Autoantibodies to tissue transglutaminase are sensitive serological parameters for detecting silent coeliac disease in patients with Type 1 diabetes mellitus.

AIMS: To investigate the clinical significance of the determination of IgA antibodies to tissue transglutaminase (tTG) for the detection of silent coeliac disease in patients with Type 1 diabetes mellitus. METHODS: A total of 520 patients with diabetes (median age 14.2 years, range 1-27) were tested for IgA antibodies to tTG (IgA anti-tTG, ELISA), endomysium (EmA, indirect immunofluoresence) and gliadin (IgA-AGA, enzyme immunometric assay) after ruling out IgA deficiency. RESULTS: The prevalence of IgA anti-tTG among patients with diabetes was 4.4% (23 of 520), and that of EmA and IgA-AGA 3.5% (18 of 520, respectively). The coefficient of agreement between IgA anti-tTG and EmA was high (Cohen's kappa = 0.87, P < 0.001). Thirteen of the 23 IgA anti-tTG-positive patients underwent duodenal biopsy. Coeliac disease was confirmed in nine of 13 patients. One of them was negative for EmA and AGA, but positive for IgA anti-tTG. Retrospective annual determinations up to 8 years in six IgA anti-tTG-positive patients showed both permanent and transient elevations of the serological markers. CONCLUSIONS: These data show that a positive IgA antibody test to tTG is a more sensitive parameter than EmA for silent coeliac disease in patients with diabetes. Confirmatory small bowel biopsy, however, remains necessary for diagnosis as some patients with positive antibodies may be without histological changes.     

Autoantibodies to human tissue transglutaminase: superior predictors of coeliac disease

BACKGROUND: Using selected sample populations, we compared sensitivity and specificity of autoantibodies to guinea pig and human tissue transglutaminase to assess if the human antigen is superior for predicting coeliac disease. METHODS: Four commercial enzyme-linked immunoassay kits using human tissue transglutaminase as antigen were used to measure autoantibody levels in serum samples from untreated adult coeliacs (n = 32). They were from a series of 130 cases diagnosed between 1997 and 1999 and chosen to bias the group towards subjects with negative autoantibodies when measured with guinea pig tissue transglutaminase as antigen. Samples from 38 control subjects (biased towards false-positive levels with guinea pig antigen) were used to compare specificity. We also assessed if human antigen kits could differentiate between levels in normal subjects and in selective IgA deficiency. RESULTS: Sensitivity for coeliac disease in this selected group using the human antigen kits ranged from 88% to 100%. Three kits showed significantly higher specificity (82%-97%, P < 0.05) than the guinea pig antigen kit (71%) for the samples studied. No kit achieved complete separation between normal autoantibody levels and lower levels in selective IgA deficiency. CONCLUSIONS: All human antigen kits showed significantly higher sensitivity for coeliac disease compared to guinea pig antigen (P < 0.001). Receiver operating characteristic curves confirmed the superior diagnostic accuracy of the human antigen kits.     

Autoantibodies of patients with coeliac disease are insufficient to block tissue transglutaminase activity

BACKGROUND AND AIMS: Coeliac disease (CD) is characterised by the presence of autoantibodies against tissue transglutaminase (tTG), the endomysial autoantigen. This study was performed to determine the effect of purified autoantibodies on the enzymatic activity of tTG. METHODS: Total IgA and IgG class antibodies and purified anti-tTG autoantibodies were isolated from sera of untreated patients with CD and controls. The inhibitory capacity of the antibodies on tTG activity was checked by a fluorometric assay based on the incorporation of monodansyl cadaverine into casein and by tTG-catalysed cross linking of biotinylated cadaverine to gliadin. RESULTS: The enriched IgA and IgG fractions of five patients with CD and three controls resulted in no significantly different inhibition of enzymatic activity. In contrast, the use of affinity purified anti-tTG autoantibodies of 12 patients with CD led to a dose dependent reduction of tTG activity, compared to control immunoglobulins (n=6). However, the remaining activity was sufficient for cross linking of cadaverine into gliadin, and enzymatic tTG activity was only blocked completely by high concentrations of a monoclonal antibody, which is directed to the active centre of tTG. CONCLUSIONS: Despite a partial inhibitory effect of isolated anti-tTG autoantibodies from patients with CD, residual enzymatic activity remains sufficiently high to cast doubt on their in vivo relevance.     

Autoantibodies to gliadin-binding 90 kDa glycoprotein in coeliac disease.

Patients with untreated coeliac disease were found to have high concentrations of circulating antibodies to 90 kDa glycoprotein, a mannose rich protein found in skin and intestinal mucosa. In contrast, patients with active Crohn's disease or ulcerative colitis had antibody concentrations within the normal range. In coeliac disease the antibody concentrations fell significantly after gluten withdrawal. 90 kDa glycoprotein bound gliadin in a carbohydrate and calcium dependent manner. The results show that circulating antibodies directed against a gliadin-binding antigen are present in coeliac disease. 90 kDa glycoprotein may be a receptor for gliadin; in susceptible subjects ligand receptor interaction may result in cytotoxicity and antibody formation.     

Prevalence of silent coeliac disease in atopics

Background. Coeliac disease sometimes runs a subclinical/silent course and is often associated with immunologic and non-immunologic diseases. Although atopy is described as one of the most frequently associated conditions, the prevalence of coeliac disease in atopics has not yet been established.

Aim. To evaluate the frequency of coeliac disease in an Italian series of atopics.

Patients and Methods. Sera from 401 consecutive atopics with no clinical evidence of malabsorption were tested for IgA antiendomysial antibodies by indirect immunofluorescence on human umbilical cord and IgA anti tissue transglutaminase by enzyme-linked immunosorbent assay.

Results. Four patients (1916) were found to be positive for both autoantibodies. Intestinal biopsy confirmed the diagnosis of active coeliac disease. One of the 4 coeliacs was also affected by Down's syndrome, autoimmune thyroiditis and coeliac hepatitis. In another case, a previously unknown severe iron deficiency was detected.

Conclusions. The present study shows, for the first time, that the prevalence of coeliac disease in atopics is 1%, which is significantly higher than that in the general Italian population. Therefore, atopy should be considered a condition at risk and atopic patients routinely screened by means of specific autoantibody testing.     

Undiagnosed coeliac disease and risk of autoimmune disorders in subjects with Type I diabetes mellitus

Aims/hypothesis. We tested the hypothesis that silent coeliac disease is more frequent than expected in both patients with Type I (insulin-dependent) diabetes mellitus and their first-degree relatives. We evaluated how the presence of other autoimmune disorders in diabetic patients and their first-degree relatives is related to silent, unrecognized coeliac disease. Methods. Sera from 491 subjects with Type I diabetes, 824 relatives and 4000 healthy control subjects were screened for anti-endomysial antibodies and all those subjects who tested positive for anti-endomysial antibodies underwent intestinal biopsy. Results. We found that the prevalence of coeliac disease was 5.7 % among the diabetic patients and 1.9 % among the relatives, values significantly higher than those found among the control subjects (p < 0.0001; p < 0.001). The prevalence of autoimmune disorders in diabetic patients with coeliac disease was significantly higher than in subjects with Type I diabetes alone (p < 0.0001). The prevalence of autoimmune disorders in the relatives with coeliac disease was significantly higher than in those who tested negative for anti-endomysial antibodies (p = 0.01). Conclusion/interpretation. This report provides further confirmation of the high prevalence of undiagnosed coeliac disease among diabetic patients and their relatives. This interesting new finding is the increased presence of other autoimmune diseases in these patients, as well as in their relatives with a delayed diagnosis for coeliac disease. Patients newly diagnosed with coeliac disease showed excellent compliance with the gluten-free diet. This should encourage policymakers to consider introducing an easy-to-use screening programme for diabetic patients and their relatives into everyday clinical practice, in order to prevent coeliac-associated symptoms and the onset of additional, more serious auto-immune disorders. [Diabetologia (2001) 44: 151–155] Received: 14 July 2000 and in revised form: 6 October 2000     

Antibodies to tissue transglutaminase C in Type I diabetes

Abstract Aims/hypothesis. Silent coeliac disease is a gluten driven autoimmune disease which is relatively frequent in patients with Type I (insulin-dependent) diabetes mellitus. To determine the extent of gluten associated autoimmunity in Type I diabetes, autoantibodies to tissue transglutaminase C, a major autoantigen in coeliac disease, were measured in patients with new-onset Type I diabetes. Methods. We measured IgG and IgA tissue transglutaminase C autoantibodies using human recombinant antigen and radio-binding assays in a cohort of 287 patients with new-onset Type I diabetes, 119 with Type II (non-insulin-dependent) diabetes mellitus and in 213 control subjects. Results. We found IgA and IgG tissue transglutaminase C antibodies in 24 (8 %) patients with Type I diabetes; 97 (33 %) patients had IgG antibodies only and 1 IgA antibodies only. Antibody concentrations were highest in those with both IgA and IgG antibodies. Only 2 (2 %) patients with Type II diabetes and 2 (1 %) control subjects had either IgG or IgA tissue transglutaminase C antibodies. Patients with HLA DRB1 ^* 04 alleles had the highest prevalence of IgG tissue transglutaminase C antibodies. Conclusion/Interpretation. These data show that almost 10 % of patients have autoimmunity typical of coeliac disease and that another 30 % have low level tissue transglutaminase C antibody binding. This high prevalence suggests either involvement of the gut in the pathogenesis of Type I diabetes or that transglutaminase is a secondary autoantigen resulting from beta-cell destruction. [Diabetologia (1999) 42: 1195–1198] Received: 15 April 1999 and in revised form: 8 June 1999     

Autoantibodies in connective tissue disease

Autoimmune connective tissue diseases are heterogeneous rheumatic diseases with the potential to affect multiple body systems. Autoantibodies are a characteristic feature of these diseases and are typically highly disease specific. In addition to aiding diagnosis, many autoantibodies have established associations with clinically important disease complications including internal organ involvement. In this chapter, we review the autoantibodies relevant to autoimmune connective tissue diseases, excluding systemic lupus erythematosus, with particular reference to the associated clinical features and how identification of such an autoantibody may inform prognosis and clinical management. We also discuss the practicalities of testing for autoantibodies along with potential difficulties and pitfalls.     

Autoantibodies associated with Type I diabetes mellitus persist after diagnosis in children

Summary To study the persistence of Type I (insulin-dependent) diabetes mellitus associated autoantibodies and their relation to genetic risk markers and clinical characteristics of the disease after clinical manifestation, serum samples were obtained from 90 children and adolescents at diagnosis and 2, 5 and 10 years later. The samples were analysed for islet cell antibodies (ICA) by immunofluorescence and for antibodies to glutamic acid decarboxylase (GADA), intracellular portion of the protein tyrosine phosphatase related IA-2 antigen (IA-2A) and insulin autoantibodies by specific radiobinding assays. Of the subjects tested 79 % were positive for IA-2A at diagnosis, 62 % for GADA, 81 % for ICA and 28 % for insulin autoantibodies, but the prevalence of IA-2A, GADA and ICA decreased substantially as a function of increasing duration of the disease (p < 0.05 or less), their levels following the same pattern (p < 0.001 for all three autoantibodies). Two thirds of the subjects still tested positive for at least one autoantibody specificity after the first 10 years of the disease and 42 % had two or three antibodies detectable. An increase over the initial antibody concentrations after the diagnosis was seen more often for GADA than for ICA (p < 0.001) or IA-2A (p < 0.05). In conclusion, autoantibodies associated with Type I diabetes appear to persist longer than expected after manifestation of the clinical disease, possibly due to small scale continuous beta-cell regeneration after diagnosis or to structural and/or functional mimicry between exogenous proteins and beta-cell antigens or both. [Diabetologia (1998) 41: 1293–1297] Received: 9 March 1998 and in revised form: 24 June 1998     

Anti tissue transglutaminase antibodies as predictors of silent coeliac disease in patients with hypertransaminasaemia of unknown origin

BACKGROUND: Unexplained hypertransaminasaemia can be regarded as an extraintestinal presentation of coeliac disease. AIM: To evaluate the reliability of immunoglobulin A anti tissue transglutaminase antibodies for identifying coeliac disease in those patients with raised transaminases of unknown origin. PATIENTS: Of 1,120 consecutive patients referred to the outpatient clinic for liver disease due to raised transaminases from September 1995 to December 1999, 110 were classified as having cryptogenic hypertransaminasaemia after the exclusion of every known cause of liver disease. METHODS: These 110 patients were tested for immunoglobulin A anti tissue transglutaminase and antiendomysial antibodies by enzyme-linked immunosorbent assay and indirect immunofluorescence, respectively. RESULTS: Ten patients resulted positive for both antibodies; in all of them duodenal biopsy showed a subtotal villous atrophy consistent with coeliac disease. They did not complain of any gastrointestinal symptom. Liver biopsy, performed in five, showed a histological picture of non-specific reactive hepatitis. CONCLUSIONS: Due to the high proportion (9.15%) of patients with cryptogenic hypertransaminasaemia affected by symptomless coeliac disease, serological screening for gluten-sensitive enteropathy must be included in the work-up of these patients. In this respect, anti tissue transglutaminase antibodies represent a valid alternative to antiendomysial antibodies with the advantage of being feasible everywhere thanks to the worldwide availability of enzyme-linked immunosorbent assay.     

Autoantibodies to DEK oncoprotein in human inflammatory disease

OBJECTIVE: To evaluate the specificity of anti-DEK antibodies for juvenile rheumatoid arthritis (JRA). METHODS: Anti-DEK autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA) using affinity-purified his6-DEK fusion protein. Sera from 639 subjects (417 patients with systemic autoimmune disease, 13 with sarcoidosis, 44 with pulmonary tuberculosis, 125 with uveitis, and 6 with scleritis, and 34 healthy control subjects) were screened. Reactivity was verified by immunoblotting and immunoprecipitation studies using baculovirus-expressed human DEK. RESULTS: Anti-DEK activity was found at the following frequencies: JRA 39.4% (n = 71), systemic lupus erythematosus (SLE) 25.1% (n = 216), sarcoidosis 46.2% (n = 13), rheumatoid arthritis 15.5% (n = 71), systemic sclerosis 36.0% (n = 22), polymyositis 6.2% (n = 16), and adult Still's disease 0% (n = 21). Autoantibodies also were detected in 9.1% of tuberculosis sera (n = 44), but were undetectable in sera from the 34 healthy controls. Western blot and immunoprecipitation assay results correlated well with the ELISA findings. In general, levels of anti-DEK autoantibodies were higher in SLE than in other patient subsets, including JRA. CONCLUSION: Anti-DEK autoantibodies are less specific for JRA than previously believed. They are produced in association with a variety of inflammatory conditions, many of which are associated with granuloma formation and/or predominant Thl cytokine production. Anti-DEK antibodies may be a marker for a subset of autoimmunity associated with interferon-gamma production rather than a particular disease subset.     

Autoantibodies to nervous tissue structures are associated with autonomic neuropathy in Type 1 (insulin-dependent) diabetes mellitus

Summary There is evidence that the immune system may play a role in the pathogenesis of autonomic neuropathy in Type 1 (insulin-dependent) diabetes mellitus. In the present study, we investigated the presence of autoantibodies to sympathetic and parasympathetic nervous structures and their correlation with other conventional autoantibodies in well-characterised diabetic populations, with or without diabetic neuropathy, and normal subjects. An indirect immunofluorescent complement-fixation technique was used, with monkey adrenal gland, rabbit cervical ganglia and vagus nerve as substrates. Of the patients with symptomatic autonomic neuropathy 33% were positive for at least one autoantibody (20% anti-sympathetic ganglia, 10% anti-vagus nerve and 13% anti-adrenal medulla). The frequency of having one or more antibodies to nervous tissues and the prevalence of anti-cervical ganglia antibodies were significantly higher in the neuropathic patients than in the diabetic control subjects with disease of similar duration and in the normal subjects (p<0.05). Of the patients without complications with diabetes of shorter duration 33% were also positive for at least one autoantibody (13% anti-ganglia, 13% anti-vagus nerve and 13% anti-adrenal medulla). No correlation was found with other tissue autoantibodies, including islet cell antibodies. Our data indicate that nervous tissue autoantibodies are associated with symptomatic autonomic neuropathy. Anti-sympathetic ganglia and anti-vagus nerve antibodies seem to be more disease-specific. Patients with diabetes of shorter duration who were positive for these autoantibodies may represent pre-neuropathic patients.     

Autoantibodies to tissue transglutaminase as predictors of celiac disease

Background & Aims: Immunoglobulin A (IgA) autoantibodies to endomysium (EMA) are highly specific and sensitive markers for celiac disease. Recently, we identified tissue transglutaminase (tTG) as the major if not sole endomysial autoantigen. Methods: An enzyme-linked immunosorbent assay (ELISA) was established to measure IgA anti-tTG titers in serum samples from 106 celiac patients with partial or subtotal villous atrophy, 43 celiac patients on a gluten-free diet, and 114 diseased and healthy controls. Results were correlated with clinical and histological data and with EMA titers. Results: In patients with biopsy-proven celiac disease consuming a normal, gluten-containing diet, 98.1% of the serum samples had elevated IgA titers against tTG, whereas 94.7% of the control sera were negative. IgA anti-tTG correlated positively with semiquantitative IgA EMA titers (r = 0.862; P < 0.0001). Conclusions: An ELISA based on tTG allows diagnosis of celiac disease with a high sensitivity and specificity. IgA anti-tTG and IgA EMA show an excellent correlation, further confirming the enzyme as the celiac disease autoantigen. Because the assay is quantitative, not subjected to interobserver variation, and easy to perform, it will be a useful tool for population screening of a hitherto underdiagnosed disease.GASTROENTEROLOGY 1998;115:1317-1321     

Prevalence of coeliac disease in siblings of patients with Type I diabetes is related to the prevalence of DQB1*02 allele

Aims/hypothesis: Coeliac disease is more prevalent among patients with Type I (insulin-dependent) diabetes mellitus and coeliac disease-related antibodies have been reported to increase in frequency in their first-degree relatives. Our aim was to find out if coeliac disease is more common among siblings of children with Type I diabetes than in the normal population. Methods: IgA endomysium antibodies were measured by indirect immunofluorescence in 550 subjects (mean age 11.8 years, range 3.1–26.9 years) with a sibling with Type I diabetes. We performed jejunal biopsy on as many subjects with positive antibodies as agreed. HLA-DQB1 genotyping was done in 427 subjects. Results: Endomysium antibodies were positive in nine subjects (1.6 %). Jejunal biopsy was diagnostic for coeliac disease in five out of seven patients. An additional patient with coeliac disease, one already on a gluten-free diet, was identified by questionnaire. The prevalence of coeliac disease was 1.1 %. Five of six patients with coeliac disease had HLA-DQB1^*02 allele, compared with 118 of 421 of those without coeliac disease (p = 0.009). The sixth patient was positive for HLA-DQB1^*0302 allele, which was also found in 241 of 421 of those without coeliac disease (p = 0.4). Conclusion/interpretation: We found the prevalence of coeliac disease among siblings of children with Type I diabetes to be similar to figures reported from recent population-based studies and to be correlated with the prevalence of coeliac disease associated HLA-DQB1 alleles. We propose that routine screening for coeliac disease among all first-degree relatives of patients with Type I diabetes is not warranted. [Diabetologia (2001) 44: 1051–1053] Received: 11 January 2001 and in revised form: 27 April 2001     

Autoantibodies to human heart conduction system in Chagas' disease

Destruction of heart tissue in chronic chagasic cardiopathy may be caused by autoimmune recognition of heart tissue. Indirect evidence suggests that there is antigenic cross-reactivity between Trypanosoma cruzi and heart tissue. The objective of this study was to determine whether seric autoantibodies against atrio-ventricular (AV) node and sinus auricular node tissues are markers of chronic cardiopathy condition. We searched for the presence of seric autoantibodies against AV node and sinus auricular node tissues in 25 sera from chronic chagasic cardiopathy patients, 20 sera from non-chagasic cardiopathy patients, 20 sera from indeterminate chagasic subjects, and 20 sera from healthy blood donors as controls. Diagnosis of dilated cardiopathy was established based on the left-ventricular end systolic dimension and cardiothoracic ratio on chest x-radiography and impaired contracting ventricle, and chagasic etiology by demonstration of circulating antibodies using ELISA and IIF. Autoantibody detection against conduction heart tissue was carried out by immunohistochemical test. The tissues were obtained from non-cardiopathy necropsy case. Human sera were diluted at 1:10 in PBS-FSB. Goat antihuman laminin was used as positive control. Autoantibodies were more frequently found in chronic chagasic cardiopathy (20%) compared to non-chagasic cardiopathy (5%) and indeterminate chagasic subjects (5%), pattern staining define interstitial and membrane targets on rich conduction system tissue. In conclusion seric autoantibodies against heart conduction system are not a good markers for chagasic cardiopathy group. Their presence showed no clear association with complex rhythm/conduction aberrations.     

Antibodies against human tissue transglutaminase and endomysium in diagnosing and monitoring coeliac disease

BACKGROUND: Coeliac disease (CD) patients often present a variety of uncharacteristic symptoms and therefore sensitive and specific screening tests are needed as an aid in making an accurate diagnosis. A recently developed ELISA, using human recombinant tissue transglutaminase (tTG) as antigen, was evaluated for its significance in the diagnosis of CD. The patient's compliance to a gluten-free diet and the serological reaction during gluten challenge were also monitored. The results were compared with IgA-endomysium antibody (EMA) results. METHODS: Sera previously collected from 365 patients (0.4-76 years) with jejunal biopsy on a gluten-containing diet and from 41 patients on a gluten-free diet or challenge were tested for IgA anti-human tTG antibodies (IgA tTG ab) with Celikey (Pharmacia Diagnostics). The study population comprised 208 CD patients and 157 controls. The diagnostic performance and cut-off for the assay were estimated with ROC analysis. EMA was analysed by indirect immunofluorescence microscopy on cryostat sections of monkey oesophagus. RESULTS: 200/208 patients with CD had positive IgA tTG ab (median >100 U/ml), while only 1/157 of the control patients were positive (median 1.67 U/ml). The area under the ROC curve was 98.3% and the sensitivity and specificity of the test were 96% and 99% for the study population. Only 4/365 patients (1%) presented discordant IgA tTG ab and EMA results, 2 of them had only IgA tTG ab and 2 only EMA. The IgA tTG ab levels and the EMA titres were closely correlated to the duration of gluten-free diet and gluten challenge, respectively. CONCLUSION: IgA tTG ab can be used as an accurate observer-independent alternative to EMA in diagnosing or monitoring CD.     

Type I diabetes as a chronic autoimmune disease

The realization that Type I diabetes is an autoimmune disease and, in particular, a chronic autoimmune disease is beginning to impact on clinical care and research directed at elucidating the cause and prevention of diabetes. For example, specialized laboratory evaluation can now be used to exclude potential renal donors who are at high risk of developing diabetes (by screening renal donor candidates who are relatives of Type 1 diabetics for cytoplasmic islet cell antibodies and evaluating first phase insulin secretion on intravenous glucose tolerance testing). The most important long-term consequence of the abllity to predict Type 1 diabetes may be the development of effective immunotherapy to prevent the disease. Finally, the realization that Type 1 diabetes is an autoimmune disease and that some of the antigens expressed by islets (e.g., specific gangliosides identified with monoclonal antibodies) are expressed by renal glomerular cells, retinal microvascular pericytes, and neurons has renewed interest in searching for immunologic factors contributing to secondary complications.