Allergy to deamidated gluten in patients tolerant to wheat: specific epitopes linked to deamidation.

BACKGROUND: Gluten proteins can be modified by deamidation to enhance their solubility and technological applications. However, severe allergic reactions have been reported after the consumption of food products containing deamidated gluten (DG) in subjects tolerant to wheat. This work aimed to characterize allergen profiles for these patients in comparison with those of patients allergic to wheat and to identify IgE-binding epitopes. METHODS: Sera were obtained from 15 patients allergic to DG and from nine patients allergic to wheat proteins (WP). IgE-binding profiles were characterized both in ELISA and in a humanized rat basophilic leukaemia (RBL) cell model. Epitopes were mapped on γ- and ω2-gliadin sequences by Pepscan, and effect of glutamine/glutamic acid substitutions was studied. RESULTS: Compared to the heterogeneous pattern of allergens detected by IgE from patients allergic to WP, responses of patients allergic to DG were homogeneous. In ELISA, all the sera displayed IgE binding to deamidated γ- and ω2-gliadins and deamidated total gliadins, frequently with high concentrations. These modified proteins induced RBL degranulation with most of the sera from DG-allergic patients. A consensus epitope was found on native γ- and ω2-gliadins (QPQQPFPQ); it was repeated several times in their sequences. The substitution of two or three glutamines of this epitope into glutamic acid at positions Q3 or Q4 and Q8 (QPEEPFPE) increased its recognition the best. CONCLUSION: Allergy to DG is a separate entity from wheat allergy. It can be evidenced by strong IgE binding to deamidated gliadins or peptides of the type QPEEPFPE.

Immunoglobulin-E-binding epitopes of wheat allergens in patients with food allergy to wheat and in mice experimentally sensitized to wheat proteins.

BACKGROUND: At present, B cell epitopes involved in food allergy to wheat are known only for a few allergens and a few categories of patients. OBJECTIVE: To characterize the epitopes of different wheat kernel allergens: α-, γ, ω2, and ω5-gliadin, a low-molecular-weight (LMW) glutenin subunit, and a lipid transfer protein (LTP1) recognized by allergic patients and by sensitized mice and provide further understanding of the role of structure in determining allergic response. METHODS: Sera were obtained from 39 patients suffering from food allergy to wheat. BALB/c mice were sensitized to gliadins or LTP1 by intraperitoneal immunizations. Continuous epitopes bound by IgE were delineated by the Pepscan technique. The response to reduced, alkylated LTP1 was compared with that of the native form to evaluate the importance of protein folding on IgE reactivity. RESULTS: Few continuous epitopes of LTP1 reacted with IgE from allergic patients and mice, but one of them was common to several patients and sensitized mice. The unfolded protein was not recognized by either patient or mouse IgE, emphasizing the major role of LTP1 folding and discontinuous epitopes in IgE-binding. In contrast, many continuous epitopes were detected by patient and mouse IgE especially for an ω5-gliadin, which is an unstructured protein, and to a lesser extent, for the other gliadins and a LMW-glutenin subunit. CONCLUSION AND CLINICAL RELEVANCE: The conformation of LTP1 appeared to have a strong impact on the type of IgE-binding epitopes elicited by this protein in both man and mouse. The responses in mice sensitized to gliadins or LTP1 were sufficiently comparable with the human response in terms of IgE-binding epitopes to provide support for the use of the mouse model in further investigations.

Calreticulin is a B cell molecular target in some gastrointestinal malignancies.

Calreticulin, upon translocation to the cell surface, plays a critical role in the recognition of tumour cells and in experimentally induced cellular anti-tumour immunity. However, less is known about anti-calreticulin antibodies and their role in malignancies. Using enzyme-linked immunosorbent assay (ELISA), we found immunoglobulin (Ig)A and/or IgG anti-calreticulin antibodies in sera of approximately 63% of patients with hepatocellular carcinoma (HCC), 57% of patients with colorectal adenocarcinoma (CRA) and 47% of patients with pancreatic adenocarcinoma (PACA), while healthy controls, patients with viral hepatitis C and with chronic pancreatitis reached only 2%, 20% and 31% seropositivity, respectively. We found significantly elevated mean levels of IgA anti-calreticulin antibodies (P < 0.001) in patients with HCC (78.7 +/- 52.3 AU, mean +/- standard deviation), PACA (66.5 +/- 30.9 AU) and CRA (61.8 +/- 25.8 AU) when compared to healthy controls (41.4 +/- 19.2 AU). Significantly elevated mean levels of IgG anti-calreticulin antibodies (P < 0.001) were detected in patients with HCC (121.9 +/- 94.2 AU), gall bladder adenocarcinoma (118.4 +/- 80.0 AU) and PACA (88.7 +/- 55.6 AU) when compared to healthy controls (56.7 +/- 22.9 AU). Pepscan analysis revealed a large number of antigenic epitopes of calreticulin recognized by both IgA and IgG antibodies of patients with HCC and PACA, indicating robust systemic immune response. Moreover, significantly elevated levels of antibodies against peptide KGEWKPRQIDNP (P < 0.001) in these patients, tested by ELISA, confirmed the distinct character of antibody reactivity against calreticulin. The high occurrence and specificity of serum anti-calreticulin autoantibodies in the majority of patients with some gastrointestinal malignancies provide the evidence for their possible clinical relevance.

[Recent aspects of antibody determination for the diagnosis of coeliac disease]. / Neue Aspekte der Antikörperbestimmung zur Zöliakiediagnostik.

Antibody assays play an important role in the diagnosis of coeliac disease (coeliac sprue, gluten-sensitive enteropathy), a condition characterized by immunological intolerance to gluten from wheat and from proteins of related cereals in genetically predisposed persons. Enhanced concentrations of IgA-antibodies against tissue transglutaminase or endomysium under gluten-containing normal diet represent an important indication for a biopsy from the small intestine. Demonstration of typical changes in the mucose of the small intestine is still required for the definitive diagnosis of coeliac disease. Recently highly specific antibodies against deamidated gliadin peptides were detected in serum. These antibodies further improve the reliability of serologic diagnosis. The new assays for IgG-antibodies against deamidated gliadin peptides have a very high diagnostic accuracy and are comparable to IgA tissue transglutaminase antibodies. Further investigations have to show whether IgG-antibodies against deamidated gliadin peptides are a reliable disease marker also in case of IgA deficiency. Prospective studies are needed to show whether antibody assays could replace biopsy in the diagnosis of coeliac disease in a substantial number of patients.

Anti-calreticulin immunoglobulin A (IgA) antibodies in refractory coeliac disease.

Refractory coeliac disease (RCD) is a very rare and dangerous form of CD, in which gluten-free diet loses its therapeutic effect and the damage of intestinal mucosa persists. Because of the adherence to the diet, serological markers of CD [immunoglobulin A (IgA) antibodies against gliadin, tissue transglutaminase (tTG) and endomysium] are often missing in RCD patients. We found substantially elevated levels of IgA anti-calreticulin (CRT) antibodies in the sera of almost all RCD patients tested. These sera were negative for IgA antibodies to gliadin and tTG and only some of them showed IgA antibodies to enterocytes. Analysis of patients' IgA reactivity to CRT fragments (quarters and halves) by Western blotting revealed differences in the specificity of IgA antibodies between RCD and CD patients. We therefore used the Pepscan technique with synthetic overlapping decapeptides of CRT to characterize antigenic epitopes recognized by serum IgA antibodies of RCD patients. Employing this method we demonstrated several dominant antigenic epitopes recognized by IgA antibodies of RCD patients on the CRT molecule. Epitope GVTKAAEKQMKD was recognized predominantly by serum IgA of RCD patients. Our results suggest that testing for serum IgA antibodies against CRT and its selected peptide could be a very useful tool in RCD differential diagnosis.

Identification of IgE-binding epitopes on gliadins for patients with food allergy to wheat.

BACKGROUND: Food allergy to wheat induces different symptoms as atopic eczema/dermatitis syndrome (AEDS), urticaria and more severe reactions as wheat-dependent exercise-induced anaphylaxis (WDEIA). Different gliadin classes are involved in this allergy but IgE-binding epitopes are known only on omega5-gliadins and for WDEIA cases. OBJECTIVES: The aim of the study was to identify IgE-binding epitopes on several gliadin classes and for several patients with different symptoms and ages. METHODS: Eleven sera were analysed by pepscan with overlapping synthetic peptides. RESULTS: Sera from five patients with anaphylaxis, urticaria or WDEIA, displayed strong IgE-binding to sequential epitopes of the repetitive domains of alphabeta, gamma, omega2 or omega5-gliadins with two immunodominant epitopes on omega5-gliadin and a consensus motif of the type QQX1PX2QQ (X1 being L, F, S or I and X2 Q, E or G). One patient allergic to deamidated wheat proteins also had IgE to a repetitive peptide of gamma and omega2-gliadins of the type QPQQPFP. Sera from four patients with AEDS detected no linear epitopes on gliadins, despite the fact that they contained specific IgE to alpha, beta, gamma or omega-gliadins. One child with AEDS recognized cysteine-containing sequences in the nonrepetitive domains of alphabeta and gamma-gliadins. CONCLUSION: B epitopes in wheat allergy were different from B epitopes of coeliac disease. Differences exist in IgE-binding epitopes between patients with food allergy to wheat. IgE from those suffering from WDEIA, anaphylaxis and urticaria detected sequential epitopes in the repetitive domain of gliadins whereas IgE from AEDS patients probably recognized conformational epitopes.

[Long-term study of patients with coeliac disease in childhood and adolescence: latent and transient coeliac disease]. / Verlaufsbeobachtung von Zöliakiepatienten im Kindes- und jungen Erwachsenenalter: latente oder transiente Zöliakie?

UNLABELLED: Coeliac disease (CD) is known as a lifelong condition of gluten intolerance. In case of some patients, the time after which gluten exposition leads to damage of the small intestinal mucosa may be very long. In addition to the florid form of CD, there are also silent (atypical mono- and oligosymptomatic with typical damage of the small intestinal mucosa) and latent forms (often asymptomatic without clear mucosal changes, antibody titres often normal). Occasionally the development of gluten tolerance is postulated (transient CD). We investigated 47 subjects diagnosed in childhood definitely as CD patients. In the age of 16.3 +/- 4.8 years, the patients started a gluten containing diet. After 6 to 9 months of gluten containing diet a small intestinal biopsy was performed in all patients. Surprisingly we found 11 patients with normal small intestinal mucosa (group 1). The other 36 patients showed a flattened mucosa as expected (group 2). The further development of group 1 was followed. In 9 patients a further biopsy was performed after more than 2 (at maximum after 8.1) years of gluten containing diet. In all patients the morphology of the mucosa was normal. In 7 of 11 cases normal numbers of intraepithelial lymphocytes were counted. Only in two patients raised titres of gliadin and endomysium antibodies were found after 4.5 and 15 years of gluten containing diet. In the other 9 patients no increase in antibody titers was found up to 10.3 years. However, in group 1, mucosal lactase activity was decreased as was also the case for group 2. CONCLUSION: A high number of adolescent coeliac patients does not respond or responds only minimally to reintroduction of gluten into the diet over a period longer than two years. These patients should regularly be further controlled serologically.

Antibody response to dietary and autoantigens in G alpha i2-deficient mice.

BACKGROUND: Mice with a targeted mutation in the G protein subunit G alpha i2 gene develop a colonic mucosal inflammation, with a highly activated B-cell response. We wanted to investigate whether this increased B-cell activity was directed against dietary antigens and/or various self tissues. METHODS: The level of antibodies specific for dietary (gliadin, soya and fish meal) antigens was measured by ELISA. Reactivity against self antigens was measured by immunohistochemistry on cryo-sectioned mouse and rat tissue. Sera and intestinal lavages were analysed from G alpha i2-/- mice before and after development of colitis and in age-matched wild type litter mates. RESULTS: Titres of antibodies against dietary antigens were significantly enhanced both in serum and in large intestinal lavages from G alpha i2-/- mice with ongoing colitis but not prior to disease, as compared to wild type mice. The autoreactivity to self tissues was significantly increased in G alpha i2-/- mice both before and after development of colitis as compared to litter mate control animals. Self tissue reactivity was directed not only against epithelial cells of the colon, small intestine and gastric glands, but also against smooth muscle cells, hepatocytes, bile duct cells, renal tubule and collecting tubule cells of the kidney. In analogy to human ulcerative colitis, autoantibodies against epithelial cells, bile duct epithelium and neutrophil granulocytes were found. CONCLUSIONS: Earlier increase in levels of autoantibodies (before onset of colitis) than of food antibodies (after onset of colitis) suggests the latter response to be a secondary phenomenon to e.g. a destroyed barrier function.

A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins.

OBJECTIVES: Antibodies that detect coeliac-toxic prolamins from wheat, barley and rye are important tools for controlling the diet of coeliac disease patients. Recently, a monoclonal antibody R5 that recognizes wheat gliadin, barley hordein and rye secalin equally was described. In this study, the epitope recognized by R5 was investigated. METHODS: Both a phage-displayed heptapeptide library and overlapping peptides spanning the sequence of alpha- and gamma-type gliadins (pepscan) were screened for binding of R5. RESULTS: Both techniques yielded comparable pentapeptide consensus sequences (phage display QXPW/FP; pepscan QQPFP). According to recent observations, this peptide stretch may be of key importance in the pathogenicity of coeliac disease. This sequence occurs repetitively in prolamins (in gamma- and omega-type prolamins more frequently than in alpha-type prolamins) together with several homologous peptide stretches, which are recognized less strongly. CONCLUSIONS: R5 seems to be a good candidate for the specific detection of putative coeliac disease-active sequences in prolamins and thus represents a valuable tool for the quality control of gluten-free food.

Callitrichid nutrition and food sensitivity.

Captive callitrichids are prone to developing intestinal problems. Their captive and natural diets differ enormously, and diet has been suggested to play a major role in wasting marmoset syndrome. Proteins in wheat, soy and milk are included in callitrichid diets of most colonies and have been linked to an immune reaction in Saguinus oedipus and Callithrix jacchus. In the present study of 23 males and females of the two species, wheat protein was tested but soy and milk products were excluded. One group had wheat and the other had rice in their diet. Blood samples and biopsies from the colon were taken. Results showed changes in the colon and an immune reaction to gliadin, a wheat protein related to coeliac disease in humans. A further immune reaction was also observed. Suggestions for further study and exclusion of cereal in the diet of these small, New World primates are discussed.

Analysis and clinical effects of gluten in coeliac disease.

The prolamin working group coordinates research on laboratory gluten analysis in food and on clinical evaluation of patient sensitivity to prolamins. As an observer organization to the Codex Alimentarius Commission, the group summarizes current data on analysis and effects of gluten in coeliac disease. All types of gliadin, the ethanol-soluble fraction of gluten, contain the coeliac-active factor. However, coeliac toxicity and immunogenicity (humoral and cellular) of various prolamins are not identical in coeliac patients. There are no conclusive data on the threshold of gluten sensitivity of coeliac patients. Information as to the long-term risk to coeliac patients exposed to small doses of gliadin is lacking. Therefore, every effort should be made to keep the diet of coeliac patients as gluten-free as possible. The prolamin group is currently evaluating a new enzyme-linked immunosorbent assay (ELISA) protocol for gluten analysis that could serve as a basis for further Codex regulations. The group recommends adherence to a single Codex limit for gluten-free foods. The current limit of 200 ppm gluten is questionable and requires reconsideration based on new information that will be available soon.

Neurological disease-associated autoantibodies against an unknown protein encoded by a RES4-22 homologous gene.

Screening a human small intestinal library with human serum yielded a clone which encoded a protein res4-22 the gene of which was highly homologous to a recently described gene located in the Huntington's disease locus. Autoantibodies against res4-22 (anti-res4-22), mainly of the immunoglobulin (Ig)A type, were detected in patients with neurological disorders at a higher frequency (18.4%) than in healthy blood donors (8.0%). In neurological patients with cerebral ischaemia anti-res4-22 was found significantly more often (47.4%) than in the total group of neurological patients. Anti-res4-22 positive sera showed significantly more frequently myelin staining in cerebellum and nerve sections than anti-res4-22 negative sera. Our findings demonstrate a new species of human autoantibodies against a newly described protein the function of which is still unknown.

Evidence for existence of coeliac disease autoantigens apart from tissue transglutaminase.

BACKGROUND: The pathogenesis of coeliac disease (CD) and of dermatitis herpetiformis (DH) is strongly associated with production of autoantibodies, defined by indirect immunohistology. Recently, tissue transglutaminase (tTG) was identified as a prominent autoantigen. It would be important to investigate if further molecules apart from tTG are involved in autoimmunity. METHODS: Tissue sections of human foetal intestine were used to compare the distribution of tTG with the autoantibody binding patterns of 14 sera samples from patients with CD or DH. Double label experiments were performed using monoclonal as well as polyclonal tTG antibodies (anti-tTG) and patient sera. The staining was investigated by using conventional light and confocal laser scanning microscopy. RESULTS: Most autoantibody binding sites were matched by tTG. Further, the binding of autoantibodies could be inhibited by preincubation with monoclonal anti-tTG. However, in nine serum samples (64%) autoantibody staining suggested a few distinct binding sites apart from tTG. In three sera (21 %) autoantibody binding fibres were detected which definitely did not match monoclonal anti-tTG signals. Distinctly stained fibres were confirmed by applying polyclonal anti-tTG. This indicates the existence of autoantigenic epitopes not related to tTG.

B cell epitopes of gliadin.

A phage displayed dodecapeptide library and synthetic octapeptides spanning the complete sequence of alpha- and gamma-type gliadin and overlapping in six amino acids (pepscan) were screened for binding to human gliadin antibodies (AGA). Phage display experiments led to four sequences recognized with significantly higher frequency by sera with raised IgA-AGA titres than by control sera. All these peptides contained the core sequence PEQ. Pepscan experiments revealed binding of AGA to five prominent regions: (i) QXQPFP (binding to IgG and IgA, X representing P, Q, and L); (ii) IPEQ (IgG) and WQIPEQ (IgA); (iii) FFQP (IgG) and QGXFQP (IgA, X representing F and S); (iv) PQQLPQ (IgG and IgA), all in alpha-type gliadin; and (v) QPQQPF (IgG and IgA) in gamma-type gliadin. In two of the sequences (QPQQPF and QQQPFP), substitution of Q by E resulting in QPEQPF and QEQPFP, respectively, increased significantly binding of AGA from sera of patients with biopsy-proven or suspected coeliac disease (CoD), all positive for endomysium antibodies (EmA). In contrast, binding of sera with high AGA titre from EmA-negative patients (CoD and dermatitis herpetiformis excluded) was not enhanced by this substitution. Thus, AGA directed against these modified epitopes can be regarded as specific for CoD. This is the first study demonstrating that deamidation of gliadin improves reactivity of AGA of CoD patients.

IgA-gliadin antibodies, IgA-containing circulating immune complexes, and IgA glomerular deposits in wasting marmoset syndrome.

BACKGROUND: Marmosets in captivity are highly susceptible to wasting marmoset syndrome (WMS), the aetiology of which is still not fully determined. METHODS: The level of IgA-gliadin antibodies (IgA-AGA), of IgA-containing circulating immune complexes (IgA-CIC), and the degree of glomerular IgA deposits were compared between marmosets suffering from WMS and animals not affected by the disorder. RESULTS: Both IgA-AGA and IgA-CIC were demonstrable in all groups of monkeys investigated. IgA-AGA and IgA-CIC were significantly higher in monkeys with WMS than in non-affected animals. There was a significant correlation between the glomerular IgA-deposition and titre of IgA-AGA. The group of marmosets strongly positive for glomerular IgA deposits comprised significantly more animals suffering from WMS than the group without deposits. In the diet of the animals a considerable amount of gliadin-like cereal proteins was assayed. CONCLUSIONS: There are several parallels between the human disorders (coeliac disease and IgA-nephropathy/Berger's disease) and the changes observed in WMS. It should be further investigated if WMS in marmosets is a suitable animal model for both human diseases.

Antibodies to human recombinant tissue transglutaminase measured by radioligand assay: evidence for high diagnostic sensitivity for celiac disease.

Celiac disease is associated with endomysial antibodies (EmA), which have recently been reported to be directed to tissue transglutaminase (tTG). To demonstrate binding of antibodies to recombinant tTG, human tTG was cloned, expressed by in vitro transcription/translation and used to develop novel radioligand assays for combined and single detection of immunoglobulin A (IgA) and G (IgG)-specific antibodies. IgA and IgG-tTGA were found in 43 (95.6%) of 45 patients with newly-diagnosed celiac disease verified by biopsy. In addition, all 30 sera from patients with gastrointestinal symptoms and positive EmA were positive for IgA-tTGA, and all but one serum (96.7%) had antibodies of the IgG class. Receiver-operating characteristic analysis including 574 sera from healthy controls revealed a specificity of 99.5%. By means of these new assays, we identified all patients with endomysial antibodies and achieved, at equal specificity, an even improved sensitivity (95.6%) as compared to EmA (91.1%) detected by the standard immunofluorescence test. Here, we have provided direct evidence that recombinant tTG is a major target of antibodies in celiac disease. Our data suggest that tTGA measured by radioligand assay have the power to overcome the limitations of the EmA-test. This new strategy may considerably facilitate large-scale screening for silent and latent celiac disease.

Primary culture and transfection of epithelial cells of human small intestine.

BACKGROUND: So far, no techniques are available for primary culture and efficient transfection of human small-intestinal enterocytes, which would provide a valuable tool to investigate intestinal function. METHODS: Human small-intestinal biopsy specimens were treated with collagenase and dispase. Resulting crypt units were cultured for several days. Using the intestinal epithelial cell lines Caco-2 and HT-29, we established optimal conditions for transfection of a control plasmid, which were then applied to primary cultured cells. RESULTS: Cells growing out of crypt units formed monolayer-like sheets and proliferated for several days. Most of the cells could be stained with antibodies against epithelial markers. Among seven different transfection reagents tested, Lipofectamine was the most potent, with transfection efficiencies up to 25% for primary enterocytes. CONCLUSIONS: An easy technique was developed providing viable small-intestinal enterocytes that can be efficiently transfected.

Use of the phage display technique for detection of epitopes recognized by polyclonal rabbit gliadin antibodies.

A random phage heptapeptide library was screened with rabbit antibodies against wheat flour proteins comprising gliadins and a small amount of low molecular weight glutenins (gli/glu). Gli/glu antibodies isolated from the sera selected different consensus sequences (CS). All CS contained tri- to pentapeptide stretches homologous to gli/glu sequences (proposed epitopes). In alpha- and gamma-type gliadins, these sequences are clustered in the N-terminal region recently suspected to be toxic for humans with celiac disease. Peptides with CS were synthesized and checked for reactivity. Only immune and no control rabbit sera reacted with synthetic peptides. One of eight human sera containing gliadin antibodies was reactive as well (4/8 peptides) but control sera were negative. Thus the phage display technique is useful for epitope screening of polyclonal antibodies even in the case of a group of homologous but diverse antigens.