Anti-gluten Antibodies Research Articles

Combination of Gluten-Digesting Enzymes Improved Symptoms of Non-Celiac Gluten Sensitivity: A Randomized Single-blind, Placebo-controlled Crossover Study

IntroductionRecently, the population of individuals with non-celiac gluten sensitivity (NCGS) who do not have celiac disease but show improved symptoms with a gluten-free diet, has increased. Enzyme replacement therapy using digestive enzymes is expected to improve the symptoms of NCGS and be sustainable, since gluten-related proteins that are indigestible by the digestive system have been considered triggers of NCGS.MethodsWe selected patients with NCGS by screening demographic interviews, as well as performing medical evaluations, anti-gluten antibody tests, and gluten challenge tests. We performed a single-blind and crossover clinical trial with these subjects using a gluten challenge with the enzyme mixture or a placebo. Our designed enzyme mixture contained peptidase, semi alkaline protease, deuterolysin, and cysteine protease derived from Aspergillus oryzae, Aspergillus melleus, Penicillium citrinum, and Carica papaya L., respectively.ResultsAdministration of the enzyme mixture significantly decreased the change in the score of the symptom questionnaire before and after the gluten challenge compared with administration of the placebo in patients with NCGS without adverse events. In particular, the changes in the score of the gluten-induced incomplete evacuation feeling and headaches were significantly improved. The serum levels of interleukin (IL)-8, tumor necrosis factor (TNF)-α, andregulated on activation, normal T cell expressed and secreted (RANTES) in subjects were not significantly changed by gluten, as expected from previous studies, and the enzyme mixture did not affect these inflammatory markers.ConclusionIn this human clinical study, we demonstrated the efficacy of the enzyme mixture derived from microorganisms and papaya in improving the symptoms of NCGS.

Characterization of Antibodies for Grain-Specific Gluten Detection.

Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation.

Evaluation of qualitative and quantitative immunoassays to detect barley contamination in gluten-free beer with confirmation using LC/MS/MS.

To meet the need for the detection and quantitation of barley gluten in beer, qualitative screening and quantitative immunoassays based on the monoclonal antigluten antibody 401/21 (Skerritt) were validated in a single laboratory. Sample replicates were tested at each stage of beer production using multiple yeast strains and methods of end-stage protein removal. Quantitation was performed using barley-specific standards based on barley flour extracts. Immunoassay results were confirmed using LC/MS/MS for barley-specific peptides. The LOD for the qualitative screening test was 5 mg/L barley gluten. Recovery for the barley-spiked worts ranged from 81 to 128% in the quantitative ELISA assay; the LOD was <1 mg/L, and the LOQ was 5 mg/L. Both screening and confirmation methods were found to be fit for the purposes of detection of low levels of barley gluten in beer.

Intraepithelial lymphocytes in celiac disease immunopathology

Celiac disease is a T cell-mediated immune disorder induced by dietary gluten that is characterized by the development of an inflammatory anti-gluten CD4 T cell response, anti-gluten antibodies, and autoantibodies against tissue transglutaminase 2 and the activation of intraepithelial lymphocytes (IELs) leading to the destruction of the intestinal epithelium. Intraepithelial lymphocytes represent a heterogeneous population of T cells composed mainly of cytotoxic CD8 T cells residing within the epithelial layer, whose main role is to maintain the integrity of the epithelium by eliminating infected cells and promoting epithelial repair. Dysregulated activation of IELs is a hallmark of CD and is critically involved in epithelial cell destruction and the subsequent development of villous atrophy. In this review, we compare and contrast the phenotype and function of human and mouse small intestinal IELs under physiological conditions. Furthermore, we discuss how conditions of epithelial distress associated with overexpression of IL-15 and non-classical MHC class I molecules induce cytotoxic IELs to become licensed killer cells that upregulate activating NKG2D and CD94/NKG2C natural killer receptors, acquiring lymphokine killer activity. Pathways leading to dysregulated IEL activation could eventually be targeted to prevent villous atrophy and treat patients who respond poorly to gluten-free diet.

The Incidence and Risk of Celiac Disease in a Healthy US Adult Population

Celiac disease (CD) is an increasingly common disease that may affect as many as 1% of the North American population. Recent population-based data suggest a substantial increase in the prevalence of CD over the last several decades. Several factors are hypothesized as possible disease triggers including intercurrent illnesses, such as gastroenteritis, surgeries, and trauma. We used the active duty US military, a unique healthy worker population with essentially complete medical diagnostic coding, as an opportunity to describe trends in CD and deployment-related risk factors. Using electronic medical encounter data (1999-2008) on active duty US military (over 13.7 million person-years), a matched, nested case-control study describing the epidemiology and risk determinants of CD (based on ≥2 ICD-9 medical encounters) was conducted. Incidence and duration of CD-related medical care were estimated, and conditional logistic regression was utilized to evaluate CD risk following infectious gastroenteritis (IGE) occurring within 3 years before CD diagnosis while controlling for other risk factors. A total of 455 incident cases of CD were identified and age, gender, and time matched to 1,820 controls. The incidence of CD increased five-fold from 1.3 per 100,000 in 1999 to 6.5 per 100,000 in 2008, with the highest rates of increase among those over 34 years of age (average annual increase of 0.8 cases per 100,000). A total of 172 IGE episodes, predominately of "viral etiology" (60.5%), were documented. In multivariate models, a significant association between IGE and CD was found (Odds ratio (OR): 2.06, 95% confidence interval (CI) 1.43, 2.97). Risk generally increased with temporal proximity to, and non-viral etiology of, exposure. Other notable risk factors for CD in multivariate models were Caucasian race (OR: 3.1, P<0.001), non-Army service (OR: 1.5, P=0.001), and greater than a high-school education (OR: 1.3, P=0.05). Incidence of CD diagnosis in the US military is increasing, particularly among those in the fourth and fifth decades of life and appears higher than other population-based estimates. An association between antecedent IGE and risk of CD was noted, but the potential for exposure misclassification cannot be ruled out and further study is needed to link pathogen-specific exposure to incident CD anti-gluten antibody development or symptom onset.

Rational Design of Combination Enzyme Therapy for Celiac Sprue

Celiac sprue (also known as celiac disease) is an inheritable, gluten-induced enteropathy of the upper small intestine with an estimated prevalence of 0.5%-1% in most parts of the world. The ubiquitous nature of food gluten, coupled with inadequate labeling regulations in most countries, constantly poses a threat of disease exacerbation and relapse for patients. Here, we demonstrate that a two-enzyme cocktail comprised of a glutamine-specific cysteine protease (EP-B2) that functions under gastric conditions and a PEP, which acts in concert with pancreatic proteases under duodenal conditions, is a particularly potent candidate for celiac sprue therapy. At a gluten:EP-B2:PEP weight ratio of 75:3:1, grocery store gluten is fully detoxified within 10 min of simulated duodenal conditions, as judged by chromatographic analysis, biopsy-derived T cell proliferation assays, and a commercial antigluten antibody test.

The Prevalence of Celiac Disease May Be Higher Than We Believe

Source: Maki M, Mustalahti K, Kokkonen J, et al. Prevalence of celiac disease among children in Finland. N Engl J Med. 2003;348:2517–2524.Using the most sensitive and specific serological tests available (detecting serum endomysial and tissue transglutaminase antibodies along with human lymphocyte antigen [HLA] typing), these Finnish investigators tested the hypothesis that assays for serum autoantibodies can be used to detect untreated celiac disease (CD), and that positive findings correlate with specific HLA haplotypes. Serum samples that were collected and frozen in 1994 for a study of risk factors for type I diabetes were re-studied. Samples from 3,654 asymptomatic students (age range, 7 to 16 years when the samples were taken in 1994) were screened in 2001 for endomysial and tissue transglutaminase antibodies. Of those 56 (1.5%) had positive serological tests. Of those, who had positive tests, 10 (18%) had become symptomatic and were diagnosed before the screening in 2001. Of the 36 asymptomatic children who underwent further testing, 27 (75%) had evidence of CD on biopsy resulting in a biopsy-proven disease prevalence of 1 in 99 school children. All but 2 of the antibody-positive subjects had either the HLA-DQ8 or the HLA-DQ2 haplotype. It was suggested that the 25% of children without evidence of disease but who have positive serologic tests will eventually show evidence of disease. The prevalence of the combination of antibody positivity and an HLA haplotype associated with CD was 1 in 67.The authors conclude that the presence of serum tissue transglutaminase and endomysial autoantibodies is predictive of small-bowel abnormalities indicative of CD. There was a good correlation between autoantibody positivity and specific HLA haplotypes.Another paper and editorial in the same issue provides a brief but very enlightening discussion of the basic pathophysiology of CD and its genetic associations. 1,2 CD represents a unique model of autoimmune disease. The enzyme tissue transglutaminase, which is expressed in the subepithelial layer of the intestine, has been identified as the target of the endomysial autoantibody. This provides a potential explanation for how and why tissue damage occurs from gluten. There is a close genetic association with HLA-DQ2 in 90–95% of cases and to HLA DQ8 in 5–10% of cases; an identifiable triggering environmental stimulus (gluten); and a highly specific humoral autoimmune response (autoantibodies against tissue transglutaminase)—all of which provide the rationale for the treatment of the disease, which is complete avoidance of gluten.2 In view of increasing evidence that there are a significant number of undiagnosed cases, the editorial raises the issue of screening for this disease and concludes that “the best epidemiologic approach to the diagnosis of CD seems to be a systematic process of case-finding in which patients with symptoms or conditions, both typical and atypical, known to be associated with the disease are targeted.”The questions that most pediatricians want answered are: who should be tested? When, and how? Anti-gluten antibodies are useless and should not be ordered. They have been supplanted by the anti-endomysial IgG and IgA antibodies and IgA antibodies to tissue transglutaminase. (A total serum IgA should accompany these tests since those that lack IgA antibodies would, even if they had CD, lack both IgA endomysial and tissue transglutaminase IgA antibodies.) The use of HLA testing seems best reserved for confusing or difficult cases. Small bowel biopsy—at least once—remains a part of the diagnostic work-up for now, although it has been suggested that it may not be necessary for all cases.3 A high index of suspicion should warrant close continued observation and, if concerns persist, serological testing.Alert your labs to get spruced up for more serologic testing for celiac disease.

Pancreatitis-associated protein in patients with celiac disease: serum levels and immunocytochemical localization in small intestine.

Since PAP is a stress protein expressed in human pancreas during pancreatitis but also constitutively synthesized in the small intestine, we looked whether its expression would be altered in patients with celiac disease. Serum PAP concentrations were determined consecutively in 54 patients with celiac disease on a free diet (group A), in 47 patients with celiac disease on a gluten-free diet (group B), in 22 patients with other intestinal pathologies but with normal intestinal mucosa (group C), in 14 patients with retarded growth, no gastrointestinal disease and normal intestinal mucosa (group D), and in 17 controls (group E). Serum PAP levels (ng/ml) were significantly higher in group A (127.3 +/- 56.8) than in the other groups (B: 47.2 +/- 20.5; C: 51.5 +/- 32.2; D: 47 +/- 22.8; E: 27.6 +/- 9.0), which were not different from each other. In group A, a positive correlation was observed between serum PAP values and antigluten antibody levels (vs. AGA IgG r = 0.58, p < 0.001; vs. AGA IgA r = 0.66, p < 0.001). Furthermore, 12 patients from group A were evaluated after 10-12 months of gluten-free diet and in all of them PAP serum concentration had decreased (mean +/- SE before the diet 122.5 +/- 36.4, after the diet 48.7 +/- 13.7, p < 0.0001). In addition, we performed an immunocytochemical study to localize PAP in the intestinal mucosa of patients from all groups except E. PAP was localized to the Paneth cells and to some globet cells, in patients with mucosal atrophy as well as in those with normal mucosa with no obvious quantitative difference. We concluded that in patients with celiac disease the active phase of the disease was accompanied by an increased serum concentration of PAP. Further studies are necessary to understand the mechanism leading to PAP elevation in the serum of patients with celiac disease.

Anti-alpha-gliadin antibodies are not predictive of celiac disease in juvenile chronic arthritis.

Some authors have recently reported an increased level of antigluten antibodies in rheumatoid arthritis, both in the adult and juvenile form. The real meaning of these antibodies is still unclear. We ascertained the levels of antigluten antibodies in a group of children with juvenile chronic arthritis to determine if these antibodies were linked with celiac disease and/or to increased intestinal permeability. In 18 of 53 patients (33.9%), the levels of antigluten antibodies (IgA or IgG) were higher than normal. No correlation was found between the increase in antigluten antibodies and the positive lactulose/mannitol test, used for determining increased intestinal permeability. In all eight patients undergoing intestinal biopsy due to abnormal levels of antigluten antibodies (IgA class), intestinal mucosa was normal. In conclusion, our study shows that in patients with juvenile chronic arthritis, immunological response to gluten is neither related to celiac disease nor to increased intestinal permeability.

The Controversy of the Use of Anti‐gluten Antibody (AGA) as a Diagnostic Tool in Celiac Disease

The Controversy of the Use of Anti‐gluten Antibody (AGA) as a Diagnostic Tool in Celiac Disease

The Controversy of the Use of Anti-gluten Antibody (AGA) as a Diagnostic Tool in Celiac Disease

The Controversy of the Use of Anti-gluten Antibody (AGA) as a Diagnostic Tool in Celiac Disease

Anti-gluten antibody: An additional immunologic abnormality in type I diabetes: [formula omitted], Z. Shmuel, I. Colins, D. Golan, Y. Kant R, S. Polack, A. Etzioni, P. Vardi.Diabetes Unit, Rambam Medical Center, Haifa, Israel

Anti-gluten antibody: An additional immunologic abnormality in type I diabetes: [formula omitted], Z. Shmuel, I. Colins, D. Golan, Y. Kant R, S. Polack, A. Etzioni, P. Vardi.Diabetes Unit, Rambam Medical Center, Haifa, Israel

Low gluten diet in the treatment of adult coeliac disease: effect on jejunal morphology and serum anti-gluten antibodies.

Treatment of patients with coeliac disease with a low gluten containing diet (LGD) remains controversial. We have studied jejunal morphology and antigluten (AG) antibody titres by ELISA in patients on a LGD of 2.5-5 g/day for three to 14 months (median six months) and compared results with patients on a strict gluten free diet (GFD) for six to 27 months (median 13 months). We found no significant difference in villous height or crypt depth (eight LGD v 10 GFD patients) or serum AG-IgA, -IgG, and IgM titres (13 LGD v 12 GFD patients). there was however, a significant increase (p less than 0.05) in intra-epithelial lymphocytes in those patients on a LGD. We conclude that adult coeliac patients can tolerate a LGD without gross morphological change and without initiating significant AG antibody responses.

Studies on the significance of the R1 anti-reticulin antibody associated with gluten sensitivity.

The R1 type anti-reticulin antibody (ARA) is closely associated with gluten-sensitive enteropathy. It disappears from the circulation within a few weeks of starting on a gluten-free diet and often reappears following gluten challenge. It is not clear how gluten ingestion leads to the production of the ARA. We have investigated four possibilities. (1) The ARA is simply a food antibody generated against meats in the diet. (2) The ARA is an anti-gluten antibody which cross-reacts with reticulin. (3) Gluten binds to gut reticulin in vivo rendering reticulin autoimmunogenic. (4) Immune complexes of gluten and anti-gluten antibody bind to reticulin (by virtue of the affinity that gluten has for reticulin) to give the appearance of an ARA in immunofluorescence tests. Our results do not support any of these possible explanations, and the significance of the ARA remains obscure.

Some characteristics of immunofluorescence tests for antibodies against gluten, using wheat grain sections or gliadin coated sepharose beads.

Antibodies against gluten and gliadin were determined by the indirect immunofluorescence technique, using frozen wheat grain sections or gliadin-coated Sepharose beads. The methodological characteristics and diagnostic usefulness of the two techniques were evaluated. The reproducibility of both was improved by introducing a fluorometric reading-off procedure. Antibody quantitations were preferably performed as end-point titrations. The two techniques had different dose-response relationships. The grain section technique was more discriminative for small variations in antibody concentration than the bead technique. The latter was, however, more reproducible. Besides antibodies against gliadin, a number of patients with gluten enteropathy had antibodies against the main septa of wheat grains. Fluorescence intensity was preferably expressed in multiples of background intensity. A reaction was visually perceived when the fluorometrically-measured fluorescence intensity reached 2.5 times the background intensity. Using this value as the limit for positive reactions, antibodies were demonstrated in 81% of the cases with verified gluten intolerance, compared with 28% in cases with other intestinal allergies and 8% in normals. The diagnostic specificity of both the grain section and Sepharose bead technique for gluten enteropathy increased with increasing antibody concentration and was apparently 100% when the fluorescence intensity produced by a 1/10 serum dilution reached a value 7 to 8 times that of the background. Antibodies against reticulin were demonstrated in 1/4 of the cases having anti-gluten antibodies but in none of those with non-gluten-induced gastro-intestinal symptoms. Antibodies of the IgG class against cow's milk were demonstrated more often and in higher titre in cases with anti-gluten antibodies than in those without them.

Gluten-sensitive enteropathy. Inhibition by cortisol of the effect of gluten protein in vitro.

Cortisol partially prevents the harmful effect of gluten on the jejunal mucosa of patients with gluten-sensitive enteropathy. To investigate further the pathogenesis of this disorder, we analyzed the effect of cortisol in cultures of jejunal specimens obtained by biopsy. Cultures were done with and without gluten or cortisol. Morphology and alkaline phosphatase activity were assessed before and after 24 hours. Biopsies from untreated patients cultured with gluten showed low enzyme values and cuboidal epithelial cells before and after culture. Biopsies cultured in a gluten-free medium showed a threefold increase in enzyme values (P less than 0.01) and morphologic improvement with change to columnar epithelial cells. Cultures with gluten plus cortisol showed rises in alkaline phosphatase and morphologic improvement indistinguishable from cultures without gluten. Cortisol and gluten had no effect on cultures from appropriate controls. Cortisol thus prevents the harmful effects of gluten on biopsies from patients with gluten-sensitive enteropathy in vitro.

The pathogenesis of gluten-sensitive enteropathy.

Gluten-sensitive enteropathy is characterized by flattening of intestinal villi and malabsorption caused by the toxic effect of gluten, a wheat protein. Gluten activates an endogenous mechanism of toxicity that may be the local mucosal immune system: local mucosal immunoglobulin and antigluten antibody production occur soon after gluten ingestion. Approximately 80% of patients with this disease possess HL-A8, a second segregant series antigen. This association also occurs in dermatitis herpetiformis, a disease with vesicular skin lesions and gluten-sensitive flattening of intestinal villi. The association suggests that the fundamental abnormality in enteropathy is a binding reaction involving gluten protein and a binding site on a cell surface, determined in part by the histocompatibility gene; this reaction then results in a local mucosal immune response to gluten. Alternatively, the fundamental abnormality may be the presence of an abnormal immune-response gene linked to the HL-A8 gene or acting in concert with it; this immune-response gene results in local mucosal production of antigluten antibody.

Predominance of histocompatibility antigen HL-A8 in patients with gluten-sensitive enteropathy.

HL-A phenotypes were determined in 24 unrelated patients with gluten-sensitive enteropathy (GSE) using a lymphocyte microcytotoxicity test. 21 of the 24 patients had HL-A8 in the second segregant series, a frequency of 0.875. In contrast, the HL-A8 frequency in 200 normal individuals was 0.215 (difference significant at P < 0.002), and in 6 patients with villous atrophy due to tropical sprue or hypogammaglobulinemia the HL-A8 frequency was 0.17 (difference from normal not significant). The HL-A types in the families of three HL-A8 positive patients with GSE indicated that the HL-A8 antigen was inherited as an autosomal dominant. Frequencies of the other HL-A antigens in the GSE group did not differ significantly from that of the normal group. These findings are compatible with the hypothesis that GSE is due to the presence of an abnormal "immune response (Ir) gene," leading to the production of pathogenic antigluten antibody or, alternatively, to the presence of a particular membrane configuration leading to the binding of gluten to epithelial cells with subsequent tissue damage.