Cereal Proteins

The cereals that are toxic for patients with celiac disease are wheat, rye, and barley; rice and maize are nontoxic and are usually used as wheat substitutes in the diet of patients with celiac disease. The toxicity of oats has been reassessed in recent years. It has in fact been shown that the use of oats as part of a gluten-free diet has no unfavorable effects on adult patients in remission and does not prevent mucosal healing in patients with newly diagnosed disease. These data have been replicated in several studies conducted in adults and children with celiac disease. Nonetheless, a few patients with celiac disease seem not to tolerate oats. Moreover, other recent studies suggest differences between oat varieties in relation to their immunogenicity for celiac patients. Nowadays, oat-based products are considered safe for celiac patients, provided that gluten contamination is avoided.

Cereal grains belong to the grass family (Gramineae); grains considered toxic for celiac patients (rye, barley) bear a close taxonomic relationship to wheat, whereas nontoxic grains (rice and maize) are taxonomically dissimilar. Wheat seed endosperm contains heterogeneous protein classes differentiated, according to their extractability and solubility in different solvents, into albumins, globulins, gliadins, and glutenins. Gliadins are monomers, whereas glutenins form large polymeric structures. The gliadins (classified according to their N-terminal amino acid sequences into alpha, gamma, and omega types) and the glutenins (subdivided into high-molecular-weight glutenins and low-molecular-weight glutenins) are the typical gluten components, and determine, respectively, the viscosity and elasticity (strength) of the dough. The wheat toxicity results from the gliadin protein fraction, and the toxicity of cereals other than wheat is most likely associated with prolamin fractions equivalent to gliadins in the grain of these other species; on the other hand, glutenin peptides have been shown to be immunogenic for mucosal T cells from patients with celiac disease. The richness in proline and glutamine confers an unusual resistance to gastrointestinal enzymes and renders prolamines a good substrate for tissue transglutaminase, with both of these phenomena favoring the reactivity with mucosal CD4 T cells. Several HLA-DQ2 restricted T-cell epitopes have in fact been found clustering in regions of gliadin that are rich in proline residues, the target of the tissue transglutaminase (TG2) deamidating activity (see Pathogenesis ). Variations in immunogenicity exist among cultivars, and work is in progress to identify wheat varieties with a reduced amount of T-cell epitopes to be used in breeding programs.

Other Environmental Factors

The high concordance rate for monozygotic twins emphasizes the strong contribution of genetic predisposition to the development of celiac disease. On the other hand, the relevance of environmental factors other than gluten in celiac disease is suggested by the significant changes in the incidence of the disease by time and place. Feeding practices in the first year of life seem to be relevant. In the 1990s, Sweden experienced an epidemic of symptomatic celiac disease in children younger than 2 years of age; the abrupt increase and decline in the incidence of the disease coincided with changes in the dietary pattern of infants. The risk of celiac disease was found to be greater when gluten was introduced in the diet in large amounts; on the contrary, it was reduced if children were still breast-fed when dietary gluten was introduced. The difference in the incidence between the two cohorts persisted after 12 years of follow-up. A favorable effect of gradual introduction of gluten in small amounts during ongoing breastfeeding has been suggested. Prospective studies have not confirmed the protective effect of breast-feeding. Still uncertain is the relevance of the age of introduction of gluten into the diets of infants. An important question is whether favorable dietary habits simply postpone the onset of celiac disease, or reduce the overall lifetime risk of the disease; a challenging possibility that requires further studies is that celiac disease might be delayed, or even prevented, with intervention in genetically susceptible individuals in dietary patterns in the first year of life.

Among other environmental factors that could play a possible role in celiac disease, infective factors have also been considered. The possible role played in pathogenesis by interferon α, and the epidemiologic evidence of increased risk in relation to the month of birth, have suggested the possible involvement of a viral infection. Infectious episodes could potentially contribute, as they might increase gut permeability with increased antigen penetration and/or may drive the immune system toward a (type 1 T helper) Th1 response. Rotavirus appears to be a good candidate. In fact, frequent rotavirus infection predicted a higher risk of celiac disease autoimmunity. Recent studies have also considered a possible role for microbiota; changes in the composition of fecal and duodenal microbiota have been reported, but their significance is still unclear.

Family Studies

Susceptibility to celiac disease is determined to a significant extent by genetic factors. This is suggested by the occurrence of multiple cases in families, the prevalence of celiac disease found among first-degree relatives being approximately 10%. Moreover, up to 75% of monozygotic twins have been found to be concordant with the disease. The concordance rate among HLA-identical siblings is about 30%, indicating that a significant part of the genetic susceptibility maps to the HLA region on chromosome 6.

Genetic Markers

The strongest association of celiac disease is with the HLA class II D region markers, with class I and class II region gene associations being secondary to linkage disequilibrium. More than 90% possess one or two copies of HLA-DQ2.5 encoded by the DQA1*05 (alpha chain) and the DQB1*02 (beta chain) genes. HLA-DQ 2.2 is highly homologous to DQ 2.5, but carries little risk because of decreased stability of bound peptides. The data available on DQ2-negative celiac patients indicate that they almost invariably are HLA DQ8 positive (DQA1*0301/DQB1*0302). A gene dosage effect has been reported for HLA alleles, allowing classification of cases in classes of risk, the highest risk being represented by DQB1*02 homozygosity. A molecular hypothesis for such a phenomenon has been proposed based on the impact of the number and quality of the HLA DQ2 molecules on gluten peptide presentation to T cells. The most likely mechanism to explain the association with HLA class II genes is, in fact, that the DQ molecule binds a peptide fragment of an antigen involved in the pathogenesis of celiac disease to present it to T cells. Less clear is the gene dosage effect on the clinical presentation of the disease: high doses are more often present in younger age at diagnosis and related to the presence of complications, such as refractory disease.

HLA explains only 35% of the disease heritability; the remaining is shared by an unknown number of non-HLA genes, the contribution of which taken singularly is modest. Genome-wide association studies (GWAS) have allowed the identification so far of 39 celiac disease–associated loci that together contain 115 different genes. They mostly control T-cell activation and recruitment. Fifty percent of associated single-nucleotide polymorphisms (SNPs) might affect the expression of other genes, very few having been found within coding sequences. Of the 39 loci, 64% were shared with at least one other autoimmune disease (e.g., type 1 diabetes), indicating parallel diseases mechanisms. The identification of non-HLA risk factors will improve the identification of high-risk individuals for a more precise diagnosis and implementation of possible prevention strategies.

Gliadin Resistance to Enzymes and Passage Through the Epithelium

Because of its high content in proline and glutamine, gliadins show an unusual resistance to gastrointestinal enzymes. It has been demonstrated that the lack of endoprolylpeptidase activity in gastric and pancreatic enzymes and in the human brush border, prevents efficient enzymatic attack of proline-rich domains in gluten proteins. It is still unclear if a primary defect of intestinal permeability exists in celiac disease. A transcellular protected transport pathway by retrotransocytosis of secretory immunoglobulin A (IgA) through the transferring receptor CD71 has been described.

Activation of the Innate Immune System

Gliadin can activate both the innate and the adaptive immune system in patients with celiac disease. There is in fact evidence that gliadin contains non-immunodominant peptide fragments (e.g., amino acid peptide 31-43 of A-gliadin), which has been shown to be resistant to gastric, pancreatic, and intestinal digestion and to be able to initiate both a stress and an innate immune response. The intestinal mucosal damage typical of celiac disease is mediated both by inflammation and by mucosal remodeling, with proliferation of crypt enterocytes. The innate immune and the proliferative responses of the celiac intestine to the peptide 31-43 involve Epidermal Growth Factor (EGF)/interleukin 15 (IL15) cooperation. The reasons that these gliadin peptides are dangerous to the small intestinal mucosa in patients with celiac disease and not to that of normal subjects are still unknown. Recently a constitutive activation of the Epidermal Growth Factor Receptor-Extracellular signal-regulated kinases (EGFR-ERK) pathway has been found in celiac disease cells (enterocytes and fibroblasts), which may represent a predisposing condition to the damaging effects of gliadins. Furthermore, recent genetic studies have revealed an association with polymorphisms of genes that are involved in actin remodeling and cell adhesion. A constitutive alteration of shape and actin cytoskeleton has been demonstrated in celiac disease cells. The toxic A gliadin peptide 31-43 is able to induce in control cells this cellular celiac disease phenotype, suggesting that this peptide is active on the same cellular pathways related to signaling, cell shape, and actin remodeling that are constitutively altered in celiac disease cells.

CD4+ T-Cell Activation in the Lamina Propria: the Adaptive Immune Response

One of the key events in the pathogenesis of celiac disease is the activation of lamina propria T cells by gliadin peptides presented with major histocompatibility complex (MHC) class II molecules HLA-DQ2 or HLA-DQ8. Gluten proteins contain a large number of peptides capable of stimulating T cells. The molecular basis of the interplay between gliadin, tissue transglutaminase 2 (TG2), HLA-DQ2 or DQ8, and T-cell receptor (TCR) is now better understood. It has been found that TG2, which may be activated in vivo by inflammation, tissue destruction, viruses, or gluten itself, enzymatically converts particular glutamine residues in gliadin to glutamic acid. This greatly increases the affinity of these peptide fragments for HLA-DQ2 or HLA-DQ8, resulting in more effective antigen presentation to naive T cells. How the TCR is selected and interacts with the gliadin–HLA complex is also being unraveled.

Cytokine Production

The pattern of cytokines produced following activation of T cells by gliadin has been characterized and shown to be Th1 predominant, with interferonγ present in the mucosa. This response is sustained by high levels of innate immune cytokine, such as IL-15 and type 1 interferons. IL-15 induces high expression of natural killer-like receptors on intraepithelial lymphocytes (IELs), resulting in enhanced cytolytic killing activity (see below). In addition, IL-21 plays an important role in interferonγ production, proliferation, and survival of natural killer (NK) and CD8+ cells. Of interest, IL-21 is downregulated in potential celiac disease (normal jejunal architecture despite the presence of celiac disease autoantibodies), this supporting its role in the development of villous atrophy. A moderate induction of IL-17 has been reported in active celiac disease, but this cytokine is apparently not produced by CD4+ T cells. The immunosuppressive and immunoregulatory cytokine IL-10 is also increased in celiac disease mucosa, but that is not sufficient to counteract the proinflammatory actions of interferonγ. It is still unclear if there is a problem of T-cell regulation. Gliadin-specific T regulatory cells have been demonstrated in the celiac disease mucosa and increased number of forkhead box P3 (FOXP3)-positive T cells counted in the active phase of the disease. Additional effector cells are recruited and activated, downstream of T-cell signaling. Increased expression of metalloproteinases, which degrade matrix structures, angiogenesis, and growth factors, contribute to the complex remodeling process that ultimately results in the classical flat mucosa of celiac disease with hypertrophic crypts.

Intraepithelial Lymphocytes

IELs represent a heterogeneous population composed primarily of TCR alpha/beta CD8+ cells, TCR gamma/delta+ cells, and NK-like cells. Their increased density, particularly of the gamma/delta+ subset, is a hallmark of CD. Mechanisms in addition to the activation of the adaptive immune response of lamina propria CD4+ T cells are required for the full-blown disease, as shown by potential celiac disease patients in whom the CD4+ response in the lamina propria is present, but villous atrophy does not occur. The second hit required is hypothesized to be innate immune signals, which allow activation of IELs. Marked alterations in the expression of major histocompatibility complex class I chain related (MIC) and HLA-E occur on the intestinal epithelium of patients with untreated celiac disease, as part of the stress response of intestinal epithelial cells, by gluten and/or infections themselves. MIC and HLA-E are ligands for NK-like receptors G2D and CD94, respectively, present on intraepithelial lymphocytes. IL-15 upregulates NK receptors on IELs, promoting T-cell-receptor–independent killing ability. Most recently, it has also been shown that CD8+ lymphocytes can directly recognize gliadin peptides, although it remains to be seen if they reside in the epithelium or in the lamina propria. Activation of intraepithelial lymphocytes, with increased FAS ligand expression, results in epithelial cell apoptosis and villous atrophy via interactions with FAS on intestinal epithelial cells. Increased perforin-granzyme production by activated IELs, which can form holes in target cell membranes, further contributes to gastrointestinal mucosal epithelial cell destruction. IL-15 may also promote the emergence of T-cell clonal proliferation due to its anti-apoptotic action on IELs.

Autoimmune Phenomena in Celiac Disease

There is significant and increasing evidence to support the categorization of celiac disease as an autoimmune disorder. Celiac disease associates with multiple other well-recognized autoimmune disorders and there are multiple autoimmune phenomena observed in this condition. The most characteristic and widely evident expression of autoimmunity in celiac disease is the presence of antibodies to tissue TG2 in patient sera. These antibodies are produced locally in the mucosa; on average, in the untreated celiac small intestine, 10% of plasma cells are TG2-specific cells. These cells have limited inhibitory activity, and their role in celiac disease pathogenesis is still unclear. IgA against extracellular forms of TG2 present in the liver, muscle, and lymph nodes, have been detected in patients with celiac disease, indicating that this TG2 is accessible to the gut-derived autoantibodies. Autoantibodies directed primarily toward TG3 or TG6, rather than TG2, are present in dermatitis herpetiformis and in neurologic manifestation of gluten sensitivity, respectively, suggesting that the heterogeneity of disease manifestations may reside in the specificity of the autoimmune response.

The mechanisms leading to autoimmunity in CD are not completely known. The upregulation and activation of TG2 observed in inflamed sites may generate additional antigenic epitopes, by cross-linking or deamidating of external or endogenous proteins. Unmasking of normally hidden epitopes in an inflamed environment, with more efficient antigen processing and presentation, has also been hypothesized as an important mechanism resulting in autoimmunity. However, the most accepted hypothesis for the observed dependence on the presence of dietary gluten for the production of anti-TG2 autoantibodies is that gluten-reactive CD4 T cells provide the required help to TG2-specific B cells in a hapten carrier-like manner by involvement of TG2–gluten complexes. It is notable that TG2 autoantibodies are not the only autoantibodies present in patients with celiac disease, as antibodies to actin and calreticulin have also been detected in the sera of celiac patients; their role in celiac disease pathogenesis is not known.

Clinical Presentation

Clinical features of celiac disease differ considerably. Intestinal symptoms are common in children diagnosed within the first 2 years of life; failure to thrive, chronic diarrhea, vomiting, abdominal distension, muscle wasting, anorexia, and irritability are present in most cases. With the shifting of the age at presentation of the disease later in childhood and with the wider and more liberal use of serologic screening tests, extraintestinal manifestations, without any accompanying digestive symptom, have increasingly been recognized, affecting almost all organs.

Short stature has probably been the first isolated extraintestinal presentation of celiac disease to be recognized; already in the early 1980s, approximately 10% of patients with isolated short stature undergoing jejunal biopsy were found to have a total villous atrophy. Nonetheless, both in children and in adults, the most frequent extraintestinal manifestation of celiac disease is iron deficiency anemia. The prevalence of celiac disease in adult patients with microcytic anemia that is unresponsive to iron therapy is as high as 8.5%. Related to the locomotor apparatus, arthritis and arthralgia as presentation symptoms of celiac disease were described by Maki et al. The majority of adults with celiac disease have metabolic osteopathy; gluten-free diet normalizes bone mass only in a proportion of subjects. However, patients whose celiac disease was diagnosed in childhood and who have since been receiving a gluten-free diet have a bone mineral density similar to that of healthy controls. The nervous system is also involved in celiac disease. An Italian report has proposed an association between celiac disease and epilepsy in patients with bilateral occipital calcifications ( Figure 34-2 ); in such patients, gluten-free diet beneficially affects the course of epilepsy only when started soon after epilepsy onset. Moreover, gluten sensitivity is proposed to be common in patients with neurologic diseases of unknown etiology, such as gluten ataxia. Peripheral neuropathies of axonal and demyelinating types have also been reported and may respond to elimination of gluten from the diet. There is no doubt that the liver is a target of gluten toxicity in celiac disease. Isolated hypertransaminasemia has been recognized as a possible presentation of celiac disease; it may be expression of chronic “cryptogenic” hepatitis resolving on a gluten-free diet. Serologic screening for celiac disease is mandatory in such patients. Patients with severe liver disease have been described in whom gluten-free diet prevented progression to hepatic failure, even in cases in which liver transplantation was considered. Patients having fertility problems may have subclinical celiac disease: unexplained infertility may be the only sign of celiac disease. Similarly, unfavorable outcomes of pregnancy such as recurrent abortions, premature delivery, or low weight at birth are more often observed in patients with undiagnosed or untreated celiac disease. Different degrees of dental abnormalities have been described in children with celiac disease; severe enamel hypoplasia is present in up to 30% of children with untreated celiac disease. Alopecia areata has been reported to be the only clinical manifestation of celiac disease.

Increased density of (A) intraepithelial lymphocytes CD3+ and (B) intraepithelial lymphocytes expressing the gamma/delta T-cell receptor, in a celiac patient with serum positive antiendomysial antibodies, but normal jejunal architecture.

A special place in this list is taken by dermatitis herpetiformis (DH), a gluten-dependent condition characterized by a symmetric pruritic skin rash with subepidermal blisters and granular subepidermal deposits of IgA in remote uninvolved skin. Most patients with DH have abnormal small intestinal biopsy pathology, histologically indistinguishable from that of celiac disease, although usually less severe. Approximately 60% of children with DH have been reported to have subtotal villous atrophy and 30% have partial villous atrophy on jejunal biopsy. The histologic changes return to normal after dietary exclusion of gluten. Therapy with dapsone usually leads to prompt clinical improvement; a strict gluten-free diet permits a reduction or discontinuation of dapsone over a period of months. Improvement of skin lesions on a gluten-free diet seems to occur also in patients with no evident mucosal abnormality; in the same patients the rash recurs with a gluten rechallenge.

Finally, there are patients clinically silent and identified during screening programs, for example in at-risk groups (first-degree relatives, patients with type 1 diabetes). Although they report no symptoms, more careful history, examination, or laboratory investigations may reveal subtle abnormalities (e.g., osteopenia).

There is no relation between severity of the clinical picture and severity or extension of the mucosal lesions. Conflicting results have been reported on the relation between HLA dose and clinical presentation. The mechanisms operating in the different manifestations of the disease may be different. Extradigestive manifestations more likely result from the intestinal damage and consequent nutritional deficiencies (e.g., anemia, osteopenia) and/or due to the deranged (auto)immune response (e.g., skin, liver, joints, central nervous system involvement).

Because of the increased attention to CD and the more diffuse screening of “at risk” subjects, an increasingly frequent problem is posed by children with positive serology for celiac disease, but with a normal intestinal mucosa; they are named “potential” celiac patients. Unlike “latent” celiac patients, they have never experienced intestinal villous atrophy, and in fact we do not know how many and which of them will develop damage of the jejunal mucosa. Anti-TG2 titers are usually lower than that showed by patients with celiac disease with atrophy; in some of them titers become negative or fluctuate during follow-up. There is no agreement on how those subjects should be managed. In most cases, they are left on a normal diet, particularly if they are clinically silent. In this case, a careful surveillance of the nutritional status of those patients, including a thorough evaluation of the bone status, is mandatory.

Associated Diseases

Some diseases, many with an autoimmune pathogenesis, are found with a higher than normal frequency in patients with celiac disease; among these are thyroid diseases, Addison’s disease, pernicious anemia, autoimmune thrombocytopenia, sarcoidosis, insulin-dependent diabetes mellitus, alopecia, and cardiomyopathies. Such associations have been interpreted as a consequence of the sharing of identical HLA haplotypes (e.g., B8, DR3). Nevertheless, the relation between celiac disease and autoimmunity is more complex. In patients with celiac disease, there is evidence that the risk of developing autoimmune diseases seems to be directly correlated to the duration of gluten exposure. However, studies in adults have challenged this concept.

An increased incidence of CD has been found in patients with Down syndrome compared with the general population. Similarly, in Turner’s syndrome and Williams’ syndrome, a higher number of cases of celiac disease was also observed. Selective IgA deficiency is also a condition associated with celiac disease. All these conditions must be actively screened (case-finding strategy). In the case of selective IgA deficiency, screening test alternatives to those based on the measurement of IgA isotype antibodies must be adopted.

Serologic Tests

Celiac disease–specific antibodies comprise autoantibodies against transglutaminase type-2 (TG2, “tissue” transglutaminase) including endomysial antibodies (EMAs), and antibodies against deamidated forms of gliadin peptides (DGPs). The positivity for anti-TG2 and/or EMA is associated with a high probability for celiac disease in children and adolescents. High concentrations of anti-TG2 antibodies in serum predict villous atrophy. Recent guidelines suggest use of IgA anti-tissue transglutaminase as the first-choice test and EMA testing if the results of IgA anti-tissue transglutaminase are equivocal. Although tests for anti-DGP antibodies performed favorably and much better than antibodies against native gliadin, their performance was inferior compared to anti-TG2 or EMA assay. Tests measuring IgG and/or IgA antibodies against deamidated gliadin peptides may be used as additional tests in children who are negative for other celiac disease–specific antibodies but in whom clinical symptoms raise a strong suspicion for celiac disease, especially if they are younger than 2 years of age. On the contrary, tests for the detection of IgG or IgA antibodies against native gliadin peptides should not be used for diagnosis of celiac disease. It is mandatory to check for IgA deficiency, particularly when the laboratory detects a low optical density on IgA anti-tissue transglutaminase test. In case of confirmed IgA deficiency, IgG anti-tissue transglutaminase and endomysium should be assessed. Finally, celiac disease antibodies are not detectable in the blood of all patients with celiac disease. However, TG2-specific antibodies may be present in small intestinal mucosa of seronegative patients where they are detectable by double immunofluorescence.