Article

Dissecting the T-cell response to hordeins in coeliac disease can develop barley with reduced immunotoxicity

CSIRO Food Futures National Research Flagship, Canberra, ACT, Australia.
Alimentary Pharmacology & Therapeutics (Impact Factor: 4.55). 11/2010; 32(9):1184-91. DOI: 10.1111/j.1365-2036.2010.04452.x
Source: PubMed

ABSTRACT Wheat, rye and barley prolamins are toxic to patients with coeliac disease. Barley is diploid with pure inbred cultivars available, and is attractive for genetic approaches to detoxification.
To identify barley hordein fractions which activated the interferon-γ (IFN-γ) secreting peripheral blood T-cells from coeliac volunteers, and compare immunotoxicity of hordeins from experimental barley lines.
To reactivate a T-cell response to hordein, volunteers underwent a 3-day oral barley challenge. Peripheral blood mononuclear cells (PBMC) were isolated from twenty-one HLA DQ2(+) patients with confirmed coeliac disease. IFN-γ ELISpot assays enumerated T-cells activated by purified prolamins and positive controls.
Hordein-specific T-cells were induced by oral barley challenge. All prolamin fractions were immunotoxic, but D- and C-hordeins were most active. Barley lines lacking B- and C-hordeins had a 5-fold reduced hordein-content, and immunotoxicity of hordein extracts were reduced 20-fold compared with wild-type barley.
In vivo oral barley challenge offers a convenient and rapid approach to test the immunotoxicity of small amounts of purified hordeins using fresh T-cells from patients in high throughput overnight assays. Barley lines that did not accumulate B- and C-hordeins were viable, yet had substantially reduced immunotoxicity. Creation of hordein-free barley may therefore be possible.

0 Followers
 · 
84 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: There is an increase in the number of people adopting a gluten-free diet, with one-fifth of Australian consumers avoiding certain food or drinks for allergy or intolerance reasons. For most consumers, the belief that a gluten-free diet is healthier than a gluten containing diet is unsupported by a formal diagnosis. However, for a small group of subjects who have gluten sensitive disorder, a lifelong, gluten-free diet is required, including avoidance of beer. Prolylendopeptidase enzymes (PEP) cleave gluten proteins after the abundant proline residues and have the potential to destroy gluten proteins and remove or minimize immunoreactive peptides from food. However, PEP treatment may also confound measurement of gluten concentration in food and beverages by destroying epitopes that are used to enumerate gluten peptides – we ask, “does PEP treatment destroy celiac reactive epitopes or merely disguise them from ELISA enumeration?” There is now sufficient data to show that treatment of gluten peptides with bacterial PEP in combination with other proteases, or the fungal Aspergillus niger PEP alone, reduces the immunoreactivity of gluten peptides, with celiac T-cells to near zero. This is also accompanied by destruction of key epitopes that are used by antibodies to enumerate gluten peptides during ELISA reactions. Thus, both immunoreactivity and ELISA measurements are reduced to near zero by PEP treatment. However, definitive evidence of the safety of treated beer for celiacs ideally requires a double-blind crossover, dietary challenge. Consuming sufficient beer to present a suitable load of hordein peptides is not possible, and presenting hordeins in the same form as encountered in treated beer is difficult. The effect of protease treatments on the safety of treated gluten for the remainder the celiac-like diseases, including gluten ataxia and dermatitis herpetiformis, and the larger spectrum of glutenrelated disorders, including gluten intolerance and the IgE-mediated allergic responses Bakers asthma, gluten allergy, WDEIA, and urticaria, cannot be definitively assessed until the epitopes involved have been defined. The gluten sensitive disorders and the gluten proteins are reviewed, and the effects of proteolysis on several archetypal gluten peptides are examined. Keywords: Beer, celiac disease, gluten, hordeins, prolylendopeptidase.
    Journal of the American Society of Brewing Chemists 02/2014; 72(1):36-50. DOI:10.1094/ASBCJ-2014-0129-01 · 0.86 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed. The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined. A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation. Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.
    PLoS ONE 02/2013; 8(2):e56456. DOI:10.1371/journal.pone.0056456 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease, an autoimmune disorder of the small intestine associated with a permanent intolerance to gluten proteins. The complete elimination of gluten proteins contained in cereals from the diet is the key to celiac disease management. However, this generates numerous social and economic repercussions due to the ubiquity of gluten in foods. The research presented in this review focuses on the current status of alternative cereals and pseudocereals and their derivatives obtained by natural selection, breeding programs and transgenic or enzymatic technology, potential tolerated by celiac people. Finally, we describe several strategies for detoxification of dietary gluten. These included enzymatic cleavage of gliadin fragment by Prolyl endopeptidases (PEPs) from different organisms, degradation of toxic peptides by germinating cereal enzymes and transamidation of cereal flours. This information can be used to search for and develop cereals with the baking and nutritional qualities of toxic cereals, but which do not exacerbate this condition.
    Nutrients 10/2013; 5(10):4250-68. DOI:10.3390/nu5104250 · 3.15 Impact Factor

Full-text

Download
60 Downloads
Available from
May 31, 2014