1. Prediction of Type 1 Diabetes (T1DM) & related Autoimmune Diseases (AD) Marco Songini, MD Diabetes Unit Azienda Ospedaliera Brotzu Cagliari (Italy)

2. Type 1 diabetes develops from the interaction between susceptibility genes and enviromental determinants. The major genetic susceptibility to type 1 diabetes is conferred by markers from HLA locus, but other genes are involved. The non genetic contribution to the disease (i.e. nutritional factors and infective agents) is even less wll-defined. This may imply aetiological heterogeneity in patients so that particular combinations of genetic susceptibility factors require exposure to specific non-genetic factors in order to initiate the disease developing process in type 1 diabetes. It is well known that immune markers (ICA, GADA, IA2, IAA) appear many years before clinical onset of type 1 diabetes. These “windows” offers the chance to pinpoint subjects at risk eventually suitable to preventive therapies. At present, intervention trials are recommended in the small subset of the population at high risk identified by genetic and immune markers.

3. Complementary strategies in the prediction of T1DM Strategy 1 AIM: TEST INTERVENTION STRATEGIES High specificity/ low sensitivity families immune markers high risk subgroup Strategy 2 AIM: REDUCE INCIDENCE OF IDDM Low specificity/ high sensitivity general population genetic + immune markers moderate risk subgroup Bingley, E. Bonifacio & E. Gale;Diabetes, vol. 42, feb. 1993

4. Preventive strategies for T1DM (1) Selective immunosuppression, using depleting or nondepleting monoclonal antibodies to lymphocyte cell surface molecules such as CD3, CD4, CD8, T cell receptor and major histocompatibility complex (MHC) antigens, or blocking peptides to T cell receptors Immunostimulation by viruses, cytokines, calcitriol, concanavalin A, bacille Calmette-Guèrin (BCG), Freund’s adjuvant or tranfusion of deficient lymphocyte subsets B-Cell rest by suppressive therapy with insulin E. Bosi & G.F. Bottazzo; Clin. Immunother. 3 (2) 1995

5. Preventive strategies for T1DM (2) Protection from oxygen radical-mediated and nitric oxide- mediated damage by nicotinamide, deferoxamine (desferrioxamine) and aminoguanidine Environmental intervention by manipulation of temperature, diet (gluten free) and hormonal milieu Induction of tolerance to B-cells by bone marrow transplantation, lymphocyte transfusion, intrathymic islet transplantation, neonatal B-cell stimulation and administration (intravenous, intrathymic, intraperitoneal or oral) of putative B-cell autoantigens such as insulin or glutamic acid decarboxylase E. Bosi & G.F. Bottazzo; Clin. Immunother. 3 (2) 1995

6. Tests to predict T1 DM & AD Autoantibodies : ICA, GADA, IA2-A, IAA, AD-Abs HLA-phenotype : DR3/DR4 (DQ2/DQ8), AD phenos HLA-genotype : Eterodimers 57Non Asp/53Arg DQ beta/DQ Alfa, AD genos ? Cell mediated markers : Alteration of lymphocyte subsets CD4/CD8, etc.

7. Immunological markers for T1DM ICA Islet Cell Abs Indirect immunofluorescence on human pancreatic cryosections Risk at 10 yrs FH+ >10 JDFU 41% >80 JDFU 80% IAA Insulin AutoAbs R.I.A. In Children first antibodies to appear Risk at 5 yrs FH+ 44% Risk at 10 yrs ICA + IAA 81%

8. GADA Glutamic Acid Decarboxilase AutoAbs Immunological markers for T1DM R.I.A. More common among adults High sensitivity low specificity IA2-A Protein Tyrosin Phosphatase AutoAbs R.I.A. More common among children High specificity low sensitivity

9. Immunological markers for T1DM Combined markers in FH+ Positivity for 3 or 4 antibodies yelds a risk of 88-100% to become diabetic in 10 years The best association of autoantibodies is: GADA + IA2-A GADA + IA2-A + IAA in young children Pastore MR et al Diabetes Care 1998, 9; 1445-50 We are able to assay GADA + IA2-A on blood spots E. Bosi, E. Bonifacio et al. Diabetes Care - March 1999

10. The preclinical stage of type 1 diabetes and related AD can last even many years These “windows” offers the chance to pinpoint subjects at risk eventually suitable to preventive therapies Background

11. HLA typing predisposing: HLA DR3-DQ2, DR4-DQ8 protective: HLA DR2-DQ6 Lernmark A Diabetes Metabolism Rev 1998, 14,3-29 Genetic markers for T1DM

12. Molecular biology of DQ chains of class second DQ A301, DQ B302, DQ B501 Alleles: 99% of diabetic patients 50% of normal people DQ B602 is fully protective for T1DM Lernmark A Diabetes Metabolism Rev 1998, 14,3-29 Gianani R et al. J Autoimmunity 1996, 9; 423-425 HLA Genetic markers for T1DM

13. 6p21 IDDM1 2,635 Davies (1994 ) 15q IDDM3 - - Field (1994) 11q13 IDDM4 1,072,5 Hashimoto (1994), Davies (1994) 6q25 IDDM5 1,165,5 Davies (1994) 2q31 IDDM7 1,134,5 Davies (1994), Copeman (1994), Owerbach and Gabbay (1995) 6q27 IDDM8 1,42 12,9 Luo (1995), Davies (1996) 18q IDDM6 1,1 3,5 Meriman (unpub.), Davies (1994) 11p21 IDDM2 1,299,4 Davies (1994), Bennet (1995) Locus s % References Genetic markers for T1DM (1)

14. Genetic markers for T1DM (2) 1,45 3q21-q25 IDDM9 1,26 8,5 Gough and Todd (unpubl.) 10p11.2-q11.2 IDDM10 13,7 Davies, Hashimoto (1994) Reed and Todd (unpubl.) 1414 95.595.5 TOT.TOT. s % 7p GCKRowe (1995) IDDM12 (CTLA-4) 2q33 Nistico (1996) 14q24.3-q31 IDDM11 Field (1996) 2q34 IDDM13 Morahan (1996) 6q21 IDDM15 Delepine (1997) Locus References

15. Identical Twins100%Tun RY, BMJ 1994 1st degree relatives (FH+)70%ICARUS Group Study Polyendocrinopathy (FH-)25%Bosi E, Diabetes 1991 Polyendocrinopathy (FH+)70%Bosi E, Diabetes 1991 High risk newborns (FH+)50%BABYDIAB (Germany) High risk newborns (gene+)50%DIPP Project (Finland) Sardinian school children (gen) 24%SSI Study (Sardinia) Natural History of T1DM 2 yrs Popul islet-related Abs+ Follow- Risk References 10 yrs 5 yrs 7 yrs 10 yrs up

16. Natural history of T1DM tt Beta cell mass 50% 25% 75% Triggers ? TYPE 1 DIABETES Auto Abs + FPIR OGTT + Time 0 Triggers ? GENES (susc)

17. Screening for pre-T1DM and related AD Schoolchildren Newborn DAISY (USA) BABYDIAB (Germany, Australia) SNI (Sardinia) DIPP (Finland) DIABFIN (Italy) France Sweden Spain Oxford Holland Estonia SSI USA Finland Germany

18. Cost of predicting T1DM Cost of insulin therapies (per year) Conventional Therapy (CT) $1450 Intensive Therapy (ICT) $ 2 x CT CSII $ 3 x CT Cost of Screening (for each enrolled case) DPT-1 $1751 DIPP (follow up=10 yrs) (newborns) $245 $733 Birth Genetic+Abs screening Counselling Abs follow up 100% 13% Birth Abs screening Abs follow up 100% Cost of DM (?AD) Hahl et al. Diabetologia (1998) 41:79-85 $ 92 billions

19. Prevention of T1DM and other related AD

20. T1DM&AD are theoretically preventable Because there are environmental causes Because we are beginning to understand the genetic and immune basis Because they develops very slowly Because we have good predictive tests Because we can stop them in animals Because we can run clinical trials

21. T1DM & AD are suitable diseases for preclinical screening and intervention Serious consequences (in USA 50 deaths yearly from DKA) Treatment following diagnosis expensive, demanding, limited effect on complications Identifiable preclinical phase also for AD Identifiable subjects “not at risk” also for AD...but as yet no preventive therapy of proved efficacy (no penicillin for prediabetes!)

22. Assigning risk Primary prevention : must be based on family history or high risk HLA - and will miss a lot of cases! Secondary prevention : immune-markers relatively stable after age 5; almost inevitable progression with multiple antibodies; excellent screening efficiency (islet imaging)

23. Setting up an intervention: in whom? Primary : Neonates with family history or high risk HLA Secondary: –Infants: HLA DR3/4 with antibodies –Children/young adults with multiple Abs (T1DM&AD) –Older adults with LADA

24. Setting up an intervention: with what? Should work: –In animal models –In newly diagnosed type 1 –In pilot trials (assessed how?) Must have: –An acceptable safety profile –Ease of administration

25. Setting up an intervention: conclusions At present trials must be large, structured, costly and long term Will depend on international collaboration We need a disciplined consensus process for evaluating and prioritizing new therapies Role of pharmaceutical industry? clinicians should have a say

26. T1DM prevention trials Primary Cow’s milk avoidance: TRIGR Gluten free diet: PREVFIN Secondary Nicotinamide: DENIS, ENDIT, New Zealand Insulin: DPT-1, EPLL SCIT; Schwabing, Brunetti 1999 Tertiary Cyclosporin: GETREM, French and Canadian studies Linomide : Franco-Swedish trial

27. Intervention trials: assumptions TrialNYr Diabetes RRR % % ENDIT5305 40:26 35 DENIS1303 30:6 80 DPT (high)3404 84:55 35 DPT (inter)4904 24:12 50 Mahon and Dupre,1997

28. Cyclosporin before onset of T1DM 6 relatives vs 9 historical controls All controls developed diabetes in 12 months 4/6 cyclosporin treated patients developed diabetes within 4 years (5, 24, 24 and 47 months) Carel et al., 1996

29. Intervention in early infancy? Level of risk? Safety of intervention? Long term data? Acceptability/compliance? Efficacy demonstrated in other AD? Can the intervention be tested effectively in this category of patient?

30. FOLLOW-UP, MONTHS 3 6 9 12 18 24TOTAL CASEIN 0/830/751/723/711/671/62 3/84 HYDROLYSATE (3.6%) p=0.06 CM-BASED0/871/846/797/786/777/76 10/89 FORMULA (11.2%) Emergence of at least one auto-Ab by the age of 2 years (n=173) The Second TRIGR Pilot Study

31. EURODIAB Sardinia (1989-98) birth seasonality N=1928, 0-29yr P<0.001 Jan-MarchApr-JuneJul-SeptOct-Dec

32. Future Directions? Surrogate end-points Safety and acceptability need to be balanced against efficacy Early “one-off” therapy would be ideal Explicit standards for performance of trials Fewer, better quality studies based on international consensus Lessons from other human autoimmune disease?