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Prevention of Type I Diabetes and Recurrent ß-Cell Destruction of Transplanted Islets¹

Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, Colorado 80262

	Abstract

Top Abstract I. Introduction II. Disease Pathogenesis III. Disease Prediction IV. Prevention of Type... V. Conclusion References

 

I. Introduction

II. Disease Pathogenesis

III. Disease Prediction

A. Genetic prediction

B. Immunological prediction

C. Metabolic prediction

IV. Prevention of Type I Diabetes

A. Trial design

B. Genetic prevention

C. Prevention: preautoimmunity

D. Prevention: postautoimmunity/prediabetes

E. New onset patients/prevention of further ß-cell destruction

F. Pancreas and islet cell transplantation/prevention of recurrent diabetes

G. Islet cell transplantation/prevention of ß-cell destruction

V. Conclusion

	I. Introduction

Top Abstract I. Introduction II. Disease Pathogenesis III. Disease Prediction IV. Prevention of Type... V. Conclusion References

 
PREVENTION of type I diabetes in animal models is a reality (1, 2, 3, 4, 5). Prevention of type I diabetes in man is currently in the realm of ongoing clinical trials (3, 6, 7). The possibility of preventing the disease is stimulating efforts to refine prediction and understand pathogenesis. A better understanding of pathogenesis is likely to be associated with elegant methods for disease prevention. At present, trials of prevention in man are primarily based upon empirical observations.

The development of type I diabetes has been divided into a series of stages (8): 1) genetic susceptibility; 2) "triggering"; 3) active autoimmunity often associated with the presence of autoantibodies; 4) loss of ß-cell function as determined by intravenous glucose tolerance testing; 5) overt diabetes in C peptide-positive individuals; and 6) total or near total ß-cell destruction with insulin dependence. Prevention of the ß-cell destruction that leads to type I diabetes can be considered at each of the major stages. The pathogenic events unique to each stage are a potential target for intervention. For example, if triggering environmental factors were identified, their removal might provide a means of disease prevention. In addition, understanding of the natural history of type I diabetes and the prognosis associated with specific markers of each of the above stages contributes to the design of clinical trials. In this review we will discuss the pathogenesis of type I diabetes, genetic, immunological, and metabolic prediction, and preventive trials.

	II. Disease Pathogenesis

Top Abstract I. Introduction II. Disease Pathogenesis III. Disease Prediction IV. Prevention of Type... V. Conclusion References

 
A large body of data indicates that type I diabetes of man, mouse, and rat is of autoimmune etiology (1, 4, 5, 9, 10, 11) and the major effector cells are T lymphocytes (11, 12, 13, 14, 15). We will utilize the term type I diabetes as synonymous with the autoimmune form of diabetes. The hallmark of type I diabetes is lymphocytic invasion of islets (16, 17, 18). Although there is one report of a lack of insulitis in children with new-onset diabetes (19), Foulis and Clark (20) found obvious insulitis in 47/60 (78%) pancreases in patients with new-onset diabetes (without serial sections), and they estimate that, given the problems of sampling errors, it is "likely that insulitis is a feature of all cases of autoimmune type I diabetes." This lymphocytic infiltration of islets is remarkable in that only islets containing insulin-secreting cells (ß-cells) are infiltrated. Islets containing only glucagon, somatostatin, and pancreatic polypeptide-secreting cells are termed pseudoatrophic islets, and these islets are free of infiltrates. Thus "insulitis" is a remarkably ß-cell-specific process. In addition, in the same section of pancreas one can often find two islets, both containing ß-cells, but only one of which is infiltrated by lymphocytes. The spottiness of this process is reminiscent of the progression of vitiligo, where melanocytes of patches of the skin are progressively destroyed over time.

Although most studies of immune pathogenesis relate to murine models and their exact relevance to human diabetes is unkown, autoimmune diabetes is relatively easy to induce by endogenous or exogenous factors that disrupt immunoregulation. This includes the lymphopenia gene of the BB rat model (21, 22), neonatal radiation of rats (23), anti-RT6 antibodies given to nonlymphopenic BB rats (24), injection of cyclophosphamide into male NOD mice or young BB rats (25, 26), infection of nonlymphopenic BB rats with Kilham rat virus (27), and the autosomal recessive gene of chromosome 21 causing the polyendocrine type I syndrome (with associated mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease) (28). For all of the former except the polyendocrine type I syndrome, genes within the major histocompatibility complex are essential for disease.

The NOD mouse strain, developed by Makino and co-workers (26), has allowed the dissection of many of the immunological events associated with pathogenesis. In particular, more than ten different genetic loci contribute to disease susceptibility (29, 30, 31, 32). Despite identification of loci, the class I and class II genes of the major histocompatibility complex are the only susceptibility genes identified to date (4, 29, 33, 34).

The NOD mouse lacks class II I-E molecules (29), which are the equivalent of human DR, and has a unique sequence of its I-A molecule (termed I-A^g7), which is the homolog of human DQ (35). The manner by which class II molecules contribute to disease susceptibility is currently unknown, but transgenic introduction into NOD mice of its missing I-E or "normal" I-A molecules prevents diabetes (25, 36, 37). I-E replacement also prevents insulitis. These class II molecules most likely influence disease by altering the T cell repertoire or the islet peptides that are presented to T cell receptors. It is easy to envision that the probability of antiislet autoimmunity (given disordered immunoregulation) may depend upon the efficiency of presentation of a specific islet peptide (e.g. a peptide of insulin) to T cells. In addition, if development of type I diabetes depends upon the utilization of a restricted T cell receptor repertoire, class II molecules may alter this repertoire. Studies of the NOD mouse have, to date, failed to find evidence for biased utilization of the ß-chain of the T cell receptor by autoreactive T cells (38, 39, 40). In contrast, we have recently discovered that NOD T cells, which react with an immunodominant peptide of insulin (see below), utilize selected V gene segments (41). Thus, regulation of the -chain repertoire is a candidate mechanism for class II effects on disease susceptibility.

Given genetic susceptibility, a number of environmental factors influence the development of diabetes in the NOD mouse. In particular, diets lacking complex proteins (42, 43, 44, 45), most viral infections, and even a single injection of Freund’s adjuvant or bacillus Calmette-Guerin (BCG) (both made from tubercular organisms) prevents disease (46, 47, 48, 49, 50, 51, 52, 53, 54, 55). Congenital rubella, but not noncongenital infection, is associated with development of type I diabetes (56). Congenital rubella infection is also associated with the development of thyroiditis (57). Congenital rubella infection may alter T cell development (58), thus creating susceptibility to a series of autoimmune disorders. An alternative hypothesis is that the rubella virus mimics an islet autoantigen leading to antiislet autoimmunity (58). Studies of the T cell receptor of T cell infiltrates have raised the possibility that a superantigen may be involved in the pathogenesis of type I diabetes (59). Skewing of T cell receptor utilization can occur after immunization (e.g. experimental autoimmune encephalitis), and it is essential to identify relevant superantigens in assessing the significance of such T cell receptor alterations.

A number of studies have implicated consumption of cows milk by infants (and specifically bovine albumin) in the pathogenesis of type I diabetes (60, 61, 62, 63, 64, 65, 66). Bovine albumin is reported to be homologous to a ubiquitous molecule, expressed by islets, termed islet cell autoantigen 69 (ICA69) (67). The epidemiological data implicating milk has primarily depended upon retrospective questioning of mothers with diabetic offspring. Recent prospective studies utilizing appearance of antiislet autoantibodies as their endpoint have failed to implicate early (first 3 months of life) ingestion of milk proteins in disease pathogenesis (68).

A series of islet proteins are targeted by the immune system, including insulin (69), a neurendocrine enzyme glutamic acid decarboxylase (GAD65 and GAD67) (70, 71, 72), membrane granule proteins with homology to tyrosine phosphatases (termed ICA512 or IA-2) (73, 74, 75, 76), and a related molecule termed phogrin, ICA512ß, or IA-2ß by different discoverers (77, 78). In addition, an islet neurendocrine ganglioside is also a target of autoantibodies (79).

The detection of T cell responses to these islet molecules in man and the NOD mouse, utilizing peripheral blood or splenocytes (NOD mouse), is a developing field (80, 81, 82, 83, 84, 85, 86, 87, 88). For example, several groups have reported T cell responses to GAD or its peptides in the NOD mouse exceeding responses seen in control strains, while other groups have been unable to reproduce these observations. This is understandable in that there are no standardized T cell assays for any autoimmune disease. It is likely that workshops will be carried out (similar to those for autoantibody assays) to resolve controversies in this field. This area is important to the understanding of pathogenesis, as specific T cell responses to islet autoantigens or ubiquitous molecules such as a heat shock proteins (89) have been reported to precede or correlate with disease pathogenesis.

T cell clones have been derived from the NOD mouse that are able to transfer disease (12, 13, 15, 82, 90, 91, 92). These include clones reacting with unknown islet antigens, GAD, and insulin. The clones reacting with insulin are currently best characterized. Wegmann and co-workers derived T cell clones directly from islets of NOD mice utilizing "islets" as the stimulating antigen (to limit in vitro selection). Suprisingly, when such clones were tested for reactivity with known islet molecules, the majority reacted with insulin (15). Of these insulin-reactive clones, more than 95% recognized a single peptide of insulin termed B2 (amino acids 9–23 of the insulin B chain) (92). To date, only CD4-positive clones have been derived, and they all require presentation of peptide by the NOD class II molecule I-Ag7. The clones not only rapidly induce diabetes in young NOD mice but can also destroy human islets when transplanted into immunodeficient NOD mice. The ability of NOD T cell clones to destroy human islets (93), which obviously cannot express mouse class II molecules, indicates that islet expression of class I or class II molecules is not required for T cell-mediated killing. Potential mechanisms for indirect T cell destruction of ß-cells include the production of lymphokines (94) and free radicals (by cells such as macrophages) under the influence of activated T lymphocytes (95, 96, 97, 98). Such molecules may then be the final effectors of ß-cell cytotoxicity. In addition, even though CD4-positive T lymphocyte clones are sufficient to destroy islets, additional effector mechanisms, such as directly cytotoxic CD8 lymphocytes (99), are likely to be involved in pathogenesis (100).

Given the complexity and chronicity of type I diabetes, multiple interventions are able to prevent the disorder in animal models (Table 1). One of the most specific preventive modalities utilizes peptides of insulin. A single injection of the B2 peptide, the whole B chain of insulin, or insulin, prevents diabetes (92, 101). In addition, the B2 peptide can be administered intranasally to prevent disease (101). We speculate that insulin is the primary autoantigen for the NOD mouse and perhaps for type I diabetes in man. Recent studies indicating that NOD mice transgenically producing proinsulin are protected from both diabetes and insulitis are consistent with this hypothesis (101a). If there is a primary autoantigen, and insulin is such an antigen, modulating the immune response to insulin may be particularly effective for disease prevention.

	III. Disease Prediction

Top Abstract I. Introduction II. Disease Pathogenesis III. Disease Prediction IV. Prevention of Type... V. Conclusion References

In contrast to the polyendocrine type I syndrome, susceptibility to type I diabetes is determined by multiple genetic loci (124, 125, 126, 127, 128). In Western countries, approximately one in 300 individuals develops type I diabetes compared with approximately one in 20 first-degree relatives of patients with type I diabetes. For unknown reasons, the risk of diabetes for offspring of mothers with type I diabetes is less than one half the risk compared with the risk for offspring of fathers with the disease (129). The risk of type I diabetes for an identical twin of a patient with type I diabetes is usually estimated at 30–50% (130, 131, 132, 133). Twin studies from Great Britain differ from those of the United States, indicating that 90% of monozygotic twins who become concordant do so within 6 yr (133). Our recent long-term follow-up of initially discordant twins indicates that the 30–50% lifetime concordance of monozygotic twins may be an underestimate (Fig. 1). With long-term follow-up (more than 30 yr of follow-up from onset of diabetes in a first twin), progression to diabetes may be as high as 70% and more than 50% of the initially discordant twins developed diabetes after 6 yr of follow-up (134). A recent population-based twin registry from Denmark also estimates a concordance rate of 70% by age 35 (135). The existence of discordant monozygotic twins has been used to argue that environmental factors contribute to the development of type I diabetes. This is a reasonable hypothesis, but the alternative hypothesis that stochastic (random) processes may determine conversion to diabetes cannot be ruled out. For example, less than 100% of NOD mice or BB rats develop type I diabetes, despite identical genes and shared environment.

View larger version (27K): [in this window] [in a new window]   Figure 1. Development of diabetes in initially discordant monozygotic twins [Derived from Ref. 134.]

The technology for determining HLA alleles has improved dramatically during the past decade. DR and DQ alleles can be rapidly assessed after PCR amplification of DNA from minute quantities of blood or buccal smears (144). Drs. Rewers, Erlich, and colleagues (145) have initiated the Diabetes Autoimmunity Study in the Young (DAISY) with the screening of newborns from a single large Denver city hospital for HLA alleles associated with diabetes risk. HLA alleles are determined utilizing cord blood samples. More than 8,000 newborns have been screened to date, with a cost of approximately $15 per sample. It is likely that this cost could be reduced with automation and screening of even larger populations. The PCR-based typing system has proven to be highly reliable, with a discordance of approximately 0.5% on repeated blinded typing of duplicate samples. More than 90% of parents consented to the screening, including various ethnic groups and socioeconomic strata. Of the newborns, 2.4% were DR3/4, DQB1¹0302, a high-risk genotype for type I diabetes. It is estimated that this 2.4% of the population will comprise 40% of all individuals developing type I diabetes, and the absolute risk for such individuals from the general population is the same as if their father had type I diabetes (6–8%). A moderate risk group, DRX/4 (DQB1¹0302), DR4/4 (DQB1¹0302), and DR3/3, comprised 16.7% of those screened and is estimated to identify an additional 40% of children who develop diabetes. The remainder of individuals are at low risk for type I diabetes (<1/300). Together, the high- and moderate-risk group comprises 80% of all children developing diabetes. As discussed in a recent DAISY publication, "screening for genetic markers associated with (but not diagnostic for) a severe and currently incurable disease, such as IDDM, raises important ethical issues" (145). At present, such screening appears to be justified only for research purposes. Given the development of preventive therapies, such genetic stratification may aid the design of general population preventive trials.

To date, HLA DQ and DR alleles (termed IDDM1) provide the bulk of prognostic genetic information. IDDM2 has been localized to a variable nucleotide tandem repeat (VNTR) 5' of the insulin gene. Polymorphisms of this region account for approximately 10% of the familial aggregation of type I diabetes. The influence of this region on diabetes risk is likely to be complex. The effect of the insulin gene polymorphism is dependent upon the parental (maternal vs. paternal) inheritance of the insulin allele (125, 146, 147, 148, 149, 150). Two studies from the United States indicate that "protective" insulin gene alleles are not protective if inherited from the mother (127, 151). This finding may be related to maternal imprinting of the insulin gene at sites other than the pancreas, in that there is no clear evidence of imprinting of insulin gene expression of islets (150). An alternative hypothesis is that the maternally imprinted IGF-II gene may contribute to diabetes susceptibility, as this gene lies next to the insulin gene (152). Because insulin is a major autoantigen associated with type I diabetes, we hypothesize that the insulin gene VNTR may influence risk by altering expression of insulin and lymphocyte development, perhaps in the thymus or yolk sac (153, 154, 155). Better identification of insulin gene polymorphisms and typing for insulin gene polymorphisms may improve the prediction of type I diabetes.

Multiple additional loci potentially associated with diabetes risk have been described (124, 146, 156, 157, 158). Each of these loci has effects less than or approximately equal to the influence of insulin gene VNTR polymorphisms; that is, each of these loci appears relatively weak. Loci were discovered by the typing of multiple DNA markers throughout the human genome in families with more than one affected sibling. The usual family studied had two affected children and two unaffected parents. A current difficulty is that studies by different investigators often do not confirm the influence of non-class II and non-insulin gene loci, although as indicated by a recent review by Risch and Merikangas (159) the definition of relevant genes by the sib pair approach may be very difficult. In contrast, there are reports of confirmation of a subset of putative loci (160). Our own bias is that with current levels of statistical significance, until there is identification of relevant mutations or polymorphisms, many reported loci may be false "positives."

Although the non-major histocompatibility complex (MHC) loci described to date have a weak influence on diabetes risk in a mixture of families, extremely potent and important regions may exist that confer diabetes risk in specific families. In particular, similar to studies of breast cancer, individual genetic loci may determine a major subset of the development of type I diabetes (10–20%) but appear weak or insignificant in sib pair analysis. Preliminary data from studies of a large Bedouin Arab family with consanguineous marriages (161) has indicated a dramatic influence of a non-MHC locus. Risk of diabetes is associated with homozygosity for a single chromosome 10 allele, in combination with one of three typical diabetes-associated HLA haplotypes. Thus, individuals homozygous for this chromosome 10 allele with DR3 or DR4 have a 40% risk of diabetes vs. <2% for nonhomozygous individuals. Much further study is necessary before a "polygenic" or threshold model for type I diabetes of man vs. an oligogenic model is accepted, and even more research is likely to be required for the identification of major disease genes of selected families. The BB rat model of type I diabetes in many cases provides an oligogenic model (MHC + lymphopenia gene) in contrast to the NOD mouse.

B. Immunological prediction
The immunological prediction of type I diabetes has greatly improved over the past 3 yr with the characterization and cloning of specific autoantigens and development of antiislet autoantibody radioassays. Cytoplasmic islet cell autoantibody testing has facilitated the identification of high-risk individuals and forms the basis for most current preventive trials. "High" titer ICA is clearly a useful marker for progression to diabetes in the general population (162).

Although there is not universal agreement concerning the continued utility of ICA testing, with testing for multiple "biochemical" autoantibodies we currently do not utilize cytoplasmic islet cell antibody testing. Rather, we routinely determine a series of three autoantibodies [antibodies reacting with GAD65, insulin, and ICA512.bdc (amino acids 256 to 979 of ICA512/IA-2) (163)]. With this panel of radioassays in our studies, prediction of risk of diabetes among first-degree relatives is not enhanced by the detection of cytoplasmic ICA (163).

Currently, the best predictor of future type I diabetes is the expression of multiple "biochemically" determined autoantibodies (163, 164, 165). Among 50 first-degree relatives of patients with type I diabetes followed to the onset of overt diabetes (including 13 cytoplasmic ICA-negative relatives), 49/50 expressed one or more autoantibodies (GAD65, insulin, and/or ICA512) exceeding the 99th percentile of normal controls. By Life Table analysis the positive predictive value after 5 yr of follow-up was 100% for relatives with three autoantibodies, 44% with two, 15% with one, and less than 0.5% for relatives expressing no autoantibodies (Fig. 2). Approximately 50% of the relatives we have followed to diabetes expressed three autoantibodies. The 100% development of diabetes within 5 yr is likely to be an overestimate, but 45 first-degree relatives expressing three autoantibodies were identified at their initial test and none remained diabetes free for more than 4.5 yr. Relatives expressing one autoantibody have a much lower risk of progression to diabetes with as long as 7 yr of follow-up. It is likely that a number of relatives [in particular those with the protective HLA allele DQB1¹0602, who express only high titer GAD autoantibodies ("restricted" or "selective" ICA)] will rarely progress to diabetes.

View larger version (25K): [in this window] [in a new window]   Figure 2. Progression to diabetes of first-degree relatives of patients with type I diabetes relative to the number of anti-islet autoantibodies (insulin, GAD65, ICA512) expressed at first screening. [Adapted with permission from C. F. Verge et al.: Diabetes 45:926–933, 1996 (163).]

Approximately 90% of children developing type I diabetes do not have a first-degree relative with the disorder. The development of "biochemical" assays for defined autoantigens is likely to also enhance the prediction of diabetes in the general population. Again the expression of multiple auto-antibodies will confer a high positive predictive value. By setting autoantibody assay cutoffs greater than the 99th percentile of normal controls, approximately 3% of individuals in the general population express a single autoantibody (GAD65, insulin, or ICA512). In contrast, of more than 300 individuals without a family history of diabetes that we have studied, none have expressed 2 autoantibodies. It is likely that approximately one in 300 individuals in the general population will express 2 autoantibodies, given a larger series. Of patients with new-onset type I diabetes, 80% express 2 of the above autoantibodies. Thus, screening the general population for 2 autoantibodies [of IAA (insulin autoantibodies, GAA (GAD65 autoantibodies), and ICA512AA] will provide high disease specificity with approximately 80% sensitivity.

Identification of a fourth readily measured autoantibody (in addition to GAD65, insulin, and ICA512 autoantibodies) will likely increase sensitivity of testing and positive predictive values. A molecule related to ICA512/IA-2, termed phogrin by Hutton and co-workers (77) and IA-2ß by Notkins and co-workers (78), has recently been cloned. In our studies to date, almost all phogrin-positive subjects are ICA512 positive, and determination of phogrin/IA-2ß positivity adds little to our ability to predict diabetes. There are other identified autoantigens, such as a GM2–1 ganglioside antigen (79, 166) and a molecule termed ICA69 (67, 167). The assays for both of these molecules are currently too cumbersome (Western blotting, staining of thin layer chromatograms) for large-scale screening. In a recent exchange of sera samples, only one of four ICA69 autoantibody assays statistically significantly distinguished sera from new-onset diabetic patients from controls (168). Until rapid and quantitative assays for autoantibodies to these molecules are developed, their importance in the prediction or diagnosis of type I diabetes will remain tentative.

We currently screen for GAD65 and ICA512 autoantibodies utilizing in vitro transcribed and translated protein. Differential labeling (³H and ³⁵S) of the two proteins with simultaneous determination, 96-well vacuum filtration, and direct 96-well Top ß-Counter determination of bound radioactivity results in a semiautomated assay with a combined sensitivity of approximately 90%. A single technician can perform more than 30,000 assays per year. In addition, only 7 µl of sera are required for the determination of both autoantibodies. This allows the screening of multiple sera samples together for the evaluation of low-risk populations (e.g. the general population). In contrast, our current insulin autoantibody assay utilizes 600 µl of sera and is not nearly as convenient. Nevertheless, in recent workshop comparisons, insulin autoantibody assays utilizing less than 600 µl of sera had sensitivities less than one half of those utilizing this larger volume of sera. An insulin assay requiring less sera, with preserved sensitivity and specificity, is needed.

A number of current studies are defining the pattern of appearance of islet cell autoantibodies among children in the first years of life (169, 170, 171, 172, 173). It is already apparent that islet autoantibodies can occur in the first year, that with prospective follow-up autoantibodies usually develop sequentially, that any autoantibody may be present early in life, and that GAD65 or insulin autoantibodies usually precede ICA512/IA-2 autoantibodies. A subset of relatives positive for autoantibodies (probably <10%) become negative without developing diabetes on prospective follow-up.

C. Metabolic prediction
The major modality for assessing metabolic risk for progression to type I diabetes is the intravenous glucose tolerance. The ICARUS study group (Islet Cell Antibody Registry of Users) has recommended a standardized protocol (174) for performance of the intravenous glucose tolerance test that has been incorporated into a number of trials for the prevention of diabetes. Low first-phase insulin secretion, usually defined as below the tenth or first percentile of normal controls, typically precedes the development of type I diabetes by 1–5 yr (9, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186). Even among relatives expressing a single biochemically determined autoantibody, relatives with insulin secretion below the first percentile have more than a 90% risk of progressing to diabetes in the next 3 yr. Among relatives expressing multiple autoantibodies, preserved insulin secretion correlate with a longer disease-free interval (163).

In addition to first-phase insulin secretion, we utilize levels of insulin autoantibodies to aid in predicting the time to onset of diabetes. The highest levels of insulin autoantibodies are found in children developing type I diabetes before age 5 (186, 187, 188, 189). Levels of insulin autoantibodies, utilizing multiple logistic regression, independent of age correlate with progression to diabetes among relatives expressing other autoantibodies.

	IV. Prevention of Type I Diabetes

Top Abstract I. Introduction II. Disease Pathogenesis III. Disease Prediction IV. Prevention of Type... V. Conclusion References

 
Progress in the identification of persons at risk for the development of type I diabetes has made possible the design of preventive trials (190). A well designed prevention trial may use as an endpoint the diagnosis of diabetes or may assess other outcomes, such as the development of autoantibodies. In addressing a particular intervention strategy, it is important to consider the duration of the prophylaxis, the effectiveness of the therapy, potential toxicity or side effects, complexity of the regimen, and expense (Table 3).

It is probably easier to prevent diabetes in animal models than in humans. This may relate to the inbred status of such animals, with the result that they are not diallelic at any genetic locus. In both the NOD mouse and the BB Wistar rat, a number of strategies have been used to prevent the onset of diabetes (4, 191, 192, 193).

It is now generally accepted that "pre-diabetes" can be accurately predicted in a select population that is at high risk. Screening and identification of high-risk individuals is justifiable because of the possibility of entry into a prevention trial. In addition, with family education and follow-up, an individual at high risk of diabetes should rarely develop ketoacidosis before outpatient institution of insulin therapy. This becomes important with the realization that approximately one in 200 children dies at diagnosis of diabetes (194). For example, approximately every 2 yr a child in Colorado (with a population of 4,000,000) dies at diabetes onset.

In 1990, the NIH convened a workshop to discuss issues surrounding immunomodulation and trials to prevent type I diabetes. In the statement issued subsequent to that meeting, the group said "The general consensus from the meeting was that indeed there is methodology that can identify with near certainty among first degree relatives of type I diabetic patients, those who will develop diabetes and that immune intervention therapy before the onset of symptoms might prevent the disease from occurring" (195). However, identification of high-risk individuals, if limited to screening of relatives of patients with diabetes, is relatively rare (1/150 relatives screened). No one large diabetes center could be expected to identify more than 20 relatives suitable to enter a prevention trial within a year. Additionally, identification of subjects is expensive (estimated at $1,200 per high-risk subject identified, including autoantibody testing and intravenous glucose tolerance testing). Trials of a number of immunosuppressive, immunomodulatory, and environmental therapies have been initiated. Several national and multinational preventive trials are now underway.

B. Genetic prevention
With the ability to assess increased risk of diabetes relative to DR and DQ alleles, it is possible to provide genetic counseling. The risk of diabetes is approximately one in 50 for offspring of a mother with type I diabetes and one in 16 for offspring of a father with type I diabetes. A decreased diabetes risk of offspring for older diabetic mothers has been reported (129) but not confirmed in all data sets (196). Larger data sets must be analyzed to address this question.

Consanguinity (usually first cousin marriages) has been reported to increase the risk of type I diabetes (197), and this is a particular consideration for a number of specific populations. We have observed three generations of a single family with multiple consanguineous marriages in which the risk of diabetes approaches that of identical twins. Nevertheless, the perceived and real benefits to specific societies of consanguineous marriages may for that society outweigh the risk of type I diabetes.

Approximately 20% of individuals carry DQ alleles (DQA1¹0102/DQB1¹0602), which provide dominant protection from type I diabetes (142, 198). Should fetal "selection" in the approximately one in five families with a diabetic parent and a DQB1¹0602 spouse be considered? Although DQA1¹0102, DQB1¹0602 provides protection from type I diabetes, it is the high-risk allele for multiple sclerosis. Understanding how DQA1¹0102, DQB1¹0602 provides protection from type I diabetes is a major research goal and with such information, practical preventive therapies will likely be devised. Selection for DQB1¹0602 in families should not be a consideration.

C. Prevention: preautoimmunity
Several large studies are underway that will better define the incidence of autoantibodies in first-degree relatives of patients with type I diabetes and in general populations. It appears that autoantibodies frequently appear before age 5 (170, 171). At present, there is only limited information concerning the youngest cohorts. One can estimate that more than 20% of DR3/4 first-degree relatives of patients with type I diabetes will be autoantibody (GAA, IAA, ICA512AA) positive before age 5, and less than 2% of DR 3/4 (DQB1¹0302) general population U.S. children will be similarly positive. The above calculations mandate large-scale screening and careful selection of individuals for trials of prevention of autoantibodies. It also must be borne in mind that autoantibodies are an imperfect surrogate marker for progression to diabetes. Nevertheless, since autoantibodies usually precede diabetes by years, they are an important surrogate marker.

Cow’s milk has been implicated as a possible antigenic trigger of the immune response that leads to ß-cell destruction in genetically susceptible hosts. Studies in both animals and humans (60, 61, 63, 64, 199) have suggested a link between cow’s milk and the development of type I diabetes. A national study to evaluate the effects of feeding cow’s milk vs. a casein hydrolysate formula for the first 6–8 months of life has been proposed in Finland. Eventually, the study may screen 6,000 infants who have a parent or sibling with type I diabetes to identify 2,000 infants who will be followed for 10 yr. Venous samples would be collected every 2 yr for ICA, GAA, ICA512AA, IAA, cow’s milk antibodies, ß-lactoglobulin antibodies, BSA antibodies, glycosylated hemoglobin, and glucose levels. The endpoint for each child will be the development of diabetes. A small pilot study of 20 HLA high-risk infants with diabetic relatives has been completed, and dietary intervention did not decrease the appearance of autoantibodies.

A single subcutaneous injection of the B chain of insulin or the immunodominant B chain peptide B9 to B23 in incomplete Freund’s adjuvant prevents diabetes in NOD mice (92, 101, 200). This form of immunological "vaccination" would be a major advance if applicable to human autoimmune disorders. Such a therapy is likely to be most effective if instituted before the development of chronic autoimmunity (as evidenced by stable expression of autoantibodies). To maximize the potential benefit of such therapy, risk of diabetes would need to be maximized. DR3/4 (DQB1¹0302) offspring of fathers with type I diabetes or siblings of patients with type I diabetes have a type I diabetes risk of approximately 20%. By age 5, more than 20% of such individuals stably express antiislet autoantibodies. Approximately 10% of offspring and siblings of patients with type I diabetes are DR3/4 (DQB1¹0302). Given these frequencies, one can estimate the numbers of relatives needed for a trial where development of autoantibodies is prevented by 50% (1- = 0.05, 1-ß = 0.80, 75% participation). Seventy-six relatives would need to be randomized (1:1 randomization); thus 1,013 such relatives would need to be HLA typed. To detect a 30% suppression of development of autoantibodies, 190 relatives would need to be randomized and 2,553 screened. With the frequent development of autoantibodies before age 5, such trials utilizing the intermediate phenotype of autoantibodies as an endpoint could be designed as 5-yr trials. In contrast, development of diabetes often extends over more than 20 yr.

D. Prevention: postautoimmunity/prediabetes
Identification of persons at risk for development of Type I diabetes now allows the design of preventive studies before hyperglycemia. Immunomodulatory agents may be used to stop islet cell destruction, thus preventing or postponing the development of insulin deficiency and clinical disease. The two agents that have been most widely studied for this purpose are nicotinamide and insulin.

The mechanism of nicotinamide action is not completely defined. Nicotinamide was originally studied because of the observation that large doses of nicotinamide blocks induction of diabetes by streptozotocin. Nicotinamide was shown to partially prevent diabetes in NOD mice as long ago as 1982 (201). In rat studies, nicotinamide has been shown to inhibit poly (ADP-ribose) synthetase and to preserve synthesis of proinsulin (202). At high concentrations nicotinamide can act as a free radical scavenger, as can thymidine (203). Nicotinamide has an effect on cellular immunity; it inhibits activated macrophage killing of ß-cells in vitro (204). It also inhibits cytokine induction of the class II MHC on ß-cells. The biological actions and therapeutic potential of nicotinamide were recently summarized in a workshop report prepared in anticipation of large multicenter studies (205). A theoretical adverse effect of nicotinamide is the development of islet tumors, in that a combination of streptozotocin plus nicotinamide can induce islet tumors in rats.

Investigators in Auckland, New Zealand, and Denver, Colorado, reported a pilot study investigating the effectiveness of nicotinamide in the prevention of diabetes (6). For this preliminary report, all of the nontreated patients came from Denver, Colorado, and different ICA assays were used in the two countries. Of 14 treated subjects, one developed diabetes after 17 months of treatment. In the same time period, all eight untreated subjects became insulin dependent. All patients were under the age of 16 at the time they were found to be ICA positive, and all had ICA levels 80 JDF (Juvenile Diabetes Foundation) units. All had first-phase insulin responses (FPIR = sum of 1 min + 3 min insulin levels) less than the fifth percentile (67 µU/ml). In further unpublished follow-up to 5 yr, three of four nicotinamide-treated Denver subjects and five of ten Auckland subjects had become insulin dependent. Another small pilot study was carried out at the Joslin Diabetes Center in Boston, and three of three nicotinamide patients developed diabetes (206).

Although these small pilot studies of nicotinamide show little efficacy, nicotinamide has the virtue of probably being harmless, is inexpensive, is available in an oral form, and is easily placebo controlled. Elliott and co-workers (207) carried out a large trial where they screened one half of the population of young children in Auckland, New Zealand, and treated the ICA-positive children with nicotinamide. A 50% "delay" of diabetes is claimed relative to the nonscreened population (207). A truly randomized trial is necessary to evaluate the efficacy of nicotinamide.

Two large randomized trials are underway to evaluate the efficacy of nicotinamide in the prevention of Type I diabetes. The first is a German national trial, DENIS (Deutsche Nicotinamide Diabetes Intervention Study), a 3-yr trial involving 74 ICA-positive relatives aged 3–12 yr, half of whom will receive nicotinamide and the other half placebo. The second is the European Nicotinamide Diabetes Intervention Trial (ENDIT). This multinational study is under the direction of Dr. Edwin Gale of London (208). It is a double-blinded, randomized, and placebo-controlled trial and will include first-degree relatives of patients with Type I diabetes who have at least two ICA-positive tests, one of which is 20 JDF units, drawn 3–12 months apart. An intravenous glucose tolerance test (IVGTT) will be performed using the ICARUS protocol (174). The IVGTT will be used for data analysis, but not as criteria for entry into the trial. An oral glucose tolerance test will be performed as a two-point test (0 min and 2 h) to rule out diabetes by World Health Organization standards. Follow-up will include a yearly IVGTT with ICA and IAA tests as well as C peptide levels to measure ß-cell function. The dose of nicotinamide will be 1.2 g/m².

Other multicenter diabetes prevention trials will evaluate insulin therapy, given parenterally or orally. The exact mechanism by which such insulin administration prevents diabetes in NOD mice or BB rats is currently unknown (209, 210). Insulin may provide a form of "ß-cell rest," protecting the ß-cell from further immune destruction. Studies in the BB rat support this hypothesis (193). Rats treated with insulin develop smaller ß-cells, with islet cells that no longer react with human sera containing anti-GAD autoantibodies (211). In contrast, in the NOD mouse model, biologically active insulin is not required for disease prevention (210). Oral insulin (101, 113, 114, 212) or "vaccination" with B chain peptides (101, 189) prevents disease. Such insulin administration is thought to induce a shift from Th1 T cells that are destructive toward a Th2 T cell response (113). Th2 T cells release "immunosuppressive" lymphokines. A rationale for the use of insulin is that only insulin, of all the identified autoantigens, is ß-cell-specific. Anti-insulin-specific T cells are found in humans and in NOD mouse islets. Treatment of NOD mice and BB rats with parenteral insulin prevented both diabetes and insulitis (209, 213). Immunization with insulin ß-chain prevents diabetes but not insulitis in NOD mice. Thus, intact insulin may have synergistic effects, producing both "ß-cell rest" and immunomodulation. Oral insulin given to NOD mice delays diabetes and generates splenocytes able to inhibit transfer of diabetes by diabetogenic NOD mouse lymphocytes.

The initial human pilot study using insulin as an immunomodulatory agent was performed at the Joslin Clinic and extended to the Joslin and Barbara Davis Center (7). Initially, five subjects, aged 7–14 yr of age, were treated with 5 days of intravenous insulin to maintain blood glucoses between 60–76 mg/dl. They then received daily insulin divided into two subcutaneous injections consisting of human regular and ultralente insulin in the morning and lente insulin at bedtime with a total daily dose of 0.2 U/kg body weight/day. After a mean of approximately 3 yr, only one of the five treated patients became diabetic as assessed by periodic oral glucose tolerance testing after discontinuing insulin for 3 days. All seven of the control subjects developed diabetes during the same period. Additional individuals have been randomly entered into the pilot study, and individuals of the initial trial continue to be followed. The first treated individual remains nondiabetic after more than 7 yr of follow-up. In total, three of nine relatives have developed diabetes on therapy vs. eight of eight untreated relatives (Fig. 3). The three who developed diabetes had very low insulin secretion at the time of entry into the trial. The above preliminary results are similar to those reported by Ziegler and co-workers and now with further follow-up where intravenous insulin is given annually and subcutaneous insulin for only 6 months. To date, six of seven untreated (randomized) high-risk relatives are diabetic vs. three of seven treated (P = 0.06 by life table analysis at 3 yr, risk = 14% treated, 71% untreated) (7).

View larger version (18K): [in this window] [in a new window]   Figure 3. Pilot trial of insulin therapy (intravenous insulin plus low dose subcutaneous insulin) for diabetes prevention of ICA-positive high-risk first-degree relatives. [Derived from Ref. 7.]

Subjects become eligible for the high-risk protocol of the study (parenteral insulin) when immunological and metabolic screening and staging indicate a greater than 50% risk of developing diabetes within a 3- to 5-yr period.

Table 4 summarizes the criteria for randomization: half of the subjects will be randomized to receive intravenous insulin for 4 days once a year for 3 to 5 yr and 0.125 U/kg body weight of ultralente insulin subcutaneously twice a day. The other half will receive no treatment but will be under close observation so that insulin therapy can be given as early as possible if diabetes develops. The major outcome parameter is development of diabetes by adult NDDG (National Diabetes Data Group) criteria with periodic oral glucose tolerance tests (93) after discontinuing insulin for 3 days in the treated group.

E. New onset patients/prevention of further ß-cell destruction
Diabetes prevention trials began with attempts to ameliorate the course of the disease once it was clinically apparent. While this approach is less appealing than that of early identification of an at-risk population and intervention before the onset of overt disease, it has taught us valuable lessons about the immune nature of type I diabetes. If successful therapies are identified, they would facilitate achieving the ideal blood glucose control necessary to reduce the risk of diabetic complications. Trials in recent-onset diabetes are based on the premise that there are still functional islet cells present at the time of clinical diagnosis. It appears from histological studies that the majority of islets have been lost at the time of diagnosis (16). Thus, the best possible outcome for these patients would be to preserve the remaining islets, prolonging the time they can produce endogenous insulin rather than actual prevention of diabetes. Because the goals of intervention are limited, so too must be the risks of therapy.

The earliest trials to preserve ß-cell function used nonspecific immunosuppressive therapies and included studies of prednisone, anti-thymocyte globulin, prednisone plus azothioprine, and Cyclosporin A. These studies demonstrated that insulin dependency could be delayed with generalized immune suppression, but metabolic remission of diabetes was lost with the withdrawal of therapy (214, 215, 216).

Cyclosporin A has been studied in several centers, but its use has been largely discontinued in the United States. It was initially studied in newly diagnosed patients with Type I diabetes in 1984 (217) and was found to indeed significantly maintain B cell function. In large French and Canadian studies the use of Cyclosporin A led to insulin-free remissions in 23% of treated subjects compared with 10% of controls. The most recent report from the French group (218) concluded that while insulin production was prolonged in the treated patients, and hypoglycemic events were fewer, by 4 yr the differences became nonsignificant. Despite continuation of cyclosporine therapy, essentially all treated patients who had a prolonged remission of their diabetes became diabetic again. The small numbers of patients who improved and the short duration of that improvement did not warrant the risks of cyclosporine therapy.

Use of insulin as an immunomodulatory agent after diagnosis has also been studied. Using a Biostator machine, a group in Florida (107) placed newly diagnosed patients on an insulin clamp that tightly regulated their blood glucose levels for a 14-day period. They reported lower glycosylated hemoglobins and higher levels of C peptide 1 yr after diagnosis in this treated group when compared with a group receiving standard insulin therapy. There have been no independent reports confirming the "benefits" of such therapy and no randomized study.

When NOD mice are stimulated immunologically with allogeneic cells or BCG, half failed to develop diabetes or insulitis (219). One nonrandomized preliminary pilot study reported that BCG administration was associated with remission of diabetes (55). A larger study is currently under way at the Barbara Davis Center in Denver exploring the possibility that BCG vaccination immediately after diabetes onset might reduce insulin requirement and increase C peptide production. The study is double-blinded with entry restricted to subjects aged 5–18 yr who are within 8 weeks of the clinical onset of diabetes or within 16 weeks if they are ICA positive. Enrollment into this study has been completed, and approximately 30 individuals have reached 2 yr of follow-up. If there is any benefit of this therapy, it is unlikely to be large. Thus, we would not recommend BCG therapy until the completion of controlled trials.

F. Pancreas and islet transplantation/prevention of recurrent diabetes
While human trials of islet cell transplantation are few and results have been disappointing, pancreas transplantation is becoming an important therapy for diabetic individuals receiving a kidney transplant. As illustrated by the recent report by Tyden and co-workers (220), a subset of patients who receive a pancreas transplant, despite immunosuppression, develop selective ß-cell destruction and insulitis, and one patient developed anti-islet autoantibodies. It is thus likely that recurrent autoimmune ß-cell destruction will be a barrier for islet and pancreatic transplantation (221).

Pancreatic transplantation has been studied since 1967 (222), and there have been thousands of recipients. Pancreas transplantation often accompanies renal transplantation in diabetic and uremic patients. Recently, there have been an increasing number of transplants of pancreas alone. By 1994 there had been some 4,000 pancreas transplants (223). The failure rate is higher than that found in either heart or kidney transplants. In a review of 2,103 pancreas transplants in the United States (223), 71% were functional at 1 yr; 66% were functional at 2 yr, and 59% were functional at 3 yr.

Pancreas transplantation alone is considered to be applicable as a treatment of extremely labile and life-threatening diabetes (224). It requires immunosuppression with an increased risk of malignancy and infection. Azothioprine is associated with myelosuppression, Cyclosporin with nephrotoxicity, and steroids with gastrointestinal bleeding, hypertension, aseptic necrosis of the hip, and alteration of lipids. A comparison of successfully transplanted patients with those unsuccessfully transplanted revealed no difference in retinopathy at 3 yr (225). Renal function actually deteriorates more rapidly after transplantation (226, 227), perhaps due to the nephrotoxic effect of Cyclosporin (228). On the other hand, there appear to be neurological benefits bestowed by successful pancreas transplantation (229) in sensory, motor, and autonomic function. Among 175 patients with abnormal cardiorespiratory reflexes, those with a functional graft had improved 7-yr survival rates (230). Quality of life as measured by the recipient is often dramatically improved with pancreatic transplantation.

Combined pancreas and kidney transplants are now common. The International Pancreas Transplant Registry documents a 1-yr survival rate of 49% for pancreas transplant alone vs. 75% when pancreas and kidney are transplanted together. Indices of kidney rejection usually occur before pancreatic rejection, allowing earlier recognition and treatment of rejection. To prevent early rejection, patients are given anti-lymphocyte globulin or OKT3 as induction in the first 14 posttransplant days. In a small series at the Mayo Clinic ten of 18 developed cytomegalovirus infection vs. two of 18 subjects who had a kidney transplant alone (231).

There are demonstrable benefits to combined pancreas/kidney transplants. In a review of 253 patients in Wisconsin, immediate insulin independence was achieved in virtually all patients. There was normalization of glycosylated hemoglobin and cholesterol levels were lower. The Stockholm group (232) reports that pancreas transplants prevented nephropathy in the cotransplanted kidney. Earlier reports showed deterioration in retinopathy after transplantation, but the Wisconsin group in a larger recent series (225) demonstrated improvement in 43%, and deterioration in only 7% of those transplanted. The Minnesota group (233) studied 113 patients who had achieved insulin independence for more than 1 yr and found normal glucose levels (86, 87, 88, 89, 90, 91), normal hemoglobin A1C levels (5.3–5.8%), and normal response to arginine and glucose stimulation.

Investigators continue to search for safer immunosuppressive agents. FK506, whose mechanism of action is similar to cyclosporine A, is now used in some centers and may be associated with a lower rate of graft loss when used for induction and a higher rate of graft salvage when used for rescue or rejection therapy (234). Ricordi and co-workers (242) are studying combined bone marrow infusions and immunosuppression. Utilization of Cell-Sept rather than azothioprine may enhance engrafment and allow maintenance, immunotherapy with a much lower dose of glucocorticoids (221). This is likely to translate into lower morbidity and mortality and improved long-term quality of life. The field of transplantation for both islets and pancreas appears to be advancing progressively. Pancreas transplantation is likely to become more accepted, particularly when combined with a kidney transplant. As therapies that prevent autoimmune diabetes are improved, the success of islet transplantation will also likely improve.

G. Islet cell transplantation/prevention of ß-cell destruction
The possibility of islet cell transplantation as a therapy for type I diabetes was mentioned by Lazarow (235) in his 1973 Banting lecture. His group had shown that fetal pancreas could be incubated in tissue culture and that the islets of the pancreas developed preferentially in vitro. Early studies in rats demonstrated the feasibility of islet transplantation (236), although injection of islets into the portal vein failed (237) and islets were usually rejected (238). Indeed, radiation, antilymphocyte serum, and transplantation into immunoprivileged sites also failed (239).

Over time, the success of islet transplantation in rodent models has improved. In particular, treatment of islet tissue by a variety of methods designed to reduce their immunogenicity can prolong graft function and, in some strain combinations, lead to long-term function.

The isolation of human islet cells has also improved over time. Simple digestion of pancreas with collegenase, which is effective in rodents, does not work well with the more densely organized human pancreas. In the early 1980s a technique was devised in which collegenase was injected into pancreatic ducts, followed by gentle mechanical dissociation and discontinuous gradient purification (240), Currently a three-step method is used. First, collegenase is infused via the pancreatic duct. Next, mechanical dissociation of the tissue is achieved. Finally, Ficoll density gradient purification concentrates islets (241). Automatic digestion (242), preparation of Ficoll gradients in hypothermic preservation solutions (243), and automatic purification protocols (244) permit mass islet cell recovery.

In the absence of autoimmunity and allograft immunity (such as islet isografts in patients after pancreatectomy), islet transplants are usually successful. Recently Bretzel and his group (245) in Germany proposed minimal requirements for quality control in islet cell transplant tissue: 1) islet mass of more than 8,000 islets/kg in Type I diabetic subjects; 2) more than 80% of total cell volume are islets; 3) more than 80% of the islets are viable; 4) islet cells function in vitro as documented by a biphasic response to glucose challenge and return to baseline; 5) exclusion of bacterial or other contamination.

Before 1990, islet cell function after transplantation was almost nonexistent. Transplanted islets did not produce insulin as measured by C peptide levels. By 1990, improvement in the quality and quantity of transplanted islets, as well as immunological induction with monoclonal or polyclonal antibodies, had improved insulin secretion [80% of recipients had C peptide levels that exceeded 1 ng/ml for greater than 1 month (246)]. Since 1990, more than 150 patients worldwide have received islet transplants. Few have achieved insulin independence. Nevertheless, the islets have often produced enough insulin to make diabetes control easier and to allow lower glycosylated hemoglobin levels (247).

A difficulty in envisioning large-scale islet cell transplantation has been the availability of islets themselves. Over the past decade investigators have improved the technique of cryopreservation, which will allow collection and storage of viable human islets in tissue banks to allow pooled human grafts. Cryopreservation became available for rodent and canine islets in 1983 and subsequently for adult human islets (248) and human fetal islets (249). The current process involves addition of a cryoprotective additive (dimethylsulfoxide), freezing and thawing of tissue, and return to a physiological medium. The best results have been achieved with slow cooling to -40 C in combination with rapid thaw from -196 C (250).

Using combinations of cryopreserved pooled islets and fresh islets, researchers at the University of Alberta treated five patients (251). One of their subjects, treated with 10,000 islet equivalents/kg became insulin independent at 69 days and has remained so for 2.5 yr.

The concept of immunoisolation of islet cells was first investigated in the late 1970s when Lacy (247) implanted hollow fibers containing rat islets into the peritoneal cavities of streptozotocin-treated diabetic mice. These animals became euglycemic, but by 10 days had again become hyperglycemic. In 1980 Lim and Sun (252) suspended islets in an alginate gel. Using this technique, mice have remained euglycemic for as long as 1 yr, and studies demonstrated viable islets. However, attempts in larger animals failed because the membranes proved mechanically unstable and stimulated fibrosis of the capsules (253, 254). Capsules have now been formulated with purified alginate, and isolated successful transplantation has been reported in rats (255, 256) and dogs (257, 258). In 1994 Soon-Shiong and colleagues (259) implanted an adult male with 678,000 islet equivalents in the intraperitoneal space. Despite the large number of islets used, insulin therapy only decreased from 0.7 U/kg/day to 0.2 U/kg/day. The huge number of islets used and minimal long-term success of the transplant, with intraperitoneal injections of tissue and alginate, limit enthusiasm for this procedure as described. However, the overall approach, especially with retrievable devices, is promising and requires additional basic research.

	V. Conclusion

Top Abstract I. Introduction II. Disease Pathogenesis III. Disease Prediction IV. Prevention of Type... V. Conclusion References

 
Relatives of patients with type I diabetes who are beginning to develop diabetes can now be readily identified with immunological and metabolic assays. It is likely, although not proven, that similar screening can now identify high-risk individuals from the general population. With such prediction is likely to come a flurry of "immunomodulatory" trials of disease prevention. In the long term, immunological "vaccination" for the prevention of type I diabetes is a possibility. With further understanding of the pathogenesis of ß-cell destruction, prevention of type I diabetes coupled with islet transplantation for those already diabetic offers hope to the many families afflicted with this disorder.

	Footnotes

 
Address reprint requests to: George S. Eisenbarth, M.D., Ph.D., Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Box B140, Denver, Colorado 80262.

¹ Research supported by grants from the NIH (DK-32083) and from the American Diabetes Association, the Blum-Kovler Foundation, and the Children’s Diabetes Foundation.

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