Springer Semin Immunopathol (2002) 24:273–295 DOI 10.1007/s00281-002-0113-z © Springer-Verlag 2002

O R I G I N A L PA P E R

Are we ready to predict and prevent endocrine/organ specific autoimmune diseases? Jochen Seissler, Werner A. Scherbaum German Diabetes Center at the Heinrich-Heine University, Auf’m Hennekamp 65, 40225 Duesseldorf, Germany

Introduction Endocrine autoimmune-mediated disorders (EADs) are characterised by an infiltration of the target organ with mononuclear cells such as macrophages, dendritic cells and lymphocytes. According to the predominant pathogenic mechanisms that contribute to the disease development, EADs can be roughly divided into cell- and humoral-mediated diseases. Cell-mediated disorders include among others, Hashimotos’s thyroiditis (HT), autoimune Addison’s disease (AD) and type 1 (insulin-dependent) diabetes (T1D) [42, 127, 145]. For each of these disorders, cellular immunity is associated with progressive tissue destruction, either via direct cytotoxicity by autoreactive CD8+ T cells or by indirect cytotoxic mechanisms through several components of the infiltrating cells (proinflammatory cytokines IL-1β, IFN-γ, TNF-α, free radicals) [96, 115]. In autoimmune diabetes both CD4+ and CD8+ T cells specific to β-cell antigens were sufficient to transfer diabetes in the non-obese diabetic (NOD) mouse model [40, 165], suggesting that autoantibodies play a minor role in the pathogenesis of βcell destruction. Indeed, for human T1D we observed the case of a boy who developed autoreactive T cells and T1D despite the presence of severe hereditary B lymphocyte deficiency [97]. In contrast to T1D, in Graves’disease (GD) autoantibodies can induce pathology by stimulating the function of the thyroid-stimulating hormone (TSH) receptor resulting in diffuse thyroid hyperplasia and hyperthyroidism [98]. Epidemiological studies have shown that genetic as well as environmental factors trigger the development of autoimmune diseases. It is believed that various susceptibility genes are necessary for the induction and potentiation of the autoimmune process, but the genetic susceptibility alone would not be sufficient for disease expression. The 30–50% concordance rate in monozygotic twins [92] documents that various environmental factors, such as virus infections and several dietary components, may have a major influence on the appearance and quality of the autoimmune re-

Correspondence to: J. Seissler

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Fig. 1. Schematic illustration of different states during development of autoimmune diabetes and possible intervention strategies. Pancreas sections demonstrate Langerhans’ islets from NOD mice. By combination of genetic and environmental factors an autoimmune response is initiated, which is characterised by migration of mononuclear cells around the islet (periinsulitis). In this phase the autoimmune response is relatively benign (dominated by Th2 cells) indicated by low-rate β-cell destruction. When further trigger factors repeatedly activate the immune system, the tolerance against other autoantigens is lost and the Tcells convert from Th2 to proinflammatory Th1 cells, which are associated with progressive, complete destruction of the β-cells and development of diabetes

sponse [7]. The repeated activation of the immune system by environmental factors may play an important role in the sequential loss of tolerance, which finally results in the progression to overt disease (Fig. 1). However, it is still unclear to what extent each factor – the genetic susceptibility, the environment and the regulation of the immune response itself – contributes to the irreversible loss of tolerance and the subsequent progressive complete destruction of endocrine glands. Several prospective studies have shown that the autoimmune process precedes the onset of autoimmune endocrine disorders by many months or years. In the past 10 years, a number of organ-specific autoantigens have been cloned and characterised. The new insights into the pathogenesis and the natural course of the EADs results in the improvement of current strategies to detect subjects at risk in an early preclinical state. In addition, novel experimental immunotherapies were developed to prevent disease manifestation, some of which have already been tested in clinical phase I/II trials. Autoantigens in endocrine autoimmune disorders EADs are characterised by the appearance of various cytoplasmic organ-specific autoantibodies. These antibodies were first demonstrated by indirect immunofluorescence tests on cryostat sections of human thyroid, adrenal gland or pancreas [24] and were the major tools for the identification and cloning of recombinant autoantigens. It is intriguing that target antigens of EADs often involve proteins essential for hormone synthesis. In autoimmune thyroid diseases (ATDs) these include thyroglobulin (TG), thyroid peroxidase (TPO) and the TSH receptor (TSH-R). TPO is a common antigen in both HT (95%) and GD (80–90%) [42, 101]. Autoantibodies to the TSH-R

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are a characteristic feature of GD and have been described in up to 99% of newly diagnosed patients using a second generation assay [37, 124]. In autoimmune AD autoantibodies are directed against three components of the steroidogenic P450 enzymes, 21-hydroxylase (210H), 17α-hydroxylase (17α-OH) and the side chain cleavage enzyme (SCC). Among patients with AD 64–86% were found positive for 21OH antibodies [16, 32, 53]. Some of the patients also have antibodies to 17α-OH and SCC, but these antibodies are more frequent in sera from patients with autoimmune polyendocrine syndrome type I (APS I) and type II (APS II) [16, 44, 135]. Among the increasing number of autoantigens described in T1D three major islet autoantigens have been shown to be most relevant for the diagnosis and prediction. These include insulin, the enzyme glutamic acid decarboxylase (GAD), and the tyrosine phosphatase-like protein IA-2. Other candidate autoantigens such as glycolipids, GLIMA 38, imogen 38, ICA69, carboxypeptidase H and heat shock proteins (HSP) are either not specific (ICA69, carboxypeptidase H, HSP), provide controversial results due to the lack of assay standardisation (imogen 38, glycolipids) or can not be used for large scale antibody detection because of their unknown identity (glycolipids, GLIMA 38) [93, 127]. Thus far, insulin is the only known autoantigen which is exclusively expressed in the pancreatic β-cells. Insulin autoantibodies (IAA) occur with greater frequency and at higher levels in young newly diagnosed patients below 5 years (60–80%) as compared to patients above 15 years of age (20–30%) [159, 170]. GAD, which catalyses the formation of the inhibitory neurotransmitter γ-amino-butyric acid (IGABA), is expressed in two distinct isoforms with molecular masses of 65 and 67 kDa in pancreatic islets and neurons [11]. Autoantibodies directed against GAD65 (GADA) are found in 70–80% of patients with T1D. In contrast, GAD67 is only recognised by a minority (10–20%) of sera from diabetic patients [62, 131]. Autoimmunity to GAD67 seems to be more closely associated with polyendocrine autoimmunity or stiff man syndrome [100, 132]. By immunoscreening of islet cell cDNA libraries two members of the tyrosine phosphatase-like proteins, termed IA-2 and the highly homologous molecule IA-2β, were identified [66, 89, 114]. Both antigens are transmembrane proteins localised in the insulin-secretory granula in islet cells and in neurosecretory vesicles in the brain [142]. Autoantibodies to IA-2 (IA-2A) were detected in 30–70% of patients with T1D of whom a subpopulation also had antibodies to IA-2β. Similar to IAA, IA-2A are more frequently observed in young patients (60–70%) than in adults (30–40%) [134, 167]. In contrast to the large numbers of studies on autoantibody reactivities in EADs, little information is available with regard to human antigen-specific T lymphocyte reactivities. Experimental studies in the NOD mouse model clearly demonstrate that T cells are directly involved in the destruction of the β-cells. There is evidence for diabetogenic CD4+ and CD8+ T cells, which recognise insulin and peptides consisting of amino acids 9–23 and 15–23 of the B chain, respectively [41, 166]. In addition, GAD and IA-2 T cell reactivities were described which are able to transfer autoimmune diabetes [75, 77, 149]. It is proposed that the cellular immunity is directly involved in tissue destruction. Therefore, it would be of great interest to measure the number and the activity of autoantigen-specific, human T cells for prediction of T1D, AD and HT, respectively. In diabetic and prediabetic subjects autoreactive human T-cells with reactivity either to insulin, GAD65 or IA-2 have been described [5, 8, 52, 67, 94, 95]. Numerous studies

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have shown that T cell immunity directed to TPO can be detected in HT and GD [6, 26, 76, 109]. Direct evidence for T cell-mediated destruction in AD is still missing. Data from T cell workshops suggest that there are problems with the purity of the proteins (e.g. endotoxins), the standardisation of the assays and the discrimination between pathogenic T cells and the normal T cell repertoire due to the low frequency of specific T cells in the peripherial blood [112, 120]. Recently, promising results were reported on insulin B peptide 9-23- and GAD peptide-reactive human T cells using the tetramer assays technology [117]. However, the practical and technical problems of the currently available T cell assays for the measurement of the cellular arm of the immune responses has led to the conclusion that T cell assays are as yet not useful for prediction of T1D. Although standardisation workshops on the detection of T cell reactivities against thyroid and adrenal specific antigens are lacking, it is assumed that there may be the same problems as reported from studies in T1D. Genetics in endocrine autoimmunity There is significant familial clustering of EADs in the Caucasian population. Large scale, genome-wide scans conducted by several groups provided evidence that autoimmune endocrine diseases are polygenic disorders [154]. A number of susceptibility loci have been identified that can confer increased genetic risk or protection from disease development. However, the strongest genetic associations by far are linked with the human leukocyte antigen (HLA) class Il region. Other susceptibility loci provide only a modest contribution. The prevalence of T1D is 5–10% in siblings and 30–50% in monozygotic twins as compared to 0.1–0.7% in the general population. Risk for T1D is associated with HLA-DR3 or -DR4 antigens but the susceptibility is mostly conferred by alleles of the HLA-DQ locus in the class II region. The HLA-DQA and DQB loci, encoding variants of two heterodimers (α and β chain) involved in peptide presentation on antigen-presenting cells to CD4+ T cells, were found to be most strongly associated with T1D. In Caucasians, DQA1*0301, DQB1*0302 and DQA1*0501, DQB1*0201 alleles have the strongest association with diabetes, whereas the DQB1*0602 haplotype confers dominant protection from the development of T1D [138, 152, 153, 198] (Table 1). HT is related with DQA1*0301, DQB1*0301, while GD is associated with DQA1*0501 and DQA1*0201 is protective. In AD susceptibility is conferred by DQA1*0501-DQB1*0201 and DQA1*0301-DQB1*0302. The DQA1*0101DQB1*0501 haplotype has been shown to be protective for AD [10, 33, 70, 104, 139, 169]. Since HLA genes provide about 40–50% of the inheritable risk for EADs, Table 1. Association between autoimmune endocrine diseases and HLA DQ haplotypes in Caucasians Disorder

Positive HLA association

Negative HLA assoeiation

Addison’s disease

DQA1*0501, DQB1*0201 DQA1*0301, DQB1*0302 DQA1*0301, DQB1*0301 DQA1*0501, DQB1*0201 DQA1*0501, DQB1*0201 DQA1*0301, DQB1*0302

DQA1*0101, DQB1*0501

Hashimoto’s thyroiditis Grave’s disease Type 1 diabetes

DQA1*0201 DQB1*0602

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genotyping may provide useful information on whether or not there is a genetic background for the development of a given disease, but it will not exceed the predictive strength observed in identical twins. However, genetic screening will be able to improve our ability to predict disease risk and contribute to the design of primary or secondary intervention trials. Predictive factors The rationale to predict the development of EADs is to identify markers that allow accurate selection of high-risk groups of subjects who may take benefit from primary or secondary intervention. The current strategy to identify these subjects is either screening for markers indicating autoimmunity or assessment of genetic predisposition. Further improvement of risk estimation may be reached by the combination of both markers (autoantibodies and gentic markers) and/or the addition of a metabolic test to assess early organ dysfunction. Autoantibodies Several prospective studies in first-degree relatives of patients with EADs have shown that organ-specific autoantibodies can be detected many months or even years before the onset of disease and are indicative for a highly increased risk for future development of endocrine disorders [13, 22, 119, 126]. Nowadays, antibody screening with the time-consuming indirect immunofluorescence test is replaced by the combined detection of GADA and IA-2A or GADA and IAA (T1D), 21OH antibodies (AD) or TPO and TSH-R antibodies (HT, GD) using highly sensitive and specific radioligand assays, which fulfill all criteria to perform population-based screening programmes on a large scale [37, 49, 53, 133, 167, 168]. HLA genotyping The strong linkage of autoimmune endocrine disorders with HLA DR and DQ alleles makes it possible to use HLA genotyping as a first line marker to distinguish between subjects carrying risk alleles and those individuals who are at low risk for endocrine autoimmunity. Due to the high frequency of genetic risk markers in the normal population, the sensitivity and positive predictive value of genotyping is low. However, in populations with high disease incidence the first line genetic screening early in life may have the advantage of excluding low-risk subjects from further follow-up investigations. In addition, children with a high genetic risk can be followedup at regular intervals to analyse the natural course of autoimmunity. Early metabolic markers for latent organ dysfunction In a given individual the detection of immunological and genetic markers provides a statistical risk for disease development within a time period. If immunological and genetic markers indicate a high risk, it will be important to estimate whether or not it is

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likely that there may be a rapid progression to overt disease by the additional analysis of the hormonal state. Latent dysfunction of the thyroid may be easily detected by the measurement of basal TSH in combination with free T4. In patients at risk for AD, cortisol response to ACTH (250 µg i.v.) can be measured to assess normal adrenal function defined by at least 10 µg/100ml increase of cortisol above the baseline or a maximum level exceeding 18 µg/100ml. Since fasting blood glucose levels or an oral glucose tolerance test (OGTT) provides insufficient information, a rather complex functional test, the intravenous GTT (ivGTT), has to be performed in prediabetes. The first phase insulin response (FPIR) defined by the sum of the 1- and 3-min insulin levels after glucose infusion can serve as indirect marker for the residual β-cell mass [160]. It is important to note that the ivGTT has several limitations. The FPIR glucose infusion varies according to age, body weight and pubertal status [3, 141]. In addition, a wide intra-subject variation after i.v. glucose infusion has been reported, which makes it difficult to compare follow-up investigations and results obtained in different studies [17]. However, when antibody-positive individuals lose the FPIR, it is unlikely that the β-cell function may ever recover. Among other independent risk factors, the loss of FPIR is most strongly associated with rapid progression to diabetes [19, 161]. A decreased FPIR, however, is also the first metabolic sign of type 2 diabetes, so that this test is only valid for the prediction of T1D when one of the above-mentioned antibody tests is positive. Current strategies for prediction There are currently two approaches for the identification of subjects at risk for EADs. First, the screening for autoantibodies and subsequent analysis of genetic markers or metabolic testing. Second, the primary screening for genetic predisposition markers followed by autoantibody determination. It is only in selected groups of patients, e.g. patients with one organ-specific autoimmune disease who are at high risk to develop a second or third EAD (5–30%), that it can be justified to screen for predictive markers to make an early diagnosis. In other subjects prediction should only be performed in the context of studies aiming at the improvement of screening procedures or the participation in a prevention trial. It is important to note that the predictive value of all screening markers is about five- to tenfold higher in high-risk populations (family members) as compared to subjects with low disease prevalence (general population). Rational screening programmes should also bear in mind the incidence of disorders and the age distribution of the clinical manifestations. GD and HT are most typically diseases of adult women in the 30–60 year age group. Onset of AD is also usually seen in middle life (peak 30 years of age) with a predominance of the female sex. Thus, first screening for these disorders may be performed in young adults. This is in contrast to TID which peaks in childhood, but also occurs in about 10% of adult diabetic patients. Relatives of patients with an endocrine autoimmune disease Prediction in childhood During the last decade the prediction of EADs has been considerably improved by the detection of autoantibodies to recombinant antigens. Comparison of different

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Table 2. Prevalence of autoantibodies in first degree relatives of patients with type 1 diabetes, autoimmune thyroid disease and Addison’s disease and in subjects without a family history of an endocrine autoimmune disorders Prevalence of autoantibodies First degree relatives

General population

2.5–3.7% 4.6–7.0% 2.1–5.3% 10.7–12.5% 2.0–6.2%

1.5–3.9% 0.3–3.0% 0.1–2.4% 1.0–9.4% 0.1–0.8%

16–27% 2.1–3.8%

3–20% 0.4–1%

Unknown

0.1–0.6%

Diabetes IAA GAD-Ab IA-2 Ab ≥ 1 antibody ≥2 antibody Thyroid disease TPO Ab TSH-R Ab Addison’s disease 21OH Ab

EADs revealed that antibodies are frequently observed in first degree relatives. Among siblings and offsprings of patients with autoimmune thyroid disorders 16–27% had TPO antibodies and 2.1–3.8% were found positive for TSH-R antibodies [28, 34, 129]. The appearance of anti-thyroid antibodies in children and adolescents was shown to indicate a moderately increased risk (5–6%) for progression to overt thyroid dysfunction during a period of 4–5 years [28, 99]. This is in contrast to diabetes-specific autoantibodies which are more strongly associated with disease development. Several prospective studies demonstrate that the risk for T1D is not associated with a particular antibody specificity, but strongly depends on the number of positive antibodies directed to distinct autoantigens. Screening in young first-degree relatives revealed the presence of at least one antibody specificity in 10–13% and multiple autoantibodies in 2–6%, respectively (Table 2). GADA were most frequently detected, followed by IAA and IA-2A. [20, 36, 49, 84, 111, 161, 171]. When a first degree relative was antibody negative or positive for only one antibody (ICA, GADA, IAA or IA-2A) the risk for the future development of T1D was extremely low (0–1%). However, when two or more antibodies against recombinant antigens were present the 5- to 10-year risk varies between 62–100% (Table 3). Data from the Diabetes Autoimmunity Study in Young (DAISY) and the Finnish Diabetes Prediction and Prevention (DIPP) Study show that screening based on HLA genotyping can also be used as first line marker to assess risk for T1D. By typing of HLA DQ genes at birth up to 75% of children who developed diabetes before the age of 5 years can be identified [87, 118]. To answer the question concerning the age at which screening should be performed, three studies were designed to characterise the development and natural course of β-cell autoimmunity, starting at birth – the German BABY-DIAB, the DAISY Study and the DIPP Study. These ongoing studies reported on the appearance of diabetes-specific autoantibodies in high frequencies among offsprings of parents with T1D early in life [87, 118, 171]. In the BABY-DIAB study the sequential appearance of IAA, ICA, GADA and IA-2A was studied in 1,353 offsprings from birth

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Table 3. Diagnostic sensitivity for the development of type 1 diabetes among antibody positive first degree relatives during a follow-up of 5–10 years Antibody

GAD65 IA-2 Insulin 2 antibodies 3 antibodies

Sensitivity

64–90% 3l–64% 24–76% 54–81% 52–61%

Positive predictive value First degree relatives

General population

42–52% 55–81% 29–59% 62–100% 64–100%

3–21% 5–19% 4–9% 10–25% 46–80%

up to the 8th year of life. At the age of 2 years 11% of children from diabetic parents had one of these antibodies and 3.5% of the children had multiple antibodies. IAA were most frequently detected (5.7%), followed by GADA (2.0%), IA-2A (1.6%) and ICA (1.2%). The sequential appearance of individual antibodies was inconsistent. Some children had autoantibodies to a single antigen (most frequently IAA) followed by the development of one or two additional antibodies during follow-up. Other individuals developed multiple antibodies simultaneously [171]. In the DAISY study about 6% of the offspring developed one or more autoantibodies within the first 5 years after birth. The DIPP Study analysed the development of autoimmunity in subjects carrying a genetic predisposition. Within the first 2 years 3.3% of the children with high-risk genotype became autoantibody positive as compared to only 1.6% in the moderate-risk group [79]. These studies conclusively demonstrate that the children often develop an immune response against two or three antigens before the development of diabetes. Children with two or more autoantibodies had a cumulative risk of 50% to develop T1D by the age of 5 years [171] and a 76.5% risk within the first 10 years of life, respectively, whereas children with only one antibody before the age of 6 years remained diabetes free [79]. The early development of autoantibodies and diabetes in childhood suggests that the first screening should be performed at the age of 2 years. However, the time schedule for repeating antibody testing is as yet unclear. Since several studies described seroconversion to one or multiple autoantibodies in older children and adolescents, a repeat of antibody screening at the ages of 5 and 10 years may be appropriate [23, 36, 84, 133]. Prediction in adults It is a common finding in EADs that children with positive antibodies are at higher risk for progressing to disease than adults with the same risk marker profile. In T1D there is evidence that patients who developed the disease in adulthood tend to have higher residual β-cell function at diagnosis compared with young children. A slow rate of β-cell destruction is also suggested in some patients who present with clinical features of type 2 diabetes at diagnosis, but develop absolute insulin deficiency within a few years after the onset of the disease. Because these patients are often positive for both ICA and GADA, the disease has been classified as a slowly progressing form of T1D called latent autoimmune diabetes in adults (LADA), type 1 1/2 diabetes, or slowly progressive type 1 diabetes [60, 72, 107, 134, 172]. This was supported

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by the demonstration of typical insulitis in the pancreas biopsy from a patient with LADA [140]. According to the data from the United Kingdom Prospective Diabetes Study (UKPDS) LADA diabetes, defined by the presence of ICA and/or GADA, applies to more than 35% of individuals with newly diagnosed “type 2 diabetes” aged 25–34 years, 16% of patients in the 35–45 age group and 9% of patients with diabetes onset above 55 years of age. The majority of patients with either ICA (94%) or GADA (84%) required insulin therapy after a follow-up of 6 years, whereas only few patients (14%) became insulin deficient when the antibodies were negative [157]. Preliminary data suggest that LADA patients may benefit from primary insulin therapy with respect to preserving the endogenous C-peptide secretion and improving metabolic control [81]. Women with gestational diabetes represent another subset of patients with an increased risk for T1D. In these women diabetes usually disappears after delivery. However, the risk to develop diabetes later in life is between 3% and 5% per year; 90% of these women develop type 2 diabetes and about 10% T1D. The risk for T1D can be assessed by the detection of autoantibodies (ICA, GADA, IA-2A) around delivery. When two autoantibodies are positive, the risk for the future development of T1D within 2 years is more than 60%. In the presence of ICA, GADA and IA-2A the 2-year risk for T1D increases to 84% [55]. Prediction in the general population Only about 10–15% of patients with EADs have a positive family history of the disease [39]. This suggests that screening strategies have to include the general population to reach the aim of an effective, early identification of the majority of affected individuals. The problem is that all screening tests in populations with low prevalence of the disease have a low positive predictive value. To overcome the problems primary screening should include multiple antibodies or be combined with a second independent test such as genetic screening. Prospective studies in unaffected schoolchildren have shown that ICA, IAA, GADA and IA-2A indicate ongoing β-cell autoimmunity [20, 21, 61, 80, 90, 122, 130, 144]. The cumulative risk for children to develop diabetes within 5–10 years was 3–21%, 10–25%, and 46–80% in the presence of one, two or three positive autoantibodies (Table 3) [20, 80, 86, 90, 121]. It is important to note that there is a large variability between different studies. This is explained by the low prevalence of autoantibodies in the general population so that only a limited number of prediabetic children (n=3–10) were available to calculate the predictive values of single and combined antibody positivity. Nevertheless, these screening programmes in the general population suggest that estimation of the diabetes risk can be performed in a way similar to that established in family studies. First-line screening can be performed with a combination of two autoantibodies (e.g. GADA and IA-2A or GADA and IAA) followed by the additional detection of IAA or IA-2A or HLA genotyping in antibody-positive subjects. This sequential strategy may be very efficient to assess the risk for T1D in relatives and subjects without family history of diabetes. Anti-thyroid antibodies are common in the general population, are more frequently observed in females and are positively associated with age. Moderately elevated TPO antibodies were detected in 3–5% of healthy children and in 14–20% of subjects aged above 80 years [28, 69]. In healthy subjects with positive thyroid autoanti-

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bodies, the progression to latent or overt hypothyroidism is often very slow. In a number of population studies a positive TPO antibody test was found in about 10% of middle-aged individuals [58, 155] of whom 2–3% had overt thyroid disease and 5–7% had raised TSH levels. Men had a five- to tenfold lower incidence of thyroid abnormalities. In the Wickham cohort only 27% of women with anti-thyroid antibodies developed overt hypothyroidism at the end of a 20-year-follow-up period, corresponding to a rate of progression of 2.1% per year [156, 158]. Risk of progression was increased in antibody-positive men, in subjects with high antibody levels and in older individuals (>45 years). Thyroid antibodies were also shown to be predictive in pregnant women. Postpartum thyroiditis occurs in 35–85% of TPO antibody-positive women as compared to 4–8% pregnant women overall [91, 102]. Due to the low incidence of AD, population-based studies are lacking. Several studies screened patients suffering from T1D or thyroid autoimmunity who were at increased risk to develop AD. Progression to overt AD was assocated with young age, and in one study with high antibody titer [15, 25]. Betterle et al. [14, 15] reported on the development of overt AD in nine of ten 21OH antibody-positive children and the remaining child developed latent adrenal insufficiency within a follow up of 3–121 months. In contrast, the presence of 21OH antibodies in adults indicates only a moderate risk for the development of AD. Within a mean follow-up of 4.2 years 32% of antibody-positive adults progressed to AD. This suggests that the detection of 21OH antibodies provides important information in children but is of only limited value in older individuals, supporting the hypothesis that the autoimmune response against endocrine glands is more aggressive in childhood than in adults. Combinations of risk markers Large population studies in family members and the general population have shown that immunological, genetic and metabolic tests are independent risk markers for EADs. Therefore, it was not surprising that the combination of these markers significantly improved prediction. This is particularly important in low-risk populations where a single test has only a limited predictive value. In a family study in France risk for T1D within 8 years was found to be 3–4% in siblings with either DR3 or DR4, 16% in childen with DR3/DR4, 56% when ICA were positive and 70% for those with ICA and HLA DR3/DR4 haplotype [47]. Similarly, 21OH antibody-positive subjects carrying a high genetic risk are at higher risk for progression to AD than subjects with intermediate or low genetic risk [169]. The additional detection of genetic markers in antibody-positive relatives can also serve as a useful tool for discriminating between subjects at high and intermediate risk from low-risk individuals [79, 85, 113]. In the Diabetes Prevention Trial 1 (DPT- 1), 100 of 1,376 (7.3%) ICA-positive relatives were found to have the protective haplotype HLA DQA1*0102/DQB1*0602 [59]. Although these subjects frequently have only one positive antibody, genotyping may provide information which would make it possible to exclude antibody-positive subjects carrying protective HLA haplotypes from intervention studies. Immunological and genetic markers can also be combined with the ivGTT to identify prediabetic children at risk for rapid progression to diabetes. ICA-positive relatives with a FPIR below the first percentile of normal controls have a 85–87% risk of developing T1D within 3–5 years compared to 48% when the FPIR is be-

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tween 50 and 100, and a 17% risk in those with FPIR >100 mU/ml [19, 31]. In patients with positive TPO antibodies, the measurement of TSH improves risk estimation. In the Wickham population study, there was a significantly higher proportion of subjects progressing to overt hypothyroidism when the basal TSH levels were increased. Subjects with elevated TSH and positive thyroid antibodies developed hypothyroidism in 33% and 27%, respectively. When both markers were present the rate of progression was 4.3% per year, resulting in 55% hypothyroid patients within 20 years [158]. Prevention of endocrine autoimmune disorders The ultimative aim of the current research in EADs is the development of a therapeutic strategy for the effective and safe prevention of disease development. Therapies can be divided into antigen-specific and unspecific therapies and between studies evaluating effects in patients and those including preclinical state. A general rule may be that intervention therapies will be more effective if given early, while many endocrine cells are still intact. Many attempts have been made to prevent T1D in predisposed individuals, but controlled studies in ATD or AD are lacking. Although about 80–90% of endocrine cells may be destroyed at manifestation of the disease [56], studies with newly diagnosed patients were performed with the objective of preserving the residual β-cell function. These studies have the advantage of recruiting only those subjects who have developed the disease, excluding the possibility of treating with a potentially harmful substance subjects who will perhaps never develop the disease. Experimental studies in animals There is a large body of evidence that the modulation of the immune systems can delay and prevent the development of spontaneous autoimmune diabetes in the NOD mouse and in BioBreeding (BB) rats (Table 4) [9, 38, 150]. Examples of unspecific immunosuppression include therapies with classical immunosuppressive drugs [cyclosporin A (CyA), tacrolimus, sirolimus, glucocorticoids], treatment with cytokines (IL-4, IL-10, IL-13) and monoclonal antibodies directed to T cell molecules (antiCD3, anti-CD4, anti-IL-2 receptor) [9, 150] (Table 5). These agents have the disadvantage of producing often prolonged suppression of the immune systems, which is associated with an increased risk for severe infections and malignancies. The objective of further studies is to develop a protocol that induces organ-specific tolerance after short-term treatment, and thus reduce the potential severe side effects. The aim of antigen-specific immunotherapies is to use a defined autoantigen or peptide for the specific suppression of autoreactive T cells. It has been shown that different ways of administration (intravenous, subcutaneous, oral, intranasal) of fulllength insulin or GAD can induce a state of anergy or can activate regulatory T cells which can prevent both insulitis and diabetes [75, 63, 71, 147, 148, 151, 173]. In addition, the same effects were observed when the mice were vaccinated with specific insulin, GAD or Hsp60 peptides [1 12, 41, 50]. It was also a general finding that all approaches that convert the cellular immunity from the predominance of proinflammatory autoreactive T helper 1 (Th1) cells to

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Table 4. Experimental therapies for the protection of autoimmune diabetes in non-obese diabetic (NOD) mice and/or BioBreeding (BB) rats Therapy

Animal

Effects

Immunosuppression Immunosuppressives Anti-CD4, anti-CD3

NOD and BB NOD

Unspecific immunosuppression Unspecific immunosuppression, long-term remission at onset

NOD and BB NOD and BB NOD

Unspecific immunostimulation, Thl/Th2 shift Th1 /Th2 shift Th1/Th2 shift

NOD and BB

Reduces antigen-specific autoreactivity, Th1/Th2 shift

Oral, nasal Insulin, GAD Insulin

NOD BB

Activation of T regulatory cells, Th1/Th2 shift No effect or acceleration of diabetes by Th1 cell activation

Diets Gluten-free, casein hydrolyasate

NOD and BB

Modulation of gut cytokine balance, prevention of immune dysregulation

Radical scavenging Nicotinamide

NOD

Scavenging of free radicals

Immune modulation BCG, Freud’s adjuvant cytokines (IL-4, IL-10, IL-13) Anti-IL-2, anti-IFN-γ Vaccination parenteral Insulin, GAD65, HSP60

Th2 cells were associated with protection from diabetes, while infiltration of Langerhans’ islets was still present [9, 82]. Similar observations were made in animal models on other Th1-mediated autoimmune diseases, e.g. multiple sclerosis and rheumatoid arthritis [46, 106]. Although these results have important implications for immunoprevention in humans, it must be kept in mind that even in the inbred NOD mouse studies have to be performed under stringent experimental conditions to maintain a high incidence of diabetes. Therefore, it remains to be shown whether these protocols can be transferred to humans and, most importantly, whether they can safely be administered to healthy children at risk for T1D. Clinical trials in newly diagnosed patients with type 1 diabetes The rationale to treat patients with new-onset diabetes is to prolong the time in which insulin can be secreted by β-cells and test the potential efficiency of a given immunotherapy before it is administered to healthy prediabetic individuals. Patients with new-onset diabetes would benefit from these treatments, which would simplify blood glucose control, lower glycosylated hemoglobin levels, and reduce the risk of diabetes-specific complications. This may, therefore, be of special interest for patients with slow β-cell destruction, such as LADA patients. The first controlled trials in patients with T1D were performed using antigen-nonspecific immunosuppressive treatments, which were effective in the NOD mouse (Table 5). The administration of CyA demonstrates that the continuous destruction of β-cells before and after manifestation of the disease can be delayed by immunosuppression [27]. However, after withdrawal of immunosuppression, progressive β-cell

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Table 5. Clinical trials in pre-type 1 diabetes Therapy

Results

Primary intervention Exclusion of cow milk proteins

Decrease of autoantibodies (results expected in 2005)

Secondary intervention Nicotinamide DENIS ENDIT

No effect Results expected in 2003

Insulin Parenteral (DPT-1) Oral Nasal

No effect Recruitment phase Recruitment phase

destruction reappeared resulting in complete insulin deficiency as observed in untreated patients [30, 143]. Even when CyA was continued for 3 years, the patients became insulin dependent, suggesting that the β-cell mass after onset of diabetes is insufficient for long-term glucose control. As CyA is nephrotoxic, the administration of CyA alone in T1D or prediabetic subjects is not recommended. These and other clinical trials were also important because it was observed that intensive insulin treatment from the onset of the disease effectively preserves endogenous insulin secretion in about 20%, and insulin independency in 10% of patients, respectively, through the first year after diagnosis [136, 146]. The effect of insulin therapy has been explained by an inactive state of β-cells, which results in both a reduced expression of autoantigens and an increase in repair mechanisms, and a modulation of the autoimmune response against insulin. Recently, a clinical trial was performed using humanized monoclonal antibodies directed against the CD3 molecule of the T cell receptor. Treatment with these antibodies for 14 days was well tolerated (no severe cytokine-release syndromes) and induced an increased preservation of C-peptide secretion [68]. The effect of novel immunosuppressive regimens developed in patients receiving β-cell transplants (anti-IL-2 monoclonal antibodies + rapamycin + low dose FK506; myophenolate mofetil) remains to be tested in patients with new-onset diabetes [137]. A different approach tested in humans is to stimulate the immune system by an unspecific agent, such as bacillus Calmette-Guérin (BCG). The aim is to modulate the disturbed immune system in such a way that the balance between Th1 and Th2 cells is recovered. While some studies reported on some benefit, others observed no difference from placebo vaccines [4]. A series of novel antigen- and peptide-based immunotherapies for the prevention and therapy of autoimmunity are being studied in patients at the onset of diabetes. In one trial, vaccination with the GAD65 molecule will be evaluated in patients with LADA. Similarly, HSP60 peptide 277 (p277) is being studied in patients with T1D and LADA. Preliminary results in T1D patients suggest a significantly higher C-peptide secretion 10 months after three vaccinations with 1 mg peptide p277 as compared to placebo-treated patients [116].

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Trials in pre-type 1 diabetes Unspecific immunosuppression used in patients with T1D or after transplantation clearly has severe side effects that are not acceptable for the treatment of subjects who carry only a statistical risk for disease development within a given period. To treat these healthy individuals a safe and effective therapy is needed where the autoimmune reaction is down-regulated in an antigen-specific way without severe disturbance of the normal immune response. Three distinct approaches are currently being explored in large trials. First, in an attempt to exclude trigger factors for autoimmunity a Finnish group is analysing the effect of breast feeding versus supplemental feeding with and without bovine milk proteins. Second, two trials are using treatment with nicotinamide in children and adults with positive diabetes-specific autoantibodies. Third, vaccination with insulin is being tested using parenteral, oral or nasal administration (Table 5). Primary intervention Exclusion of cow milk in infants Several studies reported an increased risk for T1D in infants after early introduction of supplemental feeding as compared to breast feeding. Although the overall associations with infant feeding are weak (odds ratio about 1.5) [57, 162], cow milk proteins are considered as one factor that can trigger the appearance of β-cell autoimmunity in genetically predisposed children [163]. Protection from diabetes in breast-fed children could be explained by protection from infections and/or a lower weight associated with decreased insulin secretion and autoantigen expression. Alternatively, cow milk proteins are thought to induce cross-reaction with β-cell antigens by molecular mimickry and trigger humoral and cellular immunity against bovine insulin [74, 110]. This was the rationale for designing a large study in which infants carrying a HLA-risk haplotype are fed with hydrolyzed casein-based or cow milk formula were investigated [79]. Preliminary results suggest that early feeding with cow milk formula is associated with an increase in the appearance of β-cellspecific autoantibodies and the induction of humoral and cellular immunity towards bovine insulin [110]. Secondary intervention Nicotinamide Nicotinamide is a free radical scavenger and inhibits poly (ADP-ribose) synthetase. It has been shown to delay diabetes in NOD mice, inhibits β-cell damage after exposure to diabetogenic cytokines and protects β-cells after treatment with the diabetogenic agents alloxan or streptozocin [83]. After promising results were reported from pilot trials [29, 51], the effect of nicotinamide was tested in two double-blinded, randomized, and placebo-controlled trials, the Deutsche Nicotinamide Intervention Study (DENIS) and the European Nicotinamide Diabetes Intervention Trial (ENDIT). Thus far, data are available from DENIS, which observed no difference in dia-

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betes development within a period of 3 years in ICA-positive children aged 3–12 years treated with nicotinamide (1.2 g/m2 per day) versus placebo [88]. The ENDIT study also recruited older children and antibody-positive adults, and the final conclusions of this study remain to be seen probably available by 2003. Hormone therapy There are conclusive data from NOD mice that the administration of insulin can delay and prevent diabetes. Insulin is thought to exert a protective effect by reducing the metabolic activity, which allows the β-cells to down-regulate autoantigen expression and up-regulate defense mechanisms against the attack of antigen-presenting cells and autoreactive lymphocytes [123]. Suppression of metabolic activity by hormonal therapy would also be applicable in autoimmune disorders of the thyroid (administration of thyroxine) or adrenal gland (administration of cortisol). In one study of 78 pregnant women with Graves’ disease, it was shown that levothyroxine administration decreases the postpartum recurrence of hyperthyroidism from 32% to 5% during the first years after delivery [64]. Other studies observed no significant effect of levothyroxine on the preservation of the thyroid function in patients with HT or the prevention of postpartum thyroiditis [73, 103, 128]. In AD a single report suggests that glucocorticoid therapy in patients with anti-adrenal autoantibodies leads to loss of autoantibodies and disease prevention [43]. T1D may be an exception in hormone replacement therapy because insulin also represents an autoantigen, suggesting that insulin injections may modulate the autoimmune response. Evidence for the latter hypothesis came from studies in NOD mice where the injection of insulin as well as insulin B peptide 9–23 induced regulatory T cells, which were able to prevent transfer of diabetes by diabetogenic T cells [40, 63, 71]. In humans, pilot studies suggest that twice daily injection of low-dose insulin can delay the development of diabetes in siblings at risk for T1D [78]. Based on these studies, a large multicentre trial, DPT-1, was designed to evaluate intravenous and subcutaneous insulin administration. First-degree relatives aged 4–45 years and second degree relatives aged 4–20 years were included when immunological (ICA>10 JDF units), genetic (not DQA1 0102, DQB1 0602) and metabolic (ivGTT) criteria indicated a predictive 5-year diabetes risk of more than 50%. Among 84,228 screened individuals 339 subjects were randomized to receive either intravenous insulin for 4 days once a year and 0.25 U/kg body weight of subcutaneous ultralente insulin twice daily, or no treatment. After a median follow-up of 3.7 years, it clearly appears that insulin treatment does not delay or prevent T1D [48]. It is important to note that due to the hypoglycemic effects, the dose of daily insulin was considerably lower than the effective dose in NOD mice. Nevertheless, these data also suggest that the effects of insulin are complex and that data obtained from the NOD mouse are difficult to transfer to humans. In a second arm of this trial, the effect of oral tolerance induction by daily oral insulin treatment, is being investigated in subjects with a predicted 25–50% risk for diabetes within 5 years. Results concerning whether or not oral insulin is effective are expected in 2005. In a novel study in Finland the effect of nasal insulin versus placebo is being evaluated in ICA-positive infants (DIPP study) [87].

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Conclusions During the last decade, considerable progress has been made in the identification of autoantigens and the development of strategies to predict EADs. There are consistent findings in T1D, ATDs and AD which show that the appearance of organ-specific autoantibodies indicates an increased risk for future development of the disease. The combination of immunological with genetic and/or metabolic risk markers further improves risk estimation and allows reliable and precise prediction of EADs. In T1D the currently available technique already fulfills the requirements for population screening. The detection of multiple autoantibodies directed to the recombinant antigens insulin, GAD and IA-2 allows sensitive and specific screening for subjects at risk in populations with and without family history of T1D. Since diabetes represents a major medical problem (increasing incidence, high rate of diabetes-specific complications), most intervention studies have focused on the prevention of T1D. Justification for disease prediction comes from pilot studies designed for developing protocols to prevent diabetes. Three large clinical trials are currently in progress including primary intervention by exclusion of cow milk protein in infants, and secondary intervention by treatment with nicotinamide and parenteral or oral administration of insulin. The data from the DPT-1 and the DENIS studies suggest that parenteral insulin, at the dose tested, and nicotinamide do not delay or prevent T1D. Preliminary results from other pilot trials using autoantigen or peptide vaccinations give hope that we can look forward to the day when diabetes can be prevented. Experience with the DPT-1 study, however, emphasizes that the development of autoimmunity and immunoregulation is rather complex in humans and each therapy needs to be tested in large controlled studies. Until the first intervention strategy has shown a definite success, risk assessment and in particular treatment of risk subjects should only be performed within the scope of prospective studies to further improve diabetes prediction or to evaluate novel immunotherapies.

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