. Results: TGAA were positive in 46 of 439 relatives, EMA in 38; intestinal lesions related to CD were present in 40 subjects. We also found two immunodeficient fathers with duodenal villous atrophy. In three serology-positive subjects, permission for intestinal biopsy was refused; for another three serology-positive cases, duodenal mucosa was normal. Thus, the strict CD prevalence resulted 9.5%, the enlarged prevalence 10.9%. The DQ2/DQ8 heterodimers were carried in 231 of 364 subjects and in 38 of 40 biopsy-proven celiac patients. Three DQ2-positive parents became positive to the serology during a long-lasting follow-up. Conclusions: On the basis of a carefully conducted study, CD prevalence in our series was seen as very high. These data suggest an accurate algorithm to select candidates for intestinal biopsy among CD high-risk subjects. First, an evaluation of the sensitive RIA TGAA and of total IgA (in IgA deficiency RIA IgG anti-tissue transglutaminase assay) should be performed. Then, an evaluation of the TGAA and the genetic study would be advisable 2 to 3 years later in negative subjects. Those carrying the DQ2/DQ8 heterodimers should continue the serologic follow-up; the others need a clinical follow-up.

Identification of a new coeliac disease subgroup: antiendomysial and anti-transglutaminase antibodies of IgG class in the absence of selective IgA deficiency

Abstract. Picarelli A, Di Tola M, Sabbatella L, Mastracchio A, Trecca A, Gabrielli F, Di Cello T, Anania MC, Torsoli A (University of Rome “La Sapienza”, Rome, Italy). Identification of a new coeliac disease subgroup: antiendomysial and anti‐transglutaminase antibodies of IgG class in the absence of selective IgA deficiency. J Intern Med 2001; 249: 181–188.

Atypical Celiac Disease as Cause of Increased Need for Thyroxine: A Systematic Study

OBJECTIVE Replacement T4 dose in hypothyroid patients bearing both chronic autoimmune thyroiditis and atypical celiac disease (CD) has been analyzed. DESIGN Replacement T4 dose has been analyzed in 35 hypothyroid patients with Hashimotos thyroiditis (HT) and atypical CD, as defined by the American Gastroenterological Association. We have evaluated the ability of the same dose of T4 to reach target TSH in 21 patients before and during gluten-free diet (GFD). In the remaining 14 patients, noncompliant with GFD, we analyzed replacement T4 dose and compared it with that in a similar group consisting of 68 patients with hypothyroid HT but no evidence of celiac sprue or other conditions interfering with T4 absorption. RESULTS In patients with isolated HT, the desired serum TSH (median=1.02 mU/liter) was reached in all patients after 5±2 months of treatment at a median T4 dose of 1.31 μg/kg·d. After a similar period and dose of T4, higher levels of TSH (median=4.20 mU/liter) were observed in patients with HT and CD. In 21 CD patients, target TSH (median TSH=1.25 mU/liter) has been attained after 11±3 months of GFD without increasing T4 dose (1.32 μg/kg·d). In the remaining 14 patients, who were noncompliant with GFD, target TSH has also been achieved but at a higher T4 dose (median=1.96 μg/kg·d; +49%; P=0.0002) than in hypothyroid patients without CD. CONCLUSIONS Atypical CD increases the need for T4. The effect was reversed by GFD or by increasing T4 dose. Malabsorption of T4 may provide the opportunity to detect CD that was overlooked until the patients were put under T4 therapy.

31-43 Amino Acid Sequence of the a-Gliadin Induces Anti-Endomysial Antibody Production during in Vitro Challenge

BACKGROUND Wheat gliadin is the culprit antigen of coeliac disease (CD). Two short sequences of NH2-terminal portion of gliadin seem to be responsible for CD. Antiendomysial antibodies (EMA), highly sensitive and specific for CD, are detectable in the culture media from treated CD patients, after in vitro challenge with peptic-tryptic (PT) digest of gliadin. In this study we detected EMA production after in vitro challenge with 31-43 peptide. We used 56-68 peptide, lacking toxic sequences, as a negative control. METHODS Duodenal samples from 11 treated CD patients and 9 control patients were cultured with 31-43 and 56-68 peptides and PT gliadin. Indirect immunofluorescence analysis was used for EMA detection. RESULTS EMA were detected in culture media of 10 of 11 specimens challenged with PT-gliadin and in the media of all specimens challenged with 31-43 peptide. No EMA were detectable in any treated patients cultured with 56-68 peptide or with medium alone. No EMA were observed in cultures of control specimens. DISCUSSION The ability of the 31-43 sequence of the alpha-gliadin to induce EMA production suggests its involvement in the pathogenesis of CD. Furthermore, it may be a more useful antigenic substance than PT gliadin for both in vitro and in vivo studies of CD.

Imaging active lymphocytic infiltration in coeliac disease with iodine-123-interleukin-2 and the response to diet

Abstract. Coeliac disease is diagnosed by the presence of specific antibodies and a jejunal biopsy showing mucosal atrophy and mononuclear cell infiltration. Mucosal cell-mediated immune response is considered the central event in the pathogenesis of coeliac disease, and untreated coeliac patients show specific features of T-cell activation in the small intestine. Here we describe the use of iodine-123-interleukin-2 scintigraphy in coeliac patients as a non-invasive tool for detection of lymphocytic infiltration in the small bowel and its use for therapy follow-up, and we demonstrate the specificity of binding of labelled-IL2 to activated lymphocytes by ex-vivo autoradiography of jejunal biopsies. 123I-IL2 was administered i.v. [74 MBq (2 mCi)], and gamma camera images were acquired after 1 h. Ten patients were studied with 123I-IL2 scintigraphy at diagnosis and seven were also investigated after 12–19 months of gluten-free diet. Results were expressed as target-to-background radioactivity ratios in six different bowel regions before and after the diet. At the time of diagnosis all patients showed a significantly higher bowel uptake of 123I-IL2 than normal subjects (P<0.003 in all regions). A significant correlation was found between jejunal radioactivity and the number of IL2R+ve lymphocytes per millimetre of jejunal mucosa as detected by immunostaining of jejunal biopsy (r2=0.66; P=0.008). Autoradiography of jejunal biopsies confirmed that labelled-IL2 only binds to activated T-lymphocytes infiltrating the gut mucosa. After 1 year of the diet, bowel uptake of 123I-IL2 significantly decreased in five out of six regions (P<0.03), although two patients still had a positive IL2 scintigraphy in one region. We conclude that 123I-IL2 scintigraphy is a sensitive non-invasive technique for assessing in vivo the presence of activated mononuclear cells in the bowel of patients affected by coeliac disease. Unlike jejunal biopsy, this method provides information from the whole intestine and gives a non-invasive measure of the effectiveness of the gluten-free diet.

Presence of anti-"tissue" transglutaminase antibodies in inflammatory intestinal diseases: an apoptosis-associated event?

Presence of anti-‘tissue’ transglutaminase antibodies in inflammatory intestinal diseases: an apoptosis-associated event?