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 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.     

Cellular response to alpha-gliadin in untreated coeliac disease.

An improved technique for the detection of alpha-gliadin sensitised mononuclear cells in the peripheral blood of untreated coeliac patients is described. This method is a modification of the direct LMIF assay, and involves exposure of lymphocytes to alpha-gliadin and the assay of the resultant lymphokine produced using normal leucocytes as indicator cells. All untreated coeliac patients, 14 of 15 treated patients, and two of 28 controls responded to alpha-gliadin. The direct LMIF assay in comparison is less sensitive, and detected sensitivity to alpha-gliadin in only four out of eight patients with untreated coeliac disease. Use of the indirect LMIF technique demonstrates that in untreated as well as treated coeliac patients there are cells sensitised to alpha-gliadin circulating in the peripheral blood. These findings may have pathogenic and diagnostic significance.     

Partial lipodystrophy in coeliac disease.

The association of coeliac disease and partial lipodystrophy is described. The patient also had deficiencies of serum IgA and C3 complement (the latter associated with partial lipodystrophy). In addition, there was subclinical dermatitis herpetiformis confirmed by skin biopsy. The facial wasting of fully developed partial lipodystrophy may be misinterpreted as a sign of malabsorption but the facial, upper limb, and truncal lipodystrophy contrasts with normal pelvic and lower limb appearances.     

Cystic pneumatosis in coeliac disease.

This report presents a patient with proximal small bowel cystic pneumatosis associated with poorly controlled coeliac disease and pseudo-obstruction. Cystic pneumatosis is rare in the proximal small bowel and we can find no report of its occurrence in association with coeliac disease.     

Arthritis and coeliac disease.

We report six patients with coeliac disease in whom arthritis was prominent at diagnosis and who improved with dietary therapy. Joint pain preceded diagnosis by up to three years in five patients and 15 years in one patient. Joints most commonly involved were lumbar spine, hips, and knees (four cases). In three cases there were no bowel symptoms. All were seronegative. X-rays were abnormal in two cases. HLA-type A1, B8, DR3 was present in five and B27 in two patients. Circulating immune complexes showed no consistent pattern before or after treatment. Coeliac disease was diagnosed in all patients by jejunal biopsy, and joint symptoms in all responded to a gluten-free diet. Gluten challenge (for up to three weeks) failed to provoke arthritis in three patients tested. In a separate study of 160 treated coeliac patients attending regular follow up no arthritis attributable to coeliac disease and no ankylosing spondylitis was identified, though in a control group of 100 patients with Crohn's disease the expected incidence of seronegative polyarthritis (23%) and ankylosing spondylitis (5%) was found (p less than 0.01). Arthritis appears to be a rare manifestation of coeliac disease. This relationship may provide important clues to the role of gastrointestinal antigens in rheumatic diseases.     

Infertility and coeliac disease.

BACKGROUND: Coeliac women may suffer from gynaecological and obstetric complications. It is possible that these complications are the first symptom of coeliac disease. AIMS: To investigate the occurrence of subclinical coeliac disease in patients with infertility or recurrent miscarriages. SUBJECTS: Women of reproductive age who were attending the hospital because of either primary or secondary infertility, or two or more miscarriages. Women undergoing sterilisation served as control subjects. METHODS: The diagnostic investigation for infertility included the endocrine status, diagnostic laparoscopy, investigation of tubal patency, postcoital test, and semen analysis of the partner. Circulating antibodies against IgA class reticulin and gliadin were used in screening for coeliac disease. In positive cases, the diagnosis was confirmed by small bowel biopsy specimens. RESULTS: Four (2.7%) of 150 women in the infertility group, and none of the 150 control subjects were found to have coeliac disease (p = 0.06). All four women with coeliac disease suffered from infertility of unexplained origin. Altogether 98 women had no discoverable reason for infertility. Thus, in this subgroup the frequency of coeliac disease was 4.1% (four of 98), the difference from the control group being statistically significant (p = 0.02). None of the coeliac women had extensive malabsorption, but two had iron deficiency anaemia. One women with coeliac disease has had a normal delivery. None of the 50 women with miscarriage had coeliac disease. CONCLUSION: Patients having fertility problems may have subclinical coeliac disease, which can be detected by serological screening tests. Silent coeliac disease should be considered in the case of women with unexplained infertility.     

Cellular hypersensitivity to gluten derived peptides in coeliac disease.

Wheat gluten derived antigens have been tested for their ability to inhibit the migration of leucocytes from healthy subjects and patients with coeliac disease. Three preparations of a water soluble fraction (Frazer's fraction III, FIII) of partial peptic tryptic digests of wheat gluten had different effects in a direct (one stage) assay. Subfractions B and B2 caused migration inhibition of leucocytes from patients with treated coeliac disease but not of leucocytes from healthy volunteers or patients with Crohn's disease or ulcerative colitis. This migration inhibition seems to be specific for gluten fractions because maize zein fraction B, beta-lactoglobulin and ovalbumin did not cause it. The sensitivity of coeliac leucocytes to fraction B is not related to factors present in coeliac serum as the migration of leucocytes from healthy individuals preincubated with coeliac sera was not inhibited. Puromycin diminished inhibition by fraction B, which was active at 1.2 micrograms/ml in an indirect (two stage) migration inhibition assay; this is consistent with a process involving elaboration of lymphokine(s). More highly purified fractions of B2, P1-P4 were prepared by reverse phase high performance liquid chromatography (HPLC) and showed differing potency in direct and indirect assays, with P4 being the most active fraction. Inhibition of migration by gluten derived peptides appears to result from the release of lymphokine by leucocytes specifically from coeliac patients.     

Pseudopolycythaemia and coeliac disease.

We report two patients in whom introduction of a gluten free diet for coeliac disease was associated with the development of pseudopolycythaemia.     

Autoantibodies against a 210kDa glycoprotein of the nuclear pore complex as a prognostic marker in patients with primary biliary cirrhosis

It has been reported that the presence of anti-nuclear antibody against a 210kDa glycoprotein of nuclear pore complex (anti-gp210) is highly speci?c for primary biliary cirrhosis (PBC). The aim of the present study was to investigate the signi?cance of anti-gp210, especially as a prognostic marker. The presence of anti-gp210 was ascertained in 113 patients with PBC and 162 controls by indirect immuno?uorescence assay using HepG2 cells and immunoblotting analysis using nuclear extracts from HeLa cells. Anti-gp210 was detected in 25 of the 113 (22.1%) patients. None of the 162 controls was positive for anti-gp210. The appearance and titre of anti-gp210 in the patients with PBC did not vary from the time of diagnosis and through their clinical course. Anti-mitochondrial antibodies (AMA), including antibodies against pyruvate dehydrogenase complex, branched chain α-ketoacid dehydrogenase complex and α-ketoglutarate dehydrogenase complex, were not detected by enzyme-linked immunosorbent assay in ?ve of the 113 (4.4%) patients with PBC. However, anti-gp210 alone was positive in one of these ?ve patients. The difference in prognosis was statistically signi?cant; patients with PBC positive for anti-gp210 died from hepatic failure more frequently than those who were negative (P < 0.01), although there were no statistically signi?cant differences in the frequency of jaundice and the histological stage at the time of diagnosis between the two groups. We suggest that the presence of anti-gp210 is one of the independent prognostic markers able to predict, at the time of diagnosis, a poor outcome in patients with PBC.     

Intestinal permeability in patients with coeliac disease and relatives of patients with coeliac disease.

The functional integrity of the small bowel is impaired in coeliac disease. Intestinal permeability, as measured by the sugar absorption test probably reflects this phenomenon. In the sugar absorption test a solution of lactulose and mannitol was given to the fasting patient and the lactulose/mannitol ratio measured in urine collected over a period of five hours. The sugar absorption test was performed in nine patients with coeliac disease with an abnormal jejunum on histological examination, 10 relatives of patients with coeliac disease with aspecific symptoms but no villous atrophy, six patients with aspecific gastrointestinal symptoms but no villous atrophy, and 22 healthy controls to determine whether functional integrity is different in these groups. The lactulose/mannitol ratio (mean (SEM) is significantly higher in both coeliac disease (0.243 (0.034), p < 0.0001)) and relatives of patients with coeliac disease (0.158 (0.040), p < 0.005)) v both healthy controls (0.043 (0.006)) and patients with aspecific gastrointestinal symptoms (0.040 (0.011)). The lactulose/mannitol ratio in relatives of coeliac disease patients was significantly lower than in the coeliac disease patient group (p = 0.04). The lactulose/mannitol ratio was the same in healthy controls and patients with aspecific gastrointestinal symptoms. It is concluded that the sugar absorption test is a sensitive test that distinguishes between patients with coeliac disease and healthy controls. The explanation for the increased permeability in relatives of patients with coeliac disease is uncertain. Increased intestinal permeability may be related to constitutional factors in people susceptible to coeliac disease and may detect latent coeliac disease. The sugar absorption test may therefore be helpful in family studies of coeliac disease.     

Serum carnitine concentrations in coeliac disease.

Carnitine is essential for muscle energy production and is required for the transport of long chain fatty acids and acyl co-enzyme A derivatives across the inner mitochondrial membrane. Recently, an absorptive transport mechanism was discovered at the small bowel level suggesting the possibility of a carnitine deficient state in patients with mucosal damage. Therefore, this study investigated carnitine concentrations in serum of patients with coeliac disease. Serum samples were obtained from 12 patients with active coeliac disease and seven with non-active disease, and compared with serum samples of 17 children with gastrointestinal symptoms but with a small bowel normal on biopsy examination and 33 normal controls. Total serum carnitine concentration was significantly lower in the patients with coeliac disease compared with the other two groups and to reference values. When the degree of atrophy of coeliac intestinal mucosa was numerically graded, serum carnitine concentrations did not correlate to the degree of the intestinal lesion but were significantly lower in the damaged intestine compared with the group with normal mucosa. It is suggested that coeliac disease should be considered as a potential cause of secondary carnitine deficiency.     

Lymphocyte subpopulations in adult coeliac disease.

Rosetting techniques were used to estimate T and B cell subpopulations in the peripheral blood in patients with treated and untreated adult coeliac disease and in control subjects. In patients with untreated coeliac disease, T cell numbers were significantly lower than in controls or treated patients, although there was no difference in total lymphocyte counts. There was no significant difference in B cell numbers between treated and untreated patients, and the subpopulation which increased to replace the T cells in untreated patients comprised cells not identified by B or T cell markers. Total lymphocyte counts and lymphocyte subpopulations were affected by splenic atrophy. It is suggested that these effects might be caused by the loss of lymphocytes from the gastrointestinal tract in untreated coeliac disease.