presentazione sulla celiachia e le ultime acquisizioni riguardanti la malattia autoimmune più nota e usata come modello date le sue peculiarità.

1. Gliadin, Intestinal Permeability, and Celiac Disease: From Innate Immunity to Autoimmunity: Alessio Fasano, M.D. Mucosal Biology Research Center University of Maryland School of Medicine

3. Normal small bowel Celiac disease Gluten Gluten-free diet

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

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

8. The Clinical Manifestations of Celiac Disease are More Heterogeneous Than Previously Appreciated A. Fasano, N Engl J Med 2003;348:2568-70.

9. What is so Special About Gluten? Gluten (gliadin+glutenin) Gliadin Glutenin

10. CD Pathogenesis Celiac disease Genetics Gluten NECESSARY CAUSES Gender Infant feeding Infections Others RISK FACTORS Pathogenesis ?

11. Proposed CD Pathogenesis ?????

12. The Paracellular Pathway … Tight junctions are a ‘dark horse’ implicated in a host of disease states, ranging from acute injury to chronic inflammation and autoimmune diseases

13. DISCOVERY OF ZONULIN Coomassie Western blot 1 2 3 4 1: Tissue lysate 2: Sephacryl-S300 3: Q-sepharose 4: Immuoaffinity 1 2 3 4 PURIFICATION PROTOCOL FROM HUMAN INTESTINE

14. Pillole di Saggezza Pillola #1:

15. Zonulin Signaling: How Does It Work?

16. A Way to Activate the Zonulin Innate Immunity PAthway Bacteria-Gut Interaction:

17. Studies on Cell Lines: Time 00:00 Drago et al Scand J Gastroenterol 2006; 41:408-19.

18. Double staining immunofluorescence experiments on IEC-6 cells Time 00:05: Zonulin packing GLIADIN CONTROL CASEIN Drago et al Scand J Gastroenterol 2006; 41:408-19. Actin Zonulin Nucleus

19. Studies on Cell Lines: Time 00:15: Zonulin Release Control Bacteria/gliadin Drago et al Scand J Gastroenterol 2006; 41:408-19.

20. Studies on Cell Lines: Time 00:20: Zonulin Binding PT-gliadin Caco 2 T84 Control IEC6 Drago et al Scand J Gastroenterol 2006; 41:408-19.

21. Gliadin Co-Localizes with CXCR3 100X Gliadin FITC Nucleus DAPI CXCR3 TRITC Merge

22. In Situ IFM of Biopsies from Either Celiac Disease Patients or Healthy Controls Showed Apical Expression of CXCR3 Celiac disease Non-celiac control CXCR3 Actin Nucleus

23. Proposed Zonulin Mechanism of Action Fasano et al, J Clin Invest 1995;96:710-20; el Asmar et al Gastroenterology 2002;123:1607-15 Following Pathway Activation Resting State Freeze-Fracture Zonulin signaling

24. Celiac Disease Pathogenesis: The Continuous Stimulation by Gluten is Necessary to Perpetuate the Autoimmune Process Intestinal Lumen A. Fasano and T. Shea Donohue, Nature Clin Pract Gastroenterol Hepatol 2005;2:416-22 PRR AGA EMA TTG MyD88 dependent Submucosa Bacteria

25. 1) bacterial adhesion, 2) internal signaling, 3) signal transmission to the effector cells, 4) zonulin release on the luminal side 5) zonulin binds to its receptor, 6) cascade of events ending 7) by the TJ opening, leading to 8) water and solutes paracellular passage and 9- “wash out” effect, 10) and paracellular passage of food antigens and macromolecules, 11) GALT stimulation and subsequent undesired effects H 2 O 5 6 8 7 11 10 9 H 2 O Lymphocytes Food Antigens Macromolecules Zonulin Bacteria Zonulin 1 4 2 Zonulin Enterocytes 3 3 El Asmar et al J Gastroenterology 2002;123:1607-1615 Zonulin System – Innate Immunity

26. Innate Immune Response (Early) Adaptive Immune Response (Late) Microbial insult GOAL: Eliminate “danger” Cytokines Costimulatory Molecules Abs/CMI Cytokines Cross Talk Between Innate and Adaptive Immunity Pathways

30. 0 2.5 5 7.5 10 12.5 15 zonulin ng/mg serum protein HC CD AIH PBC T1D APS-1 MS Clemente et al. Gastroenterology 2002;122 :A15 0.2 Cut-off 3.4 2.1 3.0 1.3 0.3 0.5 * p < 0.0001 * * * * Serum Zonulin in Autoimmune Disorders

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32. Potential Therapeutic Applications

34. AT-1001, the Zonulin Inhibitor Di Pierro et al, J Biol Chem 2001;276:19160-5.

35. Celiac Disease NDA Explor. Discssn w/ GI/Coag Dvn End of Phase II Meeting; confirm Fasttrack Single Dose Safety & PK, SDSS 4 wk Mult Dose Safety Study, MDSS File Celiac IND, seek fast track Pivotal #1 Celiac Pivotal #2 Celiac Start Phase II 3 mos. Dose ranging in Celiacs & Diabetics Preclinical ADME, Tox, Biology Pivotal #2 T1D T1D NDA ↓ Intest. Perm? Reformulate Yes No File T1D IND Explor. Discssn w/ Metab/Endo Dvn Addtl PD Studies The Zonulin Clinical Trial Roadmap Pivotal #1 T1D

36. Zonulin, Gliadin, And Type 1 Diabetes Sapone et al. Diabetes 2006

37. In Vivo permeability and serum glucose levels in DP rats either untreated or treated with AT-1001 Watts et al PNAS 2005;102:2196-21 Glucose LA/MA LA/MA Ratio 0 50 100 150 200 250 300 30 37 44 51 58 65 72 Age (days) Serum glucose (mg/dl) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 50 100 150 200 250 300 30 37 44 51 58 65 72 Age (days) Serum glucose (mg/dl) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 LA/MA Ratio No Diabetes – FZI/0 Treated Diabetes ICA + (100%) ICA – (92%)

38. BB/wor DP Rat Beta Cell Rescue Study

39. Blocking the Zonulin-Dependent Increased Intestinal Permeability Aborts The Autoimmune Process Age: 52-54 days Treatment Animal flow Placebo Timeframe T 0 1 2 3 4 5 6 7 14 wks Pre-T 0 AT1001 Placebo 60% T1D (N = 10) (N = 20) (N = 30) AT1001 35% T1D T1D Sapone et al. ADA Meeting 2005: late breakthrough abstract Outcome 0 50 100 150 200 250 300 30 37 44 51 58 65 72 Age (days) Serum glucose (mg/dl) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 LA/MA Ratio ICA + (100%)

40. Diabetes Treatment Study: Treatment of Autoimmune Diabetes with AT-1001 Sapone et al. ADA Meeting 2005: late breakthrough abstract Untreated T1D Treated NO T1D 10X 40X

41. Insulin staining Glucagon staining A B C D Untreated BBDP rats that developed T1D Blocking the Zonulin-Dependent Increased Intestinal Permeability Aborts The Autoimmune Process AT1001-treated BBDP rats that DID NOT develop T1D: No insulitis Islet Immunohistochemistry

42. Insulin staining Glucagon staining A B C D 40X Blocking the Zonulin-Dependent Increased Intestinal Permeability Aborts The Autoimmune Process Islet Immunohistochemistry AT1001-treated BBDP rats that DID NOT develop T1D: Islets recovering from insulitis and islet regeneration 10X

43. Role of Gliadin in Type 1 Diabetes Pathogenesis Zonulin Intestinal Permeability Incidence of T1D BBDP rats on regular diet BBDP rats on GFD BBDP rats on regular diet BBDP rats on GFD BBDR rats on regular diet BBDP rats on regular diet BBDP rats on GFD

44. Human Studies Sapone et al. Diabetes 2006

45. Sapone et al. Diabetes 2006 Role of Zonulin in T1D in Humans T1D Relatives T1D Controls 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Serum zonulin (ng/mg protein) n= 339 89 97

46. Serum Zonulin Levels and Intestinal Permeability in T1D, Their Relatives and Controls Intestinal permeability and serum zonulin levels in T1D patients and their relatives. Both parameters were significantly higher in T1D subjects as compared to their relatives. *p=0.04; **p=0.02 Sapone et al. Diabetes 2006

47. Serum Zonulin and in vivo Permeability in Children with Type 1 Diabetes After Gluten Challenge, vs. Relatives Fig. 1. Serum zonulin levels in T1D patients (n=16) and their relatives (N=35) before and after a gluten-containing meal. *<0.05 compared to baseline. Fig. 2. Serum LA/MAin T1D patients (n=16) and their relatives (N=35) 1 hour before and 1 hour after a gluten-containing meal. *<0.0002; **<0.0005 compared to before meal LA/MA. ** 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0 1 2 3 4 5 Time (hrs) Serum zonulin (ng/mg protein) T1D relatives Gliadin-containing meal * * * 0.000 0.050 0.100 0.150 0.200 0.250 0.300 1 2 3 4 5 Time (hrs) LA/MA ratio T1D relatives *

48. PROPOSED MODEL Proposed role of aberrant intestinal permeability in Type 1 diabetes pathogenesis. Non-self antigens are present in the intestinal lumen (1) and cross the tj barriers in subjects with dysregulation of the zonulin system (2-3). Antigen peptides bind to HLA receptors present on the surface of APC (4). In turn, these peptides are presented to T lymphocytes (5). In genetically susceptible individuals, an aberrant immune response (both umoral and cell-mediated) (6) leads to the autoimmune process mainly targeting the Langherans islets with subsequent insulin deficiency typical of type 1 diabetes (7). 5 Submucosa T B GAD, IA, IA-2 Tk P 2 3 4 6 1 APC Intestinal lumen 6 7 7

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