In vitro gliadin antibody production by peripheral blood mononuclear cells from patients with coeliac disease

In vitro spontaneous IgG gliadin antibody production was shown in peripheral blood mononuclear cell (PBMC) cultures from 12/14 patients with active coeliac disease; in most cases no increase and sometimes a marked reduction of the in vitro synthesis was observed after pokeweed mitogen (PWM) addition. Lower levels of gliadin antibodies were also detected in PBMC cultures from 7/12 coeliac patients in remission; in all the cases the synthesis was increased by PWM. In vitro production was confirmed by higher levels in 7-day culture supernatants than in 0-day frozen-thawed cell pellets and by the inhibitory effect of cycloheximide. Spontaneous release of antibodies occurred within the first 3 days of culture, while PWM-induced antibody production reached a plateau after 7-9 days. The analysis of the in vitro gliadin antibody production is a promising technique to assess the regulatory mechanisms involved in the humoral immune response to gliadin.     

Lysosomal damage by gliadin and gliadin peptides; An activity not related to coeliac disease

Rat-liver lysosomes have been used to determine the toxicity of gliadin fractions in relation to coeliac disease. In this study we compared the activity in acid phosphatase release from rat-liver lysosomes by casein, gliadin and by their peptic-tryptic digests. The release of acid phosphatase is not specific for gliadin.     

Amino acid composition of gliadin fractions which may be toxic to individuals with coeliac disease

Fraction 9, prepared by chromatography of a peptic-tryptic-pancreatinic digest of gliadin on S.P. Sephadex C-25, was re-chromatographed on Q.A.E. Sephadex A-25 and subfractions 9-1 and 9-2 further purified on S.P. Sephadex C-25. Sub-fractions 9-1 and 9-2 and the purified sub-fractions 9-1B and 9-2B appeared to be toxic to patients with coeliac disease on the basis of causing a reduction in d-xylose absorption.Amino acid analysis of undigested residues from sub-fractions 9-1B and 9-2B obtained after ‘in vitro’ digestion with remission coeliac mucosa contained mainly glutamine/glutamic acid and proline with some serine, leucine, phenylalanine and glycine.Another fraction (fraction 3) of wheat gliadin prepared by peptic-tryptic digestion and ion-exchange chromatography on S.P. Sephadex, previously shown to produce a skin-reaction in adults with coeliac disease, has been further purified by ion exchange chromatography, isoelectric focusing and gel filtration. The sub-fractions were submitted to amino acid analysis and the results compared with those from the undigested residues above.Isoelectric focusing of fraction 3 of the P.T. digest and its sub-fractions showed the presence of peptides of pI approximately 4.8 and 5.6 with only small amounts of peptides on either side of this region.Mucosal digestion of fractions of peptic-tryptic-pancreatinic gliadin digests ‘in vitro’ appears to be a promising method for the elucidation of the primary structure of that section of the gliadin which may be responsible for the lesion in coeliac disease. The evaluation of higher molecular mass peptic-tryptic digests by intradermal skin tests could also be useful for the preliminary screening of fractions for feeding tests, but this approach seems less likely to indicate the toxic region of the gliadin molecule.     

Amino acid composition of peptides remaining after in vitro digestion of a gliadin sub-fraction with duodenal mucosa from patients with coeliac disease

Sub-fraction 9-2, derived from fraction 9 of a peptic-tryptic-pancreatinic digest of wheat gliadin and previously shown to be toxic to individuals with coeliac disease, was digested in vitro with duodenal mucosa from patients with coeliac disease in remission and with mucosa from normals. Digestion products from coeliac mucosa caused damage to rat liver lysosomes in contrast to digestion products from normal mucosa which had practically no effects on the lysosomes. Ion exchange chromatography of the digestion products, followed by gel permeation chromatography to remove tissue proteins and amino acids allowed the separation of small peptides. Purification of the peptide residues by reversed-phase HPLC on a C18 column resulted in four subfractions, two of which were cytotoxic to rat liver lysosomes. Amino acid analysis of these latter peptide fractions showed that they were both rich in glutamine/glutamic acid, proline, serine and tyrosine. The results support the hypothesis of defective mucosal digestion as being the aetiology of coeliac disease and suggest that the causative agents are small peptides of apparent Mr congruent to 700 Da, with amino acid analysis corresponding to (Glx)3, (Pro)2, Ser and (Glx)3, (Pro)2, Tyr. These hexapeptides are likely to be H-Pro-Ser-Glx-Glx-Glx-Pro-OH and H-Glx-Glx-Pro-Tyr-Pro-Glx-OH.     

The effect of gliadin peptides on rat-liver lysosomes in relation to the pathogenesis of coeliac disease

Several fractions of a peptic-tryptic-pancreatic digest of wheat gliadin were evaluated for their effect on rat liver lysosomes as part of a study of the aetiology and pathogenesis of coeliac disease.On incubation with lysosome suspensions, Fraction 9 caused a greater degree of disruption of the lysosomes than did any other primary fraction of the digest. Peptides remaining after incubation of Fraction 9 with homogenates of duodenal mucosa from patients with coeliac disease in remission still had an appreciable effect on lysosomal membranes, in contrast to residues obtained from digestion with homogenates from control individuals.The results lend further support to the hypothesis of an intestinal peptidase deficiency in coeliac disease and point to peptides present in Sub-fraction 2 of Fraction 9 as being the most active to lysosomes.Since it has been shown that Fraction 9 and Sub-fraction 2 are incompletely digested by remission coeliac mucosa, the disruption of lysosomal bodies may possibly be a factor in the pathogenesis of coeliac disease.     

The toxic fraction of gliadin digests in coeliac disease. Isolation by chromatography on Biogel P-10

Improvement in the fractionation of gliadin digests and in the isolation of toxic fractions was achieved using chromatography on Biogel P-10. Fraction V, one of the 11 fractions eluted from a peptic-tryptic digest of crude gliadin extracted from Cappelle wheat, significantly affected coeliac jejunal mucosa in organ culture. BV, gamma V, omega V, the corresponding fractions V from beta-, gamma- and omega-gliadins, displayed similar toxic effects. Fraction VI containing peptides with a lower molecular mass did not show any significant cytotoxic activity and, moreover, inhibited the toxicity of fraction V. Analysis of the toxic fractions V showed that they contained peptides of 7-8,000 molecular mass, rich in proline and glutamine and poor in aromatic amino acids and carbohydrates. Among the various fractions, V, beta V from beta-gliadin appeared the less heterogeneous.     

The prolamin antibody reactivity against hordein polypeptides in sera from patients with coeliac disease

The antibody reactivity against the barley prolamin, hordein, was investigated by an immunoblotting technique, in sera from patients with untreated coeliac disease, patients with other gastrointestinal diseases and healthy controls. No characteristic hordein polypeptide antibody pattern could be connected to coeliac disease, as observed in a similar study using different fractions of the wheat prolamin, gliadin. Gliadin- and hordein-immunoadsorbent column experiments demonstrated that the prolamin reactivity originates from the same population of antibodies. It is speculated that distinct antigenic epitopes characteristic for untreated coeliac disease, might reside within a N-terminal repeat region of gliadin.     

Human jejunal transglutaminase: demonstration of activity, enzyme kinetics and substrate specificity with special relation to gliadin and coeliac disease

By use of a radiometric assay transglutaminase activity was demonstrated for the first time in human jejunal mucosa. The activity is similar to that in other tissues, with a pH optimum of 9.0, an absolute requirement for Ca2+ and an apparent Km for putrescine of 0.15 mmol/l. Assay of jejunal transglutaminase activity with a variety of dietary proteins as acceptors showed high activity with gliadin, comparable with that of the standard substrate, dimethylcasein. Deamidation of the gliadin markedly reduced its acceptor activity. Collagen, ovalbumin, elastin and zein exhibited very low acceptor activities. Increased transglutaminase activity was demonstrated in jejunal biopsies from four patients with untreated coeliac disease compared with 14 control subjects and eight patients with inflammatory bowel disease. Eight patients with coeliac disease in remission, with normal levels of brush border alpha-glucosidase, showed elevated transglutaminase activities compared with those of controls. It is postulated that intestinal transglutaminase activity may be important in gliadin binding to tissues and thus in the pathogenesis of coeliac disease.     

Celiac disease: antibody recognition against native and selectively deamidated gliadin peptides.

BACKGROUND: Selective deamidation of glutamine residues by tissue transglutaminase (tTG) turns gliadin peptides into stronger activators of T cells from celiac disease (CD) patients. We examined the possibility that these modified peptides could be more specific epitopes for circulating antibodies than are native peptides. METHODS: Two native synthetic peptides and their respective modified sequences were used as antigens for ELISA assays: peptide-1, with residues 56-75 of alpha-type gliadin; and peptide-2, with residues 134-153 of gamma-type gliadin. We examined 40 CD patients [31 not being treated with a gluten-free diet (GFD) and 9 being treated with a GFD] and 30 non-CD patients. RESULTS: An enhanced response against deamidated peptides was observed in 4 (IgA) and 22 (IgG) of 31 untreated CD patients for peptide-1 and in 25 (IgA) and 29 (IgG) patients for peptide-2. Higher anti-gliadin antibody and anti-tTG IgA concentrations correlated with increased IgA reactivity to modified peptides. Among the nine treated CD patients, eight also displayed an improved IgG signal for the deamidated sequence. Deamidation of peptides did not increase the reactivity of non-CD sera. CONCLUSIONS: Selective deamidation specifically increases circulating antibody recognition of gliadin peptides in CD patients. This suggests that deamidated gliadin peptides are more specific CD B-cell epitopes than native peptides; this finding may be relevant for designing improved diagnostic tests.     

Endomysium antibodies are superior to gliadin antibodies in screening for coeliac disease in patients presenting supposed functional gastrointestinal symptoms

OBJECTIVE: To study the accuracy of IgA- and IgC-gluten antibodies and endomysium antibodies as screening tools for endoscopy with small bowel biopsy for histologic diagnosing of coeliac disease. DESIGN: Comparing serology with histologic examination--the "gold standard" for diagnosing coeliac disease. SETTINGS: 1. The municipality of Osthammar, Sweden. 2. The catchment area of the University Hospital, Uppsala, Sweden. PATIENTS: 1. A random subsample (50 with dyspepsia, 50 with irritable bowel syndrome and 50 symptomless) of a representative sample from an adult Swedish general population (20-80 years; n = 1260). 2. All patients with a diagnosis of coeliac disease admitted to the University Hospital in Uppsala, Sweden during the course of 10 months. MAIN OUTCOME MEASURES: The accuracy of IgA- and IgG-gluten antibodies and endomysium antibodies. RESULTS: There were no significant correlations between IgA-gluten antibodies and IgG-gluten antibodies, on the one hand, and symptoms or symptom severity, on the other. Using duodenal biopsy results as the gold standard, IgA-gluten antibodies had a low specificity and IgG-gluten antibodies a low sensitivity, whereas endomysium antibodies had an excellent accuracy. CONCLUSION: Endomysium antibodies seem to be the screening test of choice. The load of diagnostic upper endoscopies would be considerably decreased compared to using gluten antibodies.     

Endomysium antibodies are superior to gliadin antibodies in screening for coeliac disease in patients presenting supposed functional gastrointestinal symptoms

Objective. To assess level of cardiovascular risk factors in a non-selected, middle-aged population. To estimate the proportion target for risk intervention according to present guidelines and according to different cut-off levels for two risk algorithms. Design. Population survey, modelling study. Setting. The Norwegian Hordaland Health Study (HUSK) 1997–99. Subjects. A total of 22 289 persons born in 1950–57. Main outcome measures. Own and relatives’ cardiovascular morbidity, antihypertensive and lipid-lowering treatment, smoking, blood pressure, cholesterol. Framingham and Systematic Coronary Risk Evaluation (SCORE) algorithms. The European guidelines on CVD prevention in clinical practice were applied to estimate size of risk groups. Results. Some 9.7% of men and 7.6% of women had CVD, diabetes mellitus, a high level of one specific risk factor, or received lipid-lowering or antihypertensive treatment. Applying a SCORE (60 years) cut-off level at 5% to the rest of the population selected 52.4% of men and 0.8% of women into a primary prevention group, while a cut-off level at 8% included 22.0% and 0.06% respectively. A cut-off level for the Framingham score (60 years) of 20% selected 43.6% of men and 4.7% of women, while a cut-off level of 25% selected 25.6% of men and 1.8% of women. Conclusion. The findings illustrate how choices regarding risk estimation highly affect the size of the target population. Modelling studies are important when preparing guidelines, to address implications for resource allocation and risk of medicalization. The population share to be targeted for primary prevention ought to be estimated, including the impact of various cut-off points for risk algorithms on the size of the risk population.     

Thyroid-related autoantibodies in Tunisian patients with coeliac disease

BACKGROUND: The aim of our study was to evaluate, retrospectively, the frequency of anti-thyroid antibodies (ATA) in coeliac disease (CD) patients. METHODS: ELISA was used to determine the frequency of anti-thyroid stimulating hormone receptor antibodies, thyroperoxidase antibodies and thyroglobulin antibodies in sera of 104 adult patients with CD. Patients were divided into three groups: group I, 56 untreated patients; group II, 21 patients on a strict gluten-free diet (GFD); and group III, 27 patients who did not comply with a GFD. Sera of 189 healthy blood donors served as controls. RESULTS: Out of 104 patients with CD, five (4.8%) had ATA. The frequency of ATA found in the control group (1.6%) was not significantly different from that found in all CD patients. However, the frequency of ATA in CD patients on a GFD was significantly higher than that found in the control group (8.3% vs. 1.6%, p=0.03). The frequency of ATA in groups I, II and III was 1.8%, 9.5% and 7.4%, respectively. CONCLUSIONS: ATA were found in CD patients even on a GFD.     

Diagnostic performance of IgG anti-deamidated gliadin peptide antibody assays is comparable to IgA anti-tTG in celiac disease

BackgroundDetection of IgG antibodies against deamidated gliadin peptides (DGP) is more sensitive and more specific for celiac disease than detection of IgG antibodies against native gliadin. Our aim was to evaluate the technical performance and diagnostic accuracy of four commercial IgG anti-DGP assays.MethodsCommercial IgG anti-DGP assays from Euroimmun, Inova, Phadia and The Binding Site were evaluated and their diagnostic accuracy (sensitivity and specificity) compared to other serologic assays for celiac disease (3 IgA and 2 IgG anti-tTG assays, 1 IgA and 1 IgG anti-gliadin assay, 1 IgA anti-DGP assay). The study population consisted of 86 consecutive CD patients and 741 disease controls.ResultsThe technical performance (linearity, interference and imprecision) of the IgG anti-DGP assays was acceptable. The sensitivity of the IgG anti-DGP assays varied between 76.7% and 86.0% at the cut-off recommended by the manufacturer and between 74.4% and 86.0% at the cut-off that corresponded to a specificity of 98%. The specificity varied between 97.3% and 99.3%. The diagnostic accuracy of the IgG anti-DGP assays was comparable to the diagnostic accuracy of the IgA anti-tTG assays. The sensitivity of the IgG anti-DGP assays was significantly better than sensitivity of the IgG anti-tTG assays (p < 0.05) and the specificity was significantly better than the IgA and IgG anti-gliadin assays (p < 0.05).ConclusionsThe overall performance of the four IgG anti-DGP assays was acceptable and the diagnostic accuracy comparable to the three IgA anti-tTG assays.     

Novel type of proliferating lymphoplasmacytoid cell with a characteristic spotted immunofluorescence pattern

We examined bone marrow from myeloma patients for the presence of cells with the characteristics of the clonogenic cell in the myeloma stem cell assay. We identified a novel type of cell that contained cytoplasmic immunoglobulin of the relevant idiotype located in a cytoplasmic spot. This "spotted" Ig could be located in the rough endoplasmic reticulum. Spotted cells are highly proliferative, as evidenced by the nuclear staining with the antibody Ki67, and were found in the bone marrow from most of the myeloma patients studied. This type of cell was also present in patients with immunocytomas, in some cases of benign monoclonal gammopathy, and in patients in the state of polyclonal hypergammaglobulinemia. IgG subclass distribution of so-called spotted cells and plasma cells, found in a patient with pseudo biclonal gammopathy, indicates that spotted cells are intermediate between B cells and plasma cells. Spotted cells express the B cell-associated antigens HB4 and HB6 but do not express other B cluster of differentiation antigens or plasmacytoid antigens tested.