Journal of Diabetes 3 (2011) 4857

REVIEW ARTICLE

Prediction and prevention of Type 1 diabetes mellitus

Li ZHANG and George S. EISENBARTH
Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, Colorado, USA

Correspondence George S. Eisenbarth, Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, CO 80045, USA. Tel: +1 303 724 6847 Fax: +1 303 724 6839 Email: George.Eisenbarth@UCDENVER.EDU Received 9 August 2010; revised 20 September 2010; accepted 24 October 2010. doi: 10.1111/j.1753-0407.2010.00102.x

Abstract Type 1A diabetes mellitus (T1DM) is caused by autoimmune islet b-cell destruction with consequent severe insulin deciency. We can now predict the development of T1DM by determining four biochemically characterized islet autoantibodies, namely those antibodies against insulin, glutamic acid decarboxylase 65, insulinoma antigen (IA)-2 (ICA512) and the zinc transporter ZnT8. We can also prevent T1DM in animal models, but the nal goal is the prevention of T1DM in humans. Multiple clinical trials are underway investigating methods to prevent b-cell destruction. Keywords: immunotherapy, prediction, prevention, Type 1 diabetes.

Introduction Type 1A diabetes mellitus (T1DM) is characterized by autoimmune-mediated selective destruction of pancreatic b-cells, with consequent severe insulin deciency. There are other forms of insulin-decient diabetes, including monogenic forms.1 T1DM is commonly diagnosed in children and adolescents, but can occur at any age. T1DM presents with hyperglycemia and usually a need for immediate exogenous insulin treatment. The incidence of T1DM in many developed countries is now twice as high among children as it was in the 1980s.2,3 In north Italy, the overall incidence rate from 1984 to 2004 in the 029 years age group has been reported to be 11.3 100 000 personyears.4 Furthermore, both the 014 and 1529 year age groups had a 60% higher risk in 20002004 compared with the period 19841989.5 During the past two decades, improved understanding of the natural history and pathogenesis of T1DM have led to the ability to predict the disease in humans and to prevent it in animal models. Clinical trials for the prevention of T2DM in humans are underway. Prediction of diabetes Anti-islet autoimmunity precedes the development of T1DM (usually by years) and is associated with
48

progressive metabolic abnormalities. The ability to predict the development of T1DM has been improved markedly with the combined use of genetic, islet autoantibody and metabolic testing.6 Genetic prediction T1DM has a strong genetic component, reected by the observation that rst-degree relatives have a higher risk than the general population, siblings have a higher risk (6% vs 0.4%; sibling kS 15)7 than offspring8 and there is a high concordance rate in identical twins with diabetes (approximately 50%).9 When initially nondiabetic monozygotic twins of patients with T1DM are followed prospectively, the cumulative incidence for anti-islet autoimmunity and diabetes may exceed 70% and there is no duration of discordance at which monozygotic twins are not at risk for progression.10 Diabetes susceptibility in the non-obese diabetic (NOD) mouse model results from the interaction of multiple loci (>40),11 with the strongest predisposing effect deriving from genes within the major histocompatibility complex (MHC), namely human leukocyte antigen (HLA) in humans.12,13 Similarly, with recent genome-wide association studies, >40 loci associated with T1DM have been identied, with by far the most important determinants of disease in the MHC (HLA region of humans; Fig. 1).14,15 There are three classes

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L. ZHANG and G.S. EISENBARTH

Type 1 diabetes prediction and prevention

6.50

Odds ratio

2.25 2.00 1.75 1.50 1.25 1.00 HLA INS PTPN22 ILR2A SH2BE ERBB3 PTPN2 CLEC16A CTLA4 IL18RAP PTPN2 OCR5 IFIHI CTSH CD226 IL2RA PRKCQ IL2 BACH2 UBASH3A RGS1 IL7RA CITNF6 TNFAIP3 TNFAIP3 TAGAP

Locus
Figure 1 Loci associated with risk of Type 1 diabetes identied in genome-wide association studies.14,15 (h), insulin protection and metabolism; ( ), immunity; ( ), b-cell apoptosis protection; ( ), unknown function.

of HLA genes, with Class II HLA molecules (DP, DQ, and DR) having the strongest association with T1DM. Class II HLA genes encode molecules that participate in antigen presentation to CD4 T cells. Polymorphic variants of Class II HLA genes determine 4060% of genetic susceptibility.1618 DQ molecules are formed by two polymorphic genes (a and b) and each amino acid variant of a and b is given a four digit number preceded by the gene designation (e.g. DQB1*0302). Two of the most common high-risk DQ haplotypes (DQa and b) are DQA1*0301 with DQB1*0302 and DQA1*0501 with DQB1*0501. Many studies have veried that DQB1*0302 is a strong susceptibility allele and that the heterozygous combination of DQA1*0301DQB1*0302 on an HLA-DR4 haplotype and DQA1*0501-DQB1*0201 on an HLADR3 haplotype results in enhanced risk of T1DM.13,19 In Denver (CO, USA), 2.4% of newborns from the general population are DR3 4 heterozygote with DQB1*0302. These individuals have an absolute risk of developing T1DM of approximately 1 15 compared with a risk of 1 300 in the general population.19 The genetic screening for susceptible HLA-DQB1 alleles and the follow-up of higher-risk newborn Finnish children identied 77% of those who developed the disease before 3 years of age.20 A screening strategy based on HLA-DQA1-DQB1 typing identied four susceptible genotypes (DQ*0301-0302 DQ*0501-0201, DQ*03010302 DQ*03010302, DQ*0501-0201 DQ*0501-0201, and DQ*03010302 Y) that accounted for 9% of the population and included approximately 60% of future diabetic patients diagnosed before 40 years of age and up to 70% of those diagnosed before 5 years of age.21 Class II HLA is strongly associated with T1DM in both Asian and Caucasian populations, but haplotypes

associated with T1DM are markedly different due to differences in the presence and absence of haplotypes in each population.2224 Standard HLA molecular typing is too costly for population screening at this time, but simplied molecular testing is possible. A rapid test to identify DR3 4-DQ8 subjects analyzed two single nucleotide polymorphisms (SNPs), specically rs2040410 and rs7454108, which are associated with DR3-DQB1*0201 and DR4DQB1*0302.25 These SNPs were analyzed in samples from 143 HLA-typed children who participated in the Diabetes Autoimmunity Study of the Young (DAISY), as well as in 5019 subjects from the Type 1 Diabetes Genetic Consortium (T1DMGC). In the T1DMGC samples, the two SNPs were found to have a sensitivity of 98.5% (1173 1191) and a specicity of 99.7% (3815 3828) for DR3 4-DQB1*0302. In the DAISY population, the test was 100% sensitive (69 69) and 100% specic (74 74). Lavant and Carlson26 have developed a high-throughput HLA-typing method that accurately distinguishes risk alleles. This method allows screening of several thousand samples per week, consuming 32 ng DNA template, low reagent volumes, and minimal time for data review.26 Autoantibody prediction The presence of autoantibodies against multiple islet antigens with specic assays (specicity 99%) predicts the development of T1DM. In the 1970s, using frozen sections of human pancreas, antibodies to islet cells (ICA) was identied as the rst autoantibody.27 Following the discovery of ICA, multiple biochemical antibodies were identied before or at the onset of diabetes and used to diagnose diabetes, as well as in research settings to predict diabetes. Standard well-characterized autoantibodies react with insulin [i.e. anti-insulin antibodies (IAA)], the tyrosine phosphatases insulinoma antigen (IA)-2 and IA-2b, and glutamic acid decarboxylase (GAD).2830 More recently, antibodies against the zinc transporter (ZnT8) were discovered and are now used for the prediction and diagnosis of diabetes.31 Table 1 summarizes basic information concerning the autoantibodies used to predict diabetes. Prospective population-based studies, such as DAISY,32 the German BabyDIAB study,33 and the Finnish diabetes prediction and prevention (DIPP) study,34,35 have established that the autoantibody markers appear months or years before the onset of diabetes and can occur as early as in the rst year of life.32,36 The presence of a single islet autoantibody is associated with relatively low risk on long-term follow-up (<5%), whereas individuals expressing two
49

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Table 1 Major autoantibody markers for type 1 diabetes Sensitivity (%) 70

Autoantibody ICA

Description Multiple and variable target molecules, major antigen specicity: GAD and IA-2 IA-2b Proteins of 65 kDa 67 kDa anchored within membranes of small synaptic-like microvesicles of islet cells or synaptic vesicles of neurons The only b-cell-specic antigen so far identied Highly evolutionarily conserved protein 40-kDa tryptic fragment of IA-2, a member of the protein tyrosine phosphatase-like proteins Expressed in secretory granules of b-cells and neuroendocrine cells Islet zinc transporter

Implication in diagnosis prediction High ICA-titer correlated with increased risk of diabetes in relatives Difcult to standardize pancreatic sections Major antigenic region: middle and carboxy-terminal regions of GAD65 Epitope recognition conformationally dependent Prevalence of GADA increase with age IAA rst to appear during insulitis in young children

GADA

6575

mIAA

4992

IA-2

Anti-ZnT8

Immunodominant region: intracellular domain Epitope recognition conformationally dependent Presence of IA-2A in multiple antibody-positive relatives predicts rapid progression to diabetes Present in most patients with Type 1 diabetes There are two major polymorphic variants of ZnT8, one with a tryptophan and the other with an arginine at position 325 of the molecule Most patients have autoantibodies recognizing both variants

74

6575

GAD, glutamic acid decarboxylase; GADA, antibodies to GAD; IA-2, insulinoma antigen 2; ICA, antibodies to islet cells; mIAA, microinsulin autoantibody.

or more of the autoantibodies almost always progress given long enough follow-up. It is not clear whether those with single islet autoantibodies who do not progress have islet-directed autoimmunity or simply the development of an antibody that cross-reacts with an islet autoantigen. Given screening for autoantibodies reacting with four islet autoantigens, and specicities of 99%, approximately 4% of normal controls express a single islet autoantibody whereas <1 in 300 would express two or more autoantibodies. The antigens recognized by ICA staining of pancreatic sections include GAD, IA-2 and ZnT8, but are not limited to these. In some studies of general populations, the predictive value of ICA was low (13%).37,38 The large Diabetes Prevention Trial Type 1 (DPT-1) tested autoantibodies in relatives of patients with T1DM.39 The occurrence of T1DM was only 3.9% in relatives expressing ICA only and negative for biochemical autoantibodies [IAA, autoantibodies to GAD (GADA), and ICA512 (IA-2)]. Among those with single positivity for GAD65 and ICA, increased incidence of T1DM was related to higher antibody titer. By 7 years of follow-up, ICA-positive schoolchildren and relatives (i.e. those with ICA titers 10 Juvenile Diabetes Foundation units) had similar progression to diabetes (45% vs 43%).40 In most studies, ICA has been combined with other autoantibodies to predict diabetes. The data from the large DPT-1 study39 demonstrated that ICA autoantibodies, as well as GAD and
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IA-2 autoantibodies, in relatives were relatively stable, with 7585% of all positivity (9095% with high levels) conrmed during follow-up. Given the difculty of standardizing the immunouorescent islet cell antibody assay that uses frozen sections of normal human pancreas, this assay is no longer commonly used. IA-2, also known ICA512, is a transmembrane protein localized to the secretory vesicles of both endocrine and neuroendocrine tissues. T1DM-associated autoantibodies to IA-2 (IA-2A) react with the intracellular section of the protein. In particular, it has been reported that sera from T1DM patients recognize either amino acids 771979 of the carboxyl terminal domain or the amino terminal part of the intracellular domain.41,42 Recently, it was reported that antibodies reactive with the extracellular domain of IA-2 (1577) are associated with faster T1DM progression.42 In the Childhood Diabetes in Finland (DiMe) study, siblings who progressed to clinical diabetes had IA-2 antibodies specic to the juxtamembrane region more often than siblings who did not progress to T1DM.43 These ndings suggest that juxtamembrane region-specic IA-2 antibodies are associated with an increased risk of progression to overt T1DM, whereas an IgE response to IA-2 confers relative protection against clinical disease.43 Makinen et al.44 compared epitope- and isotype-specic IA-2 antibodies between children with a humoral immune response restricted to IA-2 and children with a broad response including

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Type 1 diabetes prediction and prevention

IAA and GADA in addition to IA-2A. Children with newly diagnosed T1DM, who test positive to IA-2A only of the three molecular antibodies predictive of T1DM, had a broader IA-2-specic isotype response and stronger association with the high-risk HLA haplotype than did those children testing positive for all three molecular antibodies. GADA, a 64-kDa antigen,45 is found in approximately 70% of newly diagnosed T1DM patients.46 GAD has two isoforms. Both human and rat islets predominantly express GAD65, whereas GAD67 is the major GAD isoform in mouse islets.47 The presence of anti-GAD65 antibodies along with anti-IA-2 antibodies is a highly predictive marker for the development of T1DM in humans. The prevalence of anti-GAD65 and anti-IA-2 antibodies is approximately 90% in newly diagnosed T1DM patients and prediabetic individuals.48 LaGasse et al.37 recommended the combined use of GADA, IA2-A, and IAA for screening, but ZnT8 autoantibodies should now also be considered to complete the panel of well-characterized assays. A combination of GADA and IA-2A for primary screening has been described to be as sensitive as the combination of IAA and GADA.49 In 1983, Palmer et al.30 discovered the presence of IAA in patients with new-onset T1DM prior to the administration of exogenous insulin. Further studies revealed the presence of IAA in rst-degree relatives of diabetic patients.5052 The insulin antibody afnity and epitope specicity for insulin autoantibodies can predict which children are at high risk of progressing to diabetes. Children with high genetic risk who develop insulin antibodies (IAAs) early in life often subsequently develop multiple antibodies and eventually diabetes29 and insulin autoantibodies are usually the rst autoantibody to appear in young children developing T1DM.51,53,54 In the BabyDIAB study,51 a high percentage of children who went on to develop multiple anti-islet autoantibodies and or progress to diabetes expressed high-afnity autoantibodies (>109 L mol). The levels of insulin autoantibodies are inversely correlated with the age at which T1DM develops. Thus, levels >2000 nU mL are almost exclusively found in patients who progress to T1DM prior to 5 years of age; less than half the individuals developing T1DM after 15 years of age have anti-insulin autoantibodies levels exceeding those in controls.51 To some extent, the levels of these antibodies are genetically inuenced and are associated with DR4 and DQ8.51 There are data suggesting that the levels of anti-insulin autoantibodies among ICA-positive rst-degree relatives are correlated with the rate at which individuals progress to overt diabetes.55 However, those relatives

who only express anti-insulin autoantibodies progress to overt diabetes infrequently.51 The islet b-cell zinc cation efux transporter ZnT8 (Scl30A8) is a newly dened target of islet autoantibodies.31 This transporter was discovered as an autoantigen because it is specically expressed in islet b-cells, where it is associated with the regulated pathway of insulin secretion. ZnT8 facilitates the transportation of Zn2+ from the cytoplasm into the insulin secretory granule, where zinc is complexed with insulin, forming insulin crystals. Autoantibodies reacting with ZnT8 are present in most patients with T1DM and assay specicity and sensitivity are similar to those for GAD65 autoantibodies.31 For at-risk populations, such as relatives of patients with T1DM and individuals with high-risk HLA alleles from the general population, we measure ZnT8 autoantibodies in those expressing a single anti-islet autoantibody. If ZnT8 autoantibodies are present, these individuals would have two or more anti-islet autoantibodies and thus have a much higher risk of progressing to diabetes.5658 The presence of >1 persistently positive antibody has been shown to reliably predict the development of T1DM.32,59 If four markers are measured (i.e. GADA, IA-2A, IAA, and ZnT8A), <10% of patients with T1DM are autoantibody negative, fewer than 10% have only one marker, and approximately 70% have three or four markers.60 There is a general consensus that the presence of multiple autoantibodies (i.e. two or more) is associated with a high risk of developing diabetes while the presence of single islet cell-related autoantibodies has usually a low predictive value (<1 4). The DPT-1 study demonstrated that there was a signicant association between the increasing occurrence of T1DM and increasing autoantibody number.39 The TrialNet study estimated 5-year diabetes risk by measuring GADA, ICA512A, microinsulin autoantibody (mIAA), or ICA.61 In summary, in T1DM relatives as well in the general population, the risk of developing disease increases with the number of positive autoantibodies.38,62 T1DM relatives with two or more antibodies had a 39% and 68% risk of developing T1DM within 3 and 5 years, respectively; relatives positive for three autoantibodies have an estimated >90% risk of developing T1DM within 5 years.62 For diabetes prediction, a combination of GADA and IA-2 for primary screening, followed by ICA and IAA testing, has been proposed.49,63,64 Data from DPT-1 indicate that screening based on two antibodies and testing individuals positive for one of the remaining markers results in a sensitivity of 96.7% for GADA and ICA512, 96.7% for mIAA and GADA, 93% for ICA and ICA512, and
51

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99% for the combination of GADA and ICA.65 As the assay methodology becomes simpler, we believe simply measuring all four biochemical autoantibodies will become standard. Clinical trials for the prevention of b-cell destruction Prevention of T1DM, based on the prevention of islet b-cell destruction, can be considered at different stages of the disease, from stages prior to or following the onset of hyperglycemia. More specically, primary prevention would be applied in genetically susceptible individuals (or whole populations if the modality was safe enough; for example, vaccination) prior to the presence of islet autoantibodies. This could be accomplished by eliminating exposure to triggering factors or immune intervention. The Trial to Reduce IDDM in the Genetically at Risk (TRIGR) study is evaluating the elimination of bovine milk products early in life,6669 whereas the PrePoint study is evaluating oral insulin in very high-risk children.70 Secondary prevention is performed after autoantibodies develop (at present T cell assays do not have a high enough positive predictive value to be used for prediction), but before the onset of hyperglycemia to prevent further b-cell loss and to induce immunologic remission. Tertiary prevention seeks to stop further b-cell destruction after its clinical manifestation with hyperglycemia present. Antigen-specic therapy The most extensively studied antigens used in trials for the prevention of immunologic b-cell destruction are insulin and GAD. The National Institutes of Health (NIH) Diabetes Prevention Trial (DPT) was started with the goal of nding out whether insulin-based therapies (i.e. low-dose parenteral insulin therapy to high-risk relatives or oral insulin to intermediate-risk relatives to induce oral tolerance) would prevent or delay the onset of diabetes in cytoplasmic islet autoantibody-positive relatives of patients with T1DM. Relatives were staged in terms of their expression of insulin autoantibodies, metabolic abnormalities, and the presence of the protective HLA allele DQB1*0602. In addition, GAD65 and ICA512 autoantibodies were measured, although not included as entry criteria. These large studies, as well as the European Nicotinamide Diabetes Intervention Trial (ENDIT) using nicotinamide, demonstrated the ability to predict T1DM on a relatively large scale and to assign various levels of risk for disease.7173 Unfortunately, overall none of these studies demonstrated a delay in or the
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prevention of progression to diabetes. In one subgroup of the oral insulin trial, namely those with high levels of insulin autoantibodies, oral insulin signicantly slowed progression to diabetes (by approximately 4.5 years).74 Given this subgroup analysis, the NIH TrialNet group is now seeking to conrm whether oral insulin can ameliorate progression to diabetes of relatives with multiple islet autoantibodies and high levels of insulin autoantibodies. GAD65 is a prominent autoimmune target in humans, with GAD65 autoantibodies one of the best predictors of progression to diabetes. To assess the ability of alum-formulated GAD (GAD-alum) to reverse recent-onset T1DM in patients 1018 years of age, Ludvigsson et al.75 administered 20 lg GAD-alum to 35 patients with recent-onset T1DM and examined with the autoantigen could reduce or halt the loss of residual insulin secretion. In this trial GAD-alum vaccination delayed the loss of fasting C-peptide compared with a placebo group, although there was no improvement in HbA1c or a decrease in insulin requirements.75 Conrmatory studies of GAD65 vaccination are underway in Europe and the US, including a new-onset TrialNet study, with plans for a prevention study (detailed information is available at http:// clinicaltrial.gov). Anti-CD3 The clinical use of the original anti-CD3 (OKT3) monoclonal antibody has been limited by its induction of cytokine release syndrome with, in severe cases, pulmonary edema developing within hours of treatment.76 Two specic humanized FcR non-binding anti-CD3 antibodies, namely teplizumab and otelixizumab, have been investigated in patients with recent-onset T1DM. Herold et al.76 recruited 24 subjects with new-onset T1DM. In that open-label study, the 12 subjects randomized to test therapy were given daily infusions of teplizumab for 14 days. The control group underwent the same metabolic and immunologic studies as the drug-treated group, but the patients did not receive an infusion and were not hospitalized. The treatment group had maintenance of C-peptide responses for 1 year, whereas the control group showed signicant decline.76 Longer follow-up of patients treated with anti-CD3 indicates that, after 1 year, C-peptide loss resumes in the anti-CD3-treated patients but that C-peptide secretion exceeds that in controls. In keeping with the maintained b-cell function, the treatment group had lower HbA1c levels and lower insulin requirements compared with the control group.76 In addition, decreased insulin utilization was associated

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Type 1 diabetes prediction and prevention

with decreased loss of C-peptide. In a multicenter European study of a second modied anti-CD3,77 80 patients with new-onset T1DM were randomly assigned to receive placebo or otelixizumab for six consecutive days and patients were followed for 18 months. At 6, 12, and 18 months, residual b-cell function was better maintained with otelixizumab than with placebo. Twelve of 16 patients who received otelixizumab (75%) needed minimal doses of insulin (0.25 IU kg per day) compared with none of the 21 patients who received placebo. Short-term treatment with CD3 antibody preserves residual b-cell function for over 48 months in patients with recent-onset T1DM.78 B Lymphocyte-targeted therapies Rituximab (anti-CD20) is a monoclonal antibody that is a chimeric murine human monoclonal IgG1 kappa antibody. It selectively targets and depletes human CD20-expressing B cells. Anti-CD20 has been used as therapy for B cell lymphoma and shows efcacy in multiple sclerosis and rheumatoid arthritis.79,80 A transgenic NOD mouse expressing human CD20 (hCD20) on B cells has been generated.81 In this mouse model, a single cycle of treatment with an antibody specic for hCD20 temporarily depleted B cells and signicantly delayed and or reduced the onset of diabetes.81 In addition, anti-CD20 treatment of B cell depletion prolongs syngeneic islet graft survival, delays the onset of recurrent autoimmune diabetes, and may reverse the disease in hCD20 NOD mice.82,83 TrialNet investigators conducted a randomized double-blind placebo-controlled study with a single course of antiCD20.83 At 1 year, the mean level of C-peptide was signicantly higher in the rituximab-treated group than in the placebo group. In addition, the rituximabtreated group had signicantly lower HbA1c levels and required less insulin. Between 3 and 12 months, the rate of decline in C-peptide levels in the rituximab-treated group was signicantly less than in the placebo group,83 but thereafter C-peptide loss continued. Nevertheless, further investigations are essential (including more than a single drug course) to evaluate both efcacy and safety of rituximab as a single agent in people at risk of developing diabetes, as well as in new-onset diabetes patients. Summary Given the observation that one in 300 random cadaveric donors express two or more islet autoantibodies,84,85 it is likely that approximately 1 million

individuals in the US are in the process of developing T1DM. If safe and effective preventive therapy were developed that could prevent the progression to diabetes, even currently available islet autoantibody assays could provide the basis for large-scale screening. It is very likely that assays will improve and, indeed, the Immunology of Diabetes Society, in collaboration with the US Centers for Disease Control (CDC), has a program in place to improve worldwide assay standardization, namely the Diabetes Autoantibody Standardization Program (DASP) workshops.86 In the absence of such therapy, the screening of large populations for islet autoantibodies is performed primarily for research, although the detection of islet autoantibodies does allow the prevention of life-threatening ketoacidosis at diabetes onset.87 Large Phase III trials of both GAD65 immunization and anti-CD3 infusion in patients with recent-onset diabetes are now underway. The mechanism by which either of these therapies may delay b-cell destruction in humans at this time is poorly understood, with initial evidence pointing to enhancement of regulatory T cell responses.88,89 A major goal will be the long-term (measured in years), safe prevention of b-cell loss. It is likely that none of the agents in the current trials by themselves will provide long-term protection for most patients if given as a single course. Thus, there remains a need for both the development of novel therapeutic approaches and trials of combined therapies.90,91 Acknowledgments The authors work reported herein was supported by grants from the Brehm coalition and the Childrens Diabetes Foundation. LZ is supported by an ADA postdoctoral fellowship (7-06-MN-17). Disclosure GSE has consulted for Bayhill Therapeutics, Genetech, Glaxo Smith Kline, and Eli Lilly. This manuscript has not been published or submitted for publication elsewhere. References
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