H. Julia Ellis

The toxicity of high molecular weight glutenin subunits of wheat to patients with coeliac disease

Objectives The ability of the gliadin fraction of wheat gluten to exacerbate coeliac disease is well documented. We investigated the possible toxicity of high molecular weight glutenin subunits (HMW-GS) in coeliac disease in vitro using gluten-sensitive T cells, and in vivo with challenge studies in patient volunteers. Methods A mixture of four HMW-GS was chemically separated from wheat flour and checked for purity by HPLC, SDS-PAGE and ELISA. T-cell lines, grown up from small intestinal biopsies from coeliac patients (n=17), were tested for their reactivity to HMW-GS. Adults with coeliac disease and who were on a gluten-free diet (n=3) underwent in-vivo challenges with HMW-GS. Duodenal biopsies, taken prior to the challenge and at intervals up to 6 h afterwards, were assessed for morphology, intra-epithelial lymphocyte count, and interleukin 15 (IL-15) expression, by immunohistochemistry. Results T-cell lines from 11 of 17 patients were stimulated by HMW-GS. There was a significant change in small intestinal morphology 4 h after commencing infusions with HMW-GS in all three subjects. For example villus height to crypt depth ratios were reduced in the three patients from 3.0±0.5 to 1.29±0.2, 2.53±0.7 to 0.81±0.6 and 3.0±0.7 to 1.85±0.3, P<0.0001 in all cases. There was increased expression of IL-15 in the small intestine from 2 h after the HMW-GS challenges. Conclusion Mixed HMW-GS stimulate T-cell lines from some coeliac patients and exacerbate coeliac disease in vivo, inducing expression, within 2 h, of IL-15. This suggests an innate immune response to these proteins.

Electrochemical immunosensor for detection of celiac disease toxic gliadin in foodstuff.

Celiac disease is a gluten-sensitive enteropathy that affects as much as 1% of the population. Patients with celiac disease should maintain a lifelong gluten-free diet, in order to avoid serious complications and consequences. It is essential to have methods of analysis to reliably control the contents of gluten-free foods, and there is a definitive need for an assay that is easy to use, and can be used on site, to facilitate the rapid testing of incoming raw materials or monitoring for gluten contamination, by industries generating gluten-free foods. Here, we report on the development of an electrochemical immunosensor exploiting an antibody raised against the putative immunodominant celiac disease epitope, for the measurement of gliadin content and potential celiac toxicity of a foodstuff. To develop the gliadin immunosensor, we explored the use of two surface chemistries, based on the use of dithiols, 22-(3,5-bis((6-mercaptohexyl)oxy)phenyl)-3,6,9,12,15,18,21-heptaoxadocosanoic acid (1) and 1,2-dithiolane-3-pentanoic acid (thioctic acid) (2), for anchoring of the capture antibody. The different surface chemistries were evaluated in terms of time required for formation of self-assembled monolayers, stability, susceptibility to nonspecific binding, reproducibility, and sensitivity. The thioctic acid self-assembled monolayer took more than 100 h to attain a stable surface and rapidly destabilized following functionalization with capture antibody, while the heptaoxadocosanoic acid surface rapidlyformed (less than 3 h) and was stable for at least 5 days, stored at room temperature, following antibody immobilization. Both surface chemistries gave rise to highly sensitive immunosensors, with detection limits of 5.5 and 11.6 ng/mL being obtained for 1 and 2, respectively, with nonspecific binding of just 2.7% of the specific signal attained. The immunosensors were extremely reproducible, with RSD of 5.2 and 6.75% obtained for 1 and 2 (n = 5, 30 ng/mL), respectively. Finally, the immunosensor was applied to the analysis of commercial gluten-free and gluten-containing raw and processed foodstuffs, and excellent correlation achieved when its performance compared to that of an ELISA.

Celiac disease: Management of persistent symptoms in patients on a gluten-free diet

AIM To investigate all patients referred to our center with non-responsive celiac disease (NRCD), to establish a cause for their continued symptoms. METHODS We assessed all patients referred to our center with non-responsive celiac disease over an 18-mo period. These individuals were investigated to establish the eitiology of their continued symptoms. The patients were first seen in clinic where a thorough history and examination were performed with routine blood work including tissue transglutaminase antibody measurement. They were also referred to a specialist gastroenterology dietician to try to identift any lapses in the diet and sources of hidden gluten ingestion. A repeat small intestinal biopsy was also performed and compared to biopsies from the referring hospital where possible. Colonoscopy, lactulose hydrogen breath testing, pancreolauryl testing and computed tomography scan of the abdomen were undertaken if the symptoms persisted. Their clinical progress was followed over a minimum of 2 years. RESULTS One hundred and twelve consecutive patients were referred with NRCD. Twelve were found not to have celiac disease (CD). Of the remaining 100 patients, 45% were not adequately adhering to a strict gluten-free diet, with 24 (53%) found to be inadvertently ingesting gluten, and 21 (47%) admitting non-compliance. Microscopic colitis was diagnosed in 12% and small bowel bacterial overgrowth in 9%. Refractory CD was diagnosed in 9%. Three of these were diagnosed with intestinal lymphoma. After 2 years, 78 patients remained well, eight had continuing symptoms, and four had died. CONCLUSION In individuals with NRCD, a remediable cause can be found in 90%: with continued gluten ingestion as the leading cause. We propose an algorithm for investigation.

Detection and estimation of the barley prolamin content of beer and malt to assess their suitability for patients with coeliac disease

Malt, lager, brown ale, bitter and stout were assessed for their barley prolamin (hordein) content. Monoclonal antibody immunoperoxidase staining of electrophoretically separated samples of beer revealed the presence of immunoreactive hordein. Polyclonal antibody based enzyme linked immunosorbant assays (ELISAs) revealed 1.6 mg of hordein per gram of malt and 1.5 mg per pint in the beers. Patients with coeliac disease should avoid ingestion of beer and foods that contain malt since they contain quantifiable amounts of hordein that is known to exacerbate the condition.

Targeting of gliadin peptides, CD8, α/β-TCR, and γ/δ-TCR to Golgi complexes and vacuoles within celiac disease enterocytes

Celiac disease (CD) is characterized by autodestruction of enterocytes after exposure of genetically susceptible individuals to dietary gluten. To define the transport pathways of proteins involved in the celiac immune response, we wished to determine the subcellular compartments of the intestinal mucosa where wheat gliadin peptides colocalize with receptors of T lymphocytes, including α/β‐TCR, γ/δ‐TCR, and CD8. Semithin and ultrathin frozen section of jejunal biopsies from CD patients and controls were used to perform immunofluorescence and immunogold labeling as well as in situ hybridization experiments. In patients with active CD, we detected gliadin peptides in vacuoles and Golgi complexes of enterocytes. CD8, α/β‐TCR, and γ/δ‐TCR were found in vacuoles and Golgi complexes within these gliadin‐containing enterocytes in addition to the surface of intraepithelial and mucosal T lymphocytes. In contrast, we observed that the localization of CD4, CD3, T cell‐restricted intracellular antigen (TIA), and leukocyte common antigen (LCA) was restricted to lymphocytes in CD patients. We further detected labeling signals for gliadin peptides, CD8, α/β‐TCR, and γ/δ‐TCR at the basal membrane of enterocytes that were interdigitated by extensions of lymphocytes. In situ hybridization experiments revealed that CD8 and γ/δ‐TCR were not expressed by CD enterocytes. We conclude that CD8, α/β‐TCR, and γ/δ‐TCR are targeted to Golgi complexes and vacuoles of small intestinal enterocytes in active CD. The observed process may be involved in the pathogenesis of CD enterocytes. We propose a mechanism for the uptake of CD8, α/β‐TCR, and γ/δ‐TCR by the basolateral membrane of small intestinal enterocytes.— Zimmer, K.‐P., Naim, H., Weber, P., Ellis, H. J., Ciclitira. P. J. Targeting of gliadin peptides, CD8, α/β‐ TCR, and γ/δ‐TCR to Golgi complexes and vacuoles within celiac disease enterocytes. FASEB J: 12, 1349– 1357 (1998)

Gastrointestinal Effects of Eating Quinoa ( Chenopodium quinoa Willd.) in Celiac Patients

OBJECTIVES:Celiac disease is an enteropathy triggered by dietary gluten found in wheat, rye, and barley. Treatment involves a strict gluten-free diet (GFD). Quinoa is a highly nutritive plant from the Andes that has been recommended as part of a GFD. However, in-vitro data suggested that quinoa prolamins can stimulate innate and adaptive immune responses in celiac patients. Therefore, we aimed to evaluate the in-vivo effects of eating quinoa in adult celiac patients.METHODS:Nineteen treated celiac patients consumed 50 g of quinoa every day for 6 weeks as part of their usual GFD. We evaluated diet, serology, and gastrointestinal parameters. Furthermore, we carried out detail histological assessment of 10 patients before and after eating quinoa.RESULTS:Gastrointestinal parameters were normal. The ratio of villus height to crypt depth improved from slightly below normal values (2.8:1) to normal levels (3:1), surface-enterocyte cell height improved from 28.76 to 29.77 μm and the number of intra-epithelial lymphocytes per 100 enterocytes decreased from 30.3 to 29.7. Median values for all the blood tests remained within normal ranges, although total cholesterol (n=19) decreased from 4.6 to 4.3 mmol/l, low-density lipoprotein decreased from 2.46 to 2.45 mmol/l, high-density lipoprotein decreased from 1.8 to 1.68 mmol/l and triglycerides decreased from 0.80 to 0.79 mmol/l.CONCLUSIONS:Addition of quinoa to the GFD of celiac patients was well tolerated and did not exacerbate the condition. There was a positive trend toward improved histological and serological parameters, particularly a mild hypocholesterolemic effect. Overall, this is the first clinical data suggesting that daily 50 g of quinoa for 6 weeks can be safely tolerated by celiac patients. However, further studies are needed to determine the long-term effects of quinoa consumption.

Variable activation of immune response by quinoa (Chenopodium quinoa Willd.) prolamins in celiac disease

BACKGROUND Celiac disease is an enteropathy triggered by dietary gluten found in wheat, barley, and rye. The current treatment is a strict gluten-free diet. Quinoa is a highly nutritive plant from the Andes, with low concentrations of prolamins, that has been recommended as part of a gluten-free diet; however, few experimental data support this recommendation. OBJECTIVE We aimed to determine the amount of celiac-toxic prolamin epitopes in quinoa cultivars from different regions of the Andes and the ability of these epitopes to activate immune responses in patients with celiac disease. DESIGN The concentration of celiac-toxic epitopes was measured by using murine monoclonal antibodies against gliadin and high-molecular-weight glutenin subunits. Immune response was assessed by proliferation assays of celiac small intestinal T cells/interferon-γ (IFN-γ) and production of IFN-γ/IL-15 after organ culture of celiac duodenal biopsy samples. RESULTS Fifteen quinoa cultivars were tested: 4 cultivars had quantifiable concentrations of celiac-toxic epitopes, but they were below the maximum permitted for a gluten-free food. Cultivars Ayacuchana and Pasankalla stimulated T cell lines at levels similar to those for gliadin and caused secretion of cytokines from cultured biopsy samples at levels comparable with those for gliadin. CONCLUSIONS Most quinoa cultivars do not possess quantifiable amounts of celiac-toxic epitopes. However, 2 cultivars had celiac-toxic epitopes that could activate the adaptive and innate immune responses in some patients with celiac disease. These findings require further investigation in the form of in vivo studies, because quinoa is an important source of nutrients for patients with celiac disease.

Evaluation of the safety of ancient strains of wheat in coeliac disease reveals heterogeneous small intestinal T cell responses suggestive of coeliac toxicity

BACKGROUND & AIMS Coeliac disease is a chronic small intestinal immune-mediated enteropathy triggered by dietary gluten in genetically predisposed individuals. Since it is unknown if all wheat varieties are equally toxic to coeliac patients seven Triticum accessions showing different origin (ancient/modern) and ploidy (di-, tetra- hexaploid) were studied. MATERIALS AND METHODS Selected strains of wheat were ancient Triticum monococcum precoce (AA genome) and Triticum speltoides (BB genome), accessions of Triticum turgidum durum (AABB genome) including two ancient (Graziella Ra and Kamut) and two modern (Senatore Cappelli and Svevo) durum strains of wheat and Triticum aestivum compactum (AABBDD genome). Small intestinal gluten-specific T-cell lines generated from 13 coeliac patients were tested with wheat accessions by proliferation assays. RESULTS All strains of wheat independent of ploidy or ancient/modern origin triggered heterogeneous responses covering wide ranges of stimulation indices. CONCLUSION Ancient strains of wheat, although previously suggested to be low or devoid of coeliac toxicity, should be tested for immunogenicity using gluten-specific T-cell lines from multiple coeliac patients rather than gluten-specific clones to assess their potential toxicity. Our findings provide further evidence for the need for a strict gluten-free diet in coeliac patients, including avoidance of ancient strains of wheat.

Celiac disease in the elderly

Celiac disease is a common condition that is thought to affect 1 in 200 people throughout Europe and America, with prevalence rates reaching 1:100 in Ireland. Improvements in the sensitivity and specificity of serological testing for celiac disease over the past 15 years have resulted in a larger number of diagnoses being made. Up to 34% of patients with newly diagnosed celiac disease are older than 60 years of age. The symptomatic presentation of celiac disease in elderly patients can be subtle, leading to a considerable delay in diagnosis and potential accumulation of associated secondary complications. Given that celiac disease is associated with significant morbidity and reduced life expectancy, physicians need to be aware of this condition and its occurrence in the current increasingly elderly population. Compliance with a strict gluten-free diet is as easily achieved in elderly patients as in younger patients, and has been reported to reduce the risks of cancer and lymphoma associated with celiac disease. This Review highlights age-related differences in the clinical presentation and investigation of patients with suspected celiac disease.

Immunogenicity Characterization of Two Ancient Wheat α-Gliadin Peptides Related to Coeliac Disease

The immunogenic potential of α-gliadin protein from two ancient wheats was studied with reference to coeliac disease. To this aim we investigated Graziella Ra® and Kamut® (the latter is considered an ancient relative of modern durum wheat) in comparison to four durum wheat accessions (Senatore Cappelli, Flaminio, Grazia and Svevo). ELISA and Western Blot analyses - carried out by two monoclonal antibodies raised against the α-gliadin peptides p31-49 (LGQQQPFPQQPYPQPQPF) and p56-75 (LQLQPFPQPQLPYPQPQLPY) containing a core region (underlined) reported to be toxic for coeliac patients - always showed an antibody-antigen positive reaction. For all accessions, an α-gliadin gene has also been cloned and sequenced. Deduced amino acid sequences constantly showed the toxic motifs. In conclusion, we strongly recommend that coeliac patients should avoid consuming Graziella Ra® or Kamut®. In fact their α-gliadin not only is as toxic as one of the other wheat accessions, but also occurs in greater amount, which is in line with the higher level of proteins in ancient wheats when compared to modern varieties.