Scandinavian Journal of Gastroenterology. 2015; Early Online, 1–10

REVIEW

Coeliac disease – from genetic and immunological studies to clinical applications

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KNUT E. A. LUNDIN1,2, SHUO-WANG QIAO2, OMRI SNIR2 & LUDVIG M. SOLLID2 1

Department of Gastroenterology, Oslo University Hospital-Rikshospitalet, Oslo, Norway, and 2Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway

Abstract Coeliac disease is a common and important gastrointestinal disease. It affects at least 1%, most Western European populations and in Nordic countries it is even more frequent. It is strongly associated with certain Human Leukocyte Antigen-DQ genes and triggered by ingestion of wheat gluten and related cereals from rye and barley. The diagnosis relies on a combination of clinical signs, serology and small intestinal biopsy. Work during the last couple of decades has shown that gluten-specific, Human Leukocyte Antigen-DQ-restricted T-cells in the intestinal mucosa are of paramount importance in the disease process. The gluten peptides are chemically modified by the endogenous enzyme transglutaminase 2, the same enzyme that serves as target in today’s sensitive serological tests for coeliac disease. The increasing knowledge on the disease process allows for development of improved diagnosis, patient care and new treatment modalities.

Key Words: coeliac-disease, gastroduodenal-basic, gastroduodenal-clinical, immunology, small-intestinal-disorders

Introduction Coeliac disease (CD) was known to ancient medical science as Aretaeus of Cappadocia described a clinical picture compatible with CD in the first century CE. The English paediatrician Samuel Gee gave a precise description of the disease in the second part of the 1800s. He noted that the disease could occur at all ages, and he also noted that: “if the disease could be cured, it must be by means of the diet”. He has been proven correct. However, it was not until the seminal work of another paediatrician, Willem K. Dicke, that it was definitely shown that wheat, rye and barley were responsible for the disease [1]. In a series of challenge and withdrawal experiments he showed that his children with the disease developed steatorroea and failure to thrive, associated with clinical signs of disease. Significant progress has been made in the field of research on CD during the last decades and with accelerating pace [2,3]. We have detailed knowledge

on the key players in the immune system of CD patients, we know the disease-causing antigen wheat gluten, and we are aware of a number of genes that are involved. Particular focus has been paid to the Human Leukocyte Antigen (HLA) system, as this disease shows a particularly strong association to a small number of HLA genes. However, it should also be said that we know much less about the transition of a healthy gut to a situation where a small intestinal immune response against gluten leads to villous atrophy and a number of health issues [4]. In this review, we summarize the research on CD in our laboratory as well as in several other laboratories. Emphasis will also be paid to contributions from Nordic countries. Clinical aspects, diagnosis and epidemiology The clinical signs of CD are variable [5]. The previously often seen picture with severe malabsorption is

Correspondence: Knut E. A. Lundin, MD, PhD, Department of Gastroenterology, Oslo University Hospital-Rikshospitalet, Sognsvannsveien 20, N-0372 Oslo, Norway. Tel: +4723072400. Fax: +4723072410. E-mail: [email protected]; , [email protected]

(Received 15 March 2015; accepted 15 March 2015) ISSN 0036-5521 print/ISSN 1502-7708 online
2015 Informa Healthcare DOI: 10.3109/00365521.2015.1030766

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now only rarely seen. This could be partially due to increased awareness and earlier detection, but other factors are probably also in play. Three aspects should prompt suspicion of CD: 1) iron deficiency and anemia, 2) abdominal discomfort, wind, diarrhea or constipation, 3) a feeling of being “tired at all times” [6]”. Moreover, a range of other clinical signs must also be taken into consideration. In children, failure to thrive, short stature and dental enamel defects can be seen. Hypothyreosis, type 1 diabetes and several other endocrine disorders are associated diseases [7]. Osteoporosis and even fractures are frequent [8]. However, it must be remembered for any given sign, most individuals with those sign will not have CD. Diagnosis is in many cases simple as serology is a powerful diagnostic tool, when followed up by a diagnostic gastroscopy [5]. The vast majority of patients with active CD have IgA antibodies to the enzyme transglutaminase 2 (TG2) and also IgA and IgG antibodies to so-called deamidated gliadin peptides (DGP). The biological explanation for the presence of these antibodies will be discussed later. In patients with normal serum levels, staining for IgA deposits against TG2 within the mucosa is an early and sensitive marker for CD, but rarely used outside the research setting in most countries [9]. There is continuous debate around the sensitivity and specificity as well as positive and negative predictive values [10]. It is fair to state that the sensitivity for these tests is well above 90%, whereas the specificity of the test lies well above 95%. However, when the tests are applied to general populations with relatively low pre-test probability, the positive and negative predictive values drop. The main message therefore is that a positive serology probably means CD, whereas a negative test does not entirely rule CD out. Unexplained iron or vitamin deficiency, especially with a family history of CD, should lead to further investigation. Whereas serology is supportive of CD, most investigators and recent guidelines demand a small intestinal biopsy for definite diagnosis [5,11,12]. Four biopsies or more should be taken from the second part of duodenum and scored by the pathologist according to predefined criteria. We often use the so-called Marsh criteria modified by Oberhuber and co-workers [13]. These criteria define aspects of intraepithelial lymphocytosis, villous atrophy and crypt hyperplasia. Plasma cell (PC) density in the lamina propria should also be described, as increased density is a hallmark of CD. Composite diagnosis including clinical signs, serology and duodenal histology is recommended [5]. Whereas a demonstration of overt villous atrophy earlier was a prerequisite for the diagnosis, the trend is shifting towards allowing

also minor changes [14]. Even patients with mild symptoms benefit from having a diagnosis and commencing a gluten-free diet [15]. In children, recently published guidelines from the European Society for Pediatric Gastroenterology, Hepatology and Nutrition have defined a diagnostic algorithm where clinical signs, serology and HLA typing (to be discussed later) allow the diagnosis under strict conditions to be made even without a biopsy [16]. Far from all children with CD comply with these rules. It should be stressed that both serology and small intestinal pathology normalizes when gluten is withdrawn from the diet. The epidemiology of CD is of particular interest for the Nordic countries as several important studies have been performed here. In the early 1980s, feeding habits were changed for Swedish children with later introduction of gluten coinciding with less breastfeeding. This resulted in a large increase of CD in children below the age of 2 (for ref. see [17]). This also resulted in a national screening program, and CD has become exceedingly prevalent in several age cohorts in Sweden with prevalence numbers > 3% [18]. Similar figures are found in a pediatric study from Norway [19]. Finnish scientists have likewise been in the forefront of CD epidemiological research [20] and reported that population prevalence increased from 1% in 1980 to 2% in 2000 (for ref. see [21]). In Denmark, the disease is less prevalent, but the prevalence is rising [22], and CD is grossly under-diagnosed [23]. The reasons for these developments remain obscure. Very recent studies could not show a correlation between the age of gluten introduction, breast feeding and overall risk of developing CD [24–26]. The HLA association and genetics For > 40 years we have known that CD occurs more frequently in subject with certain HLA types [27,28]. From recent genome-wide association studies (GWAS) we now know that HLA (Major Histocompatibility Complex) is the single most important genetic factor accounting for ~ 40% of the genetic variance [29]. In addition, multiple non-HLA genes are implicated, each of them with modest contributions. By GWAS, and more recently by dense fine mapping and resequencing studies, altogether 40 non-HLA loci with 58 independent signals have been reported [29–31]. These loci together explain 13.7% of the genetic variance. Many of these signals do not localize to exons where they cause variation in protein sequence. Rather many of them control transcription of immune relevant genes, particularly

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Coeliac disease immunology and clinics in T and B cells [32]. The HLA association in CD compared to many other HLA-associated diseases is unusually clear. The HLA molecule conferring the disease susceptibility is in the majority of the patients a variant of HLA-DQ2, encoded by the DQA1*05 and DQB1*02 alleles, often termed DQ2.5 (for ref. see [33]). These patients either carry these DQA1 and DQB1 alleles on the same haplotype in cis configuration (the DR3DQ2 haplotype) or they carry them in trans configuration on two different haplotypes (the DR5DQ7 and DR7DQ2 haplotypes) [2,34]. The DR7DQ2 haplotype, carrying the DQA1*02:01 and DQB1*02 alleles that encode the DQ2.2 allotype, also confers susceptibility by itself but much less than the DQ2.5 variant. In addition to HLA-DQ2.5 and HLADQ2.2, HLA-DQ8 encoded by the DQA1*03 and DQB1*03:02 alleles and being part of DR4DQ8 haplotypes, also confers susceptibility [35]. In our Scandinavian population ~ 90% of the patients are HLA-DQ2.5, 5% are HLA-DQ2.2 (and not HLA-DQ2.5) and 5% are HLA-DQ8. For all three HLA-DQ allotypes there are gene dosage effects with increased risk for individuals who are homozygous for the HLA-DQ allotypes [36]. Gluten-reactive T-cells The very strong HLA association should per se indicate that specific recognition of antigenic peptides presented by these HLA molecules should be involved in the pathogenesis. However, before the 1990s a number of different theories were proposed including lectin activity of gluten, or genetic lack of digestive enzymes as important. We took advantage of observations in professor Per Brandtzaeg’s group showing that T-cells within small intestinal biopsies of CD patients can be activated ex vivo when confronted with gluten [37]. Thus we applied the technique of positive selection of activated T-cells by magnetic bead coated with antibodies to interleukin-2 receptors. T-cells were further cultured in the absence of further gluten re-stimulation. We succeeded in culturing gluten-reactive T-cells from CD patients, but not healthy individuals [38]. Somewhat to our surprise, all the intestinal T-cells did only recognize gluten when presented by the diseaseassociated HLA-DQ2.5 molecules, and not by any of the other HLA molecules present in the patient’s mucosal immune system. Very soon thereafter, a patient being heterozygous DR4-DQ7/DR4-DQ8 was studied, and a preferential recognition of gluten antigen in the context of DQ8 was seen [39]. As these seminal works, our group has substantiated these finding in a number of other experiments [40]. Other groups in the Netherlands and Italy made

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similar observations [41–43]. They all support the initial findings that gluten reactive T-cell lines and T-cell clones can be generated from the vast majority of CD patients. This includes treated and untreated CD patients, it can be seen after stimulation of biopsies as well as cell suspensions from cell biopsies with gluten and can be done even in the absence of stimulation ex vivo with gluten [44]. Failure to expand and establish gluten reactive T-cell lines from such patients are more likely due to technical laboratory aspects than the immunological profile of the patients. Furthermore, the T-cells from the intestinal lesion are invariably restricted by the CD-associated HLADQ2 (this is so both for HLA-DQ2.5 (ref [38]), HLA-DQ2.2 (ref [45]) and HLA-DQ8 molecules, and none of the other HLA class II molecules also expressed in the intestinal immune system. This is not due to any functional defect of these HLA molecules in this compartment, as for example astrovirus-specific, intestinal T-cells clearly can recognize foreign antigen presented by HLA-DR molecules [46]. HLA-DR restriction is otherwise the most frequent situation in immune responses in peripheral blood against many foreign antigens including gluten [47,48]. HLA class I restricted T-cells have also been reported [49], but it still remains uncertain which role these cells have in the pathogenesis of CD. Gluten reactive, intestinal T-cells have not been found in healthy individuals [40]. This applies both to effector as well as possible regulatory T-cells. Thus, the exclusive presence in the CD patients as well as the very narrow HLA-restriction patterns clearly suggest that these T-cells are of major importance in the disease pathogenesis. T-cell epitopes Strictly speaking, gluten represents the proteins of wheat only. However, as it is similar to storage proteins of other grains that also precipitate CD, gluten is often used as a collective term for avenin (oat), secalin (rye) and hordein (barley). Wheat gluten is a complex mixture of hundreds of proteins and is divided into gliadin (alcohol-soluble fraction) that is further divided into a-, g- and w-gliadins, and glutenin (alcohol-insoluble) that is subdivided into high-molecularweight and low-molecular-weight glutenins [50]. By systematically testing in vitro cultured glutenreactive CD4+ T-cells obtained from duodenal biopsies of CD patients against fractions of gluten proteins [51], we identified the first HLA-DQ2.5-restricted gluten T-cell epitope in 1998 from g-gliadin [52]. Since then, we and others have identified > 30 different gluten epitopes [53]. These gluten epitopes are recognized by the aforementioned CD4+ T-cells

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only when bound to HLA-DQ2.5, HLA-DQ8, or HLA-DQ2.2, i.e. the allotypes that are associated with CD. The majority of these T-cell epitopes are derived from wheat gluten and restricted by HLADQ2.5. Without exception, all gluten epitopes contain at least one glutamic acid residue that results from TG2-mediated deamidation. These glutamic acid residues are positioned in pockets P4 and P6 (rarely also in P7) in epitopes that are HLADQ2.5-restricted, as negatively charged amino acids are preferred in these positions. Similarly, in HLADQ8-restricted epitopes, the glutamic acid residues are placed in pockets P1 and P9 matching the binding preference of HLA-DQ8. The common denominator of all these peptide epitopes, are that they bind with good to very good affinity to the presenting HLA-DQ molecules [45,54,55]. The nomenclature of gluten epitopes was recently standardized in a joint article published by the groups that have been involved in characterizing them [53]. The epitope names hereafter contain both the HLA class II restriction element and the protein the sequence is derived from. Among the many epitopes, a handful of these are considered to be more important because they are as a rule recognized by CD4+ T-cells from almost every CD patient. They are known as the immunodominant gluten epitopes. For the vast majority of CD patients that are HLADQ2.5, the immunodominant wheat gluten epitopes are DQ2.5-glia-a1, DQ2.5-glia-a2, DQ2.5-glia-w 1 and DQ2.5-glia-w2. Transglutaminase 2 TG2 is a ubiquitously expressed protein. It is found mostly intracellularly where its main function is GTPbinding and hydrolysis. Extracellular TG2, on the other hand, has the potential to be enzymatically active in the presence of Ca2+ and catalyse 1) transamidation between an e-amino group of a lysine residue and a g-carboxamide group of glutamine residue; or 2) deamidation of a glutamine residue (Q), in particular in the Q-X-P motif [56] to glutamic acid; or 3) hydrolysis of the isopeptide bond created in transamidation. Due to the spectrum of reactions it can catalyze and the abundance of the enzyme, it is not surprising to find that extracellular TG2 is inactive under steady state, but can be activated during injury or inflammation [57]. We have found that the TG2 activity is tightly controlled by the redox potential, where TG2 is activated specifically by thioredoxin-mediated reduction [58,59]. In CD pathogenesis, it is not known when and where the TG2-mediated deamidation takes place. One hypothesis is that before CD is established, TG2 is

activated by intestinal inflammation for example as a result of a gastrointestinal infection, and thus creating deamidated gluten peptides that can bind HLA-DQ2 or HLA-DQ8 and prime CD4+ T-cells. In support of this view, epidemiological studies from Sweden have shown that children born in the summer are more at risk of developing CD, presumably because the introduction of dietary gluten coincides with more gastrointestinal infections in the winter season [60]. Whence CD is initiated, the TG2 activity is maintained by the ongoing inflammation. It is intriguing that the TG2 that mediates important posttranslational modification for increasing the antigenicity of gluten [48] is also the target to which highly disease-specific autoantibodies are made. TG2-specific autoantibodies are only produced in untreated CD patients, showing that the autoantibody production is gluten-dependent. We have proposed a possible mechanism for this link [61]. It hypothesized that the TG2-specific B cells can take up TG2-gluten complexes through its B-cell receptor and subsequently present the gluten antigen to gluten-reactive CD4+ T-cells. A hapten-carrier mechanism for generation of autoantibodies in CD was also proposed by Mäki [62]. We are currently in the process of generating both in vitro and in vivo models to test this hypothesis. B cells in CD As was mentioned above antibodies to TG2 and DGP are found in virtually all CD patients. The production of such antibodies is entirely dependent on gluten uptake; i.e. they disappear from patients’ sera when patients are treated with gluten-free diet and reappear when patients are challenged with gluten [63], and they are only found in individuals that are HLADQ2.2, HLA-DQ2.5 and HLA-DQ8. The role, however, of anti-TG2 and anti-DGP antibodies in the pathogenesis of the disease is currently unknown. High numbers of PCs that are reactive either with TG2 or DGP are found in the duodenal lamina propria of CD patients when the disease is active and their frequency drops when gluten-free diet is strictly followed, similar to the levels of antibodies in serum. In two separated studies we have cloned and expressed panels of human monoclonal antibodies (hmAbs) that are specific to either TG2 or DGP from intestinal PCs that were single-sorted from patients with active CD [64,65]. Intriguingly, the two types CD-associated antibodies have less mutations than antibodies of other gut IgA PCs. Reversion of a panel of anti-TG2 antibodies to presumed germline sequence lead to reduced reactivity, suggesting

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Coeliac disease immunology and clinics that the somatic hypermutations (SHM) had been gained by affinity maturation. [63]. The fact that the antiboides are produced in individuals with CD-assocciated HLA allotypes also suggest that the T-cell help is involved in the generation of the antibodies. There is a biased usage of VH and VL gene segments in both antibodies specific for TG2 or DGP. IGVH5-51 was most frequently used by a panel of hmAbs specific for TG2, and nearly all VL gene segments belonged to the IGVK1 gene family. IGVH3 family largely dominated the antibody response against DGP. IGVH3-15 and IGVH3-23 were predominately used and the latter was commonly combined with IGVL4-69. We are currently extending our B-cell studies in CD, using the advantage of high throughput sequencing (HTS) to monitor thousands of intestinal PCs that are reactive either with TG2 or DGP as well as memory B cells from a larger number of CD patients. Our analysis confirmed that such PCs acquire lower number of SHM in their Ig VH gene region. These features also characterized TG2 reactive PCs that were found in treated CD patients. In addition to IGVH5-51 other VH gene segments, primarily from the IGVH3 and IGVH4, were found to be overexpressed by TG2 reactive PCs. Four major epitopes were recently identified on TG2, which were clustered in the N-terminal half of the enzyme [66]. Interestingly, the reactivity of anti-TG2 hmAbs to this epitopes was associated and group according to their VH gene usage. IGVH5-51 antibodies chiefly targeted epitope 1, whereas epitope 2 and 3 were targeted by IGVH3 and IGVH4 anti-TG2 hmAbs. Our current analyses show that PCs that are reactive with either TG2 or DGP are clonally expanded and have low level of diversity. The antibody responses are dominated by large PC clones that seed and widely disseminate in the duodenum lamina propria, which is further evidenced by the high frequency of clonally related TG2-reactive PCs that are found in distinct biopsies from the small intestine (Di Niro et al., submitted for publication). Using HTS we further revealed low levels of memory B cells in peripheral blood that clonally related to PCs that are reactive with TG2 or DGP. The memory cells were predominantly IgAs and could be found in CD patients who have been treated with gluten-free diet over long period of time (Snir et al., submitted for publication). Hence, immune responses towards TG2 and DGP in CD are long-lived and have an established memory that can be detected in periphery. Cells are likely to proliferate and evolve elsewhere before seeding to the PCs niches in lamina propria, were they continuously produced anti-TG2 and antiDGP antibodies.

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Tetramers and T-cell receptors The instrumental role of the complexes between HLA-DQ2 molecules and gliadin peptides suggest that such complexes could be used for staining and following disease relevant T-cells in CD. We therefore made recombinant soluble HLA-DQ molecules bound with various gluten T-cell epitope peptides to mimic the interaction between the gluten-reactive T-cell receptor (TCR) and the peptide-HLA-DQ complex [67]. Upon multimerization, these gluten– DQ complexes, also known as tetramers, will bind antigen-specific TCRs. After challenge with regular bread for 3 days, a flux of gluten-specific T-cells can be detected in peripheral blood of CD patients as detected by antigen-specific ELISA [68,69] or by our HLA-DQ2-gliadin peptide tetramers [70,71]. However, as such a challenge is inconvenient, bothersome and sometimes not acceptable for the patients, we recently refined our staining method to e sensitivity level where we can visualize glutenreactive CD4+ T-cells in blood directly ex vivo [72]. We have shown that the circulating glutenDQ2.5 tetramer-positive effector memory T-cell population is invariably expanded in all CD patients, including those that are on a gluten-free diet. In contrast, healthy HLA-DQ2.5-positive individuals have only naive gluten-DQ2.5 tetramer-positive in their blood, and very few or none gluten-reactive effector memory cells (unpublished results). Based on these proof-of-principle findings, we are currently exploiting the enumeration of gluten-reactive T-cells by gluten-DQ tetramer-staining as a novel diagnostic tool for CD. This test is principally different from all the other existing diagnostic tools, and it has the potential to diagnose CD patients who are maintaining a gluten-free diet without the need for glutenchallenge or gastroduodenoscopy. Each T-cell owes its exquisite antigen (pMHC) specificity to the TCR it expresses, encoded by the TCRa and TCRb genes that are unique for each progenitor T-cell. Over the years, we have sequenced thousands of different gluten-reactive TCRs. Our initial interpretation was that no correlation between TCR sequences and gluten specificity exist [38]. This later proved to be wrong. We have found that T-cells that recognize the DQ2.5-glia-a2 epitope share very similar TCR (TRAV26-1 paired with TRBV7-2) sequences across many patients [73]. Other groups have found similar public TCR bias in T-cells reactive to other gluten epitopes. They have found that DQ8-glia-a1-reactive T-cells preferably use TRBV9 paired with TRAV26-2 (ref [74]) and that DQ2.5-glia-a1-reactive T-cells show a biased use of TRBV20-1 and TRBV29-1 (ref [75]). In addition, our

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own unpublished data indicate that also within the DQ2.5-glia-w2-reactive TCR repertoire, there is some degree of biased V-gene use, both in the TCRa and TCRb chains. Importantly, our data indicate that the biased V-gene usage is antigen-driven because they are only observed in the expanded effector memory T-cell subset but not among naive T-cells with the same antigen specificity. Despite the existence of several crystal structures of glutenreactive TCRs in complex with gluten-DQ molecules [74,75], our understanding of the molecular basis of the biased V-gene use remains incomplete. Nevertheless, we plan to exploit direct identification of signature gluten-reactive TCR sequences by highthroughput sequencing as another novel blood-based diagnostic method. Gluten-related disorders Gluten as a part of human nutrition has been around since ~ 9500 years B.C. Wheat has a range of benefits, it can be grown in large crops, and it is stable, contains valuable amino acids and trace elements and can be baked to give palatable products. However, from a digestive point of view, wheat poses some problems. The gluten proteins are notoriously difficult to digest, leaving larger peptide fragments that are targets for intestinal T-cells in CD. Wheat, rye and barley also contain relatively high levels of fermentable carbohydrates known as Fermentable Oligo- Di- Monosaccharides And Polyols [76]. Until the last two–three decades, gluten intolerance was thought to be restricted to CD and some cases of wheat allergy. It is only recently that we have come to recognize a clinical picture of gluten sensitivity without CD, also termed non-celiac gluten sensitivity (NCGS) [5,77,78]. This clinical entity lacks objective criteria. There are no blood tests that prove the disease, although increased levels of IgG to gluten is found in 50% of the cases [79]. The mucosa is normal with the frequent exception of intraepithelial lymphocytosis [80,81]. It may not come to a surprise to anyone that the condition is discusses heavily; nevertheless the number of persons in several Western populations avoiding gluten has reached the level of 5% or more. The corresponding gluten-free market in the USA has been estimated to be in the order of 5–10 billion USD! A large patient series from Italy suggested prevalence of NCGS around 3–4% in patients referred to gastroenterology clinics [82]. We investigated a cohort of self-instituted gluten-free persons and found that few of them turned out to have CD [71]. We could not find signs of psychosomatic disorders [83], and we found evidence of mucosal immune activation

in the patients after gluten challenge [81]. The objective clinical evaluation of these individuals is difficult but some recent evaluation algorithms have been published [84]. It is, however, not certain that wheat gluten is involved. Other non-gluten proteins may also play a role [85]. The condition has a number of clinical aspects suggesting a close connection to functional bowel disorders. It is further possible that the causative agent is not gluten as such but rather the Fermentable Oligo- Di- Monosaccharides And Polyols content of the wheat [86]. There is clearly a need for further research on this topic. Translation of basic science to patient care There are several fields where basic research has had and will become to have a major impact on the care of CD patients. The clear definition of the HLA association was shown in the late 1980s. For years it was mainly used as for its overwhelming negative predictive value – without HLA-DQ2 or HLA-DQ8 you simply do not develop CD (with very, very few exceptions) [87]. Interestingly, HLA typing was recently incorporated into the diagnostic algorithm for pediatric CD [16], but not (yet) for adult CD [5]. Furthermore, the demonstration in the 1990s of TG2 as the antigen for the previously used endomysium test allowed for development of excellent ELISA tests based on recombinant TG2 [63,88]. This test has replaced the out-dated anti-gliadin ELISA test and the endomysium test. However, the finding that the T-cells in CD recognize deamidated and not native gliadin peptides prompted investigations on the antibody repertoire in CD. This led to potent serology tests based on recognition of DGPs, tests that are about equivalent to antiTG2-based tests [10]. We have recently taken another approach and isolated and cloned human monoclonal anti-gliadin antibodies from the lesion itself. When used as a serological test these antibodies give hope of even better specificity and sensitivity values [65]. The use of oats in the diet of CD patients has for years been controversial. A seminal study from Finland provided strong evidence that oats are not toxic to CD patients [89]. A follow-up of this study and a large study on Swedish children confirmed that almost all patients with CD tolerate oats [90,91]. In contrast, we found signs of oats intolerance during a feeding study [92] although oats intake did not increase levels of IgA against oats [93]. In selected patients, oats intolerant CD patients mount an intestinal T-cell response against oats [94]. Interestingly,

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Coeliac disease immunology and clinics urine nitric oxide level measurement suggests that some children are indeed intolerant to oats, and follow-up is recommended [95]. The development of HLA-DQ2-gliadin peptide tetramers also gives hope for improved diagnosis of CD. Gluten challenge of CD patients for 3 days gives a flux of gluten specific T-cells into the blood stream [68–71]. This test has potential to become a valid test for CD, at least in the case that persons have adopted a gluten-free diet in the absence of proper diagnostics with serology and/or biopsy. Our group has recently refined the test further and can find such diseaserelevant T-cells in the majority of CD patients well treated with gluten-free diet [72]. Thus, it might be possible to find signs of CD even in the absence of gluten challenge. Treatment of CD in addition to or as a replacement for gluten-free diet is a further interesting topic. Gluten is, as mentioned, difficult to digest [96], but this can be done by enzymes derived from bacteria or cereals [97]. Such enzymes can be added to food to reduce gluten toxicity in the CD patient [98]. The role of this treatment is yet to be proven in the general CD population. The same is true for recent proposals to use intradermal injection of peptides representing immunodominant T-cell epitopes to regulate the intestinal immune system [99].

Conclusion The rapid development of scientific insight into the disease processes that are operable in CD allows for better diagnosis and patient care. We still do not fully understand what initiates the disease process, and we do not have any effective strategies for primary prevention. The elucidation of the disease processes allows for development of new and improved treatment options for the disease.

Acknowledgements Work in the authors’ laboratory is supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 179573/ V40, European Research Council advanced grant (FP/2007–2013/ERC grant 2010-268541), SouthEastern Norway Regional Health Authority and the Norwegian ExtraFoundation for Health and Rehabilitation through EXTRA funds. The authors have scientific and financial collaboration with ImmusanT and Regeneron Inc. We are grateful to all the CD patients who willingly donate biopsies and other

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biological material to research and to colleagues and co-workers for their invaluable support. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

References [1] Dicke WK, Weijers HA, Van De Kamer JH. Coeliac disease. II. The presence in wheat of a factor having a deleterious effect in cases of coeliac disease. Acta Paediatr 1953;42:34–42. [2] Sollid LM, Lundin KE. Celiac disease. In Rose N, Mackay I, editors. The autoimmune diseases. 5th ed. Elsevier; 2014. pp 855–71. [3] Lundin KE, Sollid LM. Advances in coeliac disease. Curr Opin Gastroenterol 2014;30:154–62. [4] Sollid LM, Jabri B. Triggers and drivers of autoimmunity: lessons from coeliac disease. Nat Rev Immunol 2013;13: 294–302. [5] Ludvigsson JF, Bai JC, Biagi F, Card TR, Ciacci C, Ciclitira PJ, et al. Diagnosis and management of adult coeliac disease: guidelines from the British Society of Gastroenterology. Gut 2014;63:1210–28. [6] Hin H, Bird G, Fisher P, Mahy N, Jewell D. Coeliac disease in primary care: case finding study. Bmj 1999;318:164–7. [7] Collin P, Kaukinen K, Valimaki M, Salmi J. Endocrinological disorders and celiac disease. Endocr Rev 2002;23:464–83. [8] Hjelle AM, Apalset E, Mielnik P, Bollerslev J, Lundin KE, Tell GS. Celiac disease and risk of fracture in adults– review. Osteoporos Int 2014;25:1667–76. [9] Kaukinen K, Peraaho M, Collin P, Partanen J, Woolley N, Kaartinen T, et al. Small-bowel mucosal transglutaminase 2-specific IgA deposits in coeliac disease without villous atrophy: a prospective and randomized clinical study. Scand J Gastroenterol 2005;40:564–72. [10] Leffler DA, Schuppan D. Update on serologic testing in celiac disease. Am J Gastroenterol 2010;105:2520–4. [11] Bai JC, Fried M, Corazza GR, Schuppan D, Farthing M, Catassi C, et al. World Gastroenterology Organisation global guidelines on celiac disease. J Clin Gastroenterol 2013;47: 121–6. [12] Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA; American College of G. ACG clinical guidelines: diagnosis and management of celiac disease. Am J Gastroenterol 2013;108:656–76; quiz 77. [13] Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol 1999;11:1185–94. [14] Kurppa K, Collin P, Viljamaa M, Haimila K, Saavalainen P, Partanen J, et al. Diagnosing mild enteropathy celiac disease: a randomized, controlled clinical study. Gastroenterology 2009;136:816–23. [15] Kurppa K, Paavola A, Collin P, Sievanen H, Laurila K, Huhtala H, et al. Benefits of a gluten-free diet for asymptomatic patients with serologic markers of celiac disease. Gastroenterology 2014;147:610–17; e1. [16] Husby S, Koletzko S, Korponay-Szabo IR, Mearin ML, Phillips A, Shamir R, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr 2012;54:136–60.

Scand J Gastroenterol Downloaded from informahealthcare.com by Kainan University on 04/08/15 For personal use only.

8

K. E. A. Lundin et al.

[17] Ivarsson A, Myleus A, Norstrom F, van der Pals M, Rosen A, Hogberg L, et al. Prevalence of childhood celiac disease and changes in infant feeding. Pediatrics 2013;131:e687–94. [18] Myleus A, Ivarsson A, Webb C, Danielsson L, Hernell O, Hogberg L, et al. Celiac disease revealed in 3% of Swedish 12-year-olds born during an epidemic. J Pediatr Gastroenterol Nutr 2009;49:170–6. [19] Stordal K, Bakken IJ, Suren P, Stene LC. Epidemiology of coeliac disease and comorbidity in Norwegian children. J Pediatr Gastroenterol Nutr 2013;57:467–71. [20] Maki M, Mustalahti K, Kokkonen J, Kulmala P, Haapalahti M, Karttunen T, et al. Prevalence of Celiac disease among children in Finland. N Engl J Med 2003; 348:2517–24. [21] Lohi S, Mustalahti K, Kaukinen K, Laurila K, Collin P, Rissanen H, et al. Increasing prevalence of coeliac disease over time. Aliment Pharmacol Ther 2007;26:1217–25. [22] Dydensborg S, Toftedal P, Biaggi M, Lillevang ST, Hansen DG, Husby S. Increasing prevalence of coeliac disease in Denmark: a linkage study combining national registries. Acta paediatr 2012;101:179–84. [23] Horwitz A, Skaaby T, Karhus LL, Schwarz P, Jorgensen T, Rumessen JJ, et al. Screening for celiac disease in Danish adults. Scand J Gastroenterol 2015. [Epub ahead of print]. [24] Vriezinga SL, Auricchio R, Bravi E, Castillejo G, Chmielewska A, Crespo Escobar P, et al. Randomized feeding intervention in infants at high risk for celiac disease. N Engl J Med 2014;371:1304–15. [25] Lionetti E, Castellaneta S, Francavilla R, Pulvirenti A, Tonutti E, Amarri S, et al. Introduction of gluten, HLA status, and the risk of celiac disease in children. N Engl J Med 2014;371:1295–303. [26] Aronsson CA, Lee HS, Liu E, Uusitalo U, Hummel S, Yang J, et al. Age at gluten introduction and risk of celiac disease. Pediatrics 2015;135:239–45. [27] Stokes PL, Asquith P, Holmes GK, Mackintosh P, Cooke WT. Histocompatibility antigens associated with adult coeliac disease. Lancet 1972;2:162–4. [28] Solheim BG, Ek J, Thune PO, Baklien K, Bratlie A, Rankin B, et al. HLA antigens in dermatitis herpetiformis and coeliac disease. Tissue Antigens 1976;7:57–9. [29] Trynka G, Hunt KA, Bockett NA, Romanos J, Mistry V, Szperl A, et al. Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease. Nat Genet 2011;43:1193–201. [30] Dubois PC, Trynka G, Franke L, Hunt KA, Romanos J, Curtotti A, et al. Multiple common variants for celiac disease influencing immune gene expression. Nat Genet 2010;42: 295–302. [31] Hunt KA, Mistry V, Bockett NA, Ahmad T, Ban M, Barker JN, et al. Negligible impact of rare autoimmunelocus coding-region variants on missing heritability. Nature 2013;498:232–5. [32] Kumar V, Gutierrez-Achury J, Kanduri K, Almeida R, Hrdlickova B, Zhernakova DV, et al. Systematic annotation of celiac disease loci refines pathological pathways and suggests a genetic explanation for increased interferon-gamma levels. Hum Mol Genet 2015;24:397–409. [33] Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E. Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer. J Exp Med 1989;169:345–50. [34] Abadie V, Sollid LM, Barreiro LB, Jabri B. Integration of genetic and immunological insights into a model of celiac disease pathogenesis. Annu Rev Immunol 2011;29:493–525.

[35] Spurkland A, Sollid LM, Polanco I, Vartdal F, Thorsby E. HLA-DR and -DQ genotypes of celiac disease patients serologically typed to be non-DR3 or non-DR5/7. Hum Immunol 1992;35:188–92. [36] Karell K, Louka AS, Moodie SJ, Ascher H, Clot F, Greco L, et al. HLA types in celiac disease patients not carrying the DQA1*05-DQB1*02 (DQ2) heterodimer: results from the European Genetics Cluster on Celiac Disease. Hum Immunol 2003;64:469–77. [37] Halstensen TS, Scott H, Fausa O, Brandtzaeg P. Gluten stimulation of coeliac mucosa in vitro induces activation (CD25) of lamina propria CD4+ T cells and macrophages but no crypt-cell hyperplasia. Scand J Immunol 1993;38: 581–90. [38] Lundin KE, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O, et al. Gliadin-specific, HLA-DQ(alpha 1*0501,beta 1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med 1993;178: 187–96. [39] Lundin KE, Scott H, Fausa O, Thorsby E, Sollid LM. T cells from the small intestinal mucosa of a DR4, DQ7/DR4, DQ8 celiac disease patient preferentially recognize gliadin when presented by DQ8. Hum Immunol 1994;41:285–91. [40] Molberg O, Kett K, Scott H, Thorsby E, Sollid LM, Lundin KE. Gliadin specific, HLA DQ2-restricted T cells are commonly found in small intestinal biopsies from coeliac disease patients, but not from controls. Scand J Immunol 1997;46:103–9. [41] Vader W, Kooy Y, Van Veelen P, De Ru A, Harris D, Benckhuijsen W, et al. The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterology 2002;122:1729–37. [42] van de Wal Y, Kooy YM, van Veelen PA, Pena SA, Mearin LM, Molberg O, et al. Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin. Proc Natl Acad Sci USA 1998;95:10050–4. [43] Camarca A, Anderson RP, Mamone G, Fierro O, Facchiano A, Costantini S, et al. Intestinal T cell responses to gluten peptides are largely heterogeneous: implications for a peptide-based therapy in celiac disease. J immunology 2009;182:4158–66. [44] Bodd M, Raki M, Bergseng E, Jahnsen J, Lundin KE, Sollid LM. Direct cloning and tetramer staining to measure the frequency of intestinal gluten-reactive T cells in celiac disease. Eur J Immunol 2013;43:2605–12. [45] Bodd M, Kim CY, Lundin KE, Sollid LM. T-cell response to gluten in patients with HLA-DQ2.2 reveals requirement of peptide-MHC stability in celiac disease. Gastroenterology 2012;142:552–61. [46] Molberg O, Lundin KE, Nilsen EM, Scott H, Kett K, Brandtzaeg P, et al. HLA restriction patterns of gliadinand astrovirus-specific CD4+ T cells isolated in parallel from the small intestine of celiac disease patients. Tissue Antigens 1998;52:407–15. [47] Gjertsen HA, Sollid LM, Ek J, Thorsby E, Lundin KE. T cells from the peripheral blood of coeliac disease patients recognize gluten antigens when presented by HLA-DR, -DQ, or -DP molecules. Scand J Immunol 1994;39:567–74. [48] Molberg O, McAdam SN, Korner R, Quarsten H, Kristiansen C, Madsen L, et al. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med 1998;4: 713–17. [49] Gianfrani C, Troncone R, Mugione P, Cosentini E, De Pascale M, Faruolo C, et al. Celiac disease association

Coeliac disease immunology and clinics

[50] [51]

[52]

Scand J Gastroenterol Downloaded from informahealthcare.com by Kainan University on 04/08/15 For personal use only.

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62] [63]

[64]

[65]

[66]

with CD8+ T cell responses: identification of a novel gliadinderived HLA-A2-restricted epitope. J Immunol 2003;170: 2719–26. Wieser H. Chemistry of gluten proteins. Food Microbiol 2007;24:115–19. Lundin KE, Sollid LM, Anthonsen D, Noren O, Molberg O, Thorsby E, et al. Heterogeneous reactivity patterns of HLADQ-restricted, small intestinal T-cell clones from patients with celiac disease. Gastroenterology 1997;112:752–9. Sjostrom H, Lundin KE, Molberg O, Korner R, McAdam SN, Anthonsen D, et al. Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition. Scand J Immunol 1998;48:111–15. Sollid LM, Qiao SW, Anderson RP, Gianfrani C, Koning F. Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules. Immunogenetics 2012;64:455–60. Arentz-Hansen H, Korner R, Molberg O, Quarsten H, Vader W, Kooy YM, et al. The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med 2000;191:603–12. Tollefsen S, Arentz-Hansen H, Fleckenstein B, Molberg O, Raki M, Kwok WW, et al. HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease. J Clin Invest 2006; 116:2226–36. Fleckenstein B, Qiao SW, Larsen MR, Jung G, Roepstorff P, Sollid LM. Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. J Biol Chem 2004;279:17607–16. Siegel M, Strnad P, Watts RE, Choi K, Jabri B, Omary MB, et al. Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury. PLoS One 2008;3:e1861. Stamnaes J, Pinkas DM, Fleckenstein B, Khosla C, Sollid LM. Redox regulation of transglutaminase 2 activity. J Biol Chem 2010;285:25402–9. Jin X, Stamnaes J, Klock C, DiRaimondo TR, Sollid LM, Khosla C. Activation of extracellular transglutaminase 2 by thioredoxin. J Biol Chem 2011;286:37866–73. Lebwohl B, Green PH, Murray JA, Ludvigsson JF. Season of birth in a nationwide cohort of coeliac disease patients. Arch Dis Child 2013;98:48–51. Sollid LM, Molberg O, McAdam S, Lundin KE. Autoantibodies in coeliac disease: tissue transglutaminase – guilt by association? Gut 1997;41:851–2. Maki M. editor. Sixth International Symposium on Coeliac Disease. Dublin, Ireland: Oak Tree Press; 1992. Sulkanen S, Halttunen T, Laurila K, Kolho KL, KorponaySzabo IR, Sarnesto A, et al. Tissue transglutaminase autoantibody enzyme-linked immunosorbent assay in detecting celiac disease. Gastroenterology 1998;115:1322–8. Di Niro R, Mesin L, Zheng NY, Stamnaes J, Morrissey M, Lee JH, et al. High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat Med 2012;18:441–5. Steinsbo O, Henry Dunand CJ, Huang M, Mesin L, Salgado-Ferrer M, Lundin KE, et al. Restricted VH/VL usage and limited mutations in gluten-specific IgA of coeliac disease lesion plasma cells. Nat Commun 2014;5:4041. Iversen R, Di Niro R, Stamnaes J, Lundin KE, Wilson PC, Sollid LM. Transglutaminase 2-specific autoantibodies in

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78] [79]

[80]

[81]

[82]

9

celiac disease target clustered, N-terminal epitopes not displayed on the surface of cells. J Immunol 2013;190:5981–91. Quarsten H, McAdam SN, Jensen T, Arentz-Hansen H, Molberg O, Lundin KE, et al. Staining of celiac diseaserelevant T cells by peptide-DQ2 multimers. J Immunol 2001; 167:4861–8. Anderson RP, Degano P, Godkin AJ, Jewell DP, Hill AV. In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nat Med 2000;6:337–42. Anderson RP, van Heel DA, Tye-Din JA, Barnardo M, Salio M, Jewell DP, et al. T cells in peripheral blood after gluten challenge in coeliac disease. Gut 2005;54:1217–23. Raki M, Fallang LE, Brottveit M, Bergseng E, Quarsten H, Lundin KE, et al. Tetramer visualization of gut-homing gluten-specific T cells in the peripheral blood of celiac disease patients. Proc Natl Acad Sci USA 2007;104:2831–6. Brottveit M, Raki M, Bergseng E, Fallang LE, Simonsen B, Lovik A, et al. Assessing possible celiac disease by an HLADQ2-gliadin Tetramer Test. Am J Gastroenterol 2011;106: 1318–24. Christophersen A, Raki M, Bergseng E, Lundin KE, Jahnsen J, Sollid LM, et al. Tetramer-visualized glutenspecific CD4+ T cells in blood as a potential diagnostic marker for coeliac disease without oral gluten challenge. United European Gastroenterology J 2014;2:268–78. Qiao SW, Raki M, Gunnarsen KS, Loset GA, Lundin KE, Sandlie I, et al. Posttranslational modification of gluten shapes TCR usage in celiac disease. J Immunol 2011;187: 3064–71. Broughton SE, Petersen J, Theodossis A, Scally SW, Loh KL, Thompson A, et al. Biased T cell receptor usage directed against human leukocyte antigen DQ8-restricted gliadin peptides is associated with celiac disease. Immunity 2012;37:611–21. Petersen J, Montserrat V, Mujico JR, Loh KL, Beringer DX, van Lummel M, et al. T-cell receptor recognition of HLADQ2-gliadin complexes associated with celiac disease. Nat Struct Mol Biol 2014;21:480–8. Gibson PR, Shepherd SJ. Evidence-based dietary management of functional gastrointestinal symptoms: The FODMAP approach. J Gastroenterol Hepatol 2010;25:252–8. Ludvigsson JF, Leffler DA, Bai JC, Biagi F, Fasano A, Green PH, et al. The Oslo definitions for coeliac disease and related terms. Gut 2013;62:43–52. Lundin KE, Alaedini A. Non-celiac gluten sensitivity. Gastrointest Endosc Clin N Am 2012;22:723–34. Volta U, Tovoli F, Cicola R, Parisi C, Fabbri A, Piscaglia M, et al. Serological tests in gluten sensitivity (nonceliac gluten intolerance). J Clin Gastroenterol 2012; 46:680–5. Sapone A, Lammers KM, Casolaro V, Cammarota M, Giuliano MT, De Rosa M, et al. Divergence of gut permeability and mucosal immune gene expression in two glutenassociated conditions: celiac disease and gluten sensitivity. BMC Med 2011;9:23. Brottveit M, Beitnes AC, Tollefsen S, Bratlie JE, Jahnsen FL, Johansen FE, et al. Mucosal cytokine response after short-term gluten challenge in celiac disease and non-celiac gluten sensitivity. Am J Gastroenterol 2013;108:842–50. Volta U, Bardella MT, Calabro A, Troncone R, Corazza GR; Study Group for Non-Celiac Gluten S. An Italian prospective multicenter survey on patients suspected of having non-celiac gluten sensitivity. BMC Med 2014;12:85.

Scand J Gastroenterol Downloaded from informahealthcare.com by Kainan University on 04/08/15 For personal use only.

10

K. E. A. Lundin et al.

[83] Brottveit M, Vandvik PO, Wojniusz S, Lovik A, Lundin KE, Boye B. Absence of somatization in non-coeliac gluten sensitivity. Scand J Gastroenterol 2012;47:770–7. [84] Kabbani TA, Vanga RR, Leffler DA, Villafuerte-Galvez J, Pallav K, Hansen J, et al. Celiac disease or non-celiac gluten sensitivity? An approach to clinical differential diagnosis. Am J Gastroenterol 2014;109:741–6; quiz 7. [85] Junker Y, Zeissig S, Kim SJ, Barisani D, Wieser H, Leffler DA, et al. Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4. J Exp Med 2012;209:2395–408. [86] Biesiekierski JR, Peters SL, Newnham ED, Rosella O, Muir JG, Gibson PR. No effects of gluten in patients with self-reported non-celiac gluten sensitivity after dietary reduction of fermentable, poorly absorbed, short-chain carbohydrates. Gastroenterology 2013;145:320–8; e1-3. [87] Sollid LM, Lie BA. Celiac disease genetics: current concepts and practical applications. Clin Gastroenterol Hepatol 2005; 3:843–51. [88] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3:797–801. [89] Janatuinen EK, Pikkarainen PH, Kemppainen TA, Kosma VM, Jarvinen RM, Uusitupa MI, et al. A comparison of diets with and without oats in adults with celiac disease. N Engl J Med 1995;333:1033–7. [90] Janatuinen EK, Kemppainen TA, Julkunen RJ, Kosma VM, Maki M, Heikkinen M, et al. No harm from five year ingestion of oats in coeliac disease. Gut 2002;50:332–5. [91] Hogberg L, Laurin P, Falth-Magnusson K, Grant C, Grodzinsky E, Jansson G, et al. Oats to children with newly

[92]

[93]

[94]

[95]

[96]

[97] [98]

[99]

diagnosed coeliac disease: a randomised double blind study. Gut 2004;53:649–54. Lundin KE, Nilsen EM, Scott HG, Loberg EM, Gjoen A, Bratlie J, et al. Oats induced villous atrophy in coeliac disease. Gut 2003;52:1649–52. Guttormsen V, Lovik A, Bye A, Bratlie J, Morkrid L, Lundin KE. No induction of anti-avenin IgA by oats in adult, diet-treated coeliac disease. Scand J Gastroenterol 2008;43:161–5. Arentz-Hansen H, Fleckenstein B, Molberg O, Scott H, Koning F, Jung G, et al. The molecular basis for oat intolerance in patients with celiac disease. PLoS Med 2004;1:e1. Tapsas D, Falth-Magnusson K, Hogberg L, Forslund T, Sundqvist T, Hollen E. Urinary nitric oxide metabolites in children with celiac disease after long-term consumption of oats-containing gluten-free diet. Scand J Gastroenterol 2014; 49:1311–17. Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, et al. Structural basis for gluten intolerance in celiac sprue. Science 2002;297:2275–9. Sollid LM, Khosla C. Novel therapies for coeliac disease. J Intern Med 2011;269:604–13. Lahdeaho ML, Kaukinen K, Laurila K, Vuotikka P, Koivurova OP, Karja-Lahdensuu T, et al. Glutenase ALV003 attenuates gluten-induced mucosal injury in patients with celiac disease. Gastroenterology 2014;146: 1649–58. Anderson RP, Jabri B. Vaccine against autoimmune disease: antigen-specific immunotherapy. Curr Opin Immunol 2013; 25:410–17.