researchers on celiac disease

• Open access
• Published: 23 July 2019

Celiac disease: a comprehensive current review

• Giacomo Caio   ORCID: orcid.org/0000-0002-4244-4529 1 , 2   na1 ,
• Umberto Volta 3   na1 ,
• Anna Sapone 2 , 4 ,
• Daniel A. Leffler 4 , 5 ,
• Roberto De Giorgio 1 ,
• Carlo Catassi 2 , 6   na2 &
• Alessio Fasano 2   na2  

BMC Medicine volume  17 , Article number:  142 ( 2019 ) Cite this article

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Celiac disease remains a challenging condition because of a steady increase in knowledge tackling its pathophysiology, diagnosis, management, and possible therapeutic options.

A major milestone in the history of celiac disease was the identification of tissue transglutaminase as the autoantigen, thereby confirming the autoimmune nature of this disorder. A genetic background ( HLA-DQ2/DQ8 positivity and non-HLA genes) is a mandatory determinant of the development of the disease, which occurs with the contribution of environmental factors (e.g., viral infections and dysbiosis of gut microbiota). Its prevalence in the general population is of approximately 1%, with female predominance. The disease can occur at any age, with a variety of symptoms/manifestations. This multifaceted clinical presentation leads to several phenotypes, i.e., gastrointestinal, extraintestinal, subclinical, potential, seronegative, non-responsive, and refractory. Although small intestinal biopsy remains the diagnostic ‘gold standard’, highly sensitive and specific serological tests, such as tissue transglutaminase, endomysial and deamidated gliadin peptide antibodies, have become gradually more important in the diagnostic work-up of celiac disease. Currently, the only treatment for celiac disease is a life-long, strict gluten-free diet leading to improvement in quality of life, ameliorating symptoms, and preventing the occurrence of refractory celiac disease, ulcerative jejunoileitis, and small intestinal adenocarcinoma and lymphoma.

Conclusions

The present review is timely and provides a thorough appraisal of various aspects characterizing celiac disease. Remaining challenges include obtaining a better understanding of still-unclear phenotypes such as slow-responsive, potential (minimal lesions) and seronegative celiac disease. The identification of alternative or complementary treatments to the gluten-free diet brings hope for patients unavoidably burdened by diet restrictions.

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Introduction

Celiac disease (CD) is an autoimmune condition characterized by a specific serological and histological profile triggered by gluten ingestion in genetically predisposed individuals [ 1 ]. Gluten is the general term for alcohol-soluble proteins present in various cereals, including wheat, rye, barley, spelt, and kamut [ 1 ]. In recent years, there have been significant changes in the diagnosis, pathogenesis, and natural history of this condition [ 2 ], with CD undergoing a true ‘metamorphosis’ due to the steady increase in the number of diagnoses identified, even in geriatric patients [ 2 ]. This has been mainly attributed to the greater availability of sensitive and specific screening tests, which allow identification of the risk groups for CD and led to a significant raise in diagnoses worldwide [ 2 , 3 , 4 , 5 ]. Several theories have suggested that the globalization and ubiquitous spread of ‘false’ or ‘extreme’ versions of the Mediterranean diet including the consumption of very high quantities of gluten (up to 20 g/day), has led to an increased prevalence and incidence of CD [ 3 , 4 ]. In addition, the quality of gluten itself might also play a contributory role. Indeed, the production of new grain variants due to technological rather than nutritional reasons may have influenced the observed increase in the number of CD diagnoses in recent years [ 4 , 5 ]. However, these hypotheses have not been confirmed and the real cause of the risk in CD diagnoses remains unknown. Furthermore, the epidemiological observation that similar ‘epidemics’ are reported for other autoimmune diseases in the Western hemisphere [ 6 ] suggests that environmental factors other than gluten can be at play.

In this article, we aimed to provide a thorough review on the multifaceted features of CD spanning from its epidemiological, pathogenetic, clinical, and diagnostic aspects to therapeutic strategies using a practical approach in order to help general practitioners, internal medicine physicians, and gastroenterologists in their clinical practice.

• Epidemiology

CD is one of the most common autoimmune disorders, with a reported prevalence of 0.5–1% of the general population (Table  1 ), with the exception of areas showing low frequency of CD-predisposing genes and low gluten consumption (e.g., sub-Saharan Africa and Japan) [ 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Studies have shown that most CD cases remain undetected in the absence of serological screening due to heterogeneous symptoms and/or poor disease awareness. CD prevalence is increasing in Western countries. Between the years 1975 and 2000, CD prevalence increased 5-fold in the US, for reasons that are currently unknown [ 14 ]. The prevalence of CD is higher in first-degree CD relatives (10–15%) and in other at-risk groups, particularly patients with Down syndrome, type 1 diabetes, or IgA deficiency [ 1 ].

Pathophysiology

CD is a unique autoimmune disease in that its key genetic elements (human leukocyte antigen (HLA)-DQ2 and HLA-DQ8), the auto-antigen involved (tissue transglutaminase (tTG)), and the environmental trigger (gluten) are all well defined. A major drawback in CD research has been the lack of a reliable and reproducible animal model, with the possible exception of the Irish setter dog, which may develop a gluten-related disease [ 15 ]. Nevertheless, new technologies pertinent to human gut biology and immunology are opening unprecedented opportunities for major research breakthroughs.

As with many other autoimmune diseases, we have witnessed an epidemic of CD, questioning the previous paradigm that gluten is the only key element dictating the onset of the disease in genetically at-risk subjects. Improved hygiene and lack of exposure to various microorganisms also have been linked with a steep increase in autoimmune disorders in industrialized countries during the past 40 years [ 1 , 16 ]. The hygiene hypothesis argues that the rising incidence of many autoimmune diseases may partially be the result of lifestyle and environmental changes that have reduced our exposure to pathogens. With breakthroughs in the role of the gut microbiological ecosystem [ 17 ] in dictating the balance between tolerance and immune response leading to autoimmunity, this hypothesis is under scrutiny. Regardless of whether autoimmune diseases are due to too much or too little exposure to microorganisms, it is generally accepted that adaptive immunity and imbalance between T helper 1 and 2 cell responses are key elements of the pathogenesis of the autoimmune process. Besides genetic predisposition and exposure to gluten, loss of intestinal barrier function, a pro-inflammatory innate immune response triggered by gluten, inappropriate adaptive immune response, and an imbalanced gut microbiome all seem to be key ‘ingredients’ of the CD autoimmunity recipe.

As with any other autoimmune disease, CD has a strong hereditary component as testified by its high familial recurrence (~ 10–15%) and the high concordance of the disease among monozygotic twins (75–80%) [ 18 ]. Also common to other autoimmune diseases is the relevant role of HLA class II heterodimers, specifically DQ2 and DQ8, in the heritability of CD. HLA-DQ2 homozygosis confers a much higher risk (25–30%) of developing early-onset CD in infants with a first-degree family member affected by the disease [ 19 , 20 , 21 ]. Since HLA-DQ2/HLA-DQ8 is frequent among the general population (25–35%), and only 3% of these HLA-compatible individuals will go on to develop CD [ 22 ], it is not surprising that genome-wide association studies have identified more than 100 non-HLA-related genes associated with CD [ 18 , 23 ]. The relevance of these additional genes in conferring genetic risk for CD is rather limited, but they may lead to the discovery of key pathways potentially involved in disease pathogenesis.

Gluten as an environmental trigger of CD

Introduced 10,000 years ago during the transition from a nomadic lifestyle to agricultural settlements, gluten-containing grains are a recent addition to the human diet. Moreover, gluten is one of the few digestion-resistant proteins consumed chronically in significant quantities and is constituted by several non-digestible immunogenic peptides. These two characteristics could help in breaking the tolerance to this food antigen, when the immune system is activated, as can happen during an enteric infection. Gliadins, key components of gluten, are complex proteins unusually rich in prolines and glutamines and are not completely digestible by intestinal enzymes [ 24 ]. The final product of this partial digestion is a mix of peptides that can trigger host responses (increased gut permeability and innate and adaptive immune response) that closely resemble those instigated by the exposure to potentially harmful microorganisms [ 25 , 26 , 27 , 28 ].

Gluten trafficking from lumen to lamina propria (paracellular and transcellular)

Studies from our group and others have shown that gliadin can cause an immediate and transient increase in intercellular tight junction permeability of intestinal epithelial cells [ 23 , 24 ] (Fig.  1 ). This effect has been linked to the release of zonulin, a family of molecules that increases paracellular permeability by causing tight junction disassembly [ 29 , 30 , 31 ]. Gliadin enhances zonulin-dependent increased gut paracellular permeability irrespective of disease status [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. Similarly, when tested in C57BL/6 mice duodenal tissues, gliadin caused a myeloid differentiation primary response 88-dependent increase in gut mucosa permeability [ 40 ]. We have also identified two alpha-gliadin motifs that can modulate the intestinal barrier function by binding to chemokine receptor 3, with subsequent zonulin release that causes disassembly of the interepithelial tight junction complex [ 41 ]. The involvement of the paracellular pathway for gluten trafficking in the lamina propria has also been corroborated by genetic studies identifying an association of some tight junction genes with CD [ 42 , 43 , 44 ]. There is solid evidence that gluten can also cross the intestinal barrier through the transcellular pathway once tolerance to gluten has been broken [ 45 , 46 ]. The transferrin receptor CD71, normally expressed on the basolateral side of enterocytes, is overexpressed on the luminal side of the intestinal epithelium in CD patients during the acute phase of the disease, leading to an apical-to-basal retrotranscytosis of gliadin peptides complexed with secretory IgA [ 47 ]. This retrotranscytosis of secretory IgA–gliadin complexes protects gliadin fragments from lysosomal degradation and promotes the entry of harmful gliadin peptides into the intestinal lamina propria [ 47 ], thereby perpetuating intestinal inflammation initiated by the paracellular passage of these peptides (Fig.  1 ). Because of their resistance, the gluten immunogenic peptides (GIP) can cross the defective epithelial lining, reach the blood stream (thus extending the inflammatory process), and finally be excreted with the urine [ 48 ].

Celiac disease pathogenesis. Partially digested gliadin fragments interact with chemokine receptor 3 on the apical side of epithelium (1) inducing a myeloid differentiation primary response 88-dependent release of zonulin (2). Zonulin interacts with the intestinal epithelium and triggers increased intestinal permeability (3). Functional loss of the gut barrier facilitates gliadin peptide translocation from lumen to the lamina propria (4). Gliadin peptides trigger release of IL-15, keratinocyte growth factor, and IL-8 (5), with consequent recruitment of neutrophils in the lamina propria (6). Simultaneously, alpha-amylase/trypsin inhibitors engage the Toll like receptor 4–MD2–CD14 complex with subsequent up-regulation of maturation markers and release of proinflammatory cytokines (7). Following innate immune-mediated apoptosis of intestinal cells with subsequent release of intracellular tissue transglutaminase, gliadin peptides are partially deamidated (8). Deamidated gliadin is recognized by DQ2/8 + antigen presenting cells (9) and then presented to T helper cells (10). T helper cells trigger activation and maturation of B cells, producing IgM, IgG, and IgA antibodies against tissue transglutaminase (11). T helper cells also produce pro-inflammatory cytokines (interferon γ and tumor necrosis factor α) (12), which in turn further increase gut permeability and, together with T killer cells, initiate the enteropathy. Damaged enterocytes express CD71 transporter also on their apical side, resulting in retrotranscytosis of secretory IgA-gliadin complexes (13), thus potentiating gluten trafficking from gut lumen to lamina propria. Ultimately, the interaction between CD4 + T cells in the lamina propria with gliadin induces their activation and proliferation, with production of proinflammatory cytokines, metalloproteases, and keratinocyte growth factor by stromal cells, which induces crypt hyperplasia and villous blunting secondary to intestinal epithelial cell death induced by intraepithelial lymphocytes. The hyperplastic crypts (14) are characterized by an expansion of the immature progenitor cells compartment (WNT) and downregulation of the Hedgehog signaling cascade. An increased number of stromal cells known to be part of the intestinal stem cell niche and increased levels of bone morphogenetic protein antagonists, like Gremlin-1 and Gremlin-2, may further contribute to the crypt hyperplasia present in celiac disease

The innate immune response

Innate immunity plays a critical role in initiating CD, and cytokines such as interleukin (IL)-15 and interferon α can prime the innate immune response by polarizing dendritic cells and intraepithelial lymphocyte function [ 49 ]. Recent results suggest that specific gliadin peptides may induce epithelial growth factor and an IL-15-dependent proliferation of enterocytes, structural modifications, vesicular trafficking alterations, signaling and proliferation, and stress/innate immunity activation [ 50 ]. Alpha-amylase/trypsin inhibitors – molecules conferring pest resistance in wheat – also seem to play a key role in CD innate immune response by engaging the Toll-like receptor 4–MD2–CD14 complex with subsequent up-regulation of maturation markers and release of proinflammatory cytokines in cells from CD patients [ 51 ]. These mucosal events, along with the functional breach of epithelial barrier function secondary to the gliadin-mediated zonulin release [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ], the subsequent access of toxic peptides in the lamina propria, and gliadin-induced production of high levels of the neutrophil-activating and chemoattractant chemokine IL-8 [ 26 , 52 ], cause the ‘perfect storm’ to initiate CD enteropathy (Fig.  1 ). More recently, our group showed that gliadin exerts a direct neutrophil chemoattractant effect by interacting with fMet-Leu-Phe receptor 1 [ 53 , 54 ].

The adaptive immune response

The erroneous adaptive immune response consequence of a highly specific interplay between selected gluten peptides and major histocompatibility complex class II HLA-DQ2/8-antigen restricted T cells plays a paramount role in CD pathogenesis [ 55 ]. Dependent on the post-translational deamidation of gluten peptides by transglutaminase 2 (TG2), this interplay is influenced by the initial imprinting of the innate immune system through IL-15 upregulation that promotes the CD4 + T cell adaptive immune response [ 56 , 57 ]. Presentation of gluten to CD4 + T cells carried out by dendritic cells as well as macrophages, B cells, and even enterocytes expressing HLA class II, can cause their recirculation in the lamina propria [ 58 ]. The contact of CD4 + T cells in the lamina propria with gluten induces their activation and proliferation, with production of proinflammatory cytokines, metalloproteases, and keratinocyte growth factor by stromal cells, which induces cryptal hyperplasia and villous blunting secondary to intestinal epithelial cell death induced by intraepithelial lymphocytes (IELs) [ 58 ]. Additionally, there is an overexpression of membrane-bound IL-15 on enterocytes in active CD causing over-expression of the natural killer (NK) receptors CD94 and NKG2D by CD3 + IELs [ 59 ]. CD crypt hyperplasia has been hypothesized to be the consequence of an imbalance between continuous tissue damage due to the mucosal autoimmune insult described above and inability of the stem cells to compensate. We have recently provided a more mechanistic, evidence-based explanation for hyperplastic crypts in active CD by showing that the celiac hyperplastic crypt is characterized by an expansion of the immature progenitor cell compartment and downregulation of the Hedgehog signaling cascade [ 60 ]. These data shed light on the molecular mechanisms underlying CD histopathology and illuminate the reason for the lack of enteropathy in the mouse models for CD. Indeed, lack of consistent CD-like enteropathy in humanized mice [ 61 ] supports the concept that the accelerated disruption of enterocytes secondary to the adaptive CD4 + T cell insult cannot fully explain CD pathogenesis, supporting the notion that an intrinsic defect of the stem cell compartment in subjects at risk of CD is a key element of CD enteropathy [ 60 , 62 ].

The role of the gut microbiome in the pathogenesis of CD

In Western countries, a rise in the overall prevalence of CD has been well documented, but the reasons for this ‘epidemic’ remain elusive. The combination of epidemiological, clinical, and animal studies suggests that broad exposure to a wealth of commensal, non-pathogenic microorganisms early in life are associated with protection against CD and that pre-, peri-, and post-natal environmental factors may strongly influence the gut ecosystem [ 17 ]. Therefore, the hygiene hypothesis concept can be misleading, while an ‘environment-dependent dysbiosis hypothesis’ would more closely reflect the interplay between host and environmental pressure dictating the balance between health and disease. Several studies have shown an association between CD and a change in the microbiome composition [ 63 , 64 ]. However, these associative studies do not necessarily imply causation between microbiota composition and CD pathogenesis. Many environmental factors known to influence the composition of the intestinal microbiota are also thought to play a role in the development of CD [ 19 , 21 ].

It has been reported that, compared to control infants, neonates at family risk of CD had a decreased representation of Bacteriodetes and a higher abundance of Firmicutes [ 65 ]. This study also showed that infants who developed autoimmunity had decreased lactate signals in their stools coincident with a diminished representation in Lactobacillus species in their microbiome, which preceded the first detection of positive antibodies [ 65 ]. Early microbiota alterations in infants were also suggested in a recent study comparing microbial communities between DQ2 + and DQ2 − infants [ 66 ]. However, to move from association to causation, large-scale, longitudinal studies are necessary to define if and how gut microbiota composition and metabolomic profiles may influence the loss of gluten tolerance and subsequent onset of CD in genetically susceptible subjects.

Clinical presentation

CD is diagnosed more frequently in women with a female-to-male ratio ranging from 2:1 to 3:1 [ 1 , 2 ]. However, based on serological screening, the actual female-to-male ratio is 1.5:1 [ 67 ]. The disease can occur at any age from early childhood to the elderly, with two peaks of onset – one shortly after weaning with gluten in the first 2 years of life, and the other in the second or third decades of life. The diagnosis of CD can be challenging since symptoms can vary significantly from patient to patient [ 68 ].

In 2011, the Oslo classification of CD identified the following clinical presentations: classic, non-classic, subclinical, potential and refractory [ 69 ]. Instead of the ‘classic/non-classic’ categorization, which does not fully reflect current clinical presentations, in this review, we will use a more practical terminology, i.e., intestinal/extraintestinal. These two terms better represent the main clinical phenotypes of CD, which may occur individually (i.e., intestinal vs. extraintestinal) or in combination [ 70 ].

The intestinal form of CD is more commonly detected in the pediatric population and children younger than 3 years and is characterized by diarrhea, loss of appetite, abdominal distention, and failure to thrive [ 71 ]. Older children and adults may complain of diarrhea, bloating, constipation, abdominal pain, or weight loss [ 72 ]. Nonetheless, in adults, the malabsorption syndrome with chronic diarrhea, weight loss and significant asthenia is quite rare. Despite its uncommon detection, this phenotype can cause hospitalization due to cachexia, sarcopenia, significant hypoalbuminemia, and electrolyte abnormalities. Conversely, an irritable bowel syndrome (IBS)-like presentation with constipation or alternating bowel and/or dyspepsia-like symptoms, such as nausea and sometimes vomiting, is more frequent [ 2 ].

Extraintestinal symptoms are common in both children and adults [ 2 , 72 ]. They include iron deficiency microcytic anemia, detectable in up to 40% of cases (by cause of iron malabsorption or chronic inflammation) [ 73 ] or, more rarely, macrocytic anemia due to folic acid and/or vitamin B12 deficiency (more frequent in Europe than in the US). Changes in bone mineral density, including osteopenia or osteoporosis (affecting about 70% of patients at diagnosis), are related to altered absorption of calcium and vitamin D3 [ 74 ]. In children, growth retardation and short stature can raise the suspect of an underlying CD. Other signs include tooth enamel defects, aphthous stomatitis (identified in about 20% of undiagnosed CD patients) [ 75 ], and hypertransaminasemia (40–50% of untreated patients), which can be ascribed to food and bacterial antigen translocation reaching the liver due to increased intestinal permeability [ 76 ]. A wide array of neurological symptoms, such as headache, paresthesia, neuroinflammation, anxiety and depression, can be detectable in CD patients. The clinical presentation may also include changes in reproductive function characterized by late menarche, amenorrhea, recurrent miscarriages, premature birth, early menopause, and changes in the number and mobility of spermatozoa. Notably, these manifestations can be reversed when patients start a strict gluten-free diet (GFD), although fatigue and some neurological manifestation as well as functional gastrointestinal (GI) symptoms can persist for a long period in a subgroup of CD patients [ 2 , 77 , 78 , 79 , 80 , 81 ].

The subclinical form includes patients with symptoms/signs below the clinical identification threshold and are often recognizable only after the appreciation of the beneficial effects induced by the GFD. A typical example of subclinical cases are those patients undergoing antibody screening due to being relatives of CD patients or cases identified as a result of a screening strategy in the general population [ 2 , 69 ]. The prevalence of various CD clinical phenotypes observed in our experience is reported in Fig.  2 .

Prevalence of clinical phenotypes of adult celiac disease in our experience

CD can be associated with different autoimmune and idiopathic diseases, including dermatitis herpetiformis (which, as a single manifestation, should prompt testing for CD), type 1 diabetes mellitus, Hashimoto’s thyroiditis, selective IgA deficiency, alopecia areata, Addison’s disease, connective tissue diseases (mainly Sjogren’s syndrome), chromosomal diseases (Down, Turner, and William’s syndromes), neurological diseases (cerebellar ataxia, peripheral neuropathy, epilepsy with and without occipital calcifications), hepatic autoimmune diseases (primary biliary cholangitis, autoimmune hepatitis, primary sclerosing cholangitis), and idiopathic dilated cardiomyopathy (Table  2 ) [ 2 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 ]. The importance of diagnosing CD associated with these concomitant diseases is twofold since a GFD is able to resolve symptoms, prevent complications, and improve some of the CD associated diseases [ 2 ].

The potential form of CD is characterized by positive serological and genetic markers with a normal intestinal mucosa and minimal signs of inflammation such an increase in IELs [ 69 ]. Patients with the potential form can manifest with classic and non-classic symptoms or be entirely asymptomatic. The scientific community has not universally agreed on whether or not a GFD should be prescribed for patients with potential CD.

Finally, refractory CD (RCD) is characterized by persistent symptoms and atrophy of the intestinal villi after at least 12 months of a strict GFD. RCD can lead to complications such as ulcerative jejunoileitis, collagenous sprue, and intestinal lymphoma [ 69 ].

In recent years, other forms of CD (not included in the Oslo Classification [ 69 ]), i.e., seronegative and GFD non-responsive CD, have been identified in the clinical practice. The seronegative form is characterized by the lack of demonstrable serological markers along with clinical signs of severe malabsorption and atrophy of the intestinal mucosa [ 94 ]. This form should be included in the differential diagnosis with other diseases that cause atrophy of the intestinal villi. The term non-responsive CD indicates GI symptoms that persist despite a GFD of more than 12 months [ 95 ]; however, it does not differentiate between active CD and associated conditions, which can be responsible for symptom persistence (Fig.  3 ) and alternative terminology is discussed below.

Causes of ongoing signs and/or symptoms of celiac disease (CD) despite a gluten-free diet (formerly referred to as ‘non-responsive’ CD). In this review, two clinical phenotypes have been proposed – ongoing active celiac disease (OACD), related to three main causes, and associated celiac disease conditions (ACDC), encompassing a wide array of diseases

The gold standard for CD diagnosis is represented by the combination of mucosal changes detected by duodenal biopsy and by positivity of serological tests (anti-tTG antibodies, anti-endomysium antibodies (EmA), and deamidated gliadin peptide (DGP) antibodies). Despite the progress made in serology, no antibody test currently available provides a sensitivity and specificity of 100% (Table  3 ) [ 96 , 97 ], thus requiring intestinal biopsy as a key adjunct for establishing a correct diagnosis [ 98 ]. Pediatric patients with high titers (over 10 times the cut-off) of anti-tTG antibodies, detectable EmA, HLA-DQ2/HLA-DQ8 positivity, and signs/symptoms suggestive of CD may skip duodenal biopsy as recommended by recent guidelines by the European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) [ 99 ]. Although a large multicenter European study showed diagnostic accuracy of ESPGHAN criteria in identifying CD in children [ 100 ], it should be pointed out that these criteria are not followed worldwide. In fact, in some countries such as the USA, ESPGHAN criteria are not recommended because of the poor reproducibility of the anti-tTG assays [ 101 ]. Both advantages and disadvantages exist to biopsy for children with suspected celiac disease; however, most pediatric cases, especially those with low to medium anti-tTG2 titers, require histopathological assessment to confirm celiac disease diagnosis. In a recent study, Fuchs et al. [ 102 ] showed that the combination of anti-tTG (over 10 times the cut-off), EmA, and HLA-DQ2/HLA-DQ8 positivity (triple criteria) had a good accuracy across the range of pre-test probabilities in detecting adult patients with CD. Nonetheless, duodenal biopsy still represents a pillar in the diagnosis of adult patients with suspected CD.

Current standard of care is based on the “ four out of five rule ” [ 103 ], which indicates that four out of five of the following criteria are enough to establish CD diagnosis: (1) typical signs and symptoms (diarrhea and malabsorption); (2) antibody positivity; (3) HLA-DQ2 and/or HLA-DQ8 positivity; (4) intestinal damage (i.e., villous atrophy and minor lesions); and (5) clinical response to GFD. Additionally, this rule helps physicians to identify the various subtypes of CD, i.e., seronegative CD (absence of point 2), potential CD (absence of point 4), non-classic CD (absence of point 1), and non-responsive CD (absence of point 5).

Hematology and blood biochemistry tests

Routine blood tests can lead to suspect CD [ 104 ]. Low serum levels of hemoglobin, albumin, calcium, potassium, magnesium, and phosphorus are more commonly detected in CD with a classic rather than non-classic phenotype. Most patients develop an iron deficiency microcytic anemia with low ferritin values. Normocytic, macrocytic, or dimorphic anemia is less common in CD patients with an increased variability in the size of red blood cells due to concomitant malabsorption of folate and/or vitamin B12, particularly in cases associated with autoimmune atrophic gastritis [ 73 ]. Elevated levels of bone-specific alkaline phosphatase and a significant vitamin D3 deficiency can be found in patients with CD and osteopenia/osteoporosis [ 105 ]. A cryptogenic increase of transaminases may herald the presentation of CD even in the absence of other relevant symptoms. Notably, transaminases revert to normal within 6–12 months of a GFD [ 76 ]. In a moderate percentage of adult CD patients, a blood smear can detect changes in the membrane and cytoplasm of red blood cells (i.e., Howell–Jolly bodies), whereas pitted red cells can be identified by Nomarski phase contrast microscopy; both these red blood cell abnormalities suggest an underlying hyposplenism [ 106 ]. Another sign of hyposplenism is the detection of a marked thrombocytosis in association with a small (in the most severe cases even undetectable) spleen revealed by ultrasound. Macroscopically evident or even functional (no major changes at imaging) hyposplenism is a predisposing factor for the development of infectious diseases due to encapsulated bacteria (e.g., Pneumococcus, Meningococcus), and is associated with autoimmune diseases and complications such as refractory CD, ulcerative jejunoileitis, and lymphoma [ 107 , 108 ].

Over the last 20 years, the routine use of serological tests led to a significant increase in CD diagnoses. CD-related antibodies can identify subjects with suspected CD, further confirmed by histological evaluation [ 98 ]. In the early 1980s, anti-gliadin antibodies were the first serological marker used to screen patients at risk for CD. However, due to their low specificity, this serological test has been dismissed and its role is now confined to the possible identification of a subset of cases with non-celiac gluten/wheat sensitivity [ 109 ]. Currently, the serological diagnosis of CD is based on tests that are highly predictive and widely validated, including EmA, anti-tTG, and DGP [ 97 ]. CD-related antibodies belong to IgA and IgG classes, but only those of IgA class can be regarded as highly sensitive and specific for CD [ 97 ]. The use of IgG markers (except for DGP) is often misleading due to the high percentage of false positives, and their use should be limited to patients with IgA deficiency [ 110 ]. EmA is the antibody test with the highest diagnostic accuracy since it offers an absolute specificity if tested in third-level laboratories by expert operators [ 111 , 112 ]. The sensitivity of anti-tTG IgA is higher than that of EmA IgA (97% vs. 94%), while the specificity of tTG IgA is certainly lower than that of EmA (91 and 99%, respectively) (Table  3 ) [ 96 ]. False positives for anti-tTG normally display a low antibody titer (less than twice the cut off). A transient positivity for anti-tTG IgA, not associated with duodenal mucosal damage, has been reported in patients with type 1 diabetes at onset followed by a subsequent disappearance of antibodies within 6 months of their identification [ 113 ].

Another serological marker for CD is represented by DGP [ 96 ]. Compared to native peptides, the deamidation of gliadin by tTG makes the modified gliadin peptides more immunogenic. Initial studies reported an elevated sensitivity and specificity for CD [ 96 ], although other data showed a decrease in diagnostic accuracy [ 114 ]. IgG DGP are particularly useful in identifying CD in early childhood (age < 2 years) [ 115 ]. IgA DGP have been shown to be of little usefulness in diagnosing CD and therefore are not recommended for diagnosis [ 97 ]. In adult CD, serology should include testing anti-tTG IgA along with total IgA. Should anti-tTG IgA be positive at a high titer with normal total IgA level, a duodenal biopsy can be performed without assessing EmA. With a low titer anti-tTG IgA, EmA IgA testing is necessary and, if positive, a duodenal biopsy should be recommended to confirm CD diagnosis (Fig.  4 ).

Diagnostic algorithm for celiac disease diagnosis

Strict compliance with a GFD in most CD patients leads to the disappearance or significant decrease of antibodies within 12 months (18–24 months if the antibody titer is very high) together with regrowth of the intestinal villi. IgA anti-tTG antibodies are the most commonly used test to monitor CD patients during follow-up, although their disappearance does not reflect the regrowth of intestinal villi [ 97 , 116 ]. Recent data from Choung et al. [ 117 ] demonstrated a very high specificity and sensibility of a new assay directed to identify the serum immune response to epitopes of the tTG-DGP complex. In addition to diagnosis, such markers can be useful for follow-up purposes, although further studies are eagerly needed. While waiting for the validation of a tTG–DGP complex assay, current serology is not enough for evaluating the response to GFD and the regrowth of villi [ 118 , 119 ].

Duodenal biopsy

Morphological evaluation of the duodenal biopsy is still of critical importance for confirming CD diagnosis. Histology remains the ‘gold standard’ for CD diagnosis [ 94 ]. In recent years, however, the histological criteria for CD have radically changed with the inclusion of mild villous atrophy and minimal lesions (characterized by an isolated increase in IELs) as possible expression of gluten-related intestinal damage [ 120 , 121 ]. Current recommendations are for four biopsies on the second duodenal portion and two biopsies at the bulb [ 122 ]. A fundamental principle for the correct evaluation is the orientation of biopsy samples using cellulose acetate Millipore filters [ 123 , 124 ]. The different types of CD-related lesions of the intestinal mucosa can be categorized into five stages according to the Marsh classification, modified by Oberhüber, which is currently used in all reference centers for the diagnosis of CD [ 120 ]. Type 1 and type 2 lesions, characterized by an increase in IELs (with or without crypt hyperplasia) and normal villi, compatible with, but non-specific for CD. Together with positive anti-tTG and EmA, minimal intestinal lesions indicate potential CD. In most cases, minimal lesions are attributable to other causes, including food allergies (e.g., cow milk proteins), Crohn’s disease, lymphocytic colitis, bacterial and parasitic intestinal infections, such as Giardia , common variable immunodeficiency, small intestinal bacterial overgrowth, non-steroidal anti-inflammatory drugs, and Helicobacter pylori infection (Box 1) [ 125 , 126 , 127 ].

In recent years, there has been a worrying increase in the number of diagnoses of CD incorrectly based on minimal lesions with no genetic and serological markers [ 128 ]. The IEL cytometric pattern is more accurate than subepithelial deposits of anti-TG2 IgA for identifying CD in lymphocytic enteritis [ 129 ]. The normal IEL cut-off has been established to be ≥25 lymphocytes over 100 epithelial cells. Even if it is well established that coeliac patients always display IEL counts ≥25%, a recent paper stressed the importance of a high IEL count for CD diagnosis underlining that the mean IEL count in untreated CD was 54 ± 18/100 enterocytes, whereas in non-CD patients the value was 13 ± 8 [ 130 ]. The typical lesion of CD shows villous atrophy with a change in the villi-to-crypt ratio (< 3:1 to 1:1) and an increase in IEL. This lesion, defined as type 3 in the Marsh–Oberhüber classification, is in turn subdivided into three stages depending on the severity of the atrophy, namely mild (3a), partial (3b), and subtotal atrophy (3c) [ 120 ]. Recently, Marsh et al. [ 131 , 132 ] argued against Oberhüber’s lesion III sub-division, claiming that splitting intestinal atrophy in three stages can be clinically irrelevant and sometimes misleading. In line with this theory no significant difference in IEL count was observed in mild, partial, and subtotal villous atrophy [ 130 ]. In an attempt to simplify the histopathological grading and therefore the relationship between pathologists and clinicians, Corazza and Villanacci proposed a classification from five to three stages [ 121 ]. Notably, the lesions that characterize CD were divided into two categories – non-atrophic (grade A) and atrophic (grade B) – with the latter being further subcategorized into B1, in which the villi-to-crypt ratio is less than 3:1 (with identifiable villi), and B2, in which villi are entirely atrophic. Grade A lesions, characterized by a pathological increase in the number of IELs, better identified by immunohistochemical staining for CD3, include type 1 and 2 lesions based on the Marsh–Oberhüber classification; grade B1 lesions include the 3a and 3b lesions, while grade B2 corresponds to 3c (Fig.  5 ) [ 121 ]. In some patients with more distal disease or in those with contraindication to biopsy, videocapsule endoscopy can be recommended [ 133 ].

Comparison between the two classifications for the duodenal biopsy

Classification of variants of CD

Potential cd.

In recent years, an increasing number of patients have antibody positivity (IgA EmA and anti-tTG) for CD with HLA-DQ2/HLA-DQ8 and lack of villous atrophy [ 134 , 135 ]. For this category of patients, which represents around 10% of subjects with CD, the term potential celiac disease has been adopted [ 69 ]. In patients with potential CD the intestinal mucosa may be normal (Marsh 0) or slightly inflamed (increased number of IELs, i.e., Marsh 1) [ 135 ]. Despite the absence of severe lesions in the intestinal mucosa, these patients may have GI and/or extraintestinal symptoms or be entirely asymptomatic [ 2 , 135 ]. Although the criteria for diagnosing this condition are clear, potential CD still remains a poorly studied area, with many unsettled questions and contrasting results in the studies conducted so far [ 135 , 136 , 137 , 138 , 139 , 140 , 141 ]. In children, over 80% of patients with potential CD are asymptomatic and the remaining 20% more commonly experience intestinal symptoms such as malabsorption, chronic diarrhea, and recurrent abdominal pain rather than extraintestinal signs such as iron-deficiency anemia, hypertransaminasemia, and short stature [ 137 , 138 , 141 ]. In adults, however, several studies have shown that the symptomatic phenotype in subjects with potential CD is much more common than in children, and it is primarily characterized by extraintestinal symptoms [ 135 , 136 , 139 , 140 ]. One controversial issue concerns whether subjects with potential CD should be treated by a GFD. The actual evidence suggests that a GFD should be recommended only to subjects with symptomatic potential CD. On the other hand, patients with asymptomatic potential CD are allowed to continue a gluten-containing diet while being followed-up with close clinical, serological, and histological control visits (in our experience every 6 months) [ 135 , 136 , 137 , 138 , 139 , 140 ]. Studies have reported possible fluctuation with spontaneous normalization of serological markers in patients with potential CD left on a gluten-containing diet. Few patients with potential CD consuming a gluten-containing diet develop full-blown villous atrophy [ 135 , 137 , 138 , 140 , 142 ]. In our study, only 6% of these subjects developed villous atrophy over a mean follow-up period of 3 years, whereas symptomatic subjects should be treated as they show a clear clinical improvement in symptoms with a GFD [ 135 ].

Seronegative CD

Although the specific antibodies for CD can be detected in the vast majority of patients, a small number of CD patients (around 2–3%) test negative for serological markers. In these cases, the diagnosis is closely connected to the detection of villous atrophy on the duodenal histology [ 94 , 139 , 143 ]. Performing a genetic test for CD remains a fundamental step since its negative result definitively rules out the disease and prompts physicians to seek for other causes of villous atrophy. A seronegative CD can be confirmed 1 year after the beginning of a GFD, a convenient time to demonstrate an improvement in both symptoms and histology. The diagnostic complexity of this particular variant of CD is due to the differential diagnosis with other conditions involving villous atrophy, such as parasitic infections ( Giardia lamblia ), autoimmune enteropathy, bacterial contamination of the small intestine, common variable immunodeficiency, eosinophilic gastroenteritis, drug-induced enteropathy (angiotensin II receptor antagonists, i.e., olmesartan and other sartans, non-steroidal anti-inflammatory drugs, and mycophenolate), intestinal lymphoma, Crohn’s disease, tropical sprue, HIV enteropathy, and Whipple disease (Fig.  6 ) [ 94 , 144 , 145 ]. Of all villous atrophies lacking CD antibodies, 28–45% are due to an underlying seronegative CD [ 94 , 146 , 147 ]. Seronegative CD patients display a classic clinical phenotype, characterized by diarrhea and malabsorption, a clear female gender prevalence, and have a higher risk of morbidity and mortality compared with antibody-positive CD patients [ 94 , 147 ]. Furthermore, compared to classic CD, seronegative patients have a greater association with autoimmune diseases and a higher risk of developing refractory disease. This increased morbidity could be partly due to the late diagnosis of this condition, which on average is around 50 years of age [ 94 ].

Diagnostic algorithm for seronegative villous atrophy. SIBO small intestinal bacterial overgrowth

Assessment of ongoing signs and symptoms in CD

The majority of the patients with CD exhibit a symptomatic and mucosal response to the GFD. Some patients, however, fail to have complete control of symptoms and normalization of villous structure despite attempted adherence to the GFD. These patients have traditionally been referred to as non-responsive CD [ 95 , 148 ]; however, this terminology has resulted in confusion as, in many cases, manifestations are due to associated conditions rather than CD. In light of both emerging tests for CD monitoring, such as GIPs, and emerging novel therapies for active CD, we propose updating this classification (formerly non-responsive CD). When evaluating a patient with CD on a GFD and with ongoing signs or symptoms, the initial step is the differentiation between ongoing active CD (OACD) and the presence of associated CD conditions (ACDCs). OACD can be seen in three scenarios – (1) slow response, where there is progressive improvement in symptoms and mucosal damage, but full remission does not occur for at least 1–2 years; (2) RCD, where there is ongoing severe enteropathy and malabsorptive symptoms after 6–12 months on a GFD; and (3) gluten exposure, where, despite adequate understanding of the GFD and attempted adherence, gluten avoidance is insufficient to result in symptomatic or histologic remission. This is the most frequent cause of OACD and can be due to very high sensitivity to a low level of gluten exposure or an inability of the patient to achieve standard recommended gluten restriction. Conversely, when patients with ongoing symptoms are found not to have OACD, generally when small bowel assessment shows minimal ongoing enteropathy and significant gluten exposure is excluded, investigation of possible ACDCs is recommended. ACDCs include IBS, small intestinal bacterial overgrowth, microscopic colitis, lactose intolerance, fructose intolerance, diverticular disease, Crohn’s disease, pancreatic insufficiency, and autoimmune and drug-induced enteropathy, and should be evaluated according to clinical suspicion (Fig.  3 ) [ 95 , 148 ].

CD complications

It has been widely shown that a late diagnosis of CD (after the age of 50) and/or not following a strict GFD can lead to a higher mortality compared to that of the general population [ 149 ]. Although rare (around 1% of patients diagnosed with CD) [ 150 ], the complications of CD include hyposplenism, RCD, intestinal lymphoma, small bowel adenocarcinoma, and ulcerative jejunoileitis. Complications should be suspected in all patients who, despite adherence to a GFD, complain of an unexplained persistence or re-exacerbation of symptoms (i.e., diarrhea, intestinal sub-occlusion, abdominal pain, weight loss, fever, and severe asthenia). These complications occur more commonly when a diagnosis of CD was established in elderly patients and/or in those who are homozygous for DQ2 not observing a strict GFD [ 151 ].

Hyposplenism

Anatomical or functional hyposplenism can be identified in around 30% of adult patients with CD, with prevalence increasing up to 80% in patients with complications [ 107 , 152 ]. In CD cases, the detection of a small-size spleen on abdominal ultrasound should guide physicians to confirm functional hyposplenism by evaluating Howell–Jolly bodies (on a peripheral blood smear) or pitted red cells with phase-contrast microscopy (see above) [ 107 , 152 ]. Splenic hypofunction is closely associated not only with the development of complications and other autoimmune diseases associated with CD but also encapsulated bacterial infections (i.e., Pneumococcus , Haemophilus influenzae , Meningococcus ) [ 107 ]. Because of the greater risk of developing infections (in some cases lethal or with severe sequelae) from encapsulated bacteria, anti-pneumococcal and anti-meningococcal vaccinations are recommended in this subgroup of patients [ 106 , 107 , 152 ].

Refractory CD

RCD represents about 10% of all OACD cases [ 148 ] and approximately 1–1.5% of total cases of CD [ 153 ]. This condition is characterized by symptoms of malabsorption, weight loss, and diarrhea associated with persistent villous atrophy after at least 1 year on a strict GFD, confirmed by negative CD serology [ 69 ]. Before thinking of RCD, physicians should rule out other more frequent causes of ongoing signs and symptoms of CD, as previously reported [ 95 , 148 ]. Refractory CD is in turn subdivided into two categories, primary and secondary, depending on whether the patients had a symptomatic response since the beginning of GFD, or they had a recurrence of symptoms after a more or less long period of improvement.

There are two subtypes of RCD – type 1, where the IEL population has a normal CD3 + CD8 + phenotype, and type 2, with a clonal presentation of surface CD3 − /intracytoplasmic CD3 + IELs along with monoclonal rearrangement of the gamma-chain of the T cell receptor [ 153 ]. This distinction into two subtypes is fundamental for therapeutic management and prognosis; in fact, type 2 displays a 5-year mortality rate of 55% vs. 7% for type 1 [ 154 ]. The mortality of patients with type 2 RCD is primarily due to the development of intestinal lymphoma, which appears to occur more often in male patients, although CD is more commonly detectable in female patients (female-to-male ratio 3:1) [ 155 ]. A diagnosis of RCD should always be suspected by persistent villous atrophy despite a strict, 1-year GFD, negative serology (some cases may show the persistence of low-titer CD-related antibodies), the exclusion of other causes of persistent villous atrophy, and phenotyping of the intestinal lymphocytic population aimed to confirm the presence (type 2) or absence (type 1) of a monoclonal rearrangement of T cell receptor. In all cases of type 2 RCD, it is essential to perform, at diagnosis, a computed tomography (CT) and/or magnetic resonance (MR) enterography followed by positron emission tomography (PET), capsule endoscopy, and enteroscopy in order to rule out the progression to intestinal lymphoma [ 152 , 154 ]. Due to this risk, in subjects with a diagnosis of type 2 RCD, a capsule endoscopy has been recommended once a year at the follow-up [ 156 ]. From a therapeutic perspective, the management of type 1 RCD is based on immunosuppressive therapy containing steroids, azathioprine, 6-mercaptopurine, and methotrexate, whereas type 2 therapy is based on additional medications, including cyclosporine and chemotherapy such as cladribine and fludarabine associated with anti-CD52 monoclonal antibodies (alemtuzumab). Promising results have been recently reported by treating patients with anti-IL-15 antibodies (AMG-714). In certain cases, an autologous stem cell transplantation has been attempted with promising results [ 154 , 155 , 156 ].

Intestinal lymphoma

The association between CD and cancers has been known for over 50 years [ 157 ] and a delayed diagnosis of CD exposes patients to an increased risk of developing neoplastic diseases [ 158 ]. In recent years, several studies have reported a growing incidence from 6 to 9 times higher than that of the general population for non-Hodgkin T cell intestinal lymphoma and, to a lesser extent, also B cell lymphoma [ 158 ]. In most cases, the development of intestinal lymphoma is preceded by type 2 RCD that develops into malignant disease in 33–52% of cases within 5 years from diagnosis. More rarely, intestinal lymphoma may develop from type 1 RCD, with a rate of 14% over 5 years [ 159 ]. Treatment in cases of CD-related intestinal lymphoma involves chemotherapy, i.e., high-dose ifosfamide, epirubicin, and etoposide methotrexate, followed by autologous stem cell transplantation. If lymphoma includes an elevated expression of CD30 (> 80% of the neoplasm) it is possible to use biologic therapy with anti-CD30 associated with monomethyl auristatin E (brentuximab vedotin) and a chemotherapy regimen containing cyclophosphamide–doxorubicin–prednisone followed by autologous stem cell transplantation [ 159 ]. Recent data indicate that NKp46, a NK receptor expressed by lymphocytes, can be a biomarker as well as a possible therapeutic target for T cell lymphoproliferative diseases, i.e., type 2 RCD and enteropathy-associated T cell lymphoma [ 160 ].

Small bowel adenocarcinoma

Small bowel adenocarcinoma is an extremely rare cancer in the general population (5.7 cases/1,000,000 people per year) but it is much more common in patients with CD (odds ratio reported in the literature ranges between 4.3 to 60.0), usually being detectable in the jejunum [ 161 ]. Compared to lymphomas, small bowel adenocarcinoma is rare, although increasingly detectable in the clinic. Nowadays, however, the diagnosis of this cancer occurs together with CD. Unlike intestinal lymphoma, the small bowel adenocarcinoma is not preceded by RCD and occurs more commonly in female patients [ 150 ]. The onset of a sudden intestinal (sub)/occlusion and/or anemia, particularly in patients with a late diagnosis of CD and patients who have been following a GFD for a short period of time, are clinical features suggestive of an underlying small bowel adenocarcinoma. A thorough diagnostic work-up is mandatory and requires a wide array of imaging tests (e.g., CT/MR-enterography, PET, capsule endoscopy, and enteroscopy) [ 162 ].

Follow-up for CD in adults

A well-defined follow-up strategy should be agreed by physicians and patients once CD has been diagnosed. Usually, the first follow-up visit is planned within 6 months from diagnosis and then every 12–24 months (every 3–6 months if complications occur) is adequate to confirm compliance with the GFD, rule out the onset of autoimmune diseases and metabolic changes, and, most importantly, to allow for the early diagnosis of any complications [ 163 ]. Patients should undergo a consultation with a dietician and follow-up blood tests including complete blood count, anti-tTG IgA (or IgG in case of IgA deficiency), thyroid stimulating hormone, anti-thyroidperoxidase, anti-thyroglobulin, ferritin, folate, vitamin D3, transaminases, and a metabolic profile [ 163 ]. The first follow-up should include a screening of antinuclear antibodies and non-organ-specific autoantibodies in order to rule out the presence of markers predictive of autoimmune diseases associated with CD. Should the antinuclear antibodies test reveal a high titer along with extractable nuclear antigen antibody positivity, this information might be useful to investigate for other autoimmune CD-associated disorders, e.g., primary biliary cholangitis and Sjogren syndrome [ 2 ]. In adults, a bone density scan should be performed after 12–18 months of a GFD and repeated regularly only if abnormal or in case of other indications. Subjects with osteopenia should be treated with supplements containing calcium and vitamin D, while possible treatment with bisphosphonates should be considered in cases of osteoporosis. Body weight increase may occur as a consequence of an excessive consumption of dietary products high in vegetable fats (colza, palm, and coconut oil) commonly present in GFD [ 164 ]. Therefore, nutritional counselling is advisable to prevent metabolic complications, including liver steatosis, during follow-up. On the other hand, patients who are starting GFD should be tested with an abdominal ultrasound to exclude spleen abnormality (i.e., hyposplenism) [ 165 ].

Notwithstanding a strict GFD, CD patients may experience abdominal symptoms ascribable to IBS in 30–50% of cases; these symptoms may respond to dietary recommendations (e.g., reduction of insoluble fiber intake or fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) as well as symptomatic drug therapy [ 166 ].

A self-adapted GFD, without the support of a nutritionist, can cause vitamin and trace metal deficiency, which should be supplemented if needed, particularly when patients report the onset of asthenia [ 167 ]. Additionally, constipation, which can be associated with a GFD, requires appropriate management based on non-irritant (e.g., osmotic) laxatives [ 168 ].

Should a CD-related complication occur, follow-up visits should be more frequent, i.e., every 3–6 months [ 156 ]. In these circumstances, in addition to standard tests (as previously listed), protein electrophoresis, lactate dehydrogenase, and beta-2 microglobulin testing should be included. Upper endoscopy should be performed (with new duodenal biopsies) along with abdominal ultrasound, as well as CT/MR-enterography, PET, capsule endoscopy, and enteroscopy [ 154 , 155 , 156 ].

Physicians may consider (even if not recommended by current guidelines) performing a follow-up duodenal biopsy in adults in order to check the regrowth of villi in patients on a GFD, keeping in mind that the average time to the restitutio ad integrum of the villi could take up to 3 years. A second duodenal biopsy after GFD should be recommended only in those patients with persisting symptoms and demonstrable laboratory deficiencies of micronutrients [ 133 ].

Finally, GIP assessment, a controversial test still awaiting further validation, can be performed on stool samples and may be useful for monitoring the adherence to a GFD [ 48 ].

Follow-up for CD in children

Currently, the follow-up of CD in children is lacking standardized evidence-based recommendations [ 169 ]. Children with CD should be followed up after 6 months from diagnosis and then every year in order to check symptomatic improvement, adherence to GFD, quality of life, and progressive normalization of CD-related antibodies. Laboratory tests and biochemical evaluation is crucial in these patients and should be tailored on case-by-case basis. As for adults, autoimmune thyroiditis should always be screened. Duodenal biopsy monitoring is unnecessary after a GFD has been instituted. However, should the patient have no or partial clinical response to gluten withdrawal, a careful assessment should be recommended to rule out inadvertent gluten ingestion or poor adherence to a GFD. Furthermore, in this subset of poorly responsive patients, a duodenal histopathology is advisable [ 119 , 169 ]. At variance to adults, children hardly ever develop complications, indeed only a few case reports of refractory CD have been reported [ 170 ].

Diet and new treatments

Currently, the only effective treatment available for CD is a strict GFD for life since it leads to the resolution of intestinal and extraintestinal symptoms, negativity of autoantibodies, and the regrowth of the intestinal villi. In addition, the diet offers a partial protective effect towards several complications. However, these crucial advantages are accompanied by some disadvantages, including a negative impact on quality of life, psychological problems, fear of involuntary/inadvertent contamination with gluten (as demonstrated in multicenter GIP studies) [ 48 ], possible vitamin and mineral deficiencies, metabolic syndrome, an increased cardiovascular risk, and often severe constipation [ 171 , 172 , 173 ]. Most of these CD-related drawbacks can be overcome by instructing the patient about the risks of an uncontrolled gluten-free regimen and by providing nutritional recommendations by a dietician with experience in CD. From a psychological perspective, the support a psychologist could be highly useful in accepting the disease [ 174 ].

Due to the relevant burden induced by gluten withdrawal with consequent worsening of quality of life, about 40% of CD patients are unsatisfied with their alimentary regimen and they would be keen to explore alternative treatments [ 175 ]. In recent years, researchers have attempted to meet the requests of CD patients seeking therapies different from diet [ 176 ]. Clinical trials are currently in progress, but only few have reached later clinical trial phases, namely those with larazotide acetate and gluten-specific proteases from a bacterial mix (ALV003) [ 177 , 178 , 179 , 180 ]. Larazotide acetate is a zonulin antagonist blocking tight junction disassembly, thereby limiting gluten crossing a permeable intestinal mucosal barrier [ 177 ]. Larazotide has shown efficacy in gluten-related symptom control rather than in restoring complete epithelial barrier integrity and preventing gluten from crossing the mucosal lining [ 177 ]. Taken together, the data so far published indicate that larazotide may be beneficial in allowing patients to tolerate minimal amounts of gluten such as those derived from inadvertent ingestion or probably for ‘gluten-free holidays’, i.e., a short period during which patients are allowed to eat a minimal amount of gluten. ALV003 targets gluten and degrades it into small fragments in the stomach before they pass into the duodenum [ 178 ]. This strategy has also been demonstrated to be able to ‘digest’ only small quantities of gluten and thus would be effective against contamination but not to protect patients from the effects driven by large quantities of gluten [ 178 ]. However, a recent phase 2b study by Murray et al. [ 180 ] showed that ALV003 (or latiglutenase) did not improve histologic and symptoms scores in 494 CD patients with moderate to severe symptoms versus placebo. IL-15 monoclonal antibodies (AMG 714) are being investigated in phase 2 studies in both gluten challenge and RCD type II patients, but additional safety studies are needed for the acquisition and competition of the license. Finally, vaccination (Nexvax2) is another possible therapeutic strategy aimed at desensitizing patients with CD to gliadin peptides. Although abdominal pain and vomiting were major side effects, the trial passed phase 1. Vaccines could represent a definitive cure for CD should data show actual efficacy [ 181 ].

Can CD be prevented?

Several retrospective studies have suggested that breastfeeding, modality of delivery, and time of gluten introduction in the diet of infants at risk for CD may affect the incidence of the disease. However, the data supporting the role of these factors in the risk of developing CD is limited by their retrospective design and have been criticized by alternative interpretations [ 182 , 183 , 184 ]. Two recent landmark studies [ 19 , 21 ], which prospectively screened infants with a first-degree family member with CD from birth, found that CD develops quite early in life in this risk group, demonstrating that early environmental factors may be crucial in the development of CD. However, these studies failed to identify possible targets to prevent CD, leading to the gut microbiota as the key element to scrutinize for possible innovative preventive strategies. In this line, viral (e.g., rotavirus) GI infections may potentiate subsequent development of CD. Thus, rotavirus vaccination seems to significantly decrease the risk of CD, in particular among children with early (before 6 months of age) gluten exposure [ 185 ]. The ongoing Celiac Disease Genomic, Environment, Microbiome, and Metabolomic study has been designed to identify potential primary prevention targets by establishing microbiome, metabolomic, and/or environmental factors responsible for loss of gluten tolerance, thus switching genetic predisposition to clinical outcome [ 186 ].

Although there has been a substantial increase in the number of CD diagnoses over the last 30 years, many patients remain undiagnosed [ 187 ]. The flow-chart for identifying CD in adults must always include both serology and intestinal biopsy, whereas genetics should be performed only in selected cases. Diagnostic criteria should help physicians in avoiding misdiagnosis and missing cases of CD (i.e., seronegative patients with classic symptoms not undergoing biopsy) and preserve people from an unjustified GFD. The treatment for CD is still primarily a GFD, which requires significant patient education, motivation, and follow-up. Slow response occurs frequently, particularly in people diagnosed in adulthood. Persistent or recurring symptoms should lead to a review of the patient’s original diagnosis, exclude alternative diagnoses, evaluation of GFD quality, and serologic testing as well as histological assessment in order to monitor disease activity. In addition, evaluation for disorders that could cause persistent symptoms and complications of CD, such as refractory CD or lymphoma, should be pursued. The future opens to new therapeutic and preventive strategies, which are expected to improve the patient’s quality of life and pave the way to a definitive cure for this old disease.

Box 1 Causes for the increased number of intraepithelial lymphocytes in the intestinal mucosa with normal villous architecture

Potential celiac disease

Non-celiac gluten sensitivity

Food allergies (cereals, milk proteins, soy derivatives, fish, rice, chicken)

Infectious (viral enteritis, Giardia, Cryptosporidium, Helicobacter pylori )

Bacterial contamination of the small intestine

Drugs (e.g., non-steroidal anti-inflammatory drugs)

Immune system diseases (Hashimoto’s thyroiditis, rheumatoid arthritis, systemic erythematosus lupus, type 1 diabetes mellitus, autoimmune enteropathy)

Common variable immune deficiency

Chronic inflammatory intestinal diseases (Crohn’s disease, ulcerative colitis)

Lymphocytic colitis

Availability of data and materials

Abbreviations.

Associated celiac disease conditions

Celiac disease

Computed tomography

Deamidated gliadin peptides antibodies

Anti-endomysial antibodies

European Society for Paediatric Gastroenterology Hepatology and Nutrition

• Gluten-free diet

Gastrointestinal

Gluten immunogenic peptides

Human leukocyte antigen

Irritable bowel syndrome

Intraepithelial lymphocytes

Interleukin

Magnetic resonance

Natural killer

Ongoing active celiac disease

Positron emission tomography

Refractory celiac disease

Transglutaminase 2

tissue transglutaminase

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This work was partially supported by the Fondo Incentivazione Ricerca (FIR) to R DeG, Fondi Ateneo per la Ricerca (FAR) to GC and RDe G (from University of Ferrara) and by the National Institute of Health grant R01DK104344 to AF.

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Giacomo Caio and Umberto Volta these authors share co-first authorship.

Carlo Catassi and Alessio Fasano these authors share co-last authorship.

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Department of Medical Sciences, University of Ferrara, Via Aldo Moro 8, Cona, 44124, Ferrara, Italy

Giacomo Caio & Roberto De Giorgio

Center for Celiac Research and Treatment, Massachusetts General Hospital, Boston, MA, 02114, USA

Giacomo Caio, Anna Sapone, Carlo Catassi & Alessio Fasano

Department of Medical and Surgical Sciences, University of Bologna, 40138, Bologna, Italy

Umberto Volta

Takeda Pharmaceuticals International Co, Cambridge, MA, 02139, USA

Anna Sapone & Daniel A. Leffler

Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, MA, 02115, USA

Daniel A. Leffler

Department of Pediatrics, Center for Celiac Research, Università Politecnica delle Marche, 60121, Ancona, Italy

Carlo Catassi

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Wrote the first draft of the manuscript: GC. Writing, correction and addition of fundamental insights to the manuscript: GC, UV, AS, DL, RDeG, CC, AF. All authors read and approved the final manuscript.

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Caio, G., Volta, U., Sapone, A. et al. Celiac disease: a comprehensive current review. BMC Med 17 , 142 (2019). https://doi.org/10.1186/s12916-019-1380-z

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DOI : https://doi.org/10.1186/s12916-019-1380-z

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Celiac Disease Center Research Studies

The University of Chicago Medicine Celiac Disease Center is leading the charge toward a cure for celiac disease. And we are closer than ever. Our world-renowned celiac disease experts are studying this complex autoimmune disorder from every angle to find new ways to understand, treat, and cure it.

Adult Celiac Disease Research

Our experts conduct research on a variety of topics related to celiac disease in adults.

The ILLUMINATE-062  study is looking at the effectiveness of an investigational medication designed to break down gluten in the stomach in those with celiac disease who are on a gluten-free diet. The study will evaluate how well the investigational medication reduces celiac-related symptoms and intestinal damage due to gluten exposure.

One study involves analyzing NIDDK T-cell assays . By thoroughly evaluating the immune response to gluten, we are looking to identify the cells in the small intestine and blood responsible for inflammation seen from celiac disease.

Our scientists are researching brain fog through fMRI and cognitive to test the mental impacts after gluten exposure for those with the disease.

We are also leading a longitudinal RC2 study on the healing nature of the small intestine following gluten exposure for people with celiac disease.

Pediatric Celiac Disease Research

Our pediatric celiac disease team is at the forefront of numerous studies for celiac disease in children. This includes research in areas such as:

• Celiac antibodies , and determining which antibody is the best predictor for monitoring adherence to the gluten-free diet.
• Gluten transfer from shared kitchen equipment to gluten-free food , and developing recommendations for celiac disease patients on how to avoid cross-contact with gluten.
• Video capsule endoscopy (VCE) , and evaluating the efficacy of VCE for the diagnosis of celiac disease in children.
• A gluten exposure risk assessment questionnaire , and creating a standardized tool to be used in a variety of settings for patients with celiac disease and who are following a gluten-free diet.
• A Celiac database , and collaborating with other celiac centers to examine features of our patient population and to greatly contribute to our current understanding of this disease.
• The UChicago celiac disease and type 1 diabetes population , and determining how risk factors and outcomes for these diseases vary between racial groups.
• The financial burden of a gluten-free diet , and defining the increased cost and psychosocial impact accrued by celiac patients and families treated with the diet.
• Pediatric brain fog , and studying children recently diagnosed with celiac disease to monitor neurological impacts before and after a gluten-free diet.

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Diagnosis and Management of Celiac Disease

• 1 Division of Gastroenterology and Hepatology, University of Wisconsin, Madison, Wisconsin
• 2 Department of Medicine, University of Chicago, Chicago, Illinois

Celiac disease is an immune-mediated response to gluten, a protein found in wheat, barley, and rye that affects approximately 1% of the US population. The hallmark of celiac disease is injury to the small bowel mucosa that causes villous atrophy and results in malabsorption of micronutrients, fat-soluble vitamins, iron, vitamin B 12 , and folic acid. Common signs and symptoms of celiac disease include diarrhea, abdominal bloating, abdominal discomfort, and constipation. Celiac disease is also associated with extraintestinal manifestations such as fatigue, weight loss, dermatitis herpetiformis, iron deficiency, and osteoporosis. 1 This JAMA Clinical Guidelines Synopsis focuses on the 2023 American College of Gastroenterology guidelines update on diagnosis and management of celiac disease.

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Austin K , Deiss-Yehiely N , Alexander JT. Diagnosis and Management of Celiac Disease. JAMA. Published online June 26, 2024. doi:10.1001/jama.2024.5883

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• v.28(1); 2022 Jan 7

Current guidelines for the management of celiac disease: A systematic review with comparative analysis

Alberto raiteri.

Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy

Alessandro Granito

Alice giamperoli, teresa catenaro, giulia negrini, francesco tovoli.

Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy. [email protected]

Corresponding author: Francesco Tovoli, MD, Assistant Professor, Research Fellow, Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, via Albertoni 15, Bologna 40138, Italy. [email protected]

Wheat and other gluten-containing grains are widely consumed, providing approximately 50% of the caloric intake in both industrialised and developing countries. The widespread diffusion of gluten-containing diets has rapidly led to a sharp increase in celiac disease prevalence. This condition was thought to be very rare outside Europe and relatively ignored by health professionals and the global media. However, in recent years, the discovery of important diagnostic and pathogenic milestones has led to the emergence of celiac disease (CD) from obscurity to global prominence. These modifications have prompted experts worldwide to identify effective strategies for the diagnosis and follow-up of CD. Different scientific societies, mainly from Europe and America, have proposed guidelines based on CD's most recent evidence.

To identify the most recent scientific guidelines on CD, aiming to find and critically analyse the main differences.

We performed a database search on PubMed selecting papers published between January 2010 and January 2021 in the English language. PubMed was lastly accessed on 1 March 2021.

We distinguished guidelines from 7 different scientific societies whose reputation is worldwide recognized and representative of the clinical practice in different geographical regions. Differences were noted in the possibility of a no-biopsy diagnosis, HLA testing, follow-up protocols, and procedures.

We found a relatively high concordance between the guidelines for CD. Important modifications have occurred in the last years, especially about the possibility of a no-biopsy diagnosis in children. Other modifications are expected in the next future and will probably involve the extension of the non-invasive diagnosis to the adult population and the follow-up modalities.

Core Tip: Once considered a rare condition, celiac disease (CD) is becoming a significant health issue globally. An increasing number of studies have investigated this condition. International scientific societies have proposed guidelines for the management of CD to translate this evidence into clinical practice. In this review, we critically analyse both the converging and diverging points in the current clinical guidelines of CD, focusing on the diagnostic aspects and follow-up procedures.

INTRODUCTION

Celiac disease (CD) is an immune-mediated reaction to gluten characterised by an inflammatory injury to the small bowel in genetically predisposed subjects as a result of an inappropriate T cell-mediated immune response[ 1 ].The epidemiology of CD is well known, with an estimated worldwide prevalence of 0.6%-1% of the general population[ 2 ]. However, CD remains largely underdiagnosed in developing countries and has a higher impact on children[ 3 , 4 ]. Simultaneously, the misdiagnosis of CD is becoming an emergent problem worldwide[ 5 ].

An evidence-based approach is needed to optimise diagnostic accuracy to avoid life-threatening complications (including small bowel carcinoma and lymphoma)[ 6 ] resulting from unrecognised CD on the one hand, and unnecessary cost burden and impact on the quality of life due to incorrect prescription of a life-long gluten-free diet (GFD) on the other hand.

Simultaneously, follow-up of patients with CD who are on a GFD is of critical importance to assess the responsiveness to the GFD, detect complicated CD, find associated autoimmune diseases, and identify metabolic alterations induced by the GFD[ 7 ].

Thus, an increasing number of scientific societies have proposed guidelines for diagnosing and managing CD. In our systematic review, we identified the most recent and significant national and international guidelines and compared their recommendations. We also underlined the most apparent differences among these guidelines to identify ‘hot topics’ on CD and possible future developments.

MATERIALS AND METHODS

The primary aim of this review was to identify the most recent national and international guidelines for CD by means of a systematic review and to compare their main recommendations.

We performed a database search on PubMed and selected papers published between January 2010 and January 2021 in the English language. PubMed was last accessed on 1 March 2021. The following keywords and terms were used: (1) Coeliac Diseaseor Celiac Disease ; (2) Guideline ; and (3) Management . The following string was used: (("coeliac disease"[All Fields] OR "celiac disease"[MeSH Terms] OR ("celiac"[All Fields] AND "disease"[All Fields]) OR "celiac disease"[All Fields] OR ("coeliac disease"[All Fields] OR "celiac disease"[MeSH Terms] OR ("celiac"[All Fields] AND "disease"[All Fields]) OR "celiac disease"[All Fields])) AND ("guideline"[Publication Type] OR "guidelines as topic"[MeSH Terms] OR "guideline"[All Fields] OR ("manage"[All Fields] OR "managed"[All Fields] OR "managements"[All Fields] OR "managements"[All Fields] OR "manager"[All Fields] OR "manager s"[All Fields] OR "managers"[All Fields] OR "manages"[All Fields] OR "managing"[All Fields] OR "management"[All Fields] OR "organization and administration"[MeSH Terms] OR ("organization"[All Fields] AND "administration"[All Fields]) OR "organization and administration"[All Fields] OR "management"[All Fields] OR "disease management"[MeSH Terms] OR ("disease"[All Fields] AND "management"[All Fields]) OR "disease management"[All Fields]))).

A total of 415 papers were identified with no duplicates, and, as a first step, no papers were excluded for other reasons (PRISMA flow diagram reported in Figure ​ Figure1). 1 ). However, twenty-one records were unavailable, leaving 396 papers for further evaluation. As a second step, we excluded papers that were not pertinent to any of the following criteria: (1) Clinical guidelines related to diagnosis and management of CD; and (2) Clinical guidelines published by governmental agencies and scientific associations. We included only the last version of the guidelines, excluding the previous updated versions.

PRISMA flow diagram.

According to the selection criteria, out of the 396 results of PubMed research assessed for eligibility, seven guidelines were finally included in this analysis. These guidelines strictly focus on the diagnosis and management of CD. These papers are presented in order of publication (newest to oldest): (1) European Society Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) 2020[ 8 ]; (2) European Society for the Study of Coeliac Disease (ECD) 2019[ 9 ]; (3) World Gastroenterology Organization (WGO) 2017[ 10 ]; (4) Central Research Institute of Gastroenterology, Russia, 2016[ 11 ]; (5) National Institute for Health and Care Excellence (NICE), 2015[ 12 ]; (6) British Society of Gastroenterology (BSG), 2014[ 13 ]; and (7) America College of Gastroenterology (ACG), 2013[ 14 ].

The recommendations provided by each selected guideline were systematically explored and classified into five categories: patients to be tested for CD, diagnostic tests (serology, duodenal biopsy, genetic test, no-biopsy diagnosis), potential/silent/seronegative CD, refractory/complicated CD, and follow-up. These categories represent the most discussed topics of CD.

The results are reported in different paragraphs, containing both a brief introduction to the specific topic (with references derived from the supporting evidence used by the guidelines and other relevant papers according to a narrative approach) and a comparative analysis of the guidelines’ recommendations (collected using a strictly systematic approach).

Clinical presentation and risk factors: who should be tested for CD?

CD is a diagnostic challenge as it may develop at any age (even in older adults) and with a polymorphic clinical presentation[ 15 ]. The clinical spectrum of CD includes both symptomatic and silent forms revealed only by serological screening[ 16 , 17 ]. CD-related symptoms can be both intestinal and extraintestinal, reflecting the systemic nature of the disease. These manifestations are classified as ‘classical’ and ‘non-classical’ according to the historical presentation of first described cases. Table ​ Table1 1 reports the main manifestations of CD according to their categorization[ 1 , 17 - 26 ].

Most frequent clinical manifestions of celiac disease

Some guidelines draw specific attention to some extraintestinal symptoms (Figure ​ (Figure2). 2 ). In particular, the ESsCD 2019 guidelines focus on oral-dental and neuropsychiatric manifestations[ 9 ]. CD testing is advised in cases of dental enamel defects and recurrent oral aphthae. Special attention to neurological manifestations has also been drawn by the Russian Central Research Institute of Gastroenterology[ 11 ]. These guidelines also focus on reproductive disorders, such as delayed sexual development, amenorrhea, infertility, and miscarriage[ 11 ].

Recommendations about case finding.

Despite these premises, all the guidelines agree on testing for CD in children, adolescents, and adults showing classical and non-classical symptoms of CD[ 7 - 13 ]. There is also a consensus on considering iron-deficiency anaemia and hypertransaminasemia as the most common laboratory abnormalities[ 8 - 14 ].

The high-risk group of patients did not change over time. These groups include first-degree relatives of patients with CD, patients with autoimmune conditions (such as type 1 diabetes mellitus and thyroid diseases) or genetic disorders such as IgA deficiency, Down syndrome, Turner syndrome, and Williams-Beuren syndrome[ 8 - 14 ].

There is no ‘gold standard’ for the diagnosis of CD. Clinical features, serology, or histology alone cannot provide a definitive diagnosis. Instead, the final diagnosis of CD relies on a combination of these elements. All the guidelines agree on a sequential approach to diagnosis, consisting of serology as a first-line test in high-risk patients, followed by duodenal biopsy in cases of positive serology or persistent suspicion of malabsorption (Figure ​ (Figure3). 3 ). A positive serology paired with evidence of duodenal villous atrophy indicate a definite CD diagnosis, whereas cases with discordant findings should undergo HLA testing. All the guidelines also agree that patients with discordance between serology, histology, and HLA DQ2/DQ8 positivity should be evaluated on a patient-by-patient basis in expert centres. The so-called ‘four-out-of-five rule’ has long been advocated as a standard of care[ 27 ]. According to this rule, four of the following criteria are sufficient to establish CD diagnosis: (1) Typical signs and symptoms (diarrhoea and malabsorption), (2) Antibody positivity, (3) HLA-DQ2 or HLA-DQ8 positivity, (4) Intestinal damage ( i.e. , villous atrophy and minor lesions); and (5) Clinical response to GFD. This rule also helps physicians to identify various subtypes of CD, that is, non-classic CD (absence of point 1), seronegative CD (absence of point 2), potential CD (absence of point 4), and non-responsive CD (absence of point 5). However, the ‘four-out-of-five rule’ is yet to be recognised by any guideline.

Worldwide adapted decision-making process for diagnosing celiac disease. Highly suspicious celiac disease (CD) comprises “classical presentation” ( i.e., classical symptoms in children include failure to thrive, weight loss, growth failure, vomiting, chronic diarrhea, bloating, Iron-deficiency anemia, muscle wasting, oedema due to hypoproteinemia, irritability and unhappiness; in adults, classical symptoms include chronic diarrhea, weight loss, iron-deficiency anemia, malaise and fatigue, oedema due to hypoproteinemia, and osteoporosis), frequent “non-classical presentation” ( i.e., iron deficiency and hypertransaminasemia) and “non-classical presentation” but high risk group ( i.e., CD first-degree relatives, autoimmune conditions such as type 1 Diabetes Mellitus, and thyroid disease, genetic conditions such as IgA deficiency, Down syndrome, Turner syndrome and Williams-Beuren syndrome).

We will report the guidelines’ detailed suggestions for obtaining key diagnostic elements from serology, histology, and genetic testing in the following paragraphs.

All diagnostic serological testing should be performed in patients on a gluten-containing diet[ 28 ]. Serum immunoglobulin A(IgA) anti-tissue transglutaminase antibody (anti-tTG-IgA) is widely accepted as the most sensitive test for CD diagnosis, although it suffers from low specificity, especially at low titres[ 29 - 33 ]. In contrast, IgA anti-endomysial antibodies (EMA-IgA) are nearly 100% specific for CD but are less sensitive, more expensive, and more operator-dependent than anti-tTG-IgA. Therefore, these characteristics make EMA-IgA an ideal second-line test[ 34 ]. The diagnostic performance of both anti-tTG-IgA and EMA-IgA is limited in patients with concurrent IgA deficiency. Antibodies to deamidated gliadin peptides (DGP) of the IgG class are advantageous in this setting and for younger children[ 35 , 36 ]. Even with the most recent advancements in CD serology, up to 2% of patients with CD have no circulating markers of gluten sensitivity, defining a condition of seronegative CD[ 37 ].

Currently, the guidelines are concordant and suggest anti-tTG-IgA as the initial serological test, complemented by a determination of total IgA levels to rule out concurrent IgA deficiency (Figure ​ (Figure4 4 )[ 8 - 14 ]. This initial approach was suggested for both children and adults.TheACG2013 guidelines suggest a combination of different IgA and IgG antibodies in children younger than two years of age (for instance, anti-tTG IgA and DGP-IgG)[ 14 ]. This approach is still accepted only by the WGO2017 guidelines[ 10 ]. The remaining guidelines advise against this strategy, as a combination of antibodies implies a higher sensitivity at the expense of a reduced specificity, often leading to the necessity of histological confirmation. This scenario represents an obstacle in the pursuit of a no-biopsy approach in children, for whom the anti-tTG-IgA + total IgA strategy fits better[ 8 ]. Alternatively, DGP-IgG (together with anti-tTG-IgG) maintained the unanimous recommendation as the test of choice in patients with IgA deficiency[ 8 - 14 ].

Recommendations about serology. IgA: Immunoglobulin A; IgG: Immunoglobulin G; DGP: Deamidated gliadin peptides; EMA: Anti-endomysium antibodies.

Further, EMA-IgA is considered a confirmatory test, particularly when TG2 has a low titre, i.e., < 2x the upper normal limit (UNL)[ 9 , 10 , 12 ]. A positive result is also required for a no-biopsy CD diagnosis in children with anti-tTG IgA > 10x[ 8 ]. However, the use of paired anti-tTG and EMA-IgA as the first diagnostic test is not supported by any guideline.

Currently, all of the guidelines strongly discourage urine, stool, and saliva tests in clinical practice due to their low-performances[ 8 - 14 ] and the consequent risk of initiating a GFD without a firm diagnosis, impacting the final diagnosis[ 13 ].

For a long time considered the ‘gold standard’ for diagnosing CD (ambiguously suggesting that other tests were of lesser importance), duodenal biopsies remain the mainstay of CD diagnosis, and all guidelines unanimously recognise this role. The presence of positive histology, however, was not considered CD-specific. Thus, clinical, and serological correlations are mandatory (Figure ​ (Figure5) 5 ) [ 8 - 14 ].

Recommendations about serology.

Duodenal biopsies should be obtained from all patients with suspected CD. In high-risk symptomatic patients, duodenal biopsies should be performed irrespective of serology results for CD[ 9 , 13 , 14 ]. Some authors also suggested that duodenal biopsies should be considered in any individual undergoing endoscopy because of the relatively high prevalence of CD in the general population and its polymorphic presentation[ 13 ].

Histology samples should be collected from multiple sites, given the possible patchy distribution of CD lesions. Current evidence suggests collecting four biopsies from the second duodenal portion and two biopsies from the bulb[ 38 ]. Biopsy sample orientation using cellulose acetate Millipore filters is of paramount importance to avoid artefacts, potentially leading to a false diagnosis of villous atrophy[ 39 ].

The histological findings are currently categorised according to the classification proposed by Marsh and subsequently modified by Oberhuber[ 40 ]. Pathology findings are reported as Marsh-Oberhuber 0 (normal histology), 1, 2, or 3 (subdivided into 3a, 3b, and 3c).

An increase in intraepithelial lymphocytes (IELs) without villous atrophy defines Marsh 1 Lesion. In most cases, Marsh 1 Lesions (also called minimal lesions) are attributable to other causes, including lymphocytic colitis, bacterial and parasitic intestinal infections (especially Helicobacter pylori and Giardia lamblia ), small intestinal bacterial overgrowth, Crohn’s disease, common variable immunodeficiency, and non-steroidal anti-inflammatory drugs[ 41 ]. While a Marsh 1 Lesion is not considered sufficient to diagnose CD, the BSG 2014 guidelines state that minimal lesions combined with positive serology could represent a probable CD. A trial with a GFD could be considered to support the diagnosis of CD[ 13 ]. When the increase in IELs is paired with hyperplasia of the duodenal crypts, the lesion is classified as Marsh 2. Conversely, increased IELs in combination with villous atrophy define the typical CD lesion (Marsh 3), subclassified as mild (3a), moderate (3b), or subtotal (3c)[ 40 ]. Some authors proposed a simplified histopathological grading, reducing the possible grades from five to three, thus reducing the possible inter-operator variability in the histological interpretation[ 42 ].This simplified classification is yet to be adopted by the international guidelines, which currently recommend the Marsh-Oberhuber classification[ 8 - 14 ].

At present, there is no alternative to duodenal biopsy for examining mucosal damage[ 8 - 14 ]. For instance, in children, video-capsule endoscopy (VCE) gives no indications[ 8 ], although in adults, VCE could support the diagnosis in cases of discordance between serology and biopsy[ 13 ] or if the patient is unwilling or unable to undergo traditional endoscopy[ 14 ]. VCE could also play a role in detecting CD complications ( i.e., lymphoma, adenocarcinoma, ulcerative jejunitis)[ 9 ] and in helping to differentiate extended diseases ( e.g. , CD vs proximal Crohn’s disease)[ 11 ]. Anti-actin IgA antibodies have been shown to be predictive of severe villous atrophy in CD patients at the time of diagnosis[ 43 ]. Theoretically, they may also provide indirect information about villous recovery following the introduction of the GFD; however, data are still lacking in this setting. The available information about faecal and salivary microbiome, at present, is not sufficient to allow a reliable conclusion for the diagnosis of CD[ 44 , 45 ]. Intestinal fatty-acid binding protein (I-FABP) are higher in dietary non-adherence and unintentional gluten intake and could be used as a sensible blood marker of mucosal damage[ 46 , 47 ].This exam was first mentioned in the ESsCD guidelines[ 9 ].

A repeated small intestinal biopsy, including biopsies from the jejunum, could be considered in adults with discordance between histopathology and anti-tTG-IgA results[ 13 ]. In children, re-cutting biopsies and/or a second opinion from an experienced pathologist is preferred over endoscopic repetition[ 8 ].

In adults, a gluten challenge should be proposed for patients with uncertain CD diagnosis, who have been started on a GFD[ 9 - 14 ]. In children, gluten challenge is discouraged before the age of 5 years and during puberty, and in general, it should be reserved for unusual cases[ 8 ].

Gluten challenge protocols are not homogeneous. A diet containing at least 10 g of gluten per day for 6-8 wk seems to be the most effective way to achieve disease relapse; however, the evidence is weak[ 28 ]. In shorter protocols, a diet containing at least 3 g of gluten per day for at least 2 wk seems to be sufficient for most patients[ 10 , 13 , 14 ]. Certainly, a shorter and lighter approach would fit better for highly symptomatic patients. A strategy for optimising the result would be to undergo a serology test after two weeks and, if negative, to extend the challenge to 8 wk[ 13 ].

After reintroducing gluten, physical symptoms should not be used for diagnosis in the absence of other supportive evidence[ 8 , 9 , 11 - 14 ]. A diagnosis based only on the disappearance of symptoms on GFD and relapse during gluten re-introduction can be relevant in geographic areas where serology tests are not available, as the only way to confirm the diagnosis and treat the disease[ 10 ].

Human Leukocyte Antigen testing

The strong genetic component of CD is testified by its high familial recurrence and high disease concordance among monozygotic twins (75%-80%)[ 48 ]. The presence of human leukocyte antigen (HLA) -DQ2/DQ8 is a pathogenic requisite for the development of the typical immune alterations found in CD. Simultaneously, HLA DQ2/DQ8 can be found in up to 30%-40% of the general population, so its specificity is remarkably poor[ 49 ]. In contrast, the absence of HLA DQ2/DQ8 virtually excludes CD diagnosis[ 48 , 49 ].Restricting this observation to the sole HLA DQ2 alleles, a recent systematic review of the literature confirmed that only 5.06% of patients with CD were completely lacking the HLA-DQB1*02 allelic variant[ 50 ].

Consequently, all the guidelines advise against using HLA testing as a first-line tool for the diagnosis of CD (Figure ​ (Figure6 6 )[ 8 - 14 ]. They are also concordant in allocating this resource for: (1) Patients with uncertain diagnosis of CD, already on a GFD; (2) Patients with a flat intestinal mucosa but negative serology; and (3) In patients already on a GFD, serology and histology can be inconclusive. In this context, before embarking on a so-called ‘gluten-challenge’, it is advisable to verify the presence of HLA-DQ2/DQ8[ 8 - 14 ].

Recommendations about Human Leukocyte Antigen testing.

HLA tests would be useless for patients with positive serology before a gluten-challenge because virtually 100% of those patients would be positive. Therefore, HLA typing is no longer a criterion for the ‘no-biopsy’ approach of diagnosis in children with a TGA-IgA > 10x UNL[ 8 ]. In patients with positive histology ( i.e., villous atrophy, though occasionally detected on esophagogastroduodenoscopy), and negative or questionable serology, HLA testing can exclude the diagnosis of CD[ 9 ]. In contrast, a positive result cannot confirm the diagnosis, which should be carefully evaluated on a patient-by-patient basis in expert centres.

The use of HLA typing in high-risk populations is controversial. HLA-DQ2/DQ8 can be found in more than 50% of first-degree relatives of patients with CD and in patients with other autoimmune or genetic disorders related to CD[ 14 , 49 ]. Most guidelines suggest excluding HLA-DQ2/DQ8 in CD first-degree relatives and high-risk patients, even if asymptomatic, to avoid periodic monitoring[ 9 , 10 , 13 , 14 ]. This strategy can be questioned in terms of resources and costs[ 10 , 11 , 14 ]. Some authors hav esuggested screening high-risk patients only if they complain of gastrointestinal or extraintestinal symptoms or have laboratory abnormalities[ 11 ]. In addition, a two-step genetic screening procedure starting with HLA-DQ β chains has been proposed[ 51 ].Thus, the choice of screening for symptomatic or asymptomatic first-degree relatives or high-risk patients, with or without a preliminary determination of HLA-type, remains debated, needing to take local resources and cost-benefit rates into account.

No-biopsy diagnosis

While most guidelines allow a no-biopsy diagnosis in children under strict conditions, endoscopy with duodenal biopsies is still mandatory to achieve a final diagnosis of CD in adults[ 9 - 14 ]. As the only exception, the WGO guidelines allow a diagnosis based on serology and clinical response to the GFD (Figure ​ (Figure7) 7 ) in developing countries where endoscopy may not possible or trained pathologists may not be available[ 10 ].

Recommendations about the possibility of a no-biopsy diagnosis. TGA: Anti-transglutaminase antibodies; IgA: Immunoglobulin A; EMA: Anti-endomysium antibodies; HLA: Human leukocytes antigen; CD: Celiac disease.

The ESPGHAN2012 guidelines endorsed the possibility of a no-biopsy approach in children for the first time. This possibility was limited to certain conditions, which included the presence of classic symptoms, with tTG-IgA > 10x UNL, EMA-IgA positivity, and presence of permissive HLA[ 8 ].

This approach was subsequently adopted by a plurality of international guidelines[ 9 - 12 ]. although, the ACG2013 and BSG 2014 guidelines did not include this approach[ 13 , 14 ].

The 2020 update of the ESPGHAN guidelines removed classic symptoms, EMA-IgA positivity, and HLA DQ-2 or DQ-8 as crucial criteria for a diagnosis not based on biopsy[ 7 ]. However, EMA-IgA positivity is not discouraged[ 8 , 10 ]. The increasing confidence in diagnosing CD without biopsy in children has increased so rapidly that many recent studies consider tTGA > 10x as a new possible cut-off to further reduce the need for biopsies[ 52 ].

CD diagnosis without a positive duodenal biopsy has always been discouraged in adults[ 9 - 14 ]. This choice was not dictated by the reduced reliability of the serological tests in adults. In fact, large population studies concluded that tTG-IgA>10x could accurately predict villous atrophy[ 53 ]. Rather, other considerations currently prevent the extension of paediatric criteria into the adult population. First, CD at onset can be associated with complications. In the case of primary or secondary resistance, or slow response to the GFD, the absence of baseline histology may make the diagnosis of complications difficult[ 9 ]. Index histology may also predict the risk of future complications, such as lymphoma[ 54 ]. Moreover, endoscopy may help diagnose other treatable disorders associated with CD, such as eosinophilic esophagitis, autoimmune gastritis, and lymphocytic gastritis[ 9 ].

Both complicated CD and possible differential diagnoses of CD are virtually absent in children. However, they represent a serious concern in adults, thus justifying different diagnostic algorithms according to the age of presentation of the first symptoms.

Potential, silent and seronegative CD

Potential CD is characterised by a positive serology for CD in the absence of mucosal damage at biopsy[ 1 ]. As stated above, Marsh 1 Lesions ( i.e., an increased IELs count) are not suggestive of an active CD but may increase the risk of developing villous atrophy[ 41 ].

It is widely accepted that symptomatic potential CD may benefit from a GFD, and a direct challenge would be run[ 8 - 14 ]. In adult patients with both positive TGA-IgA and EMA-IgA CD is likely, and a GFD may be initiated irrespective of symptoms[ 9 ]. A serological response after a period of approximately 12 mo confirms the diagnosis of CD[ 9 ]. In EMA-IgA negativity, HLA-typing may exclude the diagnosis before embarking on follow-up[ 9 ]. If a follow-up is started, potential CD patients should be retested after consuming a gluten-containing diet for 3-6 mo to confirm persistent seropositivity before referral for a new endoscopy (Figure ​ (Figure8 8 )[ 9 , 10 ].

Recommendations about potential, silent, and seronegative celiac disease. GFD: Gluten-free diet; HLA: Human leukocytes antigen.

Silent CD is characterised by the presence of both positive serology and histology for CD in the absence of classical or non-classical symptoms[ 1 ]. It is widely recommended to start a GFD in patients with silent CD because it is considered an active form of the disease[ 8 - 14 ].

Seronegative CD is characterised by the presence of active enteropathy and negative serology for CD, with no other causes, and with clinical and histological responses to a GFD[ 1 , 37 ]. In these cases, other causes of enteropathy should be excluded before embarking on the direct challenge of a GFD[ 37 , 55 ]. HLA-typing can also rule out the diagnosis of CD in seronegative enteropathies[ 9 , 14 , 37 ]. Finally, the direct challenge of a GFD is advised only in patients with seronegative enteropathy, positive HLA typing with no other causes. A documented histological response after 1-3 years of GFD is needed to confirm the diagnosis[ 9 , 14 , 37 ]. No major changes occurred over time in the management of seronegative CD[ 9 , 14 ].

Refractory and complicated CD

CD can be complicated by a persistent active form of the disease, independent of gluten intake, known as refractory CD (RCD)[ 1 ]. Other rare complications of CD can be neoplastic. Primarily, enteropathy-associated T-cell lymphoma (EATL) is a rare T-cell lymphoma associated with untreated CD. EATL has an abysmal prognosis and can occur primarily at diagnosis or as an evolution of RCD type 2[ 56 ]. Duodenal adenocarcinoma is possible, albeit less frequent in the CD population[ 57 ].

Refractory CD (RCD) is characterised by the persistence or recurrence of symptoms and signs of malabsorption, with documented villous atrophy, despite a strict GFD for more than 12 mo and in the absence of other causes[ 9 - 14 ]. No major changes occurred in this definition over time (Figure ​ (Figure9). 9 ).

Recommendations about refractory and complicated celiac disease. GFD: Gluten-free diet; TCR: T-cell receptor.

RCD can be primary (refractory at the time of the first diagnosis), or secondary (occurring after a period of response to the GFD)[ 1 ]. The first step in evaluating suspected RCD is to re-evaluate the initial diagnosis of CD by reviewing biopsies and serology tests obtained at the time of diagnosis[ 58 ]. The most common cause of GFD failure is inadvertent gluten ingestion[ 59 ].Therefore, evaluation by an expert dietitian should always be included[ 9 , 10 , 13 , 14 ]. Other associated or concomitant pathological conditions should be excluded before RCD diagnosis. These include lactose and fructose intolerance, small intestinal bacterial overgrowth, microscopic colitis, pancreatic insufficiency, and inflammatory bowel diseases[ 59 , 60 ]. All guidelines recommend this strategy[ 9 , 10 , 13 , 14 ].

RCD is further classified into type 1 (RCD-1) and type 2 (RCD-2)[ 1 ]. T-cell flow cytometry is the most reliable method for classifying RCDs. Aberrant T cells lose the normal surface markers CD3 and CD8 with preserved expression of intracytoplasmic CD3. In RCD-1, the percentage of aberrant T cells is below 20%, whereas in RCD-2, they represent more than 20% of the total IELs[ 58 ]. RCD-2 can be considered a pre-lymphoma or low-grade lymphoma[ 54 ]. T-cell receptor (TCR) g chain clonality analysis lacks sensitivity and specificity, and is of limited value in separating RCD-1 from RCD-2[ 54 ]. TCR analysis has been formerly indicated as a criterion for differentiating RCD-1 from RCD-2[ 11 , 13 , 14 ]. The latest ESsCD guidelines exclude TCR analysis in the RCD classification[ 9 ].

RCD-1 has an extremely high 5-year survival rate (> 90%)[ 54 , 59 , 60 ]. In RCD-1, the first-line therapy should be ‘open-capsule’ budesonide (OCB), 3 mg, 3 times a day[ 61 ]. Budesonide (open capsule or not) has been progressively accepted as the first-line therapy for RCD-1[ 9 , 11 , 13 , 14 ]. In the ACG 2013 guidelines, systemic steroids are considered the first-line therapy for RCD-1[ 14 ]. Second-line treatment for RCD-1 includes immunosuppressive drugs such as steroids (prednisone 0.5-1 mg/kg/day) and azathioprine (2-2.5 mg/kg/day)[ 60 ]. Most guidelines agree with this strategy[ 9 , 11 , 12 ]. Systemic steroids can also be considered as first-line treatment while waiting for a specialist’s advice[ 12 ]. Infliximab may be the preferred biological therapy for second-line treatment of RCD-1[ 62 ]. Evidence is still weak, and only one guideline includes infliximab as an RCD-1 treatment[ 9 ].Withdrawing of immunosuppressive therapy after 2-3 years of complete response may be considered[ 9 , 54 ].

RCD-2 is rarer than RCD-1, has a much higher mortality rate, and treatment is less well defined. Systemic steroids or open-capsule budesonide should be the first choice for milder presentations. In severe cases, cytoreductive therapies such as cladribine and fludarabine or autologous hematopoietic stem cell transplantation should be chosen[ 59 , 60 ]. Guidelines are mostly aligned with this strategy[ 9 , 13 , 14 ]. Some guidelines also report azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, and anti-TNF antibodies as possible therapies, but the data are weaker[ 11 , 13 , 14 ]. Not every guideline has raised the topic of RCD-2 treatment[ 10 - 14 ].

Transformation to enteropathy-associated T-cell lymphoma (EATL) is likely in RCD-2[ 59 ]. VCE, positron-emission tomography (PET), and magnetic resonance (MR) enterography can be useful in cases of suspected progression to EATL to assess the extent of the disease[ 63 ]. All guidelines advise the use of these tools in RCD-2 staging[ 9 - 14 ]. Severe RCD-2 and EATL may require surgery, chemotherapy, or bone marrow transplantation[ 64 ]. The former therapeutic strategies are mostly based on case reports, and only one guideline extensively discusses them[ 9 ].

Since CD is the only autoimmune disease with a known environmental trigger ( i.e., gluten), a periodical assessment of compliance to a GFD is essential[ 65 ]. Poor GFD compliance is not infrequent, and mucosal damage can persist despite negative serology and the absence of symptoms[ 66 ]. Follow-up is also essential for evaluating possible complications[ 54 ]. Osteoporosis and metabolic complications of GFD should also be evaluated during follow-up[ 67 - 69 ]. Suggested follow-up schedules are based on the frequency of complications, risk of GFD non-compliance, and reported quality of life[ 70 ].

Therefore, there is universal agreement on the necessity of long-term monitoring of patients with CD to assess the compliance and responsiveness to the GFD and allow early detection of complicated CD (Figure ​ (Figure10 10 )[ 8 - 14 ]. Follow-up evaluations should be scheduled every 3-6 mo during the first year and then every 1-2 years[ 9 - 14 ]. In children, follow-up should continue until they reach their final height[ 9 - 11 , 14 ], focusing on normal growth and development[ 9 , 10 , 14 ].

Recommendations about follow-up of celiac disease. TGA: Anti-transglutaminase antibodies; GFD: Gluten-free diet.

There is disagreement about who should oversee follow-up. While most guidelines show no preference between primary care physicians, specialists, or dietitians[ 9 - 11 , 13 , 14 ], the NICE 2015 guidelines suggest that dietitians with expertise in CD may be best suited to carry out an annual follow-up[ 12 ]. However, on a general principle, all guidelines agree that newly diagnosed patients should be referred to a dietitian[ 9 - 14 ]. Some guidelines suggest that nutritionist counselling should coincide with medical visits during follow-up[ 10 , 13 ]. The inclusion of a dietitian assessment at diagnosis and during follow-up was supported by clinical data[ 71 ]. Indeed, nutritional counselling could also help manage metabolic alterations, which frequently appear during the first years of the GFD[ 67 ].

All guidelines also provide information about the essential information that should be collected during follow-up evaluations. These evaluations should include a dietary interview, serology (TTG-IgA if normal IgA), and laboratory tests[ 9 - 14 ]. Laboratory tests should evaluate the presence of micronutrients malabsorption, including complete blood count, iron status, folate, vitamin B12, calcium, phosphate, vitamin D, and should monitor associated autoimmune conditions (thyroid-stimulating hormone and serum glucose) and liver disorders (aspartate aminotransferase/alanine aminotransferase)[ 9 - 11 , 13 , 14 ]. Normalisation of tTG-IgA levels do not predict full recovery of villous atrophy. In contrast, persistently positive serology 12 mo after GFD initiation is a strong indicator of gluten ingestion[ 72 ]. All guidelines were aligned with the interpretation of tTG-IgA levels during follow-up[ 8 - 14 ].

The inability of serology alone to predict mucosal healing automatically leads to consider the opportunity of repeating duodenal biopsies after the start of the GFD. While the general agreement is that follow-up biopsies are not mandatory in asymptomatic patients on a GFD and without an increased risk of complications[ 9 - 14 ], the guidelines diverge regarding other points. Many guidelines consider it reasonable to repeat biopsy after 2 years of GFD to assess mucosal healing[ 9 , 11 , 14 ]. Other guidelines suggest repeating biopsies only for persistent symptoms or serological abnormalities after 12 mo of GFD[ 10 , 12 , 13 ]. A growing body of literature suggests that the risk of a complicated CD is higher in patients >40 years of age at the time of diagnosis or those with a classical presentation[ 54 ]. Some guidelines agree that repeating biopsies should be of interest in these selected populations[ 13 , 14 ].

Some guidelines also provide suggestions for further examinations to be performed during follow-up. According to the ECD and Russian guidelines, bone densitometry should be offered to every patient at the time of diagnosis and should be repeated after 3 years if abnormal, or 5 years if normal[ 9 , 11 ]. Other guidelines suggest performing bone densitometry only in patients with a high risk of osteoporosis or those older than 55 years[ 12 , 13 ].

While there is a general agreement in recommending a pneumococcal vaccine[ 8 - 10 , 12 ], the WGO2017 guidelines also recommend vaccinations against Haemophilus influenzae typeB , and Meningococcus , while other guidelines state that these vaccines have a less clear indication to be given to every patient with CD[ 9 , 11 - 13 ].

Mood disorders are another common problem in patients with dietary restrictions. Anxiety, depression, and fatigue may be associated with CD before and after diagnosis and can affect the quality of life[ 73 ]. In this context, most guidelines agree on advising patients to join CD support groups and associations[ 9 , 10 , 12 , 13 ]. Some of them also suggest that psychological support provided by a specialist may be offered[ 12 , 13 ].

Gluten-free diet

Gluten is a protein with high proline and glutamine content, primarily found in wheat. Rye and barley belong to the same tribe as wheat and are known to contain gluten. In contrast, oats are derived from a different tribe and do not contain pure gluten[ 1 ].

Uncontaminated oats are safe for almost all patients with CD, but a small percentage of patients may be sensitive to some oat cultivars[ 74 ] and should be monitored[ 9 , 10 , 12 - 14 ]. Some guidelines advise the initiation of a Gluten-free diet (GFD), excluding oats, and recently introduced them[ 10 , 13 , 14 ]. The Russian guidelines (2016)are against oat consumption in patients with CD because of the high risk of contamination[ 11 ]. Even if not stated, oat consumption would be safe in many countries, though it may be discouraged in developing countries where contamination could be widespread (Figure ​ (Figure11 11 ).

Recommendations about the gluten-free diet for celiac disease.

WHO guidelines on ‘Standard for Foods for Special Dietary Use for Persons Intolerant to Gluten’ state that foods labelled as ‘gluten free’ should contain ≤ 20 parts per million (ppm) of gluten[ 75 ].

Patients should be instructed to avoid contaminating their gluten-free food by using separate cooking utensils and cooking surfaces[ 9 , 10 ]. At present, shared items can be safely used if thoroughly cleaned with soap and water between use[ 9 , 76 ].

The duration of breastfeeding and the timing of gluten introduction to the infant seem to have no impact on the risk of developing CD, even in those at high risk[ 77 ]. Therefore, there are no strict indications for gluten introduction in infant diets[ 9 ]. Formerly, it was advised to avoid either early or late gluten introduction in children at risk of CD[ 13 ].

Dermatitis herpetiformis (DH) is a bullous cutaneous disease triggered by gluten consumption like CD[ 1 ]. DH and CD often coexist and share the same treatment, GFD[ 9 , 10 , 13 , 14 ]. Interestingly, the ESsCD guidelines suggest that psoriasis could also benefit from GFD in the case of documented CD serology, even in the absence of mucosal damage[ 9 ].

Our comparative analysis of the currently adopted CD guidelines underlined differences in diagnostic aspects and the management of the follow-up. These differences mirror some relevant clinical points in both developing and developed countries.

First, the differences in the diagnostic process of CD are important. The possibility of a no-biopsy diagnosis has relevant repercussions in developing countries. Most guidelines are still cautious in this regard, with the WGO2017 guidelines being the only ones contemplating this possibility in geographical areas with a paucity of resources. As correctly underlined by these guidelines, some absolute recommendations may not be valid for developing countries where the availability of serology or endoscopy may be lacking[ 10 ]. CD seems to have a non-negligible prevalence in Asia and sub-Saharan Africa[ 77 , 78 ]. Especially in Russia and Central Asia, the prevalence of CD is very likely to be underestimated due to poor disease awareness among physicians and/or patients, limited access to diagnostic resources, inappropriate use or interpretation of the serological tests, absence of standardised diagnostic and endoscopic protocols, and insufficient expertise in histopathological interpretation[ 3 ]. Specific guidelines are lacking in these geographical areas[ 79 ]. In addition, the incidence of undiagnosed CD in children can be extremely high[ 80 ]. Knowing the high mortality and disability related to untreated CD in childhood, it would be advisable to develop specific protocols for specific geographical areas.

The no-biopsy approach has been discouraged for a long time, especially in adults[ 13 , 14 ]. In contrast, most recent guidelines have incorporated the ESPGHAN 2012 recommendations for a no-biopsy approach in children[ 9 , 10 ]. The possibility of an outright extension of these criteria into the adult population still meets key obstacles. However, in an era during which the COVID-19 pandemic has caused a staggering drop in new CD diagnoses even in industrialised countries[ 81 ], ESPGHAN released the advice to lower the TGA-IgA threshold for diagnosing CD without biopsy[ 52 ]. Moreover, retrospective data on a possible no-biopsy approach in adults are increasing[ 53 ]. Prospective data will probably lead to the integration of such an approach to future guidelines over the next decade.

Second, the differences in follow-up recommendations reflect a relatively low interest in this topic in the past. Arguably, the search for more reliable diagnostic tools was the right priority in an era characterised by a severe under-diagnosis of CD .Nowadays, significant diagnostic delays can still occur in a minority of Central European children[ 82 ], with socioeconomically deprived children being more likely to be underdiagnosed despite improved and easily available serological testing[ 4 ].

Nonetheless, the current physicians’ awareness of CD has reached fairly high levels, and the case-detection strategy has significantly contributed to the increased number of diagnoses. Consequently, the correct management of follow-up is crucial. This topic is of special interest in developed countries, in which metabolic problems possibly caused by an unbalanced GFD are particularly prevalent. Uncontrolled weight gain, metabolic syndrome, and non-alcoholic fatty liver disease are epidemic in these countries and can also be facilitated by the GFD[ 67 , 69 , 83 - 85 ]. In addition, quick detection of associated autoimmune conditions can prove highly beneficial, especially in autoimmune liver diseases[ 86 ]. Finally, early detection of complicated CD requires particular attention, as both neoplastic and non-neoplastic complications may arise years after the diagnosis[ 6 ].

We found a relatively high concordance between CD guidelines. Important modifications have occurred in recent years, especially regarding the possibility of a no-biopsy diagnosis in children. Other modifications are expected in the future and will probably involve the extension of the non-invasive diagnosis to the adult population and the follow-up modalities.

ARTICLE HIGHLIGHTS

Research background.

Celiac disease (CD) has risen from obscurity to global prominence in a few decades. These modifications have prompted experts from all over the world to identify effective strategies for the diagnosis and follow-up of CD. Different scientific societies, mainly from Europe and America regions, have proposed different guidelines.

Research motivation

CD guidelines are consistent when they deal key points in the diagnosis and follow-up of this condition. However, they differ in a number of other points.

Research objectives

To identify all of the existing guidelines across the globe and perform a comparative analysis to verify similarities and differences and, thus, discuss the most debated topics and the possible innovations in the next future.

Research methods

We searched PubMed for a complex string containing the terms “celiac disease”, “management”, and “guidelines”. The results were subsequently explored to identify the most recent versions of existing guidelines of governmental agencies and scientific societies. The recommendations provided by each selected guideline were systematically explored and classified under five categories: Patients to be tested for CD, diagnostic tests (serology, duodenal biopsy, genetic test, no-biopsy diagnosis), potential/silent/seronegative CD, refractory/complicated CD, follow-up.

Research results

We identified 7 different guidelines [European Society Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) 2020; European Society for the Study of Coeliac Disease (ECD) 2019; World Gastroenterology Organization (WGO) 2017; Central Research Institute of Gastroenterology, Russia, 2016; National Institute for Health and Care Excellence (NICE), 2015; British Society of Gastroenterology (BSG), 2014; and America College of Gastroenterology (ACG), 2013]. These guidelines were mostly concordant but differed under certain recommendation for no-biopsy diagnosis, refractory CD, and follow-up.

Research conclusions

We found a relatively high concordance between the guidelines for CD. Important modifications have occurred in the last years, especially about the possibility of a no-biopsy diagnosis in children.

Research perspectives

Modifications of the current guidelines are expected in the near future. These modification will probably regard the possibility of a no-biopsy diagnosis (especially in developing countries) and the modalities of follow-up.

Conflict-of-interest statement: The authors declare that they have no conflict of interest.

PRISMA 2009 Checklist statement: The authors have read the PRISMA 2009 Checklist, and the manuscript was prepared and revised according to the PRISMA 2009 Checklist.

Provenance and peer review: Invited article; Externally peer reviewed.

Peer-review model: Single blind

Peer-review started: March 2, 2021

First decision: July 14, 2021

Article in press: December 25, 2021

Specialty type: Gastroenterology and hepatology

Country/Territory of origin: Italy

Peer-review report’s scientific quality classification

Grade A (Excellent): 0

Grade B (Very good): B, B, B, B

Grade C (Good): C

Grade D (Fair): 0

Grade E (Poor): 0

P-Reviewer: Poddighe D, Sabelnikova EA, Sahin Y, Vasudevan A S-Editor: Wang LL L-Editor: A P-Editor: Wang LL

Contributor Information

Alberto Raiteri, Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy.

Alessandro Granito, Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy.

Alice Giamperoli, Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy.

Teresa Catenaro, Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy.

Giulia Negrini, Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy.

Francesco Tovoli, Division of Internal Medicine, Hepatobiliary and Immunoallergic Diseases, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy. [email protected] .

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The Celiac Disease Foundation Clinical Trial Finder was created to help people with celiac disease and non-celiac gluten/wheat sensitivity, and healthy controls (people who do not have the disease), participate in clinical trials to accelerate the development of drugs and treatments. With up to 50% of patients continuing to experience symptoms and/or intestinal damage while on the gluten-free diet, finding a better treatment is crucial.

The purpose of a clinical trial is to determine the most effective and safest treatment for a disease. Clinical trials are a vital component of U.S. Food and Drug Administration’s drug approval process, without which advances in therapeutics for celiac disease patients are not possible. Your participation can end the needless suffering for generations to come.

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Acute Abdomen in Adults- a Prospective Study on Emergency Department Admissions

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Longitudinal prospective multicenter Armenian registry of systemic autoimmune, autoinflammatory diseases with constitution of bio-banking.

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A Study to Assess the Safety of TPM502 in Adults With Celiac Disease

The goal of this clinical trial is to learn about the safety and the pharmacodynamic (PD) effects of TPM502 in adults with celiac disease. The main questions it aims to answer are: - if TPM502 is safe and well tolerated - if TPM502 can induce modifications in parameters indicating that it may induce tolerance to gluten Participants will: - undergo 1-day gluten challenge during screening and after administration of TPM502 or placebo. - receive 2 infusions of TPM502 or placebo, 2 weeks apart

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Bio-markers of Not-celiac Wheat Sensitivity

The aim of the investigators' study is to evaluate biochemical, immunological and histological characteristics of patients affected with the so-called "gluten (or wheat) sensitivity" who suffers from irritable bowel syndrome (IBS)-like symptoms. As it is not known what component of the cereals causes the symptoms in so called "gluten-sensitive" patients, the investigators prefer to speak of "not-celiac wheat sensitivity" (NCWS). NCWS patients may be defined as ones, neither celiac or allergic to wheat, who develop symptoms following wheat consumption, that improved on wheat/gluten free diet (GFD). For our research, we will...

Bovine Colostrum to Prevent Absorption of Gluten

To investigate the use of hyperimmune bovine colostrum to reduce gluten absorption. A double-blind, cross-over study will be performed in which persons who are following a strict gluten-free diet will be challenged with oral gluten with or without the bovine colostrum.

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Look for research institutions, hospitals, and universities actively conducting research on CeD. Search online for ongoing clinical trials or research studies related to CeD. Websites such as  ClinicalTrials.gov  or research institutions’ websites are good places to start.

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Published May 1, 2024

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When blood tests results are highly positive, a biopsy might not be needed to diagnose celiac disease.

Mildly elevated results not enough to get an accurate diagnosis for children or adults, studies suggest

By Amy Ratner, director of scientific affairs

Currently in the United States, children and adults usually need to have a positive celiac disease blood test, followed by an endoscopy and biopsy to definitively diagnose celiac disease. But scientists have been discussing whether celiac disease blood tests alone could be used for diagnosis, particularly in children.

This question was the basis of two studies presented as posters recently at Digestive Disease Week (DDW).

In one study researchers from 11 children’s medical centers in the United State and Canada set out to measure how accurate a blood test is in predicting who has celiac disease, called the positive predictive value. The second study, by researchers from six medical centers in the United States, similarly evaluated the blood test in adults.

In both studies, the tissue transglutaminase IGA (TTG-IgA) test was found to be accurate in predicting celiac disease in patients who had very high levels of TTG-IgA antibodies. However, the accuracy of the tests fell when predicting celiac disease in those whose blood tests were positive but not at extreme levels.

Highly positive blood tests results, 10 times the upper limit of normal, correctly predicted when children had celiac disease nearly 96 percent of the time, a study presented by M. Camilla Cardenas, MD, of the Mayo Clinic, found. But more than 4 percent of children with these results did not have celiac disease.

Of about 1,600 children in the study who had high levels of TTG-IgA, about 1,500 had the intestinal damage found in a biopsy that confirms a celiac disease diagnosis.  Another 73 children did not.

In the United States, guidelines from the North American Society for Pediatric Gastroenterology, Hepatology & Nutrition (NASPGHAN) call for both positive TTG-IgA tests and a biopsy showing damage to the intestine for celiac disease confirmation. Adult gastroenterological associations also recommend blood tests and a biopsy but allow in some cases for a diagnosis based on elevated levels of TTG-IgA and a positive endomysial antibody test.

European pediatric guidelines allow for a celiac disease diagnosis without a biopsy for children who meet certain criteria, including blood tests results 10 times the upper limit of normal. This has raised the question of whether the same no-biopsy approach could be used more routinely in the United States, particularly because an endoscopy with a biopsy is an invasive procedure.

During an endoscopy, a very thin, flexible tube is snaked from the mouth to the small intestine, and a small tool is used to take tissue samples, called biopsies, from the wall of the intestine. Patients are sedated, with the type and amount of sedation dependent on age and any other co-existing medical conditions.

The study also evaluated the accuracy of TTG-IgA test when all positive blood tests, not just highly positive tests, were considered.

Of about 3,800 children with any level of positive TTG-IgA, about 83 percent were confirmed to have celiac disease based on biopsy results.

Varying accuracy of TTG-IgA tests

The study’s evaluation of how well TTG-IGA tests work in positively predicting celiac disease in children found variation between tests made by different companies. That variation could lead to different results based on which test a child is given.

In children who had follow-up biopsies to confirm celiac disease, a test made by Thermo Fisher had the highest positive predictive value in both those who had a highly positive TTG-IgA result and those who had a TTG-IgA positive result. About 97 percent of children with high positive results had confirmed celiac disease and about 89 percent of those who had any positive result did as well.

This compared to results ranging from 95 percent to 90 percent in highly positive children and 81 to 76 percent in children with any positive result when tests made by Bio Rad and Inova were used.

This creates a need for standardization of tests or specific thresholds for each test if diagnosis was to be based on blood test results alone in the United States, the study concludes.

In the study that compared blood test and biopsy results, nearly 99 percent of adults who had ten times the upper limit of normal TTG-IgA were confirmed to have celiac disease. Of the 132 patients in this category, only two did not show a level of intestinal damage that indicated celiac disease.

The study included about 4,300 patients who were having an endoscopy and biopsy, with a TTG-IgA test before or shortly after the procedure. About 25 percent had some level of a positive TTG-IgA result. Overall, the positive predictive value of TTG-IgA was about 88 percent, the study presented by Claire Jansson-Knodell, MD, of the Cleveland Clinic, found.

On the flip side, when the TTG-IgA test was normal, the probability that a person did not have celiac disease was high, with a negative predictive value of about 93 percent.

“A noninvasive diagnosis of celiac disease may be accurate in selected American adults with high TTG-IgA, but caution is needed with a mildly elevated TTG-IGA-IgA,” the study concluded. In those with a mildly elevated TTG-IgA a biopsy may still be needed to confirm a celiac disease diagnosed, the study says.

DDW is the largest international gathering of physicians, researchers and academics in the fields of gastroenterology, hepatology, endoscopy and gastrointestinal surgery. Studies presented at DDW are sometimes preliminary and give an early look at investigations that are likely to include more details as they progress toward publication in a peer reviewed scientific journal. Studies selected to be presented at DDW go through a review process.

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• Published: 24 June 2024

Transcriptomic analysis of intestine following administration of a transglutaminase 2 inhibitor to prevent gluten-induced intestinal damage in celiac disease

• Valeriia Dotsenko   ORCID: orcid.org/0000-0002-0336-0533 1 ,
• Bernhard Tewes 2 ,
• Martin Hils   ORCID: orcid.org/0000-0001-5400-3600 3 ,
• Ralf Pasternack 3 ,
• Jorma Isola   ORCID: orcid.org/0000-0002-0849-5939 4 , 5 ,
• Juha Taavela   ORCID: orcid.org/0000-0003-3948-9555 1 , 6 ,
• Alina Popp 1 , 7 ,
• Jani Sarin 5 ,
• Heini Huhtala 8 ,
• Pauliina Hiltunen 9 ,
• Timo Zimmermann 2 ,
• Ralf Mohrbacher   ORCID: orcid.org/0000-0002-4976-3629 2 ,
• Roland Greinwald 2 ,
• Knut E. A. Lundin   ORCID: orcid.org/0000-0003-1713-5545 10 , 11 ,
• Detlef Schuppan   ORCID: orcid.org/0000-0002-4972-1293 12 , 13 ,
• Markku Mäki   ORCID: orcid.org/0000-0001-5053-3794 1 ,
• Keijo Viiri   ORCID: orcid.org/0000-0002-7167-5094 1 &

CEC-3 Investigators

Nature Immunology ( 2024 ) Cite this article

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• Coeliac disease

Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2 / HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.

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Gluten-containing cereals are essential foods worldwide. However, in up to 2% of individuals 1 , the ingestion of dietary gluten results in an abnormal immune response in the small intestine and the development of celiac disease (CeD). Predisposing genotypes (human leukocyte antigen (HLA), for example, HLA-DQ2 and HLA-DQ8 ) are necessary but not sufficient for the manifestation of CeD. Diarrhea, weight loss and malnutrition are classical bowel-related symptoms and signs of CeD, but anemia, osteoporosis and other autoimmune diseases, such as type 1 diabetes, are also frequent manifestations 2 , 3 , 4 .

Currently, a gluten-free diet (GFD) is the only accepted treatment option for individuals with CeD. However, the life-long strict and restrictive GFD is onerous and difficult to follow, and inadvertent gluten ingestion is common 5 , 6 , resulting in ongoing symptoms in nearly 50% of treated individuals 7 , 8 . Keeping the GFD also has a big impact on quality of life 9 . Inadvertent gluten ingestion often leads to ongoing duodenal mucosal injury, with inflammation and morphological changes 10 . Thus, even individuals on a GFD frequently have nutrient imbalances and deficiencies 11 , 12 . We have shown that despite having normal duodenal histomorphology, individuals with CeD on a GFD differ from individuals without CeD on the molecular level and display insufficient expression of micronutrient transporter genes 13 . Thus, adjunctive pharmacological therapy, together with a strict GFD, is needed to efficiently treat CeD.

The CeD autoantigen transglutaminase 2 (TG2) is expressed in the intestine, where it deamidates certain neutral glutamine residues to negatively charged glutamic acid residues in immunogenic gluten peptides 14 , 15 , 16 . These modified gluten peptides are more efficiently presented by HLA-DQ2 or HLA-DQ8 molecules on mucosal antigen-presenting cells, which leads to the activation and expansion of gluten-specific CD4 + type 1 helper T cells and the secretion of proinflammatory cytokines 17 , 18 . Eventually, this process leads to villus atrophy, crypt hyperplasia and the production of TG2 IgA.

TG2, being crucial for CeD pathogenesis, is a pertinent target for therapy, and this approach was recently tested in a phase 2, randomized, double-blind, placebo-controlled, dose-finding gluten challenge trial using the oral TG2 inhibitor ZED1227 (ref. 19 ). In this phase 2 trial, ZED1227 attenuated gluten-induced duodenal mucosal injury, both morphological deterioration and inflammation, and improved symptoms and quality of life scores in individuals with CeD 19 . Here, we report the results of the molecular histomorphometry assessment of ZED1227 efficacy along with intestinal mucosal transcriptomic analysis. Moreover, as the gene dose of HLA-DQ2 was shown to influence the severity of CeD 20 , 21 , we analyzed the efficacy parameters of ZED1227 relative to the HLA-DQ2 gene dose.

ZED1227 prevents gluten-induced transcriptomic changes

Duodenal biopsies were collected from 58 individuals with CeD before (GFD) and after a 6-week gluten challenge combined with treatment with 100 mg of the TG2 inhibitor ZED1227 per day (postgluten challenge drug (PGCd); n  = 34) or placebo (PGC placebo (PGCp); n  = 24). RNA extracted from the 116 biopsy samples was subjected to transcriptomic next-generation sequencing (NGS) analysis.

Principal component analysis (PCA) performed on all samples using DESeq2-transformed counts of all genes showed a moderate level of separation between groups (GFD drug (GFDd), GFD placebo (GFDp), PGCd and PGCp; Fig. 1a ). The PGCp group was clearly discernible, whereas the GFDd, GFDp and PGCd groups tended to cluster closer together. There was a clear cosegregation of transcriptomic profiles and mucosal morphology. Thus, a ratio of villus height to crypt depth (VH:CrD) of <1.2 separated from VH:CrD of ≥1.2 and overlapped with PGCp in the PCA (Fig. 1a ). A comparison of the PGCp versus GFDp groups detected 95 differentially expressed genes (DEGs; Fig. 1b,c ). Strikingly, only one DEG was detected when the GFDd group was compared to the PGCd group, whereas the comparison of the PGCp and PGCd groups indicated 180 DEGs (Fig. 1b,c and Supplementary Data 1 ).

a , PCA plot using DESeq2-transformed counts for all samples ( n  = 115). Green, dark green, violet and orange circles correspond to GFDd ( n  = 34), GFDp ( n  = 24), PGCd ( n  = 34), and PGCp ( n  = 23) samples, respectively. Yellow, blue and red shaded areas depict samples with a high (H; >2.5), medium (M; 1.2–2.5) and low (L; <1.2) range of VH:CrD, respectively. b , Table showing the number of DEGs (log 2  (FC) ≥ | 0.5 | and false discovery rate (FDR) ≤ 0.05) in the indicated comparisons. c , Volcano plot representations comparing DEGs as indicated. The green dots indicate DEGs (FDR ≤ 0.05) above the threshold (log 2  (FC) of ≥0.5 and ≤−0.5). The dashed horizontal line represents the FDR threshold of 0.05, and the vertical dashed lines represent the log 2  (FC) thresholds (≥| 0.5 |). d , Venn diagram illustrating the number of DEGs that are shared in the PGCp versus PGCd and PGCp versus GFDp comparisons. e , Correlation profile of all detected gene ( n  = 10,063) log 2  (FC) values between PGCp and GFDp and PGCp and PGCd comparisons. f , Pearson’s pairwise correlation heat map analyses of 220 DEGs visualizing the cross-correlations of the transcriptomic profiles of the samples (total n  = 115; GFDd n  = 34; GFDp n  = 24; PGCd n  = 34; PGCp n  = 23). Samples are organized in the ranking order of increasing VH:CrD ratio (indicated in the scatter charts above the heat map).

Source data

Because treating participants with ZED1227 eliminated the gluten-induced gene expression changes entirely, it can be assumed that the majority of the DEGs in the PGCp versus GFDp and PGCp versus PGCd comparisons were shared. Indeed, 56 of 95 (59%) DEGs after the gluten challenge were also differentially expressed, according to the comparison of the PGCp and PGCd groups (Fig. 1d ). This analysis suggests that a significant number of genes were ‘uniquely’ differentially expressed after gluten challenge (39 of 95) and between the PGCd and PGCp groups (124 of 180; Fig. 1d ). Closer inspection of both ‘uniquely expressed’ DEGs revealed that they were not uniquely differentially expressed in PGCd but, to an extent, were equivalent to those expressed in the GFD group, although this was not sufficiently statistically significant (for example, due to inadequate log (fold change) (FC) or expression level), relative to the PGCp group (Supplementary Fig. 1 ). When all detected gene log 2  (FC) values from the PGCp versus GFDp comparison were compared to those from the PGCp versus PGCd comparison, there was a positive correlation, suggesting a similar pattern of gene expression changes in both groups (Fig. 1e ). Accordingly, a Pearson’s pairwise correlation heat map analysis with the 220 selected genes showed that the GFDd, GFDp and PGCd groups had similar features, whereas the PGCp group significantly differed from all groups (Fig. 1f ). Similar to the results in Fig. 1a , ranking samples according to VH:CrD ratio made it evident that individuals with the most severe mucosal damage, that is, the lowest VH:CrD ratio, had a very different transcriptomic profile (Fig. 1f ).

ZED1227 sustains molecularly assessed intestinal functions

An analysis of the expression data of the 95 DEGs individually after the gluten challenge in the placebo group showed that the expression levels correlated with the VH:CrD ratio (Fig. 2a ). Reactome enrichment analysis showed that genes involved in the cellular response to interferon (IFN) signaling, both type 1 (IFNα/IFNβ) and type 2 (IFNγ), were upregulated and overrepresented in the gluten-induced gene expression profile (Fig. 2b , left, and Supplementary Data 2 ). Transcription motif analyses also indicated that genes harboring motifs for transcription factors transducing IFN signaling (for example, STAT1, RELA and IRF1) were significantly present (Supplementary Fig. 2 ). Notably, a reactome enrichment comparison of the DEGs in the PGCp versus PGCd groups revealed that the type 2 IFNγ signaling term was no longer statistically significant (Fig. 2b , right, and Supplementary Data 2 ). Similarly, the Gene Ontology term analyses showed that IFN-mediated inflammatory signaling was enriched in the gluten-induced gene expression profile (Fig. 2c and Supplementary Data 2 ).

a , Heat map of the 95 DEGs in the PGCp versus GFDp comparison. Samples are ordered by increasing VH:CrD ratio, as depicted in the scatter charts above the heat map (GFDd n  = 34; GFDp n  = 24; PGCd n  = 34; PGCp n  = 23). Genes are clustered according to Gene Ontology annotation. The z -score of normalized expression is plotted; OBP, other biological processes. b , Bar plot showing enriched Reactome terms of DEGs in the PGCp group relative to the GFDp and PGCd groups. Enriched terms were determined by overrepresentation analysis. P values were calculated by hypergeometric distribution (one-tailed test) and adjusted for multiple testing using the Benjamini–Hochberg method. Reactome terms with an FDR of <0.05 (–log 10  (FDR) > 1.3) were considered enriched. Green and gray dots denote significant and nonsignificant FDRs, respectively. c , Bar plots showing Gene Ontology biological process overrepresentation of DEGs in the PGCp group relative to the GFDp and PGCd groups. A Fisher’s exact overrepresentation test (one tailed) was used to find enriched categories. The obtained P values were adjusted for multiple testing using the Benjamini–Hochberg method. Gene Ontology terms with an FDR of <0.05 (–log 10  (FDR) > 1.3) were considered enriched. Green and gray dots denote significant and nonsignificant FDRs, respectively. d , GSZ score analyses were performed for categories including transit-amplifying cells, mature enterocytes, immune cells and duodenal transporters and are presented as box plots, with center lines representing the median, the box boundaries representing the interquartile range and the whiskers representing the minimum and maximum values. Values from individual participants are shown (GFDd + p n  = 58; PGCd n  = 34; PGCp n  = 23). GSZ scores were compared among groups using asymptotic P value estimation, with statistical significance defined as a P value of <0.05 (transit-amplifying cells: GFDd + p–PGCd P  = 0.3, PGCp–GFDd + p P  = 0.03, PGCd–PGCp P  = 0.004; mature enterocytes: GFDd + p–PGCd P  = 0.3, PGCp–GFDd + p P  = 0.005, PGCd–PGCp P  = 5.35 × 10 −4 ; immune cells: GFDd + p–PGCd P  = 0.73, PGCp–GFDd + p P  = 0.02, PGCd–PGCp P  = 0.03; duodenal transporters: GFDd + p–PGCd P  = 0.53, PGCp–GFDd + p P  = 0.02, PGCd–PGCp P  = 0.009).

As gluten challenge impairs enterocyte differentiation and absorptive functions and increases inflammation, we analyzed how ZED1227 protects these cellular processes. Gene sets were formed based on human duodenal single-cell RNA-sequencing data 22 . Gene set z (GSZ) scores 23 were calculated for each sample. Samples in the PGCd group demonstrated the same GSZ score levels in the categories of transit-amplifying cells, mature enterocytes, immune cells and duodenal transporters as samples in the pooled GFDd and GFDp groups (GFDd + p) group (Fig. 2d ). Importantly, the PGCp group was consistently significantly different from the PGCd group, indicating that ZED1277 efficiently sustained intestinal functions to a level similar to that observed in individuals in the GFDd + p group. Bulk RNA-sequencing deconvolution that used duodenal single-cell RNA-sequencing data as a reference revealed similar patterns in cell proportion distributions, like a decrease in enterocyte numbers accompanied with a small increase in stem and Paneth cell numbers in the PGCp group (Supplementary Fig. 3a,b ). At the same time, markers for cytotoxic intraepithelial lymphocytes (IELs) seemed to not be altered (except HLA-E) by placebo and drug treatment (Supplementary Fig. 3c ), probably because of underrepresentation of these cell types in biopsy samples.

ZED1227 can halt the IFNγ response

Reactome and Gene Ontology enrichment analyses (Fig. 2b,c ) indicated that IFN signaling was one of the most significantly affected pathways in the gluten challenge. Interestingly, a 100-mg dose of ZED1227 for 6 weeks seemed somewhat insufficient in decreasing the IFNγ response, at least according to the Reactome enrichment analysis (Fig. 2b ). We decided to set up an intestinal epithelium-specific IFNγ response gene set to assess how well ZED1227 could inhibit inflammation using an epithelial-specific IFNγ response as a gauge. Human intestinal organoids composed of pure intestinal epithelium were treated with IFNγ, and a DEG set was analyzed against the DEGs induced by gluten challenge. We found that nearly half (43 of 95) of the gluten-induced gene expression changes in CeD were associated with the epithelial response to IFNγ (Fig. 3a and Supplementary Data 3 ). The GSZ scores calculated based on these 43 genes showed that, on average, ZED1227 inhibited the epithelial IFNγ response, as participants in the PGCd group had significantly lower GSZ scores than participants in the PGCp group (Fig. 3b ). However, when the GSZ scores of the PGCd and GFDd + p groups were compared, there was a slight but statistically significant difference. This suggests that either there was a residual IFNγ response in all/many participants in the PGCd group or ZED1227 was not able to inhibit the IFNγ response completely in some individuals. When GSZ scores were calculated for each sample, it was evident that some individuals (4 of 34 participants in the PGCd group) still had an active epithelial IFNγ response even after the high-dose (100-mg) ZED1227 treatment for 6 weeks (Fig. 3c ).

a , Venn diagram of all DEGs in human duodenal organoids ( n  = 3) after a 24-h treatment with 100 U ml –1 IFNγ (violet sphere) and PGCp versus GFD (orange sphere) comparisons. b , GSZ score analyses for the epithelial IFNγ-related gene set (GFDd + p n  = 58; PGCd n  = 34; PGCp n  = 23). The box plot center lines represent the median, the box boundaries represent interquartile range, and the whisker length represents the minimum and maximum range. Values from individual participants are shown. GSZ scores were compared among groups using asymptotic P value estimation, with statistical significance defined as a P value of <0.05 (GFDd + p–PGCd P  = 0.05, P GCp–GFDd + p P  = 6.07 × 10 −6 , PGCd–PGCp P  = 1.24 × 10 −4 ). c , Bar plot of epithelial IFNγ-related GSZ scores calculated for each sample. The dashed lines represent the threshold, outside of which the gene set was considered to be ‘on’ or ‘off’. The yellow bar below illustrates the samples in which the epithelial IFNγ-related GSZ scores were on and off (GFDd n  = 34; GFDp n  = 24; PGCd n  = 34; PGCp n  = 23). d , Expression of TGM2 mRNA in the GFDd, GFDp, PGCd and PGCp groups. The box plot center lines represent the median, the box boundaries represent interquartile range, and the whisker length represents the minimum and maximum range. Values from individual participants are shown. Likelihood ratio test (LRT) P values were calculated using DESeq2, with P values representing adjusted values for multiple testing using the Benjamini–Hochberg method (FDR; GFDd n  = 34; GFDp n  = 24; PGCd n  = 34; PGCp n  = 23). e , Expression of TGM2 mRNA in human duodenal organoids ( n  = 3) treated with 100 U ml –1 IFNγ (I) or mock treated (M) for 24 h. The box plot center lines represent the median, the box boundaries represent interquartile range, and the whisker length represents minimum and maximum range. Values from individual participants are shown. LRT P values were calculated using DESeq2, with P values representing adjusted values for multiple testing using the Benjamini–Hochberg method (FDR; P  = 9.48 × 10 −17 ). f , Correlation plot for TGM2 mRNA expression and epithelial IFNγ-related GSZ scores. Each dot represents an individual participant with CeD after gluten challenge. Pearson correlation coefficient values ( R ) are presented, and the P value ( P ) was calculated based on the t- distribution under the null hypothesis of no correlation using a two-tailed test; P  = 5.57 × 10 −8 .

IFNγ has been shown to induce TG2 activity in intestinal epithelial cancer cells, and this has been suggested to contribute to CeD pathogenesis 24 . Similarly, participants in the placebo group after the gluten challenge and concomitant IFNγ response had significantly higher expression of TGM2 , whereas in participants treated with ZED1227, TGM2 was expressed at a level similar to that observed in participants in the GFDd group (Fig. 3d ). Overproduced interleukin-21 (IL-21) in CeD is known to sustain IFNγ production 25 , and we also detected an induction in the IL-21 signaling pathway in participants in the PGCp group (Supplementary Fig. 4a,b ), but this was not statistically significant. We also found that the expression of TGM2 was positively correlated ( R = 0.65) with the epithelial IFNγ response (Fig. 3f ). Direct causality was further proven by treating human intestinal duodenal organoids with IFNγ, which resulted in a significant induction of TGM2 mRNA expression (Fig. 3e ) that could not be inhibited with ZED1227 treatment (Supplementary Fig. 4c ). IFNγ treatment induced TG2 activity in Caco-2 cells, which was inhibited by ZED1227 to the level observed following mock treatment (Supplementary Fig. 4d ). These observations could be explained by ZED1227 cell impermeability 26 and its binding mainly to enterocyte luminal surfaces 27 .

ZED1227 prevents activation of gluten-induced immunological pathways

As gluten challenge caused a significant IFNγ response and concomitant upregulation of TGM2 expression and activity, we analyzed gluten challenge-induced immunological pathway alterations and how ZED1227 can inhibit them. Peroxisome proliferator-activated receptor-γ (PPARγ) has been shown to transrepress inflammatory responses 28 , 29 . PPARγ is downregulated in celiac mucosa 30 , and this has been shown to be mediated by TG2 and gliadin 31 . We also found that PPARG gene expression (Fig. 4a ) and the corresponding signaling pathway (Fig. 4b ) are significantly less active after gluten challenge in the PGCp group than in the GFD and PGCd groups. We also observed a negative correlation between the expression of TGM2 and PPARG and the expression of PPARG and IEL count (Fig. 4c ). This suggests that the mucosal inflammatory response, kept in check by PPARγ, is lifted during the gluten challenge in CeD, and this can be prevented with ZED1227 treatment.

a , Expression of PPARG mRNA in the GFDd, GFDp, PGCd and PGCp groups. LRT P values were calculated using DESeq2, with P values representing adjusted values for multiple testing using the Benjamini–Hochberg method (FDR; GFDd n  = 34; GFDp n  = 24; PGCd n  = 34; PGCp n  = 23). b , GSZ score analyses for the PPAR signaling pathway from the KEGG database gene set. GSZ scores were compared among groups using asymptotic P value estimation, with statistical significance defined as a P value of <0.05 (GFDd + p n  = 58; PGCd n  = 34; PGCp n  = 23). c , Correlation plots for TGM2 mRNA expression (top) and IEL density (number of CD3 + cells per 100 enterocytes; bottom) against PPARG mRNA expression. Each dot represents an individual participant with CeD after gluten challenge. The Pearson correlation coefficient ( R ) is presented, and the P value ( P ) was calculated based on the t -distribution under the null hypothesis of no correlation using a two-tailed test ( TGM2 mRNA expression versus PPARG mRNA expression, P  = 1.14 × 10 −5 ; IEL density versus PPARG mRNA expression, P  = 2.95 × 10 −7 ). d , Expression of NOS2 mRNA in the GFDd, GFDp, PGCd and PGCp groups. LRT P values were calculated using DESeq2, with P values representing adjusted values for multiple testing using the Benjamini–Hochberg method (FDR; GFDd n  = 34; GFDp n  = 24; PGCd n  = 34; PGCp n  = 23). e , GSZ score analyses for selected KEGG, BIOCARTA and Reactome database gene sets. GSZ scores were compared among groups using asymptotic P value estimation, with statistical significance defined as a P value of <0.05 (GFDd + p n  = 58; PGCd n  = 34; PGCp n  = 23). The box plot center lines represent the median, the box boundaries represent interquartile range, and the whisker length represents minimum and maximum range. Values from individual participants are shown. f , Heat map for selected CeD-specific immune cell marker genes detected in Atlasy et al. 58 . Samples are ordered by increasing IEL density, as depicted in the scatter charts above the heat map (GFDd n  = 34; GFDp n  = 24; PGCd n  = 34; PGCp n  = 23). The z scores of normalized expression are plotted; PC, plasma cells; Inf-MF, inflammatory macrophages.

PPARγ inhibits the expression of proinflammatory cytokines, and it also silences inducible nitric oxide (NO) synthase (iNOS/NOS2) 32 . NOS2 is induced in the mucosa of individuals with active CeD mainly in macrophages and enterocytes 33 , 34 , 35 , leading to a systemic increase of NO in the plasma 36 .

NO is needed for the responsiveness of natural killer (NK) cells to the NK cell-activating factor IL-12, which stimulates cytotoxicity and IFNγ release 37 . Our data show that ZED1227 can inhibit gluten challenge-induced NOS2 upregulation (Fig. 4d ), resulting in overrepresentation of gene sets involved in the NO–IL-12 and NK cell-mediated cytotoxicity (Fig. 4e ) pathways. Also, pathways to antigen presentation and IgA production are normalized following ZED1227 treatment (Fig. 4e ). Analysis of the expression of immunological cell gene markers showed that ZED1227 inhibits the infiltration of cell types (especially CD8 + T cells, plasma cells, NK cells and macrophages) involved in the aforementioned inflammatory responses (Fig. 4f ).

The effect of HLA-DQ genetic background on treatment outcomes

The fact that some participants treated with ZED1227 in the PGCd group still showed a significant epithelial IFNγ response (Fig. 3c ), as a sign of active residual CeD pathophysiology prompted us to study factors behind the incomplete response to treatment. To this end, we performed high-resolution genotyping for HLA class II DQ alleles using the arcasHLA tool 38 from aligned sequences obtained from genome-wide 3′ RNA-sequencing data. Five participants had too low coverage either at the HLA-DQB1 or HLA-DQA1 locus, according to RNA sequencing; thus, their allele typing was performed from blood samples collected at the on study inclusion. One participant from the placebo group, however, failed during identification. This participant is marked as ‘not identified’ in Table 1 and was excluded from subsequent analyses.

It is known that HLA-DQ2 gene dose correlates with the strength of the gluten-specific T cell response 20 ; thus, all obtained genotypes were divided into groups by their potential effectiveness in binding and presenting gliadins to T cells 39 , 40 . We were able to divide participants into three groups according to their HLA-DQ genotypes, with G1 being the high-gluten-response group and G3 being the low-gluten-response group (Table 1 ). However, one should note that the group sizes are relatively small.

When examining the changes in mean VH:CrD ratio within genotype groups over time (Fig. 5a ), it is evident that the groups exhibit different trajectories of change. Notably, the slope of the G1 group appears to deviate the most from the parallel pattern among the groups for both drug and placebo treatments.

a , The VH:CrD ratio remains higher in the drug group than in the placebo group, regardless of the genotype. Participants ( n  = 57) were divided into two groups according to the treatment received (drug or placebo). The VH:CrD ratio at PGC is shown as mean ± s.d. b , A two-way ANCOVA was performed with the VH:CrD ratio at PGC as a dependent variable, the VH:CrD ratio at GFD as a covariate and treatment (drug n  = 34 and placebo n  = 23) and HLA-DQ genotype group (G1, G2 and G3) as independent variables (ANCOVA, F 2,50  = 2.2, P  = 0.12). Post hoc pairwise multiple comparisons were performed between the drug and placebo groups among HLA-DQ genotype groups. The VH:CrD ratio at PGC is shown as estimated marginal means ± 95% confidence interval (95% CI; drug G1 n  = 6; drug G2 n  = 14; drug G3 n  = 14; placebo G1 n  = 2; placebo G2 n  = 6; placebo G3 n  = 15). c , The G1 genotype group showed weaker recovery after ZED1227 treatment as assessed by VH:CrD ratio. Participants ( n  = 34) belonging to the drug group were selected for one-way ANCOVA. The VH:CrD ratio at PGCd was used as a dependent variable, and the VH:CrD ratio at GFD was used as a covariate; HLA-DQ genotype group (G1, G2 and G3) served as independent variables (ANCOVA, F 2,30  = 5.11, P  = 0.012). Post hoc pairwise multiple comparisons were performed between HLA-DQ genotype groups, with P values adjusted by Bonferroni correction. Results are shown as estimate ± 95% CI. d , A two-way ANCOVA plot examining the effects of treatment and HLA-DQ genetic background on PGC epithelial response to IFNγ GSZ score (ANOVA, F 2,49  = 0.07, P  = 0.93). The epithelial response to IFNγ GSZ score at PGC is shown as estimated marginal means ± 95% CI (drug G3 versus placebo G3 P  = 5.50 × 10 −4 ; drug G1 n  = 6; drug G2 n  = 14; drug G3 n  = 14; placebo G1 n  = 2; placebo G2 n  = 6; placebo G3 n  = 15). e , Expression of enterocyte- ( APOB , APOA1 and TMSF4 ), proliferation- ( AGR2 , MKI67 and CENPF ) and inflammation-related ( STAT1 , GBP1 , TGM2 , CIITA , PPARG and NOS2 ) marker genes. Expression is shown as counts grouped by HLA-DQ genotype group (G1, G2 and G3) and are presented as mean (spheres) and s.d. (vertical lines; PGCd G1 n  = 6; PGCd G2 n  = 14; PGCd G3 n  = 14; PGCp G1 n  = 2; PGCp G2 n  = 6; PGCp G3 n  = 15).

The impact of treatment on VH:CrD ratio within different time points (GFD and PGC) across HLA-DQ genetic background groups (G1, G2 and G3) was assessed by fitting repeated-measures analysis of variance (ANOVA). In the placebo group, the interaction term between time point and HLA-DQ genetic groups was statistically significant ( P  = 0.003; Table 2 and Methods ), indicating that HLA-DQ genetic background has an impact on changes in VH:CrD ratio over the course of gluten challenge ( Methods ). For the drug group, however, the interaction term was not significant ( P  = 0.06; Table 2 and Methods ), suggesting that the drug appears to be effective in reducing the impact of gluten across all genotype groups. However, pairwise comparisons (Table 2 and Methods ) performed for the drug group showed that the impact of HLA-DQ genetic background is statistically significant for the G1 group ( P  = 0.05) and not significant for the G2 ( P  = 0.07) and G3 groups ( P  = 0.39).

Given the notable drop in the VH:CrD ratio after ZED1227 treatment in the high-gluten-response genotype group (G1), we analyzed the efficacy of treatments in each genotype group. A two-way analysis of covariance (ANCOVA) was performed to examine the effects of treatment and HLA-DQ genetic background on VH:CrD ratio at PGC. After adjustment for the VH:CrD ratio at GFD, there was no statistically significant interaction between treatment and the HLA-DQ genotype group on the histomorphometry parameters ( Methods ), and pairwise multiple comparisons show significant differences between the PGC VH:CrD means in all genotype groups between participants receiving drug or placebo (Fig. 5b ). This suggests that, despite a substantial decrease in VH:CrD ratio after gluten challenge in the G1 group for participants treated with drug, the VH:CrD ratio was still higher in the drug group than in the placebo group, irrespective of the genotype.

The estimated difference in the VH:CrD ratio for participants treated with drug belonging to the G3 genotype versus the G1 genotype was −0.52 (95% CI of −0.86 to −0.19) with an adjusted P value of 0.01, as assessed by fitting a one-way ANCOVA model. Other estimated differences (G3–G2 and G2–G1) were not significant but showed the tendency of group G2 having the intermediate position between G1 and G3, when judging by VH:CrD ratio (Fig. 5c ). Interestingly, the G1 high-risk genotype specifically affected VH and not CrD (Extended Data Fig. 2a,b ).

The CeD pathophysiological epithelial IFNγ response was studied with a two-way ANCOVA statistical analysis, and pairwise comparisons showed that participants in the PGCd and G1 genotype groups still had an active IFNγ response and did not statistically differ from the placebo group (Fig. 5d ). In fact, in the bar plot presenting four participants in the PGCd group with an IFNγ response in Fig. 3c , three of these participants had the high-gluten-response genotype homozygous HLA-DQ2.5 and one had homozygous HLA-DQ8 associated with an intermediate response to gluten.

The inclination of the G1 group to be highly responsive to gluten and less reactive to ZED1227 was also observed at individual gene expression levels. Reduced expression of enterocyte marker genes ( APOB , APOA1 and TM4SF4 ) and increased expression of proliferation markers ( AGR2 , MKI67 and CENPF ) were observed in participants with G1 genotypes in both the ZED1227- and placebo-treated groups (Fig. 5e ). Inflammation-related genes ( STAT1 , GBP1 and TGM2 ) showed lower expression in PGCd samples with G2 and G3 genotypes, suggesting that they were more susceptible to ZED1227 treatment. In accordance with the higher residual CeD-associated epithelial IFNγ response in participants in the PGCd and G1 groups (Figs. 3b,c and 5d ), these inflammatory genes were more highly expressed in participants treated with either placebo or drug within the genotype group G1. Furthermore, ZED1227 was less able to prevent gluten challenge-induced attenuation of PPARγ-mediated inhibition of NOS2 expression, as the expression of these genes was at the same level in G1 genotypes in the PGCd group as in the G2 and G3 genotypes in the PGCp group (Fig. 5e ). Also, the HLA class II transcriptional coactivator CIITA was more highly expressed in individuals with the G1 genotype in the PGCd group (Fig. 5e ). Moreover, the G1 group was identified as more pathognomonic when its GSZ scores for ‘transit-amplifying cells’, ‘mature enterocytes, ‘immune cells’ and ‘duodenal transporters’ were assessed (Extended Data Fig. 2c ). In addition to IFNγ signaling, molecular pathways for PPAR and lipid signaling seemed to also be affected in the G1 group (Extended Data Fig. 2c ).

Molecular histomorphometric analysis of ZED1227 efficacy

We previously created a molecular histomorphometric model to assess gluten-dependent morphological deterioration and healing in the duodenum, that is, VH:CrD, in gene transcriptomic terms 13 . This model is based on the expression of four genes ( ATP8B2 , PLA2R1 , PDIA3 and TM4SF4 ), which we showed is significantly correlated with the extent of gluten-induced histological damage 13 . Scatter plots and partial regression plots for these genes showed that the relationship between gene expression and VH:CrD ratio was linear, and participants in the PGCp group tended to separate from participants in the GFD and PGCd groups (Fig. 6a ). Moreover, a comparison of traditional and molecular histomorphometry in the regression scatter plot revealed a high coefficient of determination ( R 2  = 0.86), indicating that the previously developed molecular histomorphometric tool was able to reliably estimate VH:CrD ratios in this independent study cohort (Fig. 6b ). Finally, box plot comparisons of groups with histomorphometric and molecular histomorphometric values indicated that ZED1227 efficiently inhibited gluten-induced mucosal damage in individuals with CeD (Fig. 6c ).

a , Scatter plots and regression plots for VH:CrD prediction model genes. A linear regression with 95% CI is shown. Each dot represents an individual participant (GFDd + p n  = 58; PGCd n  = 34; PGCp n  = 23); UMI, unique molecular identifiers. b , Observed versus predicted regression scatter plot for the model predicting VH:CrD. Each dot represents an individual participant ( n  = 115). A linear regression with 95% CI is shown; R 2  = 0.86, F 1,114  = 691.6, P  < 0.001. The red dashed line represents the ideal regression case, where x  =  y ; r.m.s.d., root mean square deviation. c , Box plot comparisons of groups with histomorphometry (measured VH:CrD) values and molecular histomorphometry (regression model based on RNA expression) values. The box plot center lines represent the median, the box boundaries represent interquartile range, and the whisker length represents minimum and maximum range. Values from individual participants are shown. Two-tailed unpaired Student’s t -tests were used for the PGCd versus PGCp group comparisons (VH:CrD observed: PGCd–PGCp P  = 6.41 × 10 −4 ; VH:CrD predicted: PGCd–PGCp P  = 4.04 × 10 −5 ; GFDd n  = 34; GFDp n  = 24; PGCd n  = 34; PGCp n  = 23).

The ability of the TG2 inhibitor ZED1227 (ref. 26 ) to attenuate gluten-induced mucosal damage was previously reported in a proof-of-concept, randomized, double-blind, placebo-controlled 6-week trial with a daily 3-g gluten challenge 19 . TG2, the celiac autoantigen 14 , has a pivotal role in gluten-induced pathogenesis, leading to small intestinal mucosal injury with villus atrophy and crypt hyperplasia, the histological hallmarks of untreated CeD. Here, we sought to assess the efficacy of ZED1227 in preventing gluten-induced mucosal damage at the transcriptomic level. Remarkably, a 100-mg daily dose of ZED1227 inhibited virtually all gluten-induced transcriptomic changes (Fig. 1b,c ). Active CeD is accompanied by compromised enterocyte maturation, crypt hyperplasia due to the expansion of transit-amplifying cells 41 , 42 , 43 , immune cell infiltration 44 , 45 and decreased expression of duodenal transporters 13 , 46 , 47 . GSZ 23 scores based on published single-cell databases 22 clearly indicated that TG2 inhibition efficiently blocked all aforementioned gluten-induced intestinal manifestations in individuals with CeD (Fig. 2d ). Our recently published molecular histomorphometry regression model based on genome-wide transcriptomics analysis 13 was validated in this independent study sample. We showed a significant accordance between this new molecular tool and the traditional, more subjective biopsy-based microscopic histomorphometry reading. Overall, our transcriptomic findings strongly support the results of the clinical trial with ZED1227, which demonstrated that the inhibition of TG2 activity can efficiently and specifically prevent gluten-induced mucosal damage 19 . Our data also corroborate the previous findings that gliadin together with active TG2 induces attenuated PPARγ activity, which, together with a concomitant increase in IFNγ, lead to increased mucosal NO production and inflammation 30 , 31 , 33 , 34 , 35 , 36 . We show here that by inhibiting the gliadin deamidation activity of TG2, all these pathogenic immunological changes in CeD can be prevented (Fig. 4 ). In addition, studies have shown that gluten-derived peptides may have innate immune stimulatory properties, outside the realm of adaptive immunity, which can lead to epithelial stress in CeD 48 , 49 . Our data show, however, that halting the adaptive immunity pathway in CeD pathogenesis is sufficient to prevent gluten-induced mucosal damage, as we did not detect any molecular traces of mucosal damage remaining after ZED1227 treatment.

Gene Ontology and Reactome analyses indicated that gluten challenge most significantly affected genes related to the immune response, especially IFN-mediated defense mechanisms (Fig. 2b,c ). This is in agreement with previously published transcriptomic analyses of individuals with active CeD compared to individuals on a GFD or healthy individuals 47 , 50 , 51 . Notably, IFNγ secreted by gluten-reactive T cells in the celiac intestine induces TG2 expression and secretion and thus favors the pathogenic autoamplificatory loop of enhanced gluten deamidation by TG2, improved antigenic presentation on HLA-DQ2 or HLA-DQ8 and subsequent gluten-specific T cell activation 24 . The present study confirms the prominent role of IFN signaling in CeD pathogenesis, in line with findings that nearly half of the gluten-induced gene expression changes in duodenal biopsies can be recapitulated in human intestinal epithelial organoids treated with IFNγ (Fig. 3a ). We also detected the suggested autoamplificatory loop in our human data, as TGM2 expression positively correlated with the epithelial IFNγ response (Fig. 3f ). Notably, TGM2 expression was induced by IFNγ in human intestinal organoids ex vivo (Fig. 3e ), suggesting mutual amplification between these two key players in CeD pathogenesis. The functional relevance of this amplification loop was indeed confirmed in the clinical study in which the inhibition of TG2 activity by ZED1227 in individuals with CeD significantly inhibited both the (epithelial) IFNγ response (Fig. 3b ) and TGM2 expression (Fig. 3d ), resulting in protection from villous atrophy and intraepithelial lymphocytosis (Fig. 2d ).

However, even though TG2 inhibition exhibited significant efficacy, according to a comparison of the transcripts of the placebo/gluten challenge and the gluten challenge/ZED1227-treated group, which showed a transcriptome profile similar to that of the GFD groups, we detected heterogeneity regarding the gluten-induced and IFNγ-dependent cascade of pathogenic events among ZED1227-treated and gluten-challenged individuals with CeD. Four of these individuals still had a modestly active IFNγ response, and the majority (three of four) belonged to the HLA-DQ2.5 homozygous genotype (Fig. 3c ). HLA-DQ2.5 homozygous individuals have a fivefold higher risk of developing CeD than HLA-DQ2.5 heterozygous individuals 21 , which has been linked to the more efficient presentation of deamidated gluten peptides to gluten-specific T cells 20 . Moreover, homozygosity for HLA-DQ2 predisposes individuals to developing more rapid and severe villous atrophy 52 and is associated with malignant complications, such as refractory CeD type 2 and enteropathy-associated T cell lymphoma 53 , 54 . Along this line, we also found that individuals belonging to the high-gluten-response HLA-DQ genotype group (G1) were more sensitive to gluten, as their VH:CrD ratios dropped significantly more than individuals belonging to the mid- and low-gluten response groups (G2 and G3) during the gluten challenge, both in the placebo and drug groups (Tables 1 and 2 and Fig. 5a,b ). Thus, even after drug treatment, VH:CrD decreased significantly in the G1 versus G2 and G3 genotype groups after the gluten challenge. This was also evident when molecular histomorphometric features were assessed (Extended Data Fig. 2c ). We also discovered that PPAR signaling and lipid metabolism, previously reported to be dysregulated in CeD 30 , were less controlled in the G1 group (Extended Data Fig. 2c ). As IFNγ is known to inhibit PPAR and lipid metabolism 55 , it is conceivable that these are consequences of the overactive IFNγ response in individuals in the G1 group. Nevertheless, duodenal mucosal morphology and, especially, intraepithelial lymphocyte infiltration were significantly healthier in the ZED1227-treated group than in the placebo group, indicating that participants with G1 phenotypes may benefit from a higher dose and/or prolonged treatment with ZED1227. We suggest that the ZED1227 therapy program should include a personalized medicine approach in which HLA-DQ stratification is combined with TG2 dose adjustments, which may lead to an optimal treatment response and a more thorough abrogation of IFNγ-induced mucosal damage. According to our transcriptomic analysis of human intestinal organoids, ZED1227 does not appear to induce significant transcriptomic changes in the organoid model (Supplementary Fig. 5 ), consistent with the clinical safety observed in the phase 2 challenge study 19 .

We recognize the limitations of this study. The cohort is relatively modest and characterized by an uneven distribution of HLA-DQ genotypes. This resulted in small G1 subgroups within both the drug and placebo cohorts, which may have implications for statistical power and the generalizability of our results and warrants further corroborative studies. Additionally, we only had one dose of the drug available for this study. The transcriptomic analysis was conducted as an optional component of the study, and RNA isolation was not performed for all drug groups. This decision was made to focus our efforts on the drug group that showed the most significant improvement compared to the placebo group, allowing us to investigate potential transcriptomic changes effectively within the study’s scope.

In conclusion, the strategy to inhibit TG2 activity as a key upstream effector in gluten-induced immune activation in CeD, which has been proven efficient in the clinical study, was mechanistically buttressed by our transcriptomic analysis of the duodenal biopsies of individuals treated or not treated with ZED1227. Importantly, TG2 inhibition prominently prevented the gluten-induced IFNγ response and further downstream pathways that lead to mucosal inflammation, remodeling and villous atrophy. Our analysis also suggests that, based on HLA-DQ2.5 genetics, the dose or dose interval of ZED1227 may have to be adjusted for optimal efficacy, but larger sample sizes are required to confirm this assumption. Moreover, CeD-associated gene expression changes were observable, even on a strict GFD 13 , 56 , indicating that complete avoidance of gluten is impossible 5 , 6 . In fact, a recent meta-analysis found that 15% of foods labeled as gluten free and 28% labeled as naturally gluten free contained more than 20 mg kg –1 gluten 57 , the cutoff for qualifying as gluten free. Thus, an adjunctive TG2 inhibition-based therapy combined with a GFD would especially benefit highly gluten-sensitive individuals (possibly carrying a homozygous HLA-DQ genotype) by providing protection against intestinal damage that can occur even in a low-gluten environment.

Participants and biopsies

PAXgene-fixed and paraffin-embedded biopsies were collected from a multisite, double-blind, randomized, placebo-controlled trial aimed at dose finding and assessing the efficacy and tolerability of a 6-week treatment with ZED1227 capsules versus placebo in individuals with well-controlled CeD undergoing gluten challenge 59 . Full inclusion and exclusion criteria are published 19 . Briefly, participants who had a biopsy-proven CeD diagnosis, were on a self-reported strict GFD for at least 1 year and symptom free, showed normalized duodenal histology compared to the initial diagnostic biopsy finding (morphometrically defined as a mean VH:CrD of 1.5 or higher) and tested negative for serum anti-TG2 on study inclusion were included (GFD group; Extended Data Table 1 ). These participants then underwent a challenge with a cookie containing 3 g of gluten daily for 6 weeks (PGC group). At least 80% compliance was confirmed 19 .

Biopsy sampling was performed twice on study inclusion (denoted here as GFD) and at the final visit (denoted here as PGC; Extended Data Fig. 1 ). Duodenal forceps biopsies were immersed in PaxFPE (PAXgene fixative) and processed for paraffin block embedding using a standard formalin-free paraffin-infiltration protocol. For morphology, samples were stained with hematoxylin and eosin and measured using our validated morphometry rules separately for morphology (VH, CrD and VH:CrD) 60 .

This study used samples from two groups, placebo and the 100-mg ZED1227 group, which represented the highest dose drug group showing the most significant improvement compared to the placebo group. In total, 58 participants (drug group, n  = 34; placebo group, n  = 24; total number of biopsies = 116) of the 68 participants who had sufficient biopsy samples at both time points in the original trial 19 were included, as these exploratory (optional) studies required separate written informed consent. Demographic characteristics and duodenal histomorphometry changes in the form of VH:CrD ratio of the participants in the original cohort and in the present study are presented in Supplementary Tables 1 and 2 .

Human organoid cultures

Human duodenal tissues for establishing organoid cultures used in this study were sourced from deidentified surgical specimens ( n  = 3) of the duodenum obtained from participants who had undergone biopsy procedures unrelated to CeD at Tampere University Hospital. The protocol was approved by the Ethics Committee of Tampere University Hospital (ETL code R18082). Intestinal crypts containing stem cells were isolated following 2 mM EDTA dissociation of tissue samples for 30 min at 4 °C (ref. 61 ). Crypts were washed in PBS, and fractions enriched in crypts were collected. The supernatant was removed, and the crypt epithelial cells were seeded in 50% Matrigel (diluted with basal culture medium). Crypts were passaged and maintained in WELR500 culture medium, as previously described 62 . Organoids were treated with 100 U ml –1 IFNγ (Peprotech, 300-02) with or without 50 µM ZED1227 (Zedira) for 24 h and subjected to RNA sequencing to assess any adverse direct side effects to the intestinal epithelium (Supplementary Fig. 5 ).

Cell culture and treatments

Caco-2 colonic epithelial cells (ATCC, HTB-37; passage 22–35) were grown as standard monolayers in tissue culture flasks in complete MEM 1 g l –1 glucose medium (20% heat-inactivated fetal bovine serum, 1% nonessential amino acids, 1% penicillin–streptomycin, 1% GlutaMAX and 1% sodium pyruvate) at 37 °C in a 5% CO 2 atmosphere. Caco-2 cells were treated with 100 U ml –1 IFNγ (Peprotech, 300-02) with or without 50 µM ZED1227 (Zedira) or mock treated with DMSO for 24 h. Cells were collected by trypsinization and lysed in lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl and 1% IGEPAL) supplemented with 0.2 mM DTT and 1× Complete Protease Inhibitor Cocktail (Roche, 11836170001) and used for the transglutaminase activity assay.

RNA extraction and RNA sequencing

Total RNA was extracted from the PaxFPE-fixed biopsy specimens ( n  = 116) 63 using additional cuttings from the samples on which histomorphometry was previously assessed 19 . For extraction, an RNeasy kit (Qiagen) was used according to the manufacturer’s instructions. Library preparation and NGS were performed by the Qiagen NGS Service. A total of 10 ng of purified RNA was converted into NGS cDNA libraries. Library preparation was quality controlled using capillary electrophoresis. Based on the quality of the inserts and the concentration measurements, the libraries were pooled in equimolar ratios and sequenced on a NextSeq (Illumina) sequencing instrument according to the manufacturer’s instructions, with 100-bp read length for read 1 and 27-bp read length for read 2. The raw data were demultiplexed, and FASTQ files for each sample were generated using bcl2fastq2 software (Illumina).

RNA from the duodenal organoids was isolated using an RNeasy kit (Qiagen) following the manufacturer’s instructions. RNA purity and concentration were measured using a NanoDrop One spectrophotometer (NanoDrop Technologies). Preparation of the RNA library and transcriptome sequencing was conducted by Novogene. mRNA was purified from total RNA using poly(A) selection and subjected to library construction. Sequencing was performed on an Illumina platform, and 150-bp paired-end reads were generated.

Bioinformatic analyses

Data quality was checked using FastQC. The 3′ adapter sequences were trimmed, reads without adapters were kept, and reads with <15 bp were removed. Reads were aligned to the human genome reference consortium human build 38 (GRCh38) using the splice-aware aligner STAR. For all downstream analyses, genes with low expression (read counts that were equal to the number of samples multiplied by 5) were excluded. One sample with low total reads (1.13 million reads) was excluded, leaving 115 samples for subsequent analyses. The mean total reads for all samples were 3.51 ± 0.07 million reads. A secondary differential expression analysis involving normalization of unique molecular identifier counts and a subsequent pairwise differential regulation analysis was performed using the DESeq2 package 64 . Pre- and post-treatment samples were compared, and the paired nature of samples was included as a term in the multifactor design formula. The obtained P values were adjusted for multiple testing using the Benjamini–Hochberg method 65 . Genes with an FDR of <0.05 and | log 2  (FC) | of ≥0.5 identified by DESeq2 were assigned as differentially expressed.

Gene Ontology enrichment and Reactome enrichment analyses were performed using topGO 66 and ReactomePA 67 R packages. GSZ scores, as a particular type of overrepresentation analysis, were calculated as previously described 68 . For comparison of groups, mean GSZ score asymptotic P value calculation was applied to our datasets 69 . Gene lists for transit-amplifying cells, mature enterocytes, immune cells and duodenal transporters were retrieved from healthy human duodenal single-cell sequencing analyses published by Busslinger et al. 22 or our DEG analysis from human duodenal organoids treated with IFNγ versus mock-treated organoids. Cell-type proportions for CeD biopsy bulk RNA-sequencing data were estimated with the MuSiC analysis toolkit 70 using single-cell RNA-sequencing data from duodenal adult biopsies 71 as a reference.

Exact HLA genotypes, with a focus on DQ status ( HLA-DQA1 and HLA-DQB1 alleles), were determined in silico from RNA-sequencing data using the arcasHLA tool 38 . FASTQ files were used as input files. The minimum gene read count required for genotyping was set at 5. Due to low expression, low resolution 72 (Field1, allele group) was taken into consideration in the subsequent statistical analyses.

Statistical analysis

Statistical tests were conducted as specified in the legends of the respective figures using R version 4.3.0 (R Foundation for Statistical Computing). A repeated-measures ANOVA was used to assess the impact of treatment on VH:CrD ratio within different time points (GFD and PGC) across HLA-DQ genetic background groups (G1, G2 and G3). This analysis comprised 57 participants with identifiable HLA-DQ genotypes. Three null hypotheses were proposed: (1) VH:CrD means are equal across time points, (2) VH:CrD means are equal among HLA-DQ groups, and (3) there is no interaction between these two factors. As a post hoc analysis, multiple pairwise t -tests were used to identify differences between time points for each genotype group. To assess how the impact of the HLA-DQ genotype group on the VH:CrD outcome varies with different time points, a one-way ANOVA model was used. To address multiple testing, a Bonferroni correction was applied to P values (total tests performed = 2). Statistical significance was determined as P  < 0.05.

To assess the interaction between treatment groups and HLA-DQ genetic backgrounds on VH:CrD and epithelial response to IFNγ GSZ score at PGC, a two-way ANCOVA was conducted using these values at PGC as the dependent variable, HLA-DQ genetic background (G1, G2 and G3 genotype groups) and treatment (placebo or drug) as independent variables and baseline VH:CrD ratio and epithelial response to IFNγ GSZ score (from the GFD group), respectively, as a covariate. This analysis included 57 participants, with 1 participant from the placebo group excluded due to an unidentified allele type. The study formulated the following two null hypotheses for the two-way ANCOVA analysis: (1) no VH:CrD (epithelial response to IFNγ GSZ) difference at PCG exists between treatment groups (placebo and drug) while accounting for VH:CrD (epithelial response to IFNγ GSZ) at GFD and (2) no VH:CrD (epithelial response to IFNγ GSZ) differences at PCG exist across HLA-DQ genetic backgrounds (G1, G2 and G3 genotype groups) controlling for VH:CrD (epithelial response to IFNγ GSZ) at GFD. For the one-way ANCOVA, only participants in the drug group ( n  = 34) were selected. The null hypothesis for this analysis was that there is no significant effect of HLA-DQ genetic background (represented by HLA-DQ genotype groups) on VH:CrD within the PGCd group, while adjusting for VH:CrD at GFDd. The one-way ANCOVA regression model included VH:CrD at PGCd as the dependent variable, VH:CrD at GFDd as a covariate and HLA-DQ genotype group (G1, G2 and G3) as independent variables. The same type of approach was used for VH and CrD values. Post hoc pairwise multiple comparisons using estimated marginal means calculation (also known as least-squares means) were conducted between the drug and placebo groups for the two-way ANCOVA as well as between HLA-DQ genotype groups for the one-way ANCOVA. To address multiple testing, the Bonferroni correction was applied to P values (total tests performed = 3). Statistical significance was defined as an adjusted P value of <0.05.

Quantitative real-time PCR

Human duodenal organoids ( n  = 3) were treated with 50, 100 or 200 U ml –1 IFNγ (Peprotech) and/or 2, 25 and 50 µM ZED1227 (Zedira) for 24 h. Total RNA was isolated using TRIzol Reagent (15596018), following the manufacturer’s instructions, and 500 ng was subjected to cDNA synthesis using an iScript cDNA Synthesis kit (Bio-Rad). Real-time PCR reactions were performed with SsoFast EvaGreen Supermix (1708890, Bio-Rad) and oligonucleotides for human TGM2 (forward: 5′-TGTGGCACCAAGTACCTGCTCA-3′; reverse; 5′-GCACCTTGATGAGGTTGGACTC-3′) and GAPDH (forward: 5′-GTCTCCTCTGACTTCAACAGCG-3′; reverse: 5′-ACCACCCTGTTGCTGTAGCCAA-3′) in triplicate. The results presented were calculated as fold change to the reference sample (nontreated sample), normalized by housekeeping gene expression ( GAPDH ) as described in Schmittgen and Livak 73 . Plot whiskers represent the standard error for mean difference between three independent means.

Transglutaminase activity assays in Caco-2 cells

Transglutaminase activity was measured using a hydroxamate-based colorimetric method modified from Folk and Cole 74 . In short, each reaction contained 75 mM hydroxylammonium chloride, 30 mM Z-Gln-Gly, 10 mM CaCl 2 and 10 mM DTT in 200 mM Tris-HCl buffer (pH 8.0) mixed with cell lysate in a final volume of 100 µl. After a 2-h incubation at 37 °C, the reaction was stopped by the addition of 50 µl of stop buffer (1.67% (wt/vol) FeCl 3 , 4% (wt/vol) trichloroacetic acid and 4% (vol/vol) HCl). The reaction output was measured at 530 nm, and the activity was expressed as nanomoles of hydroxamate produced in 120 min per milligram of total protein, using l -glutamic acid γ-monohydroxamate for the standard curve.

HLA genotyping

Five participants had too low coverage either at the HLA-DQB1 or HLA-DQA1 locus according to RNA sequencing; thus, their allele typing was not performed. For four of those individuals, blood pellet samples stored at −80 °C were available. DNA was extracted from 100 µl of sample using a QIAamp DNA Blood Mini kit (51104, Qiagen) following the manufacturer’s protocol. HLA-DQB1 and HLA-DQA1 typing was performed at the Immunogenetics Laboratory at the University of Turku, and the method was based on an asymmetrical PCR and a subsequent hybridization of allele-specific probes, as previously described 75 , 76 .

Molecular histomorphometry regression model

A regression model predicting VH:CrD ratios, developed in our previous study 13 , was used on the current dataset. Models were evaluated by observed versus predicted regression.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Bulk RNA-sequencing data from participant biopsies and patient-derived intestinal organoids described in this study are available in the European Genome–Phenome Archive under accession numbers EGAS50000000337 and EGAS50000000338 . Additional data used in this paper include a full single-cell RNA-sequencing dataset of intestinal regions of adult donors ( https://www.gutcellatlas.org/ ), lists of human duodenal cell types and transporter genes expressed along the upper gastrointestinal tract downloaded from supplementary files included within Busslinger et al. 22 , lists of immune cell marker genes downloaded from supplementary files included within Atlasy et al. 58 and pathway gene sets (Reactome, KEGG and BIOCARTA) downloaded from the Human MSigDB Collections at https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp . Source data are provided with this paper. All other data are present in the article and Supplementary Information or are available from the corresponding author upon reasonable request.

Code availability

Code used in this study is freely available on GitHub at https://github.com/IntestinalSignallingAndEpigeneticsLab/Dotsenko-et-al.-2024 .

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Acknowledgements

We thank the individuals who participated for making this study possible. We also thank the expert staff for their participation in sample collection. We thank K.-L. Kolho for providing intestinal biopsies to initiate organoid cultures. This work was Dr. Falk Pharma-sponsored clinical trial supported by the Academy of Finland (310011), the Finnish Cultural Foundation, Mary och Georg C. Ehrnrooths Stiftelse, Päivikki and Sakari Sohlberg Foundation, Laboratoriolääketieteen Edistämissäätiö sr and the Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital grant. V.D. was supported by the Finnish Cultural Foundation. D.S. received project related support from the German Research Foundation (DFG) Collaborative Research Center SFB TR355/1 (490846870) project B08 (Treg in celiac disease). The funding sources played no role in the design or execution of this study or in the analysis and interpretation of the data. We acknowledge the Adult Stem Cell Organoid Facility from Tampere University for their service. Ethics approvals TUKIJA dnro 223/06.00.01/2017 and EudraCT 2017-002241-30 were obtained for the Dr. Falk Pharma-funded clinical trial. The study was conducted with deidentified data of the participants who had consented to the use of their anonymized data in research. The protocol to initiate human intestinal organoid cultures from biopsies was approved by the Ethics Committee of Tampere University Hospital (ETL code R18082).

Author information

A list of members and their affiliations appears at the end of the paper.

Authors and Affiliations

Celiac Disease Research Center, Faculty of Medicine and Health Technology, Tampere University and Tampere University Hospital, Tampere, Finland

Valeriia Dotsenko, Juha Taavela, Alina Popp, Markku Mäki & Keijo Viiri

Dr. Falk Pharma GmbH, Freiburg, Germany

Bernhard Tewes, Timo Zimmermann, Ralf Mohrbacher & Roland Greinwald

Zedira GmbH, Darmstadt, Germany

Martin Hils & Ralf Pasternack

Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland

Jorma Isola

Jilab Inc, Tampere, Finland

Jorma Isola & Jani Sarin

Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland

Juha Taavela

University of Medicine and Pharmacy ‘Carol Davila’ and National Institute for Mother and Child Health, Bucharest, Romania

Unit of Health Sciences, Faculty of Social Sciences, Tampere University, Tampere, Finland

Heini Huhtala

Department of Pediatrics, Tampere University Hospital, Tampere, Finland

Pauliina Hiltunen & Marja-Leena Lähdeaho

Norwegian Coeliac Disease Research Centre, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway

Knut E. A. Lundin

Department of Gastroenterology, Oslo University Hospital Rikshospitalet, Oslo, Norway

Institute of Translational Immunology and Celiac Center, Medical Center, Johannes-Gutenberg University, Mainz, Germany

Detlef Schuppan

Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Department of Gastroenterology, Internal Medicine Clinic, Tartu University Hospital, Tartu, Estonia

Lääkärikeskus Aava Helsinki Kamppi, Helsinki, Finland

Jari Koskenpato

Clinical Research Services Turku–CRST Oy, Turku, Finland

Mika Scheinin

Faculty of Medicine and Health Technology, Tampere University and Tampere University Hospital, Tampere, Finland

Marja-Leena Lähdeaho

Department for Gastroenterology, Infectious diseases and Rheumatology, Campus Benjamin Franklin, Charité–University Medicine Berlin, Berlin, Germany

Michael Schumann

Department of Medicine 1, Hector Center for Nutrition, Exercise, and Sports, Universitätsklinikum Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

Yurdagül Zopf

Department of Internal Medicine IV, Jena University Hospital, Friedrich-Schiller University Jena, Jena, Germany

Andreas Stallmach

I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Ansgar W. Lohse

Department of Gastroenterology, Gastrointestinal Oncology, Hepatology, Infectious Diseases and Geriatrics, University Hospital Tübingen, Tübingen, Germany

Stefano Fusco

Department for Internal and Integrative Medicine, Kliniken Essen-Mitte, Essen, Germany

Jost Langhorst

Department for Internal and Integrative Medicine, Sozialstiftung Bamberg, Medical Faculty, University of Duisburg-Essen, Bamberg, Germany

Department of Medicine II, University Hospital, LMU Munich, Munich, Germany

Helga Paula Török

University College Hospital Galway, Galway, Ireland

Valerie Byrnes

Gastroenterology Department and Institute for Digestive Research, Lithuanian University of Health Sciences, Kaunas, Lithuania

Juozas Kupcinskas

Medical Department, Institute of Clinical Medicine, Innlandet Hospital Trust, Gjøvik, Norway

Øistein Hovde

Akershus University Hospital, Lørenskog, Norway

Jørgen Jahnsen

Department of Gastroenterology and Hepatology, University Hospital Zürich, Zurich, Switzerland

Luc Biedermann & Jonas Zeitz

Swiss Celiac Center, Center for Gastroenterology, Clinic Hirslanden, Zurich, Switzerland

Jonas Zeitz

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• , Jari Koskenpato
• , Mika Scheinin
• , Marja-Leena Lähdeaho
• , Michael Schumann
• , Yurdagül Zopf
• , Andreas Stallmach
• , Ansgar W. Lohse
• , Stefano Fusco
• , Jost Langhorst
• , Helga Paula Török
• , Valerie Byrnes
• , Juozas Kupcinskas
• , Øistein Hovde
• , Jørgen Jahnsen
• , Luc Biedermann
•  & Jonas Zeitz

Contributions

V.D., K.V. and M.M. conceptualized the study. K.V. and V.D. drafted the manuscript. V.D. and K.V. performed data analysis and figure generation. H.H. assisted in statistical analyses. P.H. performed gastroscopies to obtain duodenal biopsies and organoids. B.T., M.H., R.P., J.I., J.T., A.P., J.S., T.Z., R.M., R.G., K.E.A.L. and D.S. assisted in the logistics of data collection and results interpretation. All authors read and approved the final paper.

Corresponding author

Correspondence to Keijo Viiri .

Ethics declarations

Competing interests.

V.D. and K.V. received funding from Dr. Falk Pharma to Tampere University to conduct the study. B.T., T.Z., R.M. and R.G. are employees of Dr. Falk Pharma. The data presented here are the subject of patent applications EP24173619.8 and EP24173615.6 filed by Dr. Falk Pharma, and B.T., T.Z., R.M., R.G., V.D. and K.V. are inventors on these applications. M.H. and R.P. are employees of Zedira. A.P. is a consultant for JiLab Oy. J.T. is a consultant for Jilab Oy and Dr. Falk Pharma. K.E.A.L. is a consultant for Amyra, Bioniz Pharmaceuticals, Chugai Pharmaceutical, Dr. Falk Pharma, Itrexon Actobios, TOPAS Therapeutics and Takeda California. D.S. is the data and safety monitor for Boehringer Ingelheim (Phil.) and is a consultant for the Dr. Falk Pharma, Takeda, Immunic, Sanofi and TOPAS Therapeutics. J.I. is the owner of Jilab Oy. M.M. is the founder, owner and Chair of the Board of Maki HealthTech (MHT). MHT receives Management/Advisory Affiliation fees from Dr. Falk Pharma and other funding not related to the research from Topas Therapeutics, Calypso Biotech, Vaccitech, ImmunogenX, Equillium and Immunic. MHT holds patents (patent number 7361480 (United States) and European Patent Office Number 1390753) licensed to Labsystems Diagnostics from where MHT receives royalties via Tampere University Hospital. All other authors declare no competing interests.

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Nature Immunology thanks Bana Jabri and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: S. Houston, in collaboration with the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended data fig. 1 schematic presentation of the study..

Samples ( n  = 116; n of patients = 58), in a form of PAXgene fixed and paraffin-embedded biopsies, were collected from the trial, aimed at dose-finding, and assessing the efficacy and tolerability of a 6-week treatment with ZED1227 capsules vs. placebo in subjects with well-controlled celiac disease undergoing gluten challenge. Biopsy sampling was performed twice: on study inclusion (GFDd, n  = 34; GFDp, n  = 24) and at the final visit (PGCd, n  = 34; PGCp, n  = 24). Duodenal forceps biopsies were immersed in PAXgene fixative and processed for paraffin block embedding using a standard formalin-free paraffin-infiltration protocol. Created with BioRender.com .

Extended Data Fig. 2 Histomorphometric features and molecular pathways displaying reduced control in G1 genotype.

a , Subjects (n = 34), belonging to drug group were selected for one-way ANCOVA. VH at PGCd used as a dependent variable and VH at GFD as covariate and HLA-DQ genotype group (G1, G2, G3) as independent variables. ANCOVA, F (2, 30) = 6.56, P = .004. Post-hoc pairwise multiple comparisons were performed between HLA-DQ genotype groups, with p values Bonferroni adjusted. Results demonstrated as estimate ± 95% CI. b , Subjects (n = 34), belonging to drug group were selected for one-way ANCOVA. Cr at PGCd used as a dependent variable and CrD at GFD as covariate and HLA-DQ genotype group (G1, G2, G3) as independent variables. ANCOVA, F (2, 30) = 3.6, P = .04. Post-hoc pairwise multiple comparisons were performed between HLA-DQ genotype groups, with p values Bonferroni adjusted. Results demonstrated as estimate ± 95% CI. c , Gene set Z-score was calculated for gene sets enriched in the categories of transit amplifying cells, mature enterocytes, immune cells, duodenal transporters and Reactome database pathways for patients in drug and placebo groups at PCG (PGCd (n = 34), PGCp (n = 23)). GSZ grouped by HLA-DQ genotype group (G1, G2, G3) and presented as mean (spheres) and sd (vertical lines).

Supplementary information

Supplementary information.

Supplementary Tables 1 and 2 and Figs. 1–5.

Reporting Summary

Peer review file, supplementary data 1.

List of DEGs in the PGCp versus GFDp comparison, PGCd versus GFDd comparison and PGCp versus PGCd comparison.

Supplementary Data 2

Results of the overrepresentation analysis for Reactome terms for the PGCp versus GFDp and PGCp versus PGCd comparisons and results of the overrepresentation analysis for Gene Ontology biological process terms for the PGCp versus GFDp and PGCp versus PGCd comparisons.

Supplementary Data 3

List of DEGs in the I versus M and Z versus M comparisons.

Supplementary Data 4

Enrichr results for the PGCp versus GFDp and PGCp versus PGCd comparisons (see Supplementary Fig. 2).

Supplementary Data 5

Supplementary Data for Fig. 5

Supplementary Data 6

Source data for Supplementary Tables 1 and 2 and Figs. 1–5.

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Source data fig. 3, source data fig. 4, source data fig. 5, source data fig. 6, source data extended data fig. 2, source data extended data table 1, rights and permissions.

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Dotsenko, V., Tewes, B., Hils, M. et al. Transcriptomic analysis of intestine following administration of a transglutaminase 2 inhibitor to prevent gluten-induced intestinal damage in celiac disease. Nat Immunol (2024). https://doi.org/10.1038/s41590-024-01867-0

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DOI : https://doi.org/10.1038/s41590-024-01867-0

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Managing children and adolescents with type 1 diabetes and coexisting celiac disease: Real-world data from a global survey

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Maja Raicevic , Francesco Maria Rosanio , Tiago Jeronimo Dos Santos , Agata Chobot , Claudia Piona , Laura Cudizio , Hussain Alsaffar , Katja Dumic , Sommayya Aftab , Meera Shaunak , Enza Mozzillo , Rade Vukovic; Managing children and adolescents with type 1 diabetes and coexisting celiac disease: Real-world data from a global survey. Horm Res Paediatr 2024; https://doi.org/10.1159/000540054

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Objectives: Celiac disease (CD) is among the diseases most commonly associated with type 1 diabetes (T1D). This study aimed to evaluate the worldwide practices and attitudes of physicians involved in pediatric diabetes care regarding diagnosing and managing CD in children with T1D. Methods: The 30-item survey was conducted between July and December 2023 aimed at targeting pediatricians with special interest in T1D and CD. It was shared by the JENIOUS- young investigators group of the International Society of Pediatric and Adolescent Diabetes (ISPAD) and the YES- early career group of the European Society for Pediatric Endocrinology (ESPE). Results: 180 physicians (67.8% female) from 25 countries responded. Among respondents, 62.2% expected sustaining optimal glycemic control in children with T1D and CD (T1D+CD) to be more difficult than in children with T1D alone. Majority (81.1%) agreed that more specific guidelines are needed. The follow-up routine for patients with T1D+CD differed, and one-quarter of physicians scheduled more frequent follow-up checkups for these patients. Seventy percent agreed multidisciplinary outpatient clinics for their follow-up is needed. In the multivariate ordinal logistic regression model, a statistically significant predictor of a higher degree of practice according to ISPAD 2022 guidelines was a higher level of country income (OR=3.34; p<0.001). Conclusions: These results showed variations in physicians' practices regarding managing CD in children with T1D, emphasising the need for more specific guidelines and intensive education of physicians in managing this population, especially in lower-income countries. Our data also suggest the implementation of multidisciplinary outpatient clinics for their follow-up.

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New Blood Test for Predicting Parkinson’s Disease With A.I. Shows Promise, Study Suggests

In preliminary research, scientists identified eight protein anomalies in the blood of patients with Parkinson’s, which they say can help diagnose the disease up to seven years before symptoms appear

Christian Thorsberg

Daily Correspondent

Preliminary testing of a newly developed, A.I.-enhanced blood test has shown promise in being able to accurately predict if someone will develop Parkinson’s disease in the coming years.

In research published last week in the journal Nature Communications , a team of scientists in Europe identified eight blood-based biomarkers that might alert doctors to a high potential for Parkinson’s development in a patient—up to seven years before the onset of symptoms.

The test, while still in its early stages, offers hope for a disorder that has no cure, affects nearly nine million people worldwide, kills more than 300,000 people annually and is becoming more prevalent, according to the World Health Organization . The disease is caused by protein buildup in certain dopamine-producing neurons in the brain, which ultimately kills off the cells.

Currently, treatment for Parkinson’s tends to be reactive, focused on controlling symptoms—which include tremors, slow movements, stiffness and a loss of balance—after a diagnosis is made.

“At the moment, we’re shutting the stable door after the horse has bolted,” senior author Kevin Mills , a biochemist at University College London’s Great Ormond Street Institute of Child Health, tells the Guardian ’s Ian Sample. “We need to get to people before they develop symptoms. It’s always better to do prevention rather than cure.”

With this goal in mind, the team began their work by collecting blood samples from 99 people who have Parkinson’s disease and 36 people who do not. Analyzing a selection of 70 percent of these samples, a machine learning algorithm identified eight proteins that appeared in different concentrations in the blood of those with the disease.

This pattern “could provide a diagnosis with 100 percent accuracy,” according to a statement from University College London. In a follow-up evaluation, the algorithm was given the remaining 30 percent of blood samples that it hadn’t been trained on—30 from people currently with Parkinson’s disease and 11 from people without it. The tool aced the test, correctly diagnosing every patient.

“This means that drug therapies could potentially be given at an earlier stage, which could possibly slow down disease progression or even prevent it from occurring,” Michael Bartl , a neurologist at University Medical Center Goettingen in Germany and the co-first author of the study, says in the statement .

In another trial, the researchers worked long-term with 54 people who currently have isolated rapid eye movement sleep behavior disorder (iRBD), a neurological disorder that tends to precede Parkinson’s disease, foreshadowing a diagnosis of Parkinson’s or a similar condition between 75 percent and 80 percent of the time . They took one to five blood samples from each patient and tested for the eight biomarkers.

Based on their protein patterns, the tool found that 79 percent of the iRBD patients had blood profiles consistent with someone who would go on to develop Parkinson’s disease. Following up with the patients over a ten-year period, the researchers found 16 of them have been diagnosed with Parkinson’s.

The team correctly predicted these diagnoses, on average, 3.5 years before symptoms presented themselves, with the earliest prediction coming 7.3 years before symptom onset. They’re continuing to check in with the other iRBD patients to confirm the blood test’s accuracy.

“We’ve seen tremendous progress in the development of exciting new tests for Parkinson’s in the last year alone,” Katherine Fletcher , research communications lead at the nonprofit Parkinson’s U.K. who was not involved in the study, tells Live Science ’s Michael Schubert. “We are hopeful that these new tests will start being used within the next few years.”

Still, some scientists point out the challenges that remain.

“Parkinson’s is not a single disease but a syndrome and can present in various different ways,” Ray Chaudhuri , the medical director of the Parkinson Foundation International Center of Excellence who was also not involved with the research, tells the Guardian. “As such, management differs and one size does not fit all. The prediction is unlikely to signpost these subgroups at this stage.”

What is crucial about early diagnosis, researchers say, is that it can allow patients to enroll in experimental trials of preventative treatments for Parkinson’s disease as a proactive measure.

“People are diagnosed when neurons are already lost,” Jenny Hällqvist , a biochemist at University College London and a co-author of the study, tells BBC News ’ Philippa Roxby. “We need to protect those neurons, not wait till they are gone.”

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Christian Thorsberg | READ MORE

Christian Thorsberg is an environmental writer and photographer from Chicago. His work, which often centers on freshwater issues, climate change and subsistence, has appeared in Circle of Blue , Sierra  magazine, Discover  magazine and Alaska Sporting Journal .

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