Ingrid Dahlbom

Elevation of IgG antibodies against tissue transglutaminase as a diagnostic tool for coeliac disease in selective IgA deficiency

Background: IgA serum autoantibodies against tissue transglutaminase (tTG) have an established diagnostic value in coeliac disease, and high efficacy tests are widely available for their detection. However, serological evaluation of IgA deficient subjects is still difficult. Aims: To evaluate the diagnostic potential of IgG class anti-tTG autoantibodies measured quantitatively using an enzyme linked immunosorbent assay (ELISA) compared with immunofluorescent detection of coeliac autoantibodies. Patients: We tested serum samples from 325 IgA deficient subjects, including 78 patients with coeliac disease, 73 disease controls, and 174 blood donors. Methods: IgG antibodies against human recombinant tTG were measured with an ELISA. IgG antiendomysium antibodies (EMA) were assayed by indirect immunofluorescence on human jejunum and appendix sections. Results: The IgG anti-tTG ELISA had a sensitivity of 98.7% and a specificity of 98.6%, and the correlation with IgG EMA titres was high (rs=0.91). One coeliac patient, initially negative in all autoantibody tests, displayed both IgG anti-tTG antibodies and IgG EMA during later gluten exposure. IgG anti-tTG antibodies and EMA titres showed significant decreases (p<0.001) in treated patients. The frequency of IgG anti-tTG autoantibody positivity was 9.8% among IgA deficient blood donors and 11 of the 12 positive subjects with known HLA-DQ haplotypes carried DQ2 or DQ8 alleles. Conclusions: IgG anti-tTG and IgG EMA autoantibody tests are highly efficient in detecting coeliac disease in IgA deficient patients. The high prevalence of coeliac antibodies among symptom free IgA deficient blood donors who also carry coeliac-type HLA-DQ genes indicates that all IgA deficient persons should be evaluated for coeliac disease.

Prediction of clinical and mucosal severity of coeliac disease and dermatitis herpetiformis by quantification of IgA/IgG serum antibodies to tissue transglutaminase.

Objectives: We analysed whether the quantification of autoantibodies against tissue transglutaminase could be used to predict mucosal destruction and disease severity in patients with gluten sensitivity. Patients and Methods: One hundred seventy patients with coeliac disease (CD), comprising 52 children with severe malabsorption (group I), 59 children with mild symptoms (group II), 59 adults (group III), 134 patients with dermatitis herpetiformis (DH), and 131 disease controls, were studied. Serial serum samples of patients in groups I and II on a gluten-free diet were also included. Serum levels of antibodies against recombinant tissue transglutaminase were determined with ELISA using standard curves for quantification of antibodies. Results: Immunoglobulin (Ig)A antibodies against tissue transglutaminase (IgA-TGA) were detected in all of the patients with CD and in 95% of the DH patients. The IgA-TGA and IgG-TGA levels were higher in group I (P < 0.001). The IgG-TGA levels and positivity rate in group I (100%) were higher than in group II (81%), group III (73%), and the DH group (67%). Elevated IgA-TGA and IgG-TGA levels in combination predicted a more severe small intestinal atrophy (P < 0.0001) with a specificity of 99% for Marsh IIIb–IIIc (flat) lesions. The kinetics of the IgA-TGA decrease during diet differed between groups I and II. Conclusions: High levels of IgA-TGA and IgG-TGA antibodies were associated with the grade of mucosal villous atrophy and a more severe clinical presentation. The combined measurement of IgA-TGA and IgG-TGA enables a noninvasive prediction of small intestinal villous atrophy with high accuracy, and may reduce the need for a biopsy in patients with suspected CD.

Low levels of IgM antibodies to phosphorylcholine predict cardiovascular disease in 60-year old men: Effects on uptake of oxidized LDL in macrophages as a potential mechanism

OBJECTIVE We here determine the role of IgM antibodies against phosphorylcholine (anti-PC) in prediction of cardiovascular disease (CVD) and on macrophage uptake of Oxidized LDL (OxLDL). METHODS From a screening of 4232 subjects, 60-year-old (2039 men and 2193 women), 211 incident cases of CVD (myocardial infarction, ischemic stroke, or hospitalized angina pectoris) and 633 age- and sex-matched controls were identified through a 5-7 year follow-up. Serum levels of IgM anti-PC was determined by ELISA. Anti-PC was extracted from pooled human IgM and the effect of anti-PC on the uptake of OxLDL was studied by FACScan. RESULTS Relative risks (RR) with 95% confidence intervals (CI) by quartiles of anti-PC levels with quartile 4 set as the reference value (RR = 1.0) and adjusted for smoking, BMI, type II diabetes, hypercholesterolaemia, and high blood pressure yielded an excess risk for CVD only for those within the lowest quartile of anti-PC values with an RR of 1.37 (CI 0.87-2.16). However, for men stronger associations were noted with increasing multivariately adjusted RRs from quartile 4 to quartile 1. Subjects within quartile 1 (values below 29.7 U/ml) had a significantly increased RR of 1.96 (CI 1.09-3.55). Further adjustments for hsCRP gave essentially the same results. No excess risk was noted for women. Specific anti-PC could be extracted from IgM and these antibodies inhibited macrophage uptake of OxLDL. CONCLUSIONS Low IgM anti-PC could be a novel risk marker for CVD among men. One possible mechanism could be inhibition of uptake of oxLDL in macrophages.

Recombinant Human Tissue Transglutaminase for Diagnosis and Follow-Up of Childhood Coeliac Disease

Highly discriminatory markers for celiac disease are needed to identify children with early mucosal lesions and for rapid follow-up. The aim of this study was to evaluate the potential of circulating anti-tissue transglutaminase (tTG) IgA and IgG antibodies in the diagnosis and follow-up of childhood celiac disease. An ELISA using recombinant human tTG was used to measure the levels of IgA and IgG anti-tTG antibodies in 226 serum samples from 57 children with biopsy-verified celiac disease, 29 disease control subjects, and 24 healthy control subjects. All samples were also analyzed for anti-endomysium antibodies (EMA). The levels of IgA and IgG anti-tTG antibodies correlated with the condition of the small intestinal villous structure and the serum levels of IgA EMA. All of the 25 serum samples obtained from untreated patients contained IgA anti-tTG antibodies, and 24 of 25 also had IgA EMA. Of the serum samples from 53 control children, two had IgA anti-tTG antibodies and two had IgA EMA. Children younger than 5 y of age with untreated celiac disease had the highest serum levels of both IgA and IgG anti-tTG. There was already an increase in IgA anti-tTG antibodies after 2 wk of gluten challenge (p < 0.01). Although the criteria-based diagnosis of childhood celiac disease still depends on histologic evaluation of intestinal biopsies, detection of anti-tTG antibodies provides useful complementary diagnostic information. The human recombinant tTG-based ELISA can be used as a sensitive and specific test to support the diagnosis and may also be used in the follow-up of treatment in childhood celiac disease.

Low levels of IgM antibodies against phosphorylcholine-A potential risk marker for ischemic stroke in men.

BACKGROUND Natural antibodies specific for phosphorylcholine (anti-PC) have been implicated as protective factors in atherosclerosis. We herein determined the relationship between IgM anti-PC and incidence of cardiovascular disease (CVD). METHODS We studied 349 incident cases (200 men) of first events of CVD (coronary heart disease (CHD; n=203 or ischemic stroke; n=146) and 693 age- and sex-matched controls identified through 12 years of follow-up (1991-2003) of subjects from the cardiovascular cohort within the Malmö Diet and Cancer Study. Relative risks (RR) of CVD with 95% confidence intervals (CI) of incident CVD with adjustments for age, smoking, total cholesterol and blood pressure were determined. Anti-PC-levels were measured using ELISA (Athera CVDefine). RESULTS As determined using Athera CVDefine, significant associations were attained with values of anti-PC below 17U/ml (corresponding to the lowest 9th percentile), which remained after taking confounders into account (RR: 1.79, 95% CI: 1.09-2.94, p=0.021). If men were studied separately, significance was evident at values below 17U/ml (RR: 2.01, 95% CI: 1.11-3.67, p=0.022), which was not the case among women. Furthermore, values below 17U/ml were also associated with ischemic stroke (RR=3.67, 95% CI: 1.34-10.1, p=0.01), but not with CHD. CONCLUSION Low IgM anti-PC could be a novel risk marker for development of ischemic stroke in men. Further studies are needed to establish gender and subgroup differences.

Antibody reactivity against human and guinea pig tissue transglutaminase in children with celiac disease

BACKGROUND Highly discriminatory markers for celiac disease are needed to identify children with early mucosal lesions. The purposes of this study were to evaluate the clinical potential of circulating anti-tissue transglutaminase (tTG) immunoglobulin (Ig)A antibodies in the diagnosis of childhood celiac disease and to investigate the extent of autoreactivity of these antibodies. METHODS Included in this retrospective study were samples from 22 children with biopsy-verified celiac disease, 23 control subjects with disease, and 22 healthy control subjects without any known gastrointestinal or inflammatory disorders. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum levels of IgA antibodies specific for human and guinea pig tTGs. All samples were also analyzed for antibodies to gliadin and endomysium (EMA). RESULTS The concentrations of IgA specific for human and guinea pig tTGs correlated with the small intestinal villous structure and the serum levels of IgA EMA. The tTG ELISAs exhibited a high specificity and sensitivity for detection of untreated celiac disease. The human erythrocyte IgA tTG ELISA had the highest sensitivity (100%) and a specificity of 98%. The IgA EMA method had a sensitivity of 95% and the highest specificity (100%) of all tests. CONCLUSIONS Our results provide additional support to the concept that anti-tTG IgA antibodies can be used as a highly discriminatory serologic marker for celiac disease and that measurements of these autoreactive antibodies may in the future be used as an alternative to the EMA test.

Missing endomysial and reticulin binding of coeliac antibodies in transglutaminase 2 knockout tissues

Background: Autoantibodies against transglutaminase 2 (TG2) are thought to be responsible for the endomysial (EMA), reticulin (ARA), and jejunal antibody (JEA) tissue binding of serum samples from coeliac patients but the exclusive role of TG2 in these staining patterns has not yet been established. Aims: To evaluate whether antigens other than TG2 contribute to EMA/ARA/JEA reactions. Patients: Serum samples from 61 EMA/ARA/JEA positive untreated patients with coeliac disease, 40 dermatitis herpetiformis patients, and 34 EMA/ARA/JEA negative non-coeliac controls were tested. Methods: TG2 knockout (TG2−/−) and wild-type mouse oesophagus, jejunum, liver, and kidney sections, and TG2−/− sections coated with human recombinant TG2 were used as substrates in single and double immunofluorescent studies for patient IgA binding and tissue localisation of TG2, fibronectin, actin, and calreticulin. Results: None of the patient serum samples elicited EMA, ARA, or JEA binding in TG2−/− morphologically normal tissues. In contrast, 96 of 101 gluten sensitive patient samples (95%) reacted with wild-type mouse tissues and all 101 reacted in EMA/ARA/JEA patterns with TG2−/− mouse tissues coated with human TG2. Serum IgA binding to TG2−/− smooth muscle cells was observed in low titres in 31.1%, 27.5%, and 20.5%, and to TG2−/− epithelium in 26.3%, 5.0%, and 8.8% of coeliac, dermatitis herpetiformis, and control samples, respectively. These positivities partly colocalised with actin and calreticulin but not with TG2 or fibronectin. Conclusions: EMA/ARA/JEA antibody binding patterns are exclusively TG2 dependent both in coeliac and dermatitis herpetiformis patients. Actin antibodies are responsible for some positivities which are not part of the EMA/ARA/JEA reactions.

Antigliadin Immunoglobulin A Best in Finding Celiac Disease in Children Younger Than 18 Months of Age

Objectives: The aim was to investigate age-dependent serum levels and occurrence of elevated celiac disease (CD)–related antibodies in young children, to define the optimal serological procedure when selecting for small intestinal biopsy. Patients and Methods: Included were 428 children with biopsy verified CD (median age 16 months; range 7.5 months–14 years) and 216 controls (median age 2.7 years; range 8.5 months–14.6 years). Immunoglobulin (Ig) A antibodies against gliadin (AGA-IgA), tissue transglutaminase (tTG-IgA), and endomysium (EMA-IgA) were analysed. Results: Increased serum AGA-IgA levels were found in 411 of 428 CD cases, tTG-IgA in 385 of 428, and EMA-IgA in 383 of 428. In the control group, 11 of 216 had increased levels of AGA-IgA, 5 of 216 of tTG-IgA, and 8 of 216 of EMA-IgA. In CD children younger than 18 months, elevated AGA-IgA occurred in 97% and elevated tTG-IgA and EMA-IgA were found in 83% of the cases. Conversely, in CD children older than 18 months, elevated AGA-IgA occurred in 94%, and elevated tTG-IgA and EMA-IgA were found in 99% of the cases. Conclusions: In children older than 18 months, both tTG-IgA and EMA-IgA are sufficiently accurate to be used as a single antibody marker, whereas a large proportion of younger children with CD lack these antibodies. Therefore, when selecting children for small intestinal biopsy, the detection of a combination of AGA-IgA and tTG-IgA is optimal for identifying untreated CD in children younger than 18 months.

Immunoglobulin g (IgG) anti-tissue transglutaminase antibodies used as markers for IgA-deficient celiac disease patients

ABSTRACT The role of immunoglobulin A (IgA) anti-tissue transglutaminase antibodies (IgA-tTG) as predictors of untreated celiac disease (CoD) is well documented, and the presence and levels of these antibodies are most accurately monitored with native or recombinant human antigens. However, IgA-deficient CoD patients are not identified by IgA serology, and conflicting results concerning the diagnostic validity of IgG antibodies against gliadin (IgG-AGA), endomysium (IgG-EmA), and tTG (IgG-tTG) have been reported. The aim of the present study was to evaluate the utility of IgG-tTG for the detection of CoD in IgA-deficient patients. Samples from 115 IgA-deficient and 200 IgA-sufficient subjects were collected and tested for the presence of IgA and IgG antibodies against tTG, EmA, and AGA. Antibodies against tTG were measured by an enzyme-linked immunosorbent assay based on recombinant human tTG, and antibodies against EmA were determined by immunofluorescence. The values for IgG-tTG showed a higher correlation (correlation coefficient [r] = 0.91) with those for IgG-EmA for the IgA-deficient subjects than for the IgA-sufficient subjects (r = 0.88). The overall concordance of the positive and negative results between IgG-tTG and IgG-EmA was 97%, and the IgG-tTG assay discriminated between IgG-EmA-positive and -negative subjects with IgA deficiency at a rate of 100%. Elevated levels of IgG-tTG and IgG-EmA were measured in 70% of the IgA-sufficient subjects. IgG-tTG detection with recombinant human tTG is a good alternative to IgG-EmA detection, and the addition of IgG-tTG assessment to present screening methods may improve the ability to identify IgA-deficient subjects with CoD.

A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects

The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1–2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits.