Prospective Studies on Celiac Disease

1. Prospective Studies on Celiac Disease Alessio Fasano and Carlo Catassi Center for Celiac Research University of Maryland School of Medicine

2. On the Coeliac Affection There is a kind of chronic indigestion which is met with in persons of all ages, yet is especially apt to affect children between one and five years old…. Signs of the disease are yielded by the faeces; being loose, not formed, but not watery; more bulky than the food taken would seem to account for… The causes of the disease are obscure. Children who suffer from it are not all weak in constitution. Errors in diet may perhaps be a cause, but what error? Why, out of a family of children all brought up in much the same way, should one alone suffer? To regulate the food is the main part of treatment…. The allowance of farinaceous food must be small; highly starchy food, rice, sago, corn-flour are unfit. Malted food is better, also rusks or bread cut thin and well toasted on both sides…. Gee S. On the celiac affection. St Bart Hosp Rep 1890; 24: 17-20.

3. Definition • Celiac disease is an autoimmune condition • Occurs in genetically susceptible individuals • DQ2 and/or DQ8 positive HLA haplotype is necessary but not sufficient • A unique autoimmune disorder because: • both the environmental trigger (gluten) and the autoantigen (tissue Transglutaminase) are known • elimination of the environmental trigger leads to a complete resolution of the disease

4. Pathogenesis • Genetic predisposition • Environmental triggers • Dietary • Non dietary?

5. Genetics Genes • Several genes are involved • The most consistent genetic component depends on the presence of HLA-DQ (DQ2 and / or DQ8) genes • Other genes (not yet identified) account for 60 % of the inherited component of the disease • HLA-DQ2 and / or DQ8 genes are necessary (No DQ2/8, no Celiac Disease!) but not sufficient for the development of the disease ? ? ? HLA ? + Gluten Celiac Disease

6. Be aware DR3 should now be referred to as DR17 DQ2 DQ8 DR5/DR7 DR3/DR3 DR3 { DQA1*0501 { DQA: Any DQA1*0201 DQ2 DQB1*03 DQB1*0201 Trans CIS CIS Gluten APC

7. Dietary Factors The Grass Family - (GRAMINEAE) Subfamily Tribe Festucoideae Zizaneae Oryzeae Hordeae Aveneae Festuceaea Chlorideae wild rice rice wheat oat finger millet teff (ragi) rye barley

8. The Celiac Iceberg Symptomatic Celiac Disease Manifest mucosal lesion Silent Celiac Disease Normal Mucosa Latent Celiac Disease Genetic susceptibility: - DQ2, DQ8 Positive serology

9. Option #1:Remove the genes Option #2:Remove the grains Treatment Options

10. Treatment • Only treatment for celiac disease is a gluten-free diet (GFD) • Strict, lifelong diet • Avoid: • Wheat • Rye • Barley

11. Gluten-Containing Grains to Avoid Wheat Bulgar Filler Wheat Bran Couscous Graham flour Wheat Starch Durum Kamut Wheat Germ Einkorn Matzo Flour/Meal Barley Emmer Semolina Barley Malt/ Extract Faro Spelt Rye Triticale

12. OBVIOUS SOURCES Bread Bagels Cakes Cereal Cookies Pasta / noodles Pastries / pies Rolls Sources of Gluten

13. Sources of Gluten • POTENTIAL SOURCES • Candy • Communion wafers • Cured Pork Products • Drink mixes • Gravy • Imitation meat / seafood • Sauce • Self-basting turkeys • Soy sauce

14. Gluten-Free Grains and Starches • Amaranth • Arrowroot • Buckwheat • Corn • Flax • Millet • Montina • Oats* • Potato • Quinoa • Rice • Sorghum • Tapioca • Teff • Flours made from nuts, beans and seeds *for possible cross-contamination with gluten containing grains

15. Other Items to Consider • Lipstick/Gloss/Balms • Mouthwash/Toothpaste • Play Dough • Stamp and Envelope Glues • Vitamin, Herbal, and Mineral preparations • Prescription or OTC Medications

16. Dietary Adherence:A Common Problem • Only 50% of Americans with a chronic illness adhere to their treatment regimen including: • diet • exercise • medication • Dietary compliance can be the most difficult aspect of treatment

17. Health Beliefs of Adults with Celiac Disease • Survey of 100 people in Celiac Disease support group (Buffalo, NY) • Number of people who agreed with following statements: • “If I eat less gluten I will have less intestinal damage.” –51% • “I’ve lived this long eating gluten, how much will the gluten- free diet really help me now?” –33% • “My doctor should be the one to tell me when I need follow up testing.” –26% • “Scientist/doctors still haven’t proven that gluten really hurts them.” –16%

18. Barriers to Compliance • Ability to manage emotions – depression, anxiety • Ability to resist temptation – exercising restraint • Feelings of deprivation • Fear generated by inaccurate information

19. Barriers to Compliance • Time pressure – time to plan, prepare food is longer • Planning – work required to plan meals • Competing priorities – family, job, etc. • Assessing gluten content in foods/label reading • Eating out – avoidance, fear, difficult to ensure food is safe

20. Gluten and treatment of Celiac Disease: How Much is Too Much?

21. The gluten microchallenge study Coordinator: Carlo Catassi, M.D.

22. Investigating the dose-effect relationship the gluten microchallenge • CD patients on long-term, strict GFD • Perspective study design • While the GFD is maintained throughout the study-period, a given amount of gluten/gliadin is added to the diet • Clinical, serological and biopsy evaluation before and after the microchallenge • The background noise caused by possible gluten contamination of the GFD was minimized by inclusion of a control group

23. DOSE-DEPENDENT EFFECTS OF PROTRACTED INGESTION OF SMALL AMOUNTS OF GLIADIN IN CELIAC CHILDREN • Positive linear relationship between gliadin daily dose and mucosal damage between 100 and 1000 mg/day • IEL count is the most sensitive index • Serological markers are not reliable tools for detecting minimal dietary transgressions Catassi et al, Gut 1996

24. Why performinging a new microchallenge study • Need of investigating the effects of lower gluten doses • Need of prolonging the duration of the microchallenge • Need of a control group • Need of investigating gluten rather than gliadin

25. Wheat Gluten and Giadins 200g • Gluten is the main proteic fraction in wheat (8-14 %); • The toxicity is mainly due to the gliadins (50 %), however glutenins also contribute to toxicity; • Daily intake of gluten in adults: ~ 15 g (Dautch data); • Daily consumption of flower for a typical GFD in celiac subjects: ~ 80 g; • 200 mg/Kg of gluten = 100 mg/Kg of gliadin = 100 ppm of gliadin (=2.5g of bread!) Gluten 15g Gliadin 7.5g ~52 toxic fragments

26. The new microchallenge study AIM To evaluate the consequences of the protracted ingestion of minimal daily gluten intake (either 10 or 50 mg) in a group of adult celiacs on long-term treatment with the gluten-free diet (GFD) TYPE OF STUDY Multicentre, prospective, randomized, placebo-controlled, double-blind STUDY PERIOD Years 2001-2004 SPONSOR Italian Celiac Society (AIC)

27. INCLUSION CRITERIA Patients with biopsy-proven CD on a GFD for at least 2 years EXCLUSION CRITERIA Younger than 18 yrs Poor compliance to the GFD Abnormal results at the baseline evaluation Associated selective IgA deficiency The “new” Italian microchallenge study

28. The Italian microchallenge studyStudy-Design Steps GFD GFD  2 yrs Running-in Baseline Microchallenge T1 Intervention Informed consent Strict monitoring of the GFD Clinical Serology SB Biopsy Randomization Clinical Gluten exposure Serology Monthly check SB biopsy + 50 mg gluten Patient flow + 10 mg gluten + 0 mg gluten Timeframe Pre-T0 T0 T0 1 2 3 m

29. The Italian microchallenge studyMethods • Purified gluten was used for the microchallenge study (Amygluten 110, Tate & Lyle, UK) • Gluten- or lactose (placebo) containing capsules were centrally prepared • All laboratory tests were centrally performed • Monthly monitoring of adherence to the protocol • Measurement of gluten contamination in commercially available GF food by ELISA (Ridascreen Gliadin, R-Biopharm AG, Germany) • Serum AGA (ELISA) and anti-tTG (ELISA) • Small bowel biopsy and morphometry on 10 villi, IEL count (CD3+), ab IEL count • Control biopsies from non-celiac GE patients

30. Gluten content in commercially-available gluten free products in Italy where currently food labeling policies for gluten free products are set at 20 ppm

31. The Italian microchallenge studySubjects completing the study

32. VH/CD Ratio Controls CD The Italian microchallenge study:Biopsy findings at baseline CD3+ T cells (x100 enterocytes) Controls CD

33. The Italian microchallenge studyBiopsy findings at baseline IEL count (X100 enterocytes) VH/CD Ratio

34. The Italian microchallenge studyClinical findings Symptoms Placebo 10 mg 50 mg None 6 8 7 Abdominal pain and distension 2 1 2 Anemia and/or iron deficiency 1 0 0 Loss of appetite 0 0 1 Bloating, mood changes 2 1 0 Apthous stomatitis 0 0 1 Constipation 2 0 0 Headache, abdominal distention 1 0 0 Weight loss 0 0 1

35. 10 25 8 20 6 15 IgG-AGA (U/mL) IgA anti-tTG (U/mL) 4 10 2 5 0 0 Placebo 10 mg 50 mg Placebo 10 mg 50 mg Treatment Treatment The Italian microchallenge studySerological findings

36. 3 * 2 Vh/Cd ratio 1 0 Placebo 10 mg 50 mg Treatment • 50 mg significantly different from placebo (Kruskal-Wallis test) The Italian microchallenge study Morphometry findings1

37. 45 40 35 30 25 IELs count (no. x 100 enterocytes) 20 15 10 5 0 Placebo 10 mg 50 mg Treatment The Italian microchallenge study Morphometry findings2

38. 12.5 10.0 7.5 ab cells (X100 enterocytes) 5.0 2.5 0.0 Placebo 10mg 50mg The Italian microchallenge study Morphometry findings

39. Tolerable daily intake of gluten and ppm of gluten in food for celiacs

40. Toxicity of gluten traces:the Italian study on gluten microchallenge Catassi C1,2, Fabiani E1, Mandolesi A3, Bearzi I3, Iacono G4, D’Agate C5, Francavilla R6, Corazza GR7, Volta U8, Accomando S9, Picarelli A10, De Vitis I11, Bardella MT12, Pucci A13, Fasano A2 1 Department of Pediatrics, Università Politecnica delle Marche, Ancona, Italy; 2 Center For Celiac Research, University of Maryland School of Medicine, Baltimore (USA); 3 Department of Pathology, Università Politecnica delle Marche, Ancona (Italy); 4 Department of Gastroenterology, Children Hospital, Palermo; 5 University Department of Gastroenterology, Catania; 6 University Department of Pediatrics, Bari; 7 University Department of Gastroenterology, Pavia; 8 University Department of Internal Medicine, Bologna; 9 University Department of Pediatrics, Palermo; 10 Department of Gastroenterology, “La Sapienza” University, Rome; 11 University Department of Internal Medicine, “Gemelli” University, Rome; 12 University Department of Medical Sciences, Milan; 13 Italian Celiac Society.