M.C. Anania

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.

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.

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.

Usefulness of the organ culture system in the in vitro diagnosis of coeliac disease: A multicentre study

Objective. Diagnosis of coeliac disease is based on the presence of villous atrophy which recovers following a gluten-free diet. The presence of circulating antiendomysial antibodies as well as their disappearance after a gluten-free diet supports the diagnosis. It has also been demonstrated that antiendomysial antibodies are detectable in supernatants of cultured intestinal biopsies from patients with coeliac disease. The objective of this study was to compare the histology and antiendomysial antibodies in culture supernatants of intestinal biopsies to validate the in vitro organ culture system as a future diagnostic tool for coeliac disease. Material and methods. Seventy-five antiendomysial serum-positive patients on a gluten-containing diet were evaluated. Patients underwent endoscopy with 5 biopsy fragments: 3 for histology, 1 cultured with and the other without gliadin-peptide activator. Antiendomysial antibodies were evaluated in all culture supernatants. Results. Sixty-eight patients had evidence of villous atrophy, while 73 out of 75 were positive to the organ culture system. The agreement rate between organ culture and histology results was 94%. Conclusions. As all the centres participating in the study obtained good agreement between organ culture and histology results, the new system could be considered a reliable tool for the diagnosis of coeliac disease. Nevertheless, it is possible to highlight cases with an organ culture-positive and -negative histology. This feature could be of considerable interest because, as the sensitivity of organ culture seems to be greater than the initial histology, the new system might be useful in uncertain cases where the risk of missing the diagnosis of coeliac disease is high.

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.

Celiac disease diagnosis in misdiagnosed children.

Antiendomysial antibodies (EMA) are today considered the most sensitive and specific serological marker of celiac disease (CD). The aim of the present study was to assess the occurrence of EMA of IgG isotype in EMA IgA negative children with clinical suspicion of malabsorption and their relationship with CD. Serum EMA IgG1 determination was performed on 30 EMA IgA negative children with clinical suspicion of CD. Total serum IgA levels were further investigated. Sixty children with gastroenterological diseases other than CD were used as control disease patients and 63 healthy children were evaluated as the control group. Eighteen out of 30 children in the study showed EMA IgG1 positivity in sera and a villous height/crypt depth ratio <3:1 as index of intestinal atrophy. It is noticeable that a selective IgA deficiency was present in only 9 of 18 EMA IgG1 positive children. In addition, clinical symptoms, EMA IgG1, and mucosal atrophy disappeared after 8–10 mo on a gluten-free diet. Neither EMA IgA nor EMA IgG1 were detected in the children in the control groups. The other 12 children in study group showed no histologic abnormalities and were EMA IgG1 negative. In this study, we reveal a group of EMA IgG1 CD children without IgA deficiency. The diagnosis was based on the presence of gluten-dependent typical serological and histologic features of CD. Our data suggest that EMA IgG1 determination could be a new tool in the diagnostic workup of CD, useful in avoiding possible misdiagnosis.

Antiendomysial antibody detection in fecal supernatants: in vivo proof that small bowel mucosa is the site of antiendomysial antibody production

OBJECTIVES: Serum antiendomysial antibodies (EMAs), highly sensitive and specific serological markers of celiac disease (CD), are detectable in culture media of biopsy samples from CD patients. This finding can be considered an in vitro evidence that intestinal mucosa is a site of EMA production. To confirm this finding, we investigated the presence of EMAs and of anti-tissue transglutaminase (anti-tTG), recently identified as the autoantigen of the EMA, in fecal supernatants of CD patients. METHODS: Twenty-one newly diagnosed CD patients, 10 treated CD patients on a gluten-free diet, and 14 control disease patients on a gluten-containing diet were enrolled. Twenty-four-hour stool collections and fecal supernatants were obtained from all patients in the study. Biopsy cultures were also performed. IgA EMAs were detected in sera, culture media, and fecal supernatants. IgA, IgG, IgM, and IgE anti-gliadin antibodies (AGAs) and IgA anti-tTG antibodies were measured in fecal supernatants. The weights, water content, and pHs of the 24-h stool collections were also measured. RESULTS: In all untreated CD patients EMAs were detectable in sera, culture media, and fecal supernatants. In treated CD patients, EMAs were detected only in culture media after in vitro gliadin challenge. No EMAs were detected in controls. Anti-tTG levels were higher in untreated CD patients than in treated CD patients and controls. IgA AGA levels were higher in untreated CD patients than in treated CD and control patients, whereas IgM AGAs were higher in both untreated and treated CD patients than in controls. No statistically significant differences were observed for IgG and IgE AGAs among the above-mentioned populations. Fecal weights, water content, and pHs were higher in untreated CD than in control patients. CONCLUSIONS: The presence of EMAs in fecal supernatants represents the in vivo proof that intestinal mucosa is a site of EMA production. Furthermore, EMA detection in the stools could be a simple and useful additional tool to clarify diagnosis in the patchy conditions of CD.

Prolonged analgesia induced by cathinone. The role of stress and opioid and nonopioid mechanisms

Cathinone, the active principle of Catha edulis (khat), shows long-lasting analgesic effects when the tail-flick test is used in rats. The involvement of monoamines, endogenous opioids and stress in this analgesic effect was tested. Both early (30 min) and late (24 h) analgesic effects of cathinone were prevented by reserpine or p-chlorophenylalanine, which deplete catecholamines or serotonin, respectively, and by nomifensine, which prevents neuronal uptake of biogenic amines and amphetamines. The same inhibitory effect was obtained with a high dose (4 mg/kg) of naloxone. However, rats made tolerant to morphine retained both early and late analgesic response to cathinone. The increase in plasma ACTH induced by the tail-flick test at 30 min and 24 h was significantly enhanced by cathinone, in a naloxone-reversible way. However, the analgesic responses shown at these times were not prevented by either dexamethasone or adrenalectomy. We conclude that the prolonged analgesia induced by cathinone is primarily due to an amphetamine-like activation of monoaminergic pathways, but requires the integrity of non-mu-opioid mechanisms. The involvement of the adrenohypophyseal axis in this cathinone effect is less probable.

Brief Footshock Analgesia: Long-Lasting Enhancement Induced by Cathinone, an Amphetamine-Like Agent

In view of the analgesic effects produced by cathinone (CATH), an amphetamine-like agent, and of the interaction of amphetamines with stressful environmental stimuli, the present study evaluated in rats the influence of CATH on the nonopioid analgesia induced by a brief electric footshock (FSA; 3 min of continuous 2.5 mA current). The influence of this combination on body temperature was also evaluated. CATH (5 mg/kg, i.p.) alone induced a brief and slight increase in latency during the hot plate test (HPT), but enhanced and prolonged the analgesic effect induced by FS. In addition, the presentation of the environment (shock box with unelectrified grid) where other rats received FS, caused CATH to induce a slow-rising analgesic effect for 180 min. A hyperthermic response paralleling the analgesic effect was observed in shocked and nonshocked rats receiving CATH. After 24 h, rats that had received both CATH and FS on the previous day showed prolonged latencies on the HPT before and after a 1-min presentation of unelectrified grid. These animals also showed an increased analgesic response to the subsequent application of a 15-second FS. At the same time no differences in body temperature were observed between treatment groups. These results suggest that CATH can interact with environmental stimuli to induce an analgesic effect, the time-course of which depends upon the intensity of the stimulus applied.

Usefulness of the organ culture system in the in vitro diagnosis of Celiac Disease: A multicenter study

OBJECTIVE Diagnosis of coeliac disease is based on the presence of villous atrophy which recovers following a gluten-free diet. The presence of circulating antiendomysial antibodies as well as their disappearance after a gluten-free diet supports the diagnosis. It has also been demonstrated that antiendomysial antibodies are detectable in supernatants of cultured intestinal biopsies from patients with coeliac disease. The objective of this study was to compare the histology and antiendomysial antibodies in culture supernatants of intestinal biopsies to validate the in vitro organ culture system as a future diagnostic tool for coeliac disease. MATERIAL AND METHODS Seventy-five antiendomysial serum-positive patients on a gluten-containing diet were evaluated. Patients underwent endoscopy with 5 biopsy fragments: 3 for histology, 1 cultured with and the other without gliadin-peptide activator. Antiendomysial antibodies were evaluated in all culture supernatants. RESULTS Sixty-eight patients had evidence of villous atrophy, while 73 out of 75 were positive to the organ culture system. The agreement rate between organ culture and histology results was 94%. CONCLUSIONS As all the centres participating in the study obtained good agreement between organ culture and histology results, the new system could be considered a reliable tool for the diagnosis of coeliac disease. Nevertheless, it is possible to highlight cases with an organ culture-positive and -negative histology. This feature could be of considerable interest because, as the sensitivity of organ culture seems to be greater than the initial histology, the new system might be useful in uncertain cases where the risk of missing the diagnosis of coeliac disease is high.