Edition Coeliac Disease a Review. Celikey the Key to Celiac Disease Diagnosis. Current Diagnosis for Celiac Disease.

Transcription

1 Edition n Guest Article Coeliac Disease a Review n New Parameter Celikey the Key to Celiac Disease Diagnosis n Interview Current Diagnosis for Celiac Disease

2 Contents Editorial Dear Readers, Gliadin, the gluten protein in wheat and rye flour is usually introduced into our children s diet during the first year of life, e.g. in the form of semolina, bread or pasta. If celiac disease develops, the first symptoms do usually not appear until some weeks or even months later, and a diagnosis cannot be made until the end of the first year of life, or indeed early on in the second year. Until a few years ago, celiac disease was regarded as very rare. Today, however, it is recognized that patients may exhibit a wide variety of disease manifestations ranging from no symptoms (patients detected by serological screening) to full-blown malabsorption (insufficient intake of nutritional components) with steatorrhea, fatigue and severe anemia. Because of this broad spectrum of symptoms, many cases were wrongly diagnosed, or not diagnosed at all. Early diagnosis in children is critical, however, in order to avoid the possibility of permanent growth retardation. Statistics on the occurrence of celiac disease vary greatly within Europe. It is only in the past few years that researchers have come to believe that the disease is far more prevalent than was previously assumed. On the basis of current estimates, it appears that about one person in 250, or even one in 100, is affected. Depending on the screening method, however, the information obtained differs so much that it is impossible to study incidence rates or whether there is any relationship with environmental factors. Serological antibody tests are currently used for most celiac screening. Endomysium antibodies (EMA) have proven to be the most reliable markers, with a specificity for celiac disease of %. An immunofluorescence test on monkey oesophagus is used to detect EMA. It is obvious that an alternative substrate would be preferable. In practice, the IgA-EMA test is relatively laborious and also has the inherent limitations of any test that depends on subjective scoring. In 1997 Dieterich identified tissue transglutaminase (ttg) as the antigen recognized by EMA. This was a major breakthrough in the development of alternative diagnostic methods. In all the studies to date, ELISAs for anti-ttg have proven to be excellent tools for detecting untreated celiac disease. So far, a commercially available guinea pig ttg antigen has been used for the tests. With Celikey, however, Pharmacia has succeeded in developing a commercial ELISA with human recombinant antigen. This was reason enough for us to dedicate this issue of the Elias Journal to celiac disease and the anti-ttg ELISA. Enjoy reading all about it! Your Elias Journal editorial team. 3 Guest Article Coeliac Disease a Review 7 New Parameter Celikey the Key to Celiac Disease Diagnosis 9 Interview Current Diagnosis for Celiac Disease EliA Journal is the Journal of Phadia GmbH Munzinger Straße 7, D Freiburg Editor: Contributors: Layout: Nina Olschowka Reprint: April 2007 Numbers printed: 1,000 E. Mummert, A.-M. Bürgin-Wolff, F. Hadziselimovic, R. Nieminen, M. Mäki M. Tritschler / H. Schneider

3 Guest Article Celiac Disease a Review Extensive summary from the report of the working group 2000 on celiac disease with comments Faruk Hadziselimovic, Annemarie Bürgin-Wolff C eliac disease (CD) is an autoimmune condition characterized by an immune mediated enteropathy of the small intestine. However, this disease does not fit the classical model of an autoimmune disease. The condition occurs in genetically predisposed individuals who ingest prolamins (alcohol soluble proteins rich in glutamine and proline) that are found in wheat, rye and barley. CD produces a spectrum of small intestinal mucosal changes ranging from an increase in the number of intraepithelial lymphocytes to mucosal remodeling with crypt hyperplasia and flattening of the villi. The flat intestinal mucosa is no longer the only diagnostic paradigm of CD. But rather, clinical characteristics along with anti-gliadin, anti-endomysium, and anti-human-ttg antibodies have become the new paradigm for diagnosing CD accurately and for monitoring the compliance of a gluten-free diet. Malabsorption of nutrients is a consequence of the intestinal damage and when prolonged can cause significant malnutrition. Treatment requires life-long adherence to a diet that strictly eliminates products containing wheat, rye and barley. On such a diet, complete recovery of the small intestinal damage can be expected with reversal of nutritional deficits and resolution of symptoms. The transient form of CD is still a significant challenge to diagnose accurately in 5 10 % of the children. Although they have a flat mucosa and also have anti-gliadin, anti-endomysium, and anti-human ttg anti bodies at one time, those with this form of CD will not have to maintain a life-long gluten-free diet. Fortunately, those with this varient form of CD establish tolerance in some way to gliadin. Historical Aspects Samuel Gee is credited with the first thorough description of the features in children in Dietary factors in the pathogenesis were recognized during the 1920 s, with Haas describing a clinical cure in patients maintained on a strict banana diet in During the Second World War a Dutch pediatrician, Karel Dicke, noticed an association between CD and bread consumption, as symptoms improved during the Dutch famine but relapsed after bread supplies became more plentiful. In collaboration with Van der Kamer, Dicke subsequently demonstrated a relationship between wheat protein ingestion and fat malabsorption in patients with CD, published in his thesis in With the advent of peroral intestinal mucosal biopsy in the mid 1950 s, the characterisation of small intestinal mucosal changes were recognized. This led to a realization that CD in childhood was the same condition known as non tropical sprue in adults. The first serological tests for CD were developed in Basel as early as Their use in screening various population groups has significantly altered our perception of the clinical manifestations and prevalence of CD. Epidemiology CD has been described throughout the world including Europe, Russia, the Mediterranean countries, North and South America, South Africa, India, Australia and New Zealand but there is little good data on the prevalence in many of these areas. lt is rare in black Africans and those Asians of Chinese or Japanese ancestry. Recent data from several European countries suggests the condition may occur in as many as 1 : 100 of the population. An upward shift in the age of onset of symptoms has been reported by most countries in the last two decades and with few exceptions, it is now less common for CD to present in the very young child. During this same period of time, the clinical manifestations of CD have changed significantly. Gastrointestinal complaints are still frequent, but non-gastrointestinal features are now equally common. Advances in the field of CD Serological tests for CD have been of immense value in the evolution of our understanding of the disease. The tests measure antibodies to gliadin (AGA), reticulin (ARA), endomysium (EMA) and tissue transglutaminase (ttg). They are primarily used to identify individuals requiring intesti nal biopsy for definitive diagnosis, but may also be of value in monitoring response to therapy and dietary compliance. They have played a pivotal role in studies on the pre valence of CD and have been instru mental in our appreciation of the extremely protean manifestations of the condition. Through their use it has become apparent that some individuals remain asymptomatic

4 Guest Article despite having typical changes of CD on small intestinal biopsy. Further more, there are known cases where gluten ingestion in genetically susceptable individuals has induced clinical disease without detectable mucosal damage. As stated above it is also possible that some individuals throughout life have no symptoms despite having significant mucosal changes.the therapeutic implications of this discovery are not yet known. lt is possible that the complete clinical spectrum of CD has not yet been described and novel manifestations may be recognized in the future through continued use of serological screening programs. The ultimate goal remains to develop a serological test that can replace the intestinal biopsy for definitive diagnosis. The following strategy described in the Lancet article should be an alter native approach. (Fig. 1) Measurement of antigliadin and antiendomysium antibodies in serum has effectively diminished the neces sity of a small-bowel biopsy for the histological assessment of celiac disease. If the endomysium and gliadin antibodies are present, there is % likelihood of a flat mucosa. Conversely, if the antibodies are absent, the chance of a flat mucosa is only 0,5 % in children and 2 % in adults. Therefore, the necessity of a small-bowel biopsy could be abrogated by non-invasive immunological investigations. The figure shows our alternative algo rithm for diagnosing the various forms of celiac disease. This approach is gentle, low-risk, and costeffective; all forms of the disease are diagnosed, and the antibody assay is a simple and powerful tool to estimate the compliance of patients on a strict gluten-free diet. (Lancet 1998, 351, 62 63) The recent discovery that tissue transglutaminase (ttg) is the main autoantigen recognized by celiac patients may prove to be of fundamental importance in diagnosis and in our understanding of the immunopathogenesis of CD. Comparative evaluation of serological tests for celiac disease in Europe performed by the working group on serologic screening for celiac disease concluded that interlaboratory reliability of EMA testing provided data superior (K = 0.93) to data for all other test protocols, even though the immunofluorescence reading was subjective. Consequently, there is the need for experienced and well trained laboratory personnel to read the test results in particular, to differentiate Suspected celiac disease Measurement of serum antibody (AB): IgA + IgG gliadin(aga) + IgA endomysium (EMA) All AB negative Celiac disease unlikely Normal diet Observe the patient * All AB positive Only 1 or 2 AB positive Biopsy Gluten-free diet 3 5 years Flat mucosa ** Gluten challenge All AB negative Transient celiac disease Normal diet Observe the patient * EMA and/or AGA positive Normal mucosa Latent celiac disease diet Normal Observe the patient * Biopsy Flat mucosa ** Celiac disease Life-long gluten-free diet ** Normal mucosa Transient or latent celiac disease Normal diet Observe the patient* Fig. 1: Algorithem for diagnosis of celiac disease * If clinical symptoms of celiac disease reappear or all antibodies become positive: biopsy. ** Presence of large granular lymphocytes / natural killer cells in mucosal epithelium indicates abortive celiac disease: gluten challenge after 5 10 years. (F. Hadziselimovic, A. Bürgin-Wolff, Lancet 1998, 351, 62 63)

5 Guest Article between endomysium and smooth muscle antibodies. The test protocol for EMA immunofluorescence was robust and provided the best reproducibility data. Currently available ttg-assays show some differences which are mostly due to variations in the native antigen. Recombinant human antigens tend to be very close to EMA-testing. Further, a complete understanding of the immune mechanisms involved in tissue damage is essential if alternative novel therapies for CD are to be developed. Pathogenesis of CD CD is a complex disease involving interactions between genetic and environmental factors. Evidence for a genetic component is both circumstantial and based on specific HLA types. A specific gene abnormality has not yet been identified and probably more than one gene is involved. Among the environmental factors implicated in the pathogenesis, prior ingestion of wheat, rye or barley is an absolute prerequisite to initiate disease in the susceptible individual. Previously oats were also regarded as toxic but this is now contentious. In vitro and in vivo studies have identified several specific peptides within the A-gliadin molecule that appear capable of initiating disease. The A-gliadin molecule contains a number of repetitive amino acid motifs, and it is probable that multiple peptides within wheat, rye and barley have toxic potential. The precise relationship between immunogenicity and toxicity remains undefined. Identification of the smallest peptide that is toxic for patients with CD may facilitate our understanding of the events that initiate the inflammatory process and may lead to novel therapies for CD. Gluten-sensitivity does not fit the classical model of an autoimmune disease. It is triggered by gluten ingestion and even after a lifetime s exposure IgA antibodies disappear and most patients mucosae regenerate villi when gluten is removed from diet. This contracts with the autoimmune state where irreversible destruction of the target organ is a necessary prelude to onset of symptoms. This is clearly not the case with celiac disease even though transglutaminase may be an ancillary (secondary) target antigen (M.N. Marsh 1997). An attempt to define gluten sensitivity as primary autoimmune should be viewed with great caution. Additional environmental factors have been proposed as a trigger factor for CD in some individuals. Principal among these is viral infections and sequence homology has been demonstrated between gliadin and certain viral proteins. Molecular mimicry might explain how viruses may be involved in the pathogenesis of CD. The need for additional nondietary factors as a trigger factor to elicit disease also provides an attractive explanation for the marked variation in age of onset and clinical manifestation. However, it must be emphasized that there is still no hard evidence that viruses or other nondietary factors are involved. Immunopathogenesis of CD The mucosal damage that characterizes CD is considered to be immune mediated. lt remains unknown if specific immune mechanisms are solely responsible for the tissue damage or whether they act in concert with a direct effect of gliadin on a susceptible mucosa. The precise sequence of immunological events is not known, but binding of gliadin by the HLA-DQ molecule for antigen presentation to the T cells appears to be a central event. In addition, the purported role of the innate immune system (NK cells, macrophages, dendritic cells, etc.) to induce tolerance in a few rare cases of CD should be discussed. The enzyme tissue transglutaminase (ttg) seems to play an important role in this process by modifying the gliadin molecule to increase its affinity for the DQ2 molecule peptide-binding groove. The modified gliadin also binds strongly to ttg suggesting mechanism for the gluten-dependent production of antitransglutaminase antibodies. DQ2 peptide binding induces T cell activation with production of cytokines. This process is thought to occur in discrete regions just under the intestinal mucosal epithelium in patients with CD, but the precise sequence of events culminating in mucosal remodeling remains unclear. Humoral events may also play a role in the intestinal changes. Production of antibodies to ttg has been shown to inhibit transforming growth factor.this in turn inhibits intestinal epithelial cell differentiation necessary for maintaining villus integrity. An understanding of the immune mechanisms that lead to mucosal destruction in CD is essential if alternative non-dietary therapies are to be developed in the future. Health implications of CD Short-term effects of delayed diagnosis are features such as discomfort, lassitude and weight loss adversely impacting on school and work performance. Longer term adverse effects of untreated CD include permanent growth stunting, Prof. Faruk Hadziselimovic 1970 Doctor at the University of Basel 1980 Habilitation since 1984 Head of the Department of Pediatric Gastroenterology, Universitäts-Spital Basel since 1995 Head of pediatrics at the children s hospital Kindertagesklinik Liestal, Liestal, Switzerland Dr. Annemarie Bürgin-Wolff Studies for natural sciences at the University of Basel, PhD Post doctoral training at the Institute of microbiology, University of Basel University children s hospital, Basel, Laboratory of virology and immunology Since 1992 with Prof. Dr. F. Hadziselimovic at the institute for coeliac disease in Liestal, Switzerland

6 Guest Article dental enamel defects, osteoporosis, anemia, and an increased risk for malignancies such as intestinal lymphomas. Financial and emotional costs related to CD are difficult to estimate. With delays in diagnosis such costs may include numerous visits to doctors, laboratory investigations, medications and loss of income from time off work. Therapy for CD Currently the only therapy for CD is life-long adherence to a diet (known as a gluten-free diet GFD) that eliminates all products containing wheat, rye and barley. On a GFD there is usually dramatic clinical improvement and normalization of the intestinal changes on histology within 6 months. The loss of specific serum antibodies (anti-gliadin, antiendomysium and anti-ttg) and the normaliza tion of the flat mucosa in our popula tion of children with CD required more than 6 months. But in adults, the neurological and psychic changes induced by CD may necessitate several years for the clinical symptoms to normalize. Additional dietary restrictions are occasionally required initially, including cow milk. These also include elimination of lactose because of a secondary lactase deficiency and provision of vitamin and iron supplements. Rarely cortico steroids are required for treatment of lifethreatening situations during a celiac crisis. In the future alternative therapies involving immuno modu lation may become reality. 2. Ivarsson A, Persson LA, Nystrom L, et al. (2000) Epidemic of coeliac disease in Swedish children Acta Paediatr 89, Mäki M, Collin P, (1997) Coeliac disease Lancet 349, Clot F, Gianfrani C, Babron MC, et al. (1999) HLA-DR53 molecules are associated with susceptibility to celiac disease and selectively bind gliadin-derived peptides Immunogenetics 49, Greco L, Corazza G, Babron MC, et al. (1998) Genome search in celiac disease Am J Hum Genet 62, Holopainen P, Arvas M, Sistonen P, et al. (1999) CD28/CTLA4 gene region on chromosome 2q33 confers genetic susceptibility to celiac disease A linkage and family-based association study. Tissue Antigens 53, Djilali-Saiah I, Schmitz J, Harfouch-Hammoud E, et al. (1998) CTLA-4 gene polymorphism is associated with predisposition to coeliac disease Gut 43, Sollid LM (2000) Molecular basis of celiac disease Annu Rev Immunol 18, Jabri-Bendelac B, Cellier C, Patey N, et al. (2000) Intraepithelial lymphocytes in coeliac disease In: Auricchio S, Greco L, et al. (eds.) Coeliac Disease. Naples, JG Edition (in press) 10. Visakorpi JK, Mäki M, (1994) Literature 1. Greco L, Mäki M, Di Donato F, Visakorpi JK (1992) Epidemiology of coeliac disease in Europe and the Mediterranean areas. A summary report of the multi center study by the European Society for Paediatric Gastroenterology and Nutrition In: Auricchio S, Visakorpi JK, eds. Common Food Intolerances 1: Epidemiology of Coeliac Disease. Dyn Nutr Res, Basel, Karger 1992;2:25 44 Changing clinical features of coeliac disease Acta Paediatr Suppl 83 (395), Troncone R, Greco L, Mayer M, et al. (1996) Latent and potential coeliac disease Acta Paediatr Suppl 412, Troncone R, Auricchio S, (1999) Celiac disease In: Wytlie R, Hyams JS (eds.) Pediatric Gastrointestinal Disease. 2nd ed. Philadelphia, W.B. Saunders, pp Meeuwisse GW, (1970) Diagnostic criteria in coeliac disease Acta Paediatr Scand 59, Walker-Smith JA, Guandalini S, Schmitz J, et al. (1990) Revised criteria for diagnosis of coeliac disease Arch Dis Child 65, Codex Alimetarius Commission (1998) Draft revised standard for glutenfree foods (CY,/NFSDU 98/4) 16. Janatuinen EK, Kemppainen TA, Pikkarainen PH, et al. (2000) Lack of cellular and humoral immunological responses to oats in adults with coeliac disease Gut 46, Holmes GK (1997) Celiac disease and malignancy J Pediatr Gastroenterol Nutr 24, Bürgin-Wolff A, Hadziselimovic F, (1997) Screening test for coeliac disease Lancet 349, Bürgin-Wolff A, Gaze H, Hadziselimovic F, et al. (1991) Antigliadin and antiendomysium antibody determination for coeliac disease Arch Dis Child 66, Hadziselimovic F, Bürgin-Wolff A, (1989) Celiac disease Lancet 351, Hill ID, Ghatnager S, Cameron D, et al. (2000) Report on working group of the World Congress of Pediatric Gastroenterology, Hepatology and Nutrition 2000, pp Marsh MN (1997) Transglutaminase, gluten and celiac disease. Food for thought Nat Med, 3, Stern M and the Working Group on Serologic Screening for Celiac Disease (2000) Comparative Evaluation of Serologic Tests for Celiac Disease: A European Initiative Toward Standardization J Pediatr Gastroenterol Nutr 31,

7 New Parameter Celikey the key to Celiac Disease diagnosis ESPGHAN criteria Mandatory requirements for the diagnosis of CD 1 The histological finding of flat small intestinal mucosa with hyperplastic villous atrophy while the patient is eating adequate amounts of gluten. 2 Complete clinical remission once on a strict gluten-free diet. This response should be reasonably rapid occurring within a matter of weeks rather than many months. Eckart Mummert T he diagnosis of Celiac Disease (CD) is based on the combi nation of clinical symptoms and biopsy (see ESPGHAN criteria) and, more recently, supported by a serological test like the indirect immunofluorescence test on monkey esophagus (Endomysial Antibodies: EMA). The EMA test proved to be very sensitive and specific for CD and is recognised as golden standard for the serological part of CD diagnosis. Since the anti gen recognised by the EMA was found to be tissue transglutaminase (ttg), endomysial antibodies can also be detected in ELISA using the purified protein. Anti-tTG ELISA systems, however, did not meet the performance of EMA until now, lacking both, sensitivity and specificity. This is due to the source of the antigen used in these assays. Usually the ttg is purified from guinea pig liver and thus is easy to obtain, but not quite the same as the original human antigen. To solve this problem we use recombinant techniques to express the genuine human ttg in the best system available, the baculovirus/insect cell system. The resulting protein has many advantages compared to ttg purified from native sources. As shown in Fig. 1, it is much purer than commercially available preparations from guinea pig liver, resulting in less contami nations and minimized risk for false positives. Furthermore human recombinant ttg expressed in the baculovirus/insect cell system proves to be identical to the antigen recognised by the patients antibodies in EMA, since inhibition experiments reveal that complete inhibition of antibody binding is only achieved with human recombinant and not with guinea pig ttg (Fig. 2a c). These results are also reflected in the performance of immunoassays using guinea pig or human recombinant ttg, respectively. The ELISA using human recombinant antigen shows a much better discrimination between positives and negatives and gives less false positive values (Fig. 3 a,b). The human recombinant antigen in this case even outmatches the human preparation from erythrocytes (Fig. 3c). Encouraged by these results we developed CelikeyTM, the ELISA using human recombinant ttg expressed in the baculovirus/insect cell system. The superior performance of CelikeyTM was assessed in a clinical study with 208 CD patients and 157 controls. The study showed a A 280 nm 50 µl commercial guinea pig liver ttg, 0.3 mg/ml 0.25 clinical sensitivity of the assay of 96 % and a clinical specificity of 99 %. Furthermore the CelikeyTM results almost perfectly matched the EMA results in negative and positive discrimination. These results show that CelikeyTM is a serious alternative to EMA, showing almost the same performance data, but easier to handle and less time consuming µl recombinant human ttg, 0.3 mg/ml A 280 nm ml ml Figure 1: Chromatographic profiles of guinea pig and human recombinant ttg.

8 New Parameter a Commercial guinea pig ttg b Human recombinant ttg >3 >3 >3 CD: n = 40 IBD: n = 19 C: n = 26 CD: n = 40 IBD: n = 19 C: n = CD: n = 40 IBD: n = 19 C: n = OD 492 nm OD 492 nm OD 492 nm CD IBD C Human ttg c CD IBD C 0 CD IBD C Figure 3a c Results from ELISAs using guinea pig or human recombinant ttg or ttg purified from human erythrocytes. The ELISA using human recombinant ttg shows the best discrimination between CD and IBD (inflammatory bowel disease) patients. ESPGHAN: Diagnostic Criteria for Celiac Disease The first classic description of celiac disease was published in In 1969 the diagnostic criteria for celiac disease (subsequently known as the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN)* criteria) were proposed at the Interlaken meeting of the society.1 These criteria were further enunciated at the second symposium on celiac disease in 1974, namely: Structurally abnormal jejunal mucosa when taking a diet containing gluten. Clear improvement of villous structure when taking a gluten-free diet. Deterioration of the mucosa during challenge.2 In the following years, new diagnostic tools antigliadin, antireticulin, and anti endo mysium antibodies have proven to be reliable indicators of sensitization to gluten. Further reports have been published in which these ESPGHAN criteria from 1969 (three serial biopsies) have been used in a remarkably large series of 3293 children.3 These authors, however, suggest that in most cases gluten challenge is not essential for diagnosis. The ESPGHAN workshop on Diagnostic criteria of coeliac disease in Budapest in 1989 revised the 1969 criteria.4 It was concluded, that the diagnosis of CD does not require further confirmation if the initial diagnosis is based firstly on the appearance of flat small intestinal mucosa with the histological features of hyperplastic villous atrophy while the patient is eating adequate amounts of gluten, and secondly on the unequivocal and full clinical remission after withdrawal of gluten from the diet. The finding of circulating antibodies (IgA gliadin, antireticulin, and anti endomysium) at time of diagnosis and their disappearance when the patient is taking a gluten free diet add weight to the diagnosis. * former name: European Society for Paediatric Gastroenterology and Nutrition (ESPGAN) Literature 1 Meeuwisse GW (1970) Diagnostic criteria for coeliac disease Report of a round table discussion. Acta Paediatr Scand 59, Visakorpi JK (1974) Definition of Coeliac disease in children In: Hekkens WT, Pena AS (eds) Coeliac disease. Proceedings of the Second International Coeliac Symposium Noordwijkerhout, the Netherlands, Stenfert Kroese, Leiden, Figure 2a Intense endomysial staining, no inhibition Figure 2b Weak, incomplete inhibition of endomysial staining using guinea pig ttg 3. Guandalini S, Ventura A, Ansaldi N, et al. (1989) Diagnosis of coeliac disease: time for a change? Arch Dis Child 64, Walker-Smith JA, Guandalini S, Schmitz J, et al. (1990) Revised criteria for diagnois of coeliac disease Arch Dis Child 65, Figure 2c Complete inhibition of endomysial staining using human recombinant ttg

9 Interview Current Diagnosis for Coeliac Disease Raimo Nieminen interviewed Markku Mäki C oeliac disease (CD) has been recognised as both a clinical and diagnostic challenge for more than 100 years. However, the relationship between wheat protein ingestion and genetically predisposed individuals was firstly described in the late 1950 s; where the author demonstrated a relationship between wheat protein ingestion and fat malabsorption in patients with coeliac disease. At the turn of the century medical literature described coeliac disease as a rare gastroenterological disorder. This point of view was erroneous in two senses: coeliac disease is neither an exclusively gastrointestinal disease, neither it is a rare one. It has turned out to be a relatively common condition, which was not actually confirmed by studies until recently. The regrettable fact still remains that only one in ten patients suffering from coeliac disease is confirmed and properly treated. We had the opportunity to interview Professor Markku Mäki, and question his opinion on today s understanding of the mechanism, diagnosis and treatment of coeliac disease. Mr. Mäki is the head of the Coeliac Disease Study Group at the Institute of Medical Technology of Tampere University, Finland. Professor Mäki is currently employed at the Paediatric Department of Tampere University Hospital. Coeliac disease a common are detected is a result of a number of worldwide disease causative factors which I will further Coeliac Disease is described discuss here. throughout the world, but the epidemiological data given for the This condition occurs in genetically prevalence of the coeliac disease is predisposed individuals, which very variable and in some cases explains why it is a familial disease. inaccurate. This is the case also in This genetic susceptibility is associated to HLA genes in the short Finland. According to International epidemiological data, the prevalence arm of chromosome 6. Over 90 % of clinically diagnosed coeliac of individuals diagnosed with coeliac disease is estimated to be 1:1,000 disease have the HLA DR3-DQ2 1:10,000. However, in the most recent haplotype (or DR5/7-DQ2). Some individuals with coeliac disease that screening trials in western countries, the prevalence of the disease seems are HLA-DQ2 negative express the HLA DR4-DQ8 haplotype, which is to be estimated to 1:100. This high prevalence makes it realistic to also characteristic of type-1- dia accept coeliac disease as a common betes. disease, Professor Mäki emphasises. Professor Mäki continues: Coeliac The reasons that only a fraction of disease occurs in genetically pre disposed individuals who cannot these cases perhaps 1:500 or 1:1,000 Prof. Markku Mäki, Tampere University, Finland tolerate the prolamins found in wheat, rye and barley. Prolamins are alcoholsoluble proteins rich in glutamine and proline. This intolerance leads to a typical small-bowel mucosal lesion, duodenal and jejunal villous atrophy. A characteristic alteration is the deepening of the crypts (crypt hyper plasia) and the increase of inflammatory T lymphocytes in the mucosa. This happens in both the epithelium and in the lamina propria. Professor Mäki states that: Coeliac disease does not always present with solely gastroenterological disorders; the ingestion of gluten in genetically predisposed individuals can present without gastrointestinal symptoms. Dermatitis herpetiformis is a classical example of an extraintesti nal manifestation. Damage to permanent-tooth dental enamel, osteopenia and osteoporosis, peripheral neuropathies and ataxia are manifestations of coeliac disease. Studies claim that about 15 % of patients suffering from neurological disorders of unknown causes may actually suffer from coeliac disease. Symptoms of coeliac disease One of the reasons why coeliac disease is highly under diagnosed is a result of the multi-faced nature of the disease. The gastrointestinal symptoms can vary from insignificant

10 Interview to severe and in some cases signs often arise outside the gastrointestinal tract. This spectrum of symptoms makes diagnosis difficult. The classical symptoms in children with coeliac disease are diarrhoea, malabsorption, frequent stools, loss of weight and disturbance in growth, failure to thrive. In adults the characteristic symptoms are also, diarrhoea, flatulence, loss of weight, malabsorption and anemia. However, only a minority of individuals fit in this classic picture. In most cases the symptoms are mild and unclear. There are also many false diagnoses of lactose intolerance among the undiagnosed coeliac disease patients. Some individuals may have iron deficient anaemia, osteoporosis, and joint problems. Studies indicate that a fraction of individuals (4 %) treated for infertility is actually those whose problems focus on untreated coeliac disease. There are also individuals suffering from liver and neurological disorders presenting with minor gastrointestinal complaints, so to even suspect coeliac disease would be a secondary thought for a clinician. Coeliac disease is also frequent in individuals with selective IgA deficiency and Down s syndrome. Further disease associations are type 1 diabetes, autoimmune thyroid disease and Sjögren s syndrome. Problems of differential diagnosis The unfortunate fact is that it may take years, up to ten years from the moment the patient experiences symptoms or signs of coeliac disease, until a confirmed diagnosis is made, Professor Mäki says. This is not caused only by the lack of diagnostic methods but also by the fact that in most cases the investigating clinician has not suspected coeliac disease due to the unspecific symptoms. From a differential diagnosis approach, lactose intolerance can provide an additional challenge. Untreated coeliac disease causes secondary lactose intolerance among half of the sufferers. This is caused by the decrease of lactase enzyme as the result of the destroyed duodenal epithelium. However, when coeliac disease is properly treated, the secondary lactose intolerance is normally corrected, Professor Mäki stresses. The European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) decreed diagnostic criteria in 1990 according to which a diagnosis of coeliac disease should not be purely made on the symptoms or laboratory test indications. The final confirmation of coeliac disease is made by endoscopic examination and biopsy (or suction capsule biopsy). In Finland, a typical alteration of the duodenal epithelium would indicate an untreated coeliac disease. It is, however, very important during the differential diagnosis to have in mind the following conditions: Acute enteritis Chronic inflammatory bowel disease (Crohn s disease) Milk allergy, soya allergy Eosinophilic gastroenteritis Immune deficiency Giardiasis Bacterial overgrowth syndrome Drugs, esp. NSAIDs Radiation therapy One cannot perform endoscopic examinations on all children with stomach complaints. Some of these complaints are psychosomatic; others caused by lactose intolerance. Laboratory methods for presumptive diagnosis are therefore of paramount importance to screen out those individuals whom should be further examined by endoscopy. With adult patients it is more efficient to perform an endoscopic examination because other conditions can be eliminated or diagnosed, for example Helicobacter pylori infection, reflux oesophagitis, ulcers, and dyspeptic problems, all of which can be confirmed by endoscopy. Here, I would like to add that coeliac disease has been found by chance in some 3 4 % of cases when a clinician has performed an endoscopy suspecting another condition in adults. It is not, however, feasible to carry out endoscopy for coeliac disease on every patient with mild gastrointestinal symptoms, thus giving laboratory screening methods a crucial role. Serum antibody diagnostics At our research centre, we base diagnoses on the coeliac antibody package, which comprises of three different methods, Professor Mäki tells us. The classic is the gliadin antibody method for determining IgA and IgG class antibodies. However, it is a relatively insensitive and non-specific method, thus limiting the detection of the diseases in mild or early stages. Non-specificity in this sense implies that atopy, inflammatory bowel diseases and oral mucus membrane disorders can also have elevated IgA/IgG gliadin antibody levels. Secondly, we use an endomysial antibody method employing monkey oesophagus or, in the most recent applications, human umbilical cord as the antigen. This method has replaced the reticulin antibody method. These tissue autoantibodies are highly sensitive and specific in detecting untreated coeliac disease. The drawback of this method is the need for highly trained and skilled personnel for microscopic evaluation. The third laboratory test we engage is the tissue transglutaminase autoantibody assay. Tissue transglutaminase (ttg) autoantibody assay in coeliac diagnostics Personally, I believe that the tissue transglutaminase antibody assay will enhance the diagnosis of coeliac disease in the future, continues Professor Mäki. 10

11 Interview With the introduction of new ELISA techniques, the determination of tissue transglutaminase antibodies are less ambiguous, time and cost saving and less dependent on skilled laboratory staff as in the case of the endomysial antibody test. The fact that we are employing three different methods for the diagnosis of coeliac disease is partly due to scientific interest but also because no serological test is 100 % specific to coeliac disease. We have also noticed that if one begins on a gluten-free diet (either purposely or spontaneously) before a proper diagnosis is made can obscure disease confirmation. In such cases however, the duodenal epithelium may still be abnormal and a diagnosis can be confirmed, but the serological tests are not conclusive. Professor Mäki feels, If the endo my sial and tissue trans glutami nase antibody determinations are positive it suggests that the patient has an untreated coeliac disease, irrespective of the clinical picture. This provides a sufficient base for an endoscopy to confirm the diagnosis according to the ESPGAN criteria. The treatment of coeliac disease The treatment of coeliac disease is in principle very simple, but it is not always so simple in practice. Wheat, barley and rye should be replaced by gluten-free grain. A coeliac patient should remain on a strict life-long gluten-free diet. It should also be noted that even gluten-free grain can become contaminated by wheat in some phase of production, but generally speaking, gluten-free products are clean, especially naturally gluten-free and industrially purified flour. As the individual adapts to the new diet, the symptoms diminish, and the mucosa returns to normal. A glutenfree diet tends to be very deficient in fibre; therefore the introduction of oats to the diet boosts fibre consumption. A trial diet is not a part of the diagnosis, stresses Professor Mäki. If a gluten-free trial is made before a proper diagnostic regime, diagnosis becomes difficult and the inter preta tion of the results uncertain. Correct diagnosis with biopsy should always precede any treatment, Professor Mäki points out. It is remarkable how quickly the treatment of the disease is reflected in the assays. Even the highest titres of serological tests indicate negative values within 12 months, low titres within a couple of months. If the titres remain elevated, it is more than likely that the patient is eating glutencontaining foods, either deliberately or unintentionally. Modern assays clearly indicate the efficacy of treatment and the status of the duodenal epithelium. The future For the time being, the diagnosis of coeliac disease will require more than one method employed simultaneously for disease confirmation. It is evident that endoscopy is and will be a laborious and inconvenient method, and does not always provide the complete picture. Sometimes there are no characteristic alterations in the epithelium, but coeliac disease can be raging within!! Personally, I forecast that future diagnoses will be based on the reliable serum antibody assays and determination of certain genetic markers. Endoscopy and biopsies will continue to be employed providing the clinician with additional infor mation, but I believe that they will slowly diminish from use. My opinion is that the tissue trans glutaminase assay has the makings of a specific method that may be a cornerstone in the diagnosis of coeliac disease, or coeliac trait, in the future. What is... Tissue Transglutaminase (ttg) belongs to a diverse family of calciumdependent enzymes that catalyse a cross-linking reaction between proteins or peptides. ttg is widely distributed in human organs and is found associated with fibres surrounding smooth muscle and endothelial cells in connective tissue. ttg plays a role in extracellular matrix assembly and tissue repair mechanisms. It is present in cell cytoplasm but is secreted in low amounts by mononuclear cells and endothelial cells and by fibroblasts during wound healing. In damaged tissue such as the small bowel mucosa of untreated celiac disease, the ttg levels increase. Wheat gliadins can act as a substrate for the transgluatminase reactions. IgA anti-tg antibodies are highly diseasespecific serological markers of CD and dermatitis herpetiformis. Undoubtedly, the finding of ttg (IgA) antibodies and the development of sensitive and specific in vitro assays for measurement of these antibodis have increased the possibilities for diagnosis of CD.... tissue Transglutaminase? 11

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