Validation of Antibody-Based Strategies for Diagnosis of Pediatric Celiac Disease Without Biopsy.

BACKGROUND & AIMS: A diagnosis of celiac disease is made based on clinical, genetic, serologic, and duodenal morphology features. Recent pediatric guidelines, based largely on retrospective data, propose omitting biopsy analysis for patients with concentrations of IgA against tissue transglutaminase (IgA-TTG) >10-fold the upper limit of normal (ULN) and if further criteria are met. A retrospective study concluded that measurements of IgA-TTG and total IgA, or IgA-TTG and IgG against deamidated gliadin (IgG-DGL) could identify patients with and without celiac disease. Patients were assigned to categories of no celiac disease, celiac disease, or biopsy required, based entirely on antibody assays. We aimed to validate the positive and negative predictive values (PPV and NPV) of these diagnostic procedures. METHODS: We performed a prospective study of 898 children undergoing duodenal biopsy analysis to confirm or rule out celiac disease at 13 centers in Europe. We compared findings from serologic analysis with findings from biopsy analyses, follow-up data, and diagnoses made by the pediatric gastroenterologists (celiac disease, no celiac disease, or no final diagnosis). Assays to measure IgA-TTG, IgG-DGL, and endomysium antibodies were performed by blinded researchers, and tissue sections were analyzed by local and blinded reference pathologists. We validated 2 procedures for diagnosis: total-IgA and IgA-TTG (the TTG-IgA procedure), as well as IgG-DGL with IgA-TTG (TTG-DGL procedure). Patients were assigned to categories of no celiac disease if all assays found antibody concentrations <1-fold the ULN, or celiac disease if at least 1 assay measured antibody concentrations >10-fold the ULN. All other cases were considered to require biopsy analysis. ULN values were calculated using the cutoff levels suggested by the test kit manufacturers. HLA typing was performed for 449 participants. We used models that considered how specificity values change with prevalence to extrapolate the PPV and NPV to populations with lower prevalence of celiac disease. RESULTS: Of the participants, 592 were found to have celiac disease, 345 were found not to have celiac disease, and 24 had no final diagnosis. The TTG-IgA procedure identified patients with celiac disease with a PPV of 0.988 and an NPV of 0.934; the TTG-DGL procedure identified patients with celiac disease with a PPV of 0.988 and an NPV of 0.958. Based on our extrapolation model, we estimated that the PPV and NPV would remain >0.95 even at a disease prevalence as low as 4%. Tests for endomysium antibodies and HLA type did not increase the PPV of samples with levels of IgA-TTG ≥10-fold the ULN. Notably, 4.2% of pathologists disagreed in their analyses of duodenal morphology-a rate comparable to the error rate for serologic assays. CONCLUSIONS: In a prospective study, we validated the TTG-IgA procedure and the TTG-DGL procedure in identification of pediatric patients with or without celiac disease, without biopsy. German Clinical Trials Registry no.: DRKS00003854.

Antibody Concentrations Decrease 14-Fold in Children With Celiac Disease on a Gluten-Free Diet but Remain High at 3 Months.

BACKGROUND & AIMS: Celiac disease can be identified by a serologic test for IgA against tissue transglutaminase (IgA-TTG) in a large proportion of children. However, the increased concentrations of antibody rarely normalize within the months after children are placed on a gluten-free diet (GFD). Early serologic predictors of sufficient adherence to GFD are required for optimal treatment. METHODS: In a prospective study, we observed the response to a GFD in 345 pediatric patients (67% girls; mean age, 8.4 y) who underwent duodenal biopsy to confirm or refute celiac disease from October 2012 through December 2015. Baseline serum samples were tested centrally for IgA-TTG and IgG against deamidated gliadin. Follow-up serologic analyses of children on a GFD were performed about 3 months later. RESULTS: The geometric mean concentration of IgA-TTG decreased from 72.4-fold to 5.2-fold the upper limit of normal (ULN), or by a factor of 14.0 (95% CI, 12.0-16.4). A substantial response (defined as a larger change than the typical variation in patients not on a GFD) was observed in 80.6% of the children. Only 28.1% of patients had a substantial response in the concentration of IgG against deamidated gliadin. Concentration of IgA-TTG remained above 1-fold the ULN in 83.8% of patients, and above 10-fold the ULN in 26.6% of patients with a substantial response. CONCLUSIONS: Serum concentration of IgA-TTG decreases substantially in most children with celiac disease within 3 months after they are placed on a GFD, but does not normalize in most. This information on changes in antibody concentrations can be used to assess patient response to the diet at short-term follow-up evaluations. Patients with a substantial response to a GFD often still have high antibody levels after 3 months. German Clinical Trials Registry no. DRKS00003854.

Will the Real Coeliac Disease Please Stand Up? Coeliac Disease Prevalence in the German LIFE Child Study.

OBJECTIVES: Assessing the seroprevalence and the prevalence of definite coeliac disease (CD) in the German LIFE Child Health study cohort including immunoglobulin A (IgA) antibodies against tissue transglutaminase (IgA-TTG) in addition to IgG antibodies against deamidated gliadin peptides (IgG-DGP) and human leukocyte antigen (HLA)-DQ2/8 genotyping. METHODS: Samples from children and adolescents were first screened for IgA-TTG and IgG-DGP. If IgA-TTG was above 0.5 times the upper limit of normal and/or IgG-DGP was positive, IgA antibodies against endomysium (IgA-EmA) were measured, and HLA was genotyped. In patients with only IgG-DGP positivity, total IgA was assayed. Subjects with suspicious results were followed up serologically and, in case of repeatedly positive antibody results, invited for a personal interview. Further diagnostic data were obtained independent from our study. RESULTS: We screened 2363 children's blood samples collected from 2011 to 2015. The seroprevalence, that is, IgA-TTG and/or IgA-EmA positivity or IgG-DGP positivity with IgA <0.05 g/L, was 1.57% (95% confidence interval [CI95%] 1.14-2.15). The prevalence of suspected CD, that is, seroprevalence and compatible HLA genotype with hitherto unknown mucosal damage, was 1.35% (CI95% 0.96-1.91). Definite CD, that is, seropositivity accompanied by positive intestinal biopsy or IgA-TTG ≥ 10 × upper limit of normal, was found in 0.42% (CI95% 0.22-0.80). Seven children claimed to have CD. The HLA haplotype, however, matched in only 4 of them resulting in an overall CD prevalence of at least 0.59% (CI95% 0.34-1.02). Thirteen unclear cases remained; therefore, the prevalence may even be higher. CONCLUSIONS: The prevalence of definite CD in a population-representative German cohort is higher than previously described. HLA-DQ typing is helpful to identify false-positive IgA-TTG patients negative for IgA-EmA and/or IgG-DGP under screening conditions and unmasks possible misdiagnoses of CD.

Hemolysis and IgA-antibodies against tissue transglutaminase: When are antibody test results no longer reliable?

BACKGROUND: Antibodies against tissue transglutaminase (TTG) of isotype IgA (IgA-aTTG) represent reliable diagnostic markers to confirm or exclude celiac disease (CD). Hemolysis (HL) is an important pre-analytical factor. HL can be quantified as HL index (HI) correlating with the concentration of free hemoglobin. TTG is abundant in erythrocytes and released upon HL. In immunoassays, the released TTG may interfere with binding of IgA-aTTG to the coated TTG. METHODS: We selected 17 HL-free sera from children with biopsy-confirmed CD: 7 with low-positive (1-5 multiples of upper limit of normal [×ULN]), 5 with intermediate (5-10 × ULN) and 5 with high IgA-aTTG (10-15 × ULN). Sera were spiked with hemolysates resulting in HIs ranging from 12.5 to 800 (12.5-800 mg/dL free hemoglobin). RESULTS: IgA-aTTG values were significantly decreased (>10%) after addition of hemolysates even if HL was invisible (HI <50). This effect is diagnosis-relevant if IgA-aTTG values are measured just below the cut-offs: (i) 0.4-1 × ULN at HI ≥25 (CD not excludable) and (ii) 8.5-10 × ULN at HI ≥200 (diagnosis of CD without biopsy not possible). Antibodies against deamidated gliadin were not influenced by HL. CONCLUSIONS: IgA-aTTG results in sera with HI ≥25 can yield inconclusive results. Therefore, those antibody results should be assessed only under consideration of the HI.

Determination of IgG and IgA antibodies against native gliadin is not helpful for the diagnosis of coeliac disease in children up to 2 years old.

OBJECTIVE: Assays for antibodies against native gliadin (anti-nGli) are still often assumed to perform better in the diagnosis of coeliac disease in young children than tests for antibodies to deamidated gliadin (anti-dGli), tissue transglutaminase (anti-tTG), and endomysium (EmA). We compared the performance of assays for anti-nGli, anti-dGli, anti-tTG, and EmA in this age group. METHODS: We investigated retrospectively 184 children (42 with coeliac disease under normal diet and 142 controls) up to 2 years of age. Immunoglobulin (Ig) A- and IgG-anti-dGli, IgA- and IgG-anti-nGli, IgA- and IgG-anti-tTG, and IgA-EmA were measured in serum. Areas under receiver operating characteristics curves, sensitivities, specificities, positive and negative predictive values, positive and negative likelihood ratios, as well as diagnostic odds ratios were calculated. RESULTS: From all of the tests investigated, only assays for IgG-anti-dGli, IgA-anti-tTG, and IgA-EmA had high specificity (≥ 0.96) connected with high sensitivity (≥ 0.86), with high positive predictive values (≥ 0.52 and ≥ 0.69 at pretest probabilities of 0.05 and 0.1, respectively) and negative predictive values (≥ 0.99 and ≥ 0.98 at pretest probabilities of 0.05 and 0.1, respectively). These assays also showed high positive likelihood ratio (≥ 24) at low negative likelihood ratio (≤ 0.15) and high diagnostic odds ratios (≥ 136). CONCLUSIONS: Our results do not support the use of assays of anti-nGli to diagnose coeliac disease in young children. IgA-anti-tTG, IgA-EmA, and IgG-anti-dGli perform better than anti-nGli.

Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.

Immunoassay of in vitro activated human tissue transglutaminase.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that exerts a variety of physiological functions and is involved in various pathoprocesses. To characterize the role of tTG in disease, simple assays are necessary for detection. We developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) that combines a transamidation step with immunological detection to determine tTG. This assay is based on covalent binding of in vitro activated tTG to N,N'-dimethylated casein and subsequent detection of coupled tTG by specific immunoglobulin G (IgG) antibodies directed against tTG followed by binding of a secondary biotin-labeled antibody. The assay reaches a detection limit of 0.05ng of tTG/ml. Type 1 and 3 transglutaminases and factor XIII are not detected. By use of this assay, tTG in several cell lines and the stimulatory effect of retinoic acid on tTG expression could be demonstrated. The new assay represents a promising tool for the study of tTG in normal cell physiology and under pathological conditions.

Endocytotic segregation of gliadin peptide 31-49 in enterocytes.

OBJECTIVE: Coeliac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in human leucocyte antigen (HLA)-DQ2/8-positive individuals. The molecular mechanisms relevant to oral tolerance induction towards toxic cereals such as gliadin remain poorly understood. Enterocytes, which express predominantly HLA-DR proteins, are capable of processing, transcytosing and presenting food antigens from the intestinal lumen to T lymphocytes of the lamina propria. METHODS: Epitope-specific monoclonal antigliadin antibodies are utilised to unravel the intraepithelial transport processes of gliadin peptides in human duodenal biopsy specimens from patients with CD and reconstitute the transepithelial and endocytic pathways of gliadin in intestinal epithelial HT29 cells. RESULTS: The gliadin peptide AA 31-49 is segregated from the peptides AA 56-68 and AA 229-246 along the endosomal pathway. Thus, AA 31-49 bypasses HLA-DR-positive late endosomes in intestinal cells and in biopsy specimens of patients with untreated CD. Further, it is localised in early endosomes and consequently escapes antigen presentation at the basolateral membrane, unlike peptides AA 56-68 and AA 229-246 that reach HLA-DR-positive late endosomes. Strikingly, forms of gliadin peptide AA 31-49 conjugated to cholera toxin B are sorted into late endosomes of HT29 cells. CONCLUSIONS: Endocytic segregation of gliadin peptide AA 31-49 seems to be a constitutive process. It explains why this peptide cannot stimulate gluten-sensitive T cells. Presentation of gliadin peptides by HLA-DR proteins via late endosomes within enterocytes might induce a tolerogenic effect and constitutes a potentially promising therapeutic approach for induction of tolerance towards gliadin.

IgG antibodies against deamidated gliadin peptides for diagnosis of celiac disease in patients with IgA deficiency.

BACKGROUND: Assays for IgG antibodies against deamidated gliadin (IgG-anti-dGli) are comparable in performance with tests detecting IgA antibodies against tissue transglutaminase (IgA-anti-tTG) in diagnosing celiac disease (CD). IgA-anti-tTG are absent in IgA deficiency, a condition often associated with CD. In IgA deficiency, IgG-anti-tTG, which have a lower overall diagnostic accuracy, are routinely measured. We examined whether IgG-anti-dGli would be useful for diagnosing CD in patients with IgA deficiency. METHODS: We studied 34 IgA-deficient CD patients, 185 IgA-competent newly diagnosed children with CD, 316 children without CD, 400 adult blood donors, and 6 control IgA-deficient individuals without CD. Anti-dGli and anti-tTG were measured by ELISA, and endomysium antibodies (EmA) were measured by immunofluorescence on monkey esophagus (IgA as well as IgG class for all antibodies). We calculated diagnostic sensitivity (percentage of patients above cutoff with 95% CIs) according to age-specific cutoffs for 95% diagnostic specificity and according to cutoffs proposed by the manufacturer of the assays. RESULTS: No IgA-deficient CD patients were positive for any IgA-based antibody assay. Diagnostic sensitivity of IgG-anti-tTG was 91.2% (95% CI 76.3%-97.7%) according to age-specific cutoffs and 82.4% (66.1%-92.0%) according to manufacturer cutoffs. The diagnostic sensitivity of IgG-EmA was 75.8% (58.8%-87.4%) and the sensitivity of IgG-anti-dGli was 88.2% (72.8%-95.9%) according to both cutoffs. CONCLUSIONS: IgG-anti-dGli and IgG-anti-tTG have comparable diagnostic sensitivities for IgA-deficient celiac patients. IgG-anti-dGli may be useful for diagnosing CD in IgA-deficient patients.

Antibodies against deamidated gliadin as new and accurate biomarkers of childhood coeliac disease.

OBJECTIVES: Assays of tissue transglutaminase antibodies (anti-tTG) represent the cornerstone of serological coeliac disease (CD) diagnostics. Assays of antibodies against native gliadin (anti-nGli) lost importance due to low validity. We investigated the performance of new assays for antibodies against deamidated gliadin (anti-dGli) in childhood CD. METHODS: We retrospectively compared children (142 with active CD and 160 without CD, diagnosis confirmed or excluded by intestinal biopsy) concerning (immunoglobulin [Ig] G and IgA) anti-nGli, anti-tTG, and 2 different anti-dGli assays. RESULTS: IgG-anti-dGli1, IgG-anti-dGli2, and IgA-anti-tTG performed similarly. Area under the receiver-operating characteristic curve (AUC) was 98.6%, 98.9%, and 97.9%; accuracy was 94.7%, 95.7%, and 96.7%. Anti-dGli1 and anti-dGli2 (IgG and IgA) and IgA-anti-tTG performed significantly better than IgA-anti-nGli and IgG-anti-nGli. Both IgG-anti-dGli showed higher AUC and accuracy than IgA-anti-dGli and IgG-anti-tTG. Combined evaluation of IgA-anti-tTG with one of the IgG-anti-dGli tests reduced the rate of falsely classified patients. At enhanced cutoff (specificity >99%), sensitivity was above 67% for both IgG-anti-dGli and IgA-anti-tTG. If IgA-anti-tTG assay was combined with one of the IgG-anti-dGli tests, then the fraction of patients identified with more than 99% specificity as coeliacs increased significantly above 84.5%. Combined evaluation of the 2 IgG-anti-dGli tests did not improve the performance. CONCLUSIONS: The new IgA and IgG-anti-dGli tests outperform conventional anti-nGli assays. The validity of IgG-anti-dGli cannot be distinguished from IgA-anti-tTG. It should be studied prospectively whether antibody assays could replace biopsy in diagnosis of CD in a substantial segment of children.

Specificity analysis of anti-gliadin mouse monoclonal antibodies used for detection of gliadin in food for gluten-free diet.

A gluten-free diet (GFD) is the sole effective long-lasting treatment of celiac disease. Four monoclonal antibodies (Abs) were prepared by immunization of animals kept on GFD with gliadin. The specificity of these Abs to decapeptides of alpha- and gamma-gliadin and omega-secalin was analyzed by the PEPSCAN technique. Repetitive sequences of alpha- and gamma-gliadin and omega-secalin containing the motifs QPFPXQ (X = Q, L, P) were recognized by all Abs tested. These Abs also frequently reacted with peptides containing the sequences QQSFPQQ, QQTFPQP, and QPFRPQ. On the basis of PEPSCAN results two Abs--8D4 and 7C6--were selected for the construction of a new ELISA kit for the detection of gliadin in food. The comparison of data obtained using the newly developed ELISA kit and commercially available ones indicated that Abs selection on the basis of their fine specificity to gliadin is useful for sensitive detection of gliadin in foods.

Primate liver tissue as an alternative substrate for endomysium antibody immunofluorescence testing in diagnostics of paediatric coeliac disease.

BACKGROUND: Immunofluorescence assays of antibodies against endomysium (EmA) on primate oesophagus sections represent the gold standard in serological testing for coeliac disease (CD). As alternative immunofluorescence technique, staining of primate liver tissue is in use. We compared performance and predictive power of IgA- and IgG-EmA on primate oesophagus and primate liver sections. METHODS: Sera of 298 paediatric biopsy-proven CD patients under gluten-containing diet and 574 disease controls were investigated. Samples were collected between 2004 and 2013 in six children's hospitals. The antibodies were assayed blinded to diagnoses and histological data. Sensitivity, specificity, and positive (PPV) and negative predictive values (NPV) were calculated for different assays. RESULTS: (Oesophagus vs liver): For IgA-EmA, sensitivity (0.953 vs 0.956) and specificity (0.981 vs 0.972) as well as PPV (0.963 vs 0.947) and NPV (0.976 vs 0.979) were comparable on both tissues. IgG-EmA on liver showed significantly higher sensitivity (0.520 vs 0.631; p=0.006) but significantly lower specificity (0.995 vs 0.963; p=0.002) and PPV (0.981 vs 0.899; p=0.0002) than on oesophagus. NPV on liver was higher than NPV on oesophagus, however, the difference was not statistically significant (0.799 vs 0.834; p=0.099). CONCLUSION: Primate liver can be used as alternative, equally well functioning substrate for IgA-EmA testing.

How gluten-free is gluten-free, and what does this mean to coeliac patients?

A gluten-free diet is the cornerstone treatment of coeliac disease. Until now, it is not known conclusively whether trace amounts of gluten might be allowed in the diet, as suggested by Codex Alimentarius. Gluten-free foods intended for dietary use can now be analysed reliably for residual gluten by the new R5 enzyme-linked immunosorbent assay (ELISA) system. Some major problems of gluten analysis (sensitivity, specificity, reproducibility) can be solved by the new method. Therefore, the information given by the new test system is relevant and superior to earlier methods. Further clinical studies using small dose challenges and dietary records including gluten analysis are necessary until a more meaningful discussion on standards for gluten-free foods can be started.

Production of recombinant human tissue transglutaminase using the baculovirus expression system, and its application for serological diagnosis of coeliac disease.

BACKGROUND AND OBJECTIVES: Tissue transglutaminase was identified as the main autoantigen in coeliac disease (CD) but enzyme immunoassays applying the commercially available antigen from guinea pig liver show insufficient specificity and sensitivity for diagnosis as compared with endomysium antibodies (EmA). The aim of this present study was to develop a new method for the cloning and expression of human tissue transglutaminase (hu-tTG) and to test hu-tTG in the serological diagnosis of CD. METHODS: Hu-tTG was cloned and expressed using a baculovirus system and SF9 insect cells. The enzyme carried a C-terminal His tag allowing efficient affinity purification from cell lysates. The recognition of hu-tTG by human sera was checked by using an enzyme linked immunosorbent assay (ELISA). For this, 35 patients with active CD were compared with 144 controls (18 patients with bioptically excluded CD, 89 blood donors, 30 patients with inflammatory bowel disease, and seven patients with cystic fibrosis). RESULTS: The ELISA using hu-tTG showed a sensitivity of 100% and a specificity of 98.6%. Titres of antibodies against hu-tTG (anti-hu-tTG) were positively correlated with EmA titres. All results negative for EmA were also negative for anti-hu-tTG. There were, however, EmA positive results up to a titre of 1 : 80 below the cut-off for anti-hu-tTG. For comparison, antibodies against guinea pig tissue transglutaminase (anti-gp-tTG) were determined in parallel. All patients with anti-hu-tTG below the cut-off were also negative for anti-gp-tTG. However, there were eight patients positive for anti-hu-tTG but negative for anti-gp-tTG. CONCLUSIONS: The new test reaches and even exceeds diagnostic efficiency of EmA for coeliac diagnosis.

Antibodies in the diagnosis of coeliac disease: a biopsy-controlled, international, multicentre study of 376 children with coeliac disease and 695 controls.

Diagnosis of coeliac disease (CD) relies on a combination of clinical, genetic, serological and duodenal morphological findings. The ESPGHAN suggested that biopsy may not be necessary in all cases. New guidelines include omission of biopsy if the concentration of CD-specific antibodies exceeds 10 times the upper limit of normal (10 ULN) and other criteria are met. We analysed the 10 ULN criterion and investigated multiple antibody-assays. Serum was collected from 1071 children with duodenal biopsy (376 CD patients, 695 disease-controls). IgA-antibodies to tissue transglutaminase (IgA-aTTG), IgG-antibodies to deamidated gliadin peptides (IgG-aDGL) and IgA-endomysium antibodies (IgA-EMA) were measured centrally. We considered 3 outcomes for antibody test procedures utilizing IgA-aTTG and/or IgG-aDGL: positive (≥10 ULN, recommend gluten-free diet), negative (<1 ULN, no gluten-free diet) or unclear (perform biopsy). Positive (PPV) and negative (NPV) predictive values were based on clear test results. We required that they and their lower confidence bounds (LCB) be simultaneously very high (LCB >90% and PPV/NPV >95%). These stringent conditions were met for appropriate antibody-procedures over a prevalence range of 9-57%. By combining IgG-aDGL with IgA-aTTG, one could do without assaying total IgA. The PPV of IgG-aDGL was estimated to be extremely high, although more studies are necessary to narrow down the LCB. The proportion of patients requiring a biopsy was <11%. The procedures were either equivalent or even better in children <2 years compared to older children. All 310 of the IgA-aTTG positive children were also IgA-EMA positive. Antibody-assays could render biopsies unnecessary in most children, if experienced paediatric gastroenterologists evaluate the case. This suggestion only applies to the kits used here and should be verified for other available assays. Confirming IgA-aTTG positivity (≥10 ULN) by EMA-testing is unnecessary if performed on the same blood sample. Prospective studies are needed.

Tissue transglutaminase is not a biochemical marker for Alzheimer's disease.

Typical hallmarks of Alzheimer's disease (AD) are pathologic deposits in cortical and subcortical regions consisting of self-aggregated proteins such as amyloid-beta (Aß) or tau. Tissue transglutaminase (tTG) catalyses calcium-dependent cross-linking between proteins (transamidation) resulting in protease-resistant isopeptide bonds. Because of this ability, tTG was suspected to participate in AD pathogenesis. Aß and tau can be cross-linked by tTG in vitro. In AD neocortex, messenger RNA expression of tTG is increased. However, data on transamidation in AD specimens--activity of not only tTG but also other transglutaminases--are contradictory. The aim of our study was to investigate if tTG is involved in AD development and may be useful as biomarker for AD. We studied human brain samples for tTG concentration, tTG localization, and transamidation activity and cerebrospinal fluid (CSF) for tTG content by novel sensitive and highly specific methods. Neither tTG concentration nor transamidation was increased in AD brain homogenates. Immunohistologically, we found no colocalization of tTG in neocortex sections with tau or Aß deposits but with blood vessels. Only in rare cases, tTG was detectable in CSF samples. This could be attributed to liberation from erythrocytes. Our data contradict the view that tTG is a potential biochemical marker for AD.

Similarity of fine specificity of IgA anti-gliadin antibodies between patients with celiac disease and humanized α1KI mice.

Gliadins, and primarily α-gliadins containing several sequences such as aa 31-49, aa 56-88 (33-mer), aa 57-68, and aa 69-82, are critical in the induction of immune response or toxic reaction leading to the development of celiac disease (CLD). The role of IgA anti-gliadin antibodies (IgA AGA) is unknown. To this end, we prepared several humanized monoclonal IgA AGA using transgenic α1KI mice. Employing Pepscan with overlapping decapeptides of α-gliadin we observed a robust similarity between the specificity of humanized mouse monoclonal IgA AGA and IgA AGA from patients with florid CLD. The common immunodominant region included several sequential epitopes localized in the N-terminal part of α-gliadin (QFQGQQQPFPPQQPYPQPQPFP, aa 29-50, and QPFPSQQPYLQL, aa 47-58). Notably, IgA AGA produced by clones 8D12, 15B9, 9D12, and 18E2 had significant reactivity against sequences localized in the 33-mer, LQLQPFPQPQ (aa 56-65) and PQLPYPQPQPFL (aa 69-80). Humanized mouse monoclonal IgA AGA that have a known specificity are suitable as standard in ELISAs to detect serum IgA AGA of CLD patients and for studying the AGA pathogenic role in CLD, especially for analyzing the translocation of complex of specific IgA antibodies and individual gliadin peptides through enterocyte barrier.

Use of likelihood ratios improves clinical interpretation of IgG and IgA anti-DGP antibody testing for celiac disease in adults and children.

OBJECTIVE: To evaluate whether taking into account anti-deamidated gliadin peptide (DGP) antibody concentrations improves clinical interpretation. DESIGN AND METHODS: We calculated likelihood ratios (LR) using data from two previously published studies for assays from EUROIMMUN and INOVA. RESULTS: LRs markedly increased with increasing IgG anti-DGP concentrations. LRs also increased with increasing IgA anti-DGP concentrations, although they were lower than for IgG anti-DGP. CONCLUSIONS: Use of LRs for different test result intervals improves clinical interpretation.