Handbook of Food Allergen Detection and Control. http://dx.doi.org/10.1533/9781782420217.3.367Copyright 2015 Elsevier Ltd. All rights reserved.

Detection and control of gluten as a food allergen I. Nasr 1 , J. Messing 2 , I. H. Nasr 3 , P. J. Ciclitira 4 1 Guys and St Thomas NHS Trust , London, UK ; 2 Rutgers University , Piscataway, NJ, USA ; 3 Barts Health NHS Trust , London, UK ; 4 King s College London , London, UK

19

19.1 Introduction

Celiac disease (CD) is a chronic, systemic, autoimmune disorder in genetically predisposed individuals. Gluten proteins and related prolamins found in wheat, barley, and rye trigger an autoimmune injury to the gut, skin, liver, joints, uterus, and other organs. 1 In the gut, the resulting injury leads to atrophy of the mucosal villous lining. The villous atrophy is identifi ed on small bowel biopsy, which is considered the gold standard for diagnosing this condition. False negative small bowel histology can be expected due to patchy small bowel mucosal changes.

In the past, the classical presentation of CD was symptomatic of gastrointestinal disorders including diarrhoea and weight loss. Since the 1980s, fewer patients have presented with symptomatic CD and there has been a signifi cant shift toward more patients presenting as asymptomatic adults detected at screening. 2 CD affects on average 1 % of the population. Milder clinical features and use of serology have led to an increased detection rate. 3 The age of presentation varies. It can affect children and elderly alike. The peak age of presentation is in childhood as the presenting symptoms are usually malabsorption and failure to thrive. The other peak is in women of childbearing age who present with anemia. The third peak is in the fourth to fi fth decade who present with osteoporosis. Untreated CD is associated with signifi cant mortality. Undiagnosed CD is associated with a nearly four-fold increased risk of death. 4 With increased disease awareness, newer trends for detection of the disease are important.

19.1.1 Etiology of celiac disease (CD)

Celiac disease (CD) is an intestinal disorder with multifactorial etiology. Genetic and environmental factors are recognized to be involved in the process. This complex interplay between intrinsic and extrinsic factors could explain the range of clinical presentation from asymptomatic individuals to severe malabsorption. 5 HLA and non-HLA genes together with gluten and possibly additional environmental factors predispose to CD. 6 There is a high prevalence of CD among fi rst-degree relatives of celiac patients. 7 It has been reported that high frequency of rotavirus infections may

368 Handbook of Food Allergen Detection and Control

increase the risk of CD autoimmunity in childhood in genetically predisposed individuals. 8 CD, like type 1 diabetes, rheumatoid arthritis, and multiple sclerosis, has a chronic nature where particular HLA alleles are over-represented among the patients. 9 Patients go into complete remission when they are put on a gluten-free diet, and they relapse when gluten is reintroduced into the diet. CD is in this respect unique among the chronic infl ammatory HLA-associated diseases in that a critical environmental factor has been identifi ed. 6

19.1.2 Gluten toxicity

Gluten toxicity in CD indicates a small bowel infl ammatory response. CD is confi rmed when the intestinal epithelium demonstrates villous atrophy, blunting or at least an epithelial infi ltrate of T-cells. Other signs of toxicity may be exhibited outside the intestinal territory, such as the characteristic skin rash dermatitis herpetiformis. The toxicity effects on the small bowel epithelium are potentially reversible once toxic gluten is eliminated from the diet resulting in better nutrient absorption and preventing complications such as osteoporosis 10 and small bowel lymphoma. 11

19.1.3 Classifi cation of cereal proteins involved

Wheat grains contain the outer husk (bran), the germ and the endosperm (white fl our), which accounts for approximately 70 % of the whole grain and contains the toxic components. 12 The storage proteins of cereals constitute the ethanol soluble fraction, known as prolamin because of preponderance of proline and glutamine. 13 Prolamin is known as gluten for wheat, secalins for rye, hordeins for barley, avenins for oats and zeins for maize. Gluten protein is divided according to solubility into gliadins, which are soluble in 4090 % ethanol, and glutenins, which are insoluble. Each wheat variety expresses a large number of related proteins through repeated gene duplications and mutations making biochemical studies and toxicity mapping very diffi cult. 14 All four gliadins sub-fractions ( , , and u) were shown to exacerbate CD both in vitro and in vivo . 15 Several recent studies have indicated that glutenins may be involved in the CD disease process. 1619

19.2 Pathogenesis of celiac disease (CD)

CD is strongly associated with human leukocyte antigen (HLA) class II gene (DR3, DR5/DR7 or DR4) known as HLA-DQ2 and HLA-DQ8 located on chromosome 6p21. 20 It is associated to a larger extent with class II HLA-DQ2 (haplotypes DR-17 or DR5/7) and, to a lesser extent, HLA-DQ8 (haplotype DR-4). 21 These haplotypes are located in antigen-presenting cells of the mucosa. Approximately 95 % of the individuals with CD express HLA-DQ2 heterodimer and the remainder express HLA-DQ8. 22 The absence of these molecules has a negative predictive value for CD close to 100 %. 23

Detection and control of gluten as a food allergen 369

19.2.1 Adaptive immune response

Individuals with CD, in contrast to the healthy population, exhibit a T-cell response to gluten in the small intestine, which is restricted to either HLA-DQ2 or -DQ8. 24 Gluten is the single major environmental factor that triggers CD. The requirement for DQ2 or DQ8 is a major factor in the genetic predisposition to CD. However, most people who demonstrate a positive DQ2 or DQ8 do not develop CD despite daily exposure to gluten in their diet. This corroborates the fact that additional environmental or genetic factors are required for loss of immune tolerance to dietary gluten. HLA-DQ2 or HLA-DQ8 is required to present gluten peptides to the gluten-specifi c T-cells that are present in the gut of CD sufferers. As a result, antigen-specifi c T-cell response is activated. 25 The enzyme tissue transglutaminase (tTG) has been identifi ed as the autoantigen against which the abnormal immune response is directed.

In CD, gliadin peptides resist human proteases and remain intact in the intestinal lumen and have an increased content of proline and glutamine residues. 2628 These peptides gain access to the lamina propria probably due to faulty tight junctions or endothelial cell transcytosis. 29,30 Gluten peptides are de-aminated in the submucosa by tissue transglutaminase (tTG). This creates a high-affi nity binding between these peptides and either HLA-DQ2 or HLA-DQ8 that is in the antigen-presenting cells and also activates helper T-cells. 31 A gliadin 33-mer peptide has several characteristics, suggesting it is the primary initiator of the infl ammatory response to gluten in celiac patients. 32 It stimulates interleukin-15 by dendritic cells and macrophages, which then stimulate intra-epithelial lymphocytes leading to epithelial damage. 33,34

19.2.2 Innate immune response

The innate immune response has an important role in the pathogenesis of CD and in some of its related complications. In particular, an increase in the number of intraepithelial lymphocytes (IELs) in the mucosa of the small intestine is a characteristic feature of CD, and those cells are likely to be important for the on-going pathogenesis of CD. 3539 In vitro studies of intestinal organ cultures, primary antigen-presenting cells, and epithelial and monocytic cell lines support the concept that gluten affects the innate immune system. 40 The IELs have a signifi cant role in the pathogenesis of CD complications, in particular refractory CD and the development of enteropathy-associated T-cell lymphoma (EATL). 4143 The exposure to toxic gluten in CD results in activation of / T-cell IELs. 14 Various mechanisms including the release of tumour necrosis factor (TNF- ), 44 interferon (IFN- ), 4547 and Fas ligand (FasL) system 48 are involved in mediating mucosal damage in T-cell-dependent intestinal diseases. Natural killer group 2, member D/major histocompatibility complex class I chain-related gene A (NKG2D/MICA) pathway, 27 perform with subsequent granzyme induction 49 and matrix metalloproteinase tissue remodeling 50 may also contribute. Interleukin (IL)15 also has a vital role the innate immune response. Upregulation of IL-15 expression by epithelial cells or macrophages increases MICA ligand expression on epithelial cells to facilitate the NKG2D receptor signaling pathway. 27,37

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19.3 Testing of gluten toxicity

Due to the complex heterogenicity of gluten, it is quite challenging to have a gold standard for testing. Immunological techniques such as enzyme-linked immunosorbent assay (ELISA) offer a sensitive and simple way of detecting antibodies against whole gluten digest 51 or a single peptide. 52 These techniques may miss other toxic cereals depending on the antibodies used. In addition, T cell epitopes are known to participate in disease mechanism which can be missed because antiserum recognizes B cell epitomes. Spaenij-Dekking has raised monoclonal antibodies to the proposed immunodominant epitope, in order to address this problem. 53 In addition, the majority of the immunological methods available use antibodies directed at the gliadins and miss some of the glutenins proteins that are now being implicated in disease pathogenesis 16,19 and will need to be considered when selecting reagents for measurement of gluten.

19.3.1 In vitro testing

Biopsies obtained from patients with CD are tested for the response to gluten toxicity. The small intestinal epithelium morphology from the untreated patients improves when their small intestinal biopsies are placed in an organ culture chamber for up to 24 hours in the absence of gluten. 54 This improvement can be altered by the addition of gluten. 55 Peptides can be tested in organ culture for evaluation of toxicity in CD; however, clinical in vivo testing is mandatory to confi rm the toxicity of these. 14

19.3.2 In vivo testing

A baseline small bowel epithelial biopsy is assessed for villus height to crypt depth (VH:CD), enterocyte height (ECH) and IEL count. A gluten challenge is introduced in vivo , usually with bread over a four-week period, may be indicated when there is doubt regarding the diagnosis.

19.4 Current EU labeling and codex guidelines

Tolerability of gluten varies amongst individuals. Western European dietary products labeled as gluten-free accept some amount of gluten. This is usually to improve baking qualities and palatability. Patients with CD generally show good histological and clinical response despite consuming such a wheat starch-containing diet. 56,57 However, in other parts of the world, such as Australia and the North American continent, a gluten-free diet should contain no gluten and a naturally gluten-free diet is used instead. If some gluten is included in the product, then foods are labeled as low gluten. 58 In 2012, the new European regulation for labeling products in relation to the gluten content took effect. The gluten content threshold was set in accordance

Detection and control of gluten as a food allergen 371

to the Codex Alimentarius. Regulation (EC) No 41/2009 applies to all foodstuffs with the exception of infant and follow-on formulae.

The WHO Codex Alimentarius standard proposes a gluten limit of 0.02 % (200 ppm) for gluten-free food and 0.002 % (20 ppm) for naturally gluten-free food. The Regulation (EC) 41/2009 states that gluten-free products should contain less than 20 mg/kg of gluten in the fi nished product. Very low gluten-containing products should contain less than 100 mg/kg of gluten in the fi nished product. The difference in the acceptable levels refl ects the fact that the exact tolerable limit for gluten is unknown. 59 Catassi et al . showed that less than 50 mg gluten per day is safe; however, some individuals relapsed when ingesting gluten at a much lower dose. 60 The safe maximum level of gluten remains debatable. The normal average intake of gluten by healthy individuals is approximately 13 g per day. 61

19.5 Methods for measurement of gluten contamination in foods

Gluten contamination in gluten-free products cannot be absolutely avoided and the safe threshold for gluten remains debatable. One way of detecting the toxic gluten epitopes is through ELISA, a method which uses monoclonal antibodies (MAB) specifi c for the immunotoxic peptides present in food. It is highly sensitive, specifi c and reproducible. The experience with this, however, depends on the monoclonal antibody used and whether it targets a single peptide or the whole protein. Some commercially available kits use antibodies against u-gliadin, which recognizes wheat and rye but not barley. 62 Valdes et al . 63 used an antibody that was able to target wheat, barley and rye prolamins. In this study, they were able to quantify levels as low as 1.56 ppm gliadins and 3.2 ppm gluten. Other kits can detect gliadins, hordeins and secalins to the same extent using a combination of three MABs. 64 Another issue is the fact that MABs target B-cell epitopes. As CD is a result of infl ammatory T-cell response, Spaenij-Dekking et al . 53 developed antisera that are able to detect T-cell eptitopes of the - and -gliadin. Gluten quantifi cation may not be completely accurate as the methods used rely on what is targeted and which toxic epitope is present in the food tested. Methods to test glutenins will also need to be established.

19.5.1 Malted cereals

Despite breaking down the cereals, there is no evidence that toxic gluten is disintegrated. It is known now that certain, signifi cantly cell-reactive areas of gliadin are highly resistant to enzymatic degradation. 26,28 Gliadin-like epitopes have been found in beer and malt. 65,66 Experts describe encountering patients with CD who are symptomatic following consumption of large amounts of malted products. Therefore, the question on whether malted breakfast cereals are consumed should be considered when dealing with patients with CD, who are symptomatic despite consuming a gluten-free diet.

372 Handbook of Food Allergen Detection and Control

19.5.2 Communion wafers

Wheat fl our is used for the making of these wafers. It has been demonstrated that, in addition to an otherwise gluten-free diet, consumption of a milligram of gluten from wheat fl our-based wafers leads to failed mucosal recovery after 18 months challenge. 67 Gluten-free communion wafers containing Codex wheat starch are commercially available. A list of products and suppliers is available on Coeliac UK website.

19.5.3 Oats

Early feeding experiments suggested that oats are toxic to individuals with CD and have been excluded from the gluten-free diet. The possible harmful effect of oats has been revised and subsequently studied. Janatuinen et al . 68 in a Finnish randomized trial of 92 patients with CD compared the effects of gluten-free diets with and without oats. Eleven patients withdrew from the study. Duodenal biopsies revealed no effect on the mucosa in either group. Serological normalization was not delayed. The 35 patties from the oats study groups were re-evaluated after fi ve years. 69 Twenty-three of them were consuming oats regularly with no harmful effects detected. Other smaller studies were conducted. Some concluded that oats are safe and well tolerated. 70,71 Other published data suggest the possibility that a small group of individuals with CD is intolerant to oats. 72,73

It is diffi cult to conclude whether oats are safe to use in CD. The studies show that intolerance to oats is not very frequent, but toxicity can still occur. Some patients withdrew from some of the studies, which raises the question of intolerance to oats. Patients with CD eating oats were found to have more abdominal complaints than those not eating oats. 74 Commercially available oats are frequently contaminated with cereals containing toxic gluten and therefore are not suitable to be included as part of a strict gluten-free diet. 75

19.6 Future trends and summary

The standard methods for diagnosing CD included serology testing, histological confi rmation and identifying the HLA status. In recent years, studies have looked into the use of a fi nger prick-based kit for self-testing of fi rst- and second-degree relatives of CD patients. 76 However, so far the gold standard for diagnosing CD is by histological analysis of the small bowel biopsy. Further research is needed to identify an alternative reliable method which is easily accessible to all.

CD is a gluten-sensitive small intestinal enteropathy. Treatments involve a gluten-free diet omitting wheat, rye, barley and possibly oats. In vitro and in vivo testing of sequenced gluten peptides have identifi ed a variety of cereal epitopes that exacerbate the condition. Debate continues regarding the maximum permitted amount of gluten that patients with CD can safely tolerate. Research is required to increase our knowledge of the pathogenesis of the condition and to improve therapeutic options.

Detection and control of gluten as a food allergen 373

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