Chapter 11

Prediction of Type 1A Diabetes:

The Natural History of the Prediabetic Period

Updated 09-2012

George S. Eisenbarth

Introduction

Predictive Factors in Relatives

Prediction in the General Population

Prediction/Diagnosis in Adults

Stage in Life Initiation

Are there Abnormalities that Precede Autoantibodies?

Environmental Factors?

Are Beta Cells Destroyed in a Progressive/Linear Fashion?

Conclusions

Introduction

The importance of understanding the natural history of immune mediated pre-diabetes^1-3 lies in the development of prevention strategies^4-6. Several initial randomized clinical intervention trials have concluded and the next generation of such trials will rely upon improved and simplified identification of individuals⁷ who are at high risk of progression to diabetes⁸. This is essential to ensure that trials will have sufficient statistical power to detect a given effect of the intervention (if it exists) within the time available for the study. Such understanding is also needed to avoid exposing those who will not develop diabetes to the risk of adverse effects of the intervention. In addition, it is likely that many interventions will be more effective if given early, with more extant beta cells. In addition there is accumulating evidence that at the onset of type 1A diabetes, and in a subset of patients years after the onset of diabetes (Figure 11.1) there remains islet beta cells⁹, and preservation of even low levels of insulin secretion has multiple benefits in terms of improved glycemic control and prevention of complications^10-14.

The amount of beta cell destruction at the onset of diabetes remains an open question^15-20 with one estimate that a 40% reduction in beta cell mass is sufficient for diabetes of a 20 year old while 80-90% loss may be required for children presenting with diabetes less than age 5²¹. Given individual differences in rates of progression to diabetes and insulin resistance (as well as potential function per beta cell), it is likely that the variance of beta cell loss will be large at any age. It is of interest that the slope of loss of c-peptide after diabetes onset decreased modestly from the immediate prediabetic phase²². This may reflect “synchronization” with greater rate of loss associated with presentation with diabetes. With long-term type 1 diabetes the mean beta cell loss is dramatic(Figure 10.1).

Figure 11.1. Area of insulin containing cells in long-term patients with diabetes ²³.

First-degree relatives of individuals with type 1A diabetes have an approximate 5% risk of developing the disease (independent of country ²⁴) while children without a relative with diabetes in the United States have a risk of 1/300 while in Japan the risk is less than 1/3,000. Longitudinal studies of autoantibody-positive relatives and more recently general populations^{25, 26} have provided a wealth of information on the natural history of autoimmunity during the pre-hyperglycemic phase of the disease (prediabetes). These studies have established the predictive value of age²⁷, islet cell antibodies (ICAs)²⁸, multiple “biochemical” autoantibodies ^29-32 first phase insulin release (FPIR)³³, impaired glucose tolerance, C-peptide secretion (Prediction”Scores”) ³⁴, and human leukocyte (HLA) haplotypes³⁵. Increasingly, combinations of markers are being used to better define the risk of diabetes ^36-40 Prediction is not absolute, but can be expressed as the percentage of individuals developing diabetes within a given time period. In addition the age at which islet autoantibodies first appear and levels of insulin autoantibodies (not antibodies reacting with GAD, IA-2 or ZnT8) predict approximate age of onset of type 1 diabetes^{25, 26}. This chapter will review predictive factors currently in use and discuss some of the unanswered questions on the natural history of Type 1A (immune mediated) prediabetes.

Predictive Factors in Relatives

The first large scale studies of the prediction of type 1A diabetes relied upon the detection of cytoplasmic islet cell autoantibodies (ICA, Figure 11.2). These studies framed much of our knowledge concerning progression to diabetes but suffered from the difficulty of the cytoplasmic ICA assay, including difficulty in quanitation and standardization³⁰. As will subsequently be emphasized determination of autoantibodies reacting with four major islet autoantigens, insulin, GAD65, ICA512 (IA-2), and ZnT8 ^{41, 42}has become central components of studies of the natural history of diabetes. Cytoplasmic ICA represents antibodies reacting with GAD65, ICA512, ZnT8 and other unknown antigens, but not insulin autoantibodies. High titer cytoplasmic ICA is most often associated with the presence of multiple anti-islet autoantibodies (of GAD65, ICA512, ZnT8, or insulin) and thus is associated with a high risk of progression to diabetes⁴³. It is important to emphasize that a positive ICA test (binding of antibodies to islets of human pancreas detected with indirect immunofluorescence assays) can represent antibodies to only the GAD autoantigen (Glutamic acid decarbosylase), only IA-2 autoantibodies, only non-GAD and non-IA-2 antigens, or a mixture of the above⁴⁴, as well as antibodies reacting with ZnT8 (unpublished). Thus detection of ICA positivity in addition to detection of biochemical autoantibodies usually increases risk of progression to diabetes given association with higher titer and multiple biochemical autoantibodies.

Riley and coworkers reported on the University of Florida, Gainesville, family study in 1990²⁷. At that time 3413 first-degree relatives had been screened for ICA, of whom 3.3% were positive. Positive sera were defined as those with 10 or more Juvenile Diabetes Foundation (JDF) units. ICAs are more frequent among the siblings of diabetic probands than among parents. They were also more frequent among relatives from multiplex families and among relatives less than 20 years old. After a maximum follow-up of 10 years, (median 3.5 years) 40 relatives developed type 1 diabetes. The risk of diabetes was significantly higher in relatives with ICAs of 20 or more JDF units at the time of initial screening, those aged less than 10 years (at initial screening), and those from multiplex families. Each of these risk factors was independent of the others. The presence of IAA was not an independent risk factor, after allowing for ICA. However, a later report with further follow-up came to the opposite conclusion, with evidence that IAA add independently to prediction⁴⁵. The presence of ICA in titers of less then 20 JDF units did not confer significantly increased risk of diabetes, but increasing titers above this level were associated with progressively increasing risk. Nevertheless, one third of the relatives progressing to diabetes were ICA negative on the first test and, of these, 62% remained ICA negative at the onset of diabetes. With analysis of “biochemical” autoantibodies it has become evident that the presence of ICA in the absence of GAD65 or ICA512 autoantibodies is associated with a low risk of progression to diabetes. Verge et al ³⁶ proposed the “general” rule that presence of autoantibodies reacting with >=2 of the biochemical autoantibodies (GAD65, ICA512, insulin) are associated with greatly increased risk of type 1 diabetes,and we would now add ZnT8 autoantibodies to the group⁷. The same was found with further follow up of the Gainesville natural history studies, with the concordant observation that in the absence of biochemical autoantibodies ICA alone is associated with little risk⁴⁶.

The Bart's-Windsor family study, conducted in England from 1978 onwards, also found that higher ICA titer was associated with shorter diabetes-free survival²⁸. Seven hundred and nineteen first-degree relatives were followed for up to 10.5 years. ICAs were tested every 4 to 6 months, using a sensitive assay with a detection limit of four JDF units. Detectable ICAs were found in the sera of 3.3% of the relatives at initial testing, compared with 2.2% of 540 healthy child and adult controls. However, only one control had ICA of 20 or more JDF units, compared with 10 relatives. ICAs were detected in an additional 14 relatives on follow-up samples and follow-up time survival analysis was calculated from the time of ICA detection. With increasing ICA cutoff the positive predictive value for future diabetes rose, and the sensitivity fell. The risk of diabetes within 10 years associated with ICAs of 80 or more JDF units was 100% (95% confidence interval: 52%-100%). In comparison, ICAs of 20 or more JDF units were associated with a 73% risk (95% confidence interval: 45%-100%), and ICAs of four or more JDF units with a 40% risk (95% confidence interval: 23%-57%). Utilization of biochemical autoantibody assays in these cohorts also demonstrated the importance of multiple autoantibodies to identify high-risk individuals. The cumulative risk of developing diabetes within 15 years was 47% (>=10 JDF units), 66% for >=20JDF units, but only 2.8% for those with ICA but without GAD or ICA512 (IA-2) autoantibodies versus 66% for those with ICA and either or both of GAD or ICA512 autoantibodies³⁷.

Figure 11.2

Data from the Joslin Diabetes Center family study indicate that impaired FPIR (First Phase Insulin Release, usually analyzed as the sum of insulin at 1 and 3 minutes following a bolus of intravenous glucose) is an additional risk factor³³. Thirty-five first-degree relatives with high-titer ICAs (> 40 JDF units) underwent serial intravenous glucose tolerance testing (IVGTT). The age of the subjects ranged from 2.6 to 66 years, the mean follow-up was 3.6 years from the first test, and 18 progressed to overt diabetes. The FPIR was calculated as the sum of the 1- and 3-minute insulin levels after a standard bolus of intravenous glucose (0.5 g per kg body weight, infused as a 20%-25% solution over 2 to 4 minutes). Percentiles for the FPIR were determined in 225 healthy, non-obese, control subjects. Even in control subjects the FPIR showed wide within-subject variation (discussed in detail below). Nevertheless, relatives with an initial FPIR below the first percentile (48 mU/l) had significantly reduced diabetes-free survival. Importantly, the presence of FPIR below the first percentile did not signify that diabetes was already present^47-50. For most of the relatives, oral glucose tolerance tests (OGTT) were also performed during follow-up to detect asymptomatic diabetes. For the survival analysis the onset of diabetes was defined by a diabetic OGTT or the occurrence of symptomatic hyperglycemia, or whichever came first. A number of recent studies are analyzing impaired fasting glucose, impaired glucose tolerance (2 hour glucose on oral glucose tolerance testing)⁵¹, c-peptide secretion⁵² and potential correlates of insulin resistance (e.g. HOMA-R) and there is evidence of abnormalities preceding diabetes even in the subset of individuals with relatively normal first phase insulin secretion^{53, 54}. The average time from the discovery FPIR below the first percentile to the onset of diabetes was 1.8 years. Sosenko and coworkers for DPT and Trialnet data have developed risk scores that primarily depend on quantitation of glucose tolerance testing. Though there is variability between subsequent tests, in general, as glucose increases risk increases especially in children^{53, 54}.

Updated data from the Joslin family study, with longer follow-up and larger numbers of relatives, have confirmed these findings. Among 79 relatives with high titer ICAs (> 40 JDF units), those with FPIR below the first percentile on the first test had 3-year diabetes-free survival of 13% (95% confidence interval: 0%-30%) compared with a 78% (95% confidence interval: 63%-93%) for the group with higher FPIR⁵⁵. Studies at the Barbara Davis Center have confirmed the predictive value of FPIR measurements as has studies from the Melbourne family study, studies from Finland ⁵⁶, and the DPT-1 (Diabetes Prevention Trial) North American study ⁵⁷ and analysis of the combined ICARUS database⁵⁸.

Data from the Joslin study also suggest that IAA add to the prediction of type 1 diabetes, but with a weaker effect than ICA⁵⁹. Forty-two ICA-positive (> 20 JDF units) relatives and 1670 ICA-negative relatives (representing a subset of all relatives found to be ICA negative) were tested for IAA. Among the ICA-negative relatives, 2.7% were IAA positive, whereas among ICA-positive relatives, 45% were IAA positive. IAA alone had less predictive value than ICA, but the combination of IAA and ICA was useful. The risk of diabetes within 5 years was 17% for the IAA-positive/ICA-negative group, increasing to 42% for the ICA-positive/IAA-native group and to 77% for double antibody positive relatives.

Reports that identifying ICA subtypes improves the predictive value of ICA can now be put in the general context of the rule that multiple biochemical autoantibodies are associated with high risk³⁶. The restricted ICA subtype (reacting with human and rat islets but not mouse [mouse islets express little or no GAD65] and with antibody staining restricted to beta cells of rat islets), defined according to the pattern of sustaining on pancreatic sections, confers a significantly lower risk of progression to diabetes than a non-restricted subtype⁶⁰. Preabsorption of sera with glutamate decarboxylase (GAD) blocks the ICA staining of restricted ICA-positive sera⁶¹ and reduces the ICA staining of most non-restricted ICA-positive sera⁶². This suggests that restricted ICA is due to antibodies directed against a single antigen (GAD) and is associated with lower risk.

The ICA assay is difficult to standardize, is labor intensive, and requires human pancreas³⁶. At the Barbara Davis Center, we utilize a combination of four assays employing recombinant antigens (insulin autoantibodies, anti-GAD, anti-ICA512(IA-2) and anti-ZnT8 ⁶³) and no longer determine cytoplasmic ICA. For children the ICA assay provides only marginal additional information, compared with the combination of defined-antigen assays. Relatives expressing two or more of IAA, anti-GAD, and anti-ICA512 have overall risk of diabetes within 5 years of more than 68% by life table analysis. The addition of intravenous glucose tolerance testing does improve prediction of the time to overt diabetes. Among relatives with two or more antibodies, those with an FPIR less than the first percentile have a 50% risk of diabetes within 1 year; those with higher FPIR have a 50% risk within 3 years³⁶.

Genetic factors can also be considered in assessing diabetes risk^64-68. Deschamps and coworkers examined the predictive value of HLA typing in a study of 536 siblings of diabetic probands in France⁶⁹. The risk of type 1 diabetes after 8 years, estimated by life table analysis, was 10% for siblings who were HLA identical with the probands, 3%-4% for siblings with either DR3 or DR4, and 16% for those with DR3/DR4. This compares with 56% for those with ICA greater than 4 JDF units and 70% for those with the combination of ICA and the highest risk HLA type, DR3/DR4. In addition studies by Becker and colleagues from Pittsburgh indicate a high risk with long-term follow-up (12.5 years) for autoantibody negative relatives with the DR3/4 (DQ8) genotype (approximately 25%) compared to 6% for those lacking this genotype and autoantibody negative⁷⁰. Even greater risk can be defined genetically for siblings of patients with type 1 diabetes who are DR3/4-DQ2/DQ8 and have inherited both HLA haplotypes identical by descent with their proband sibling. The risk for such children appears to be as high as 80% of activating anti-islet autoimmunity (by age 15) with most proceeding to diabetes with a several years delay from the appearance of autoantibodies (Figure 11.3)⁶⁷. In contrast siblings of patients with type 1 diabetes who are DR3/4-DQ2/8 who have inherited one or no HLA haplotype identical by descent with their proband have a risk of approximately 20% of progressing to diabetes by age 15.⁶⁷

Aly et al Extreme genetic risk for type 1A diabetes⁶⁷

Figure 11.3 Highest risk siblings in the DAISY study with DR3/4-DQ2/8 genotype progressing to expression of islet autoantibodies (left panel) and diabetes (right panel).

In other studies, molecular typing has revealed that the HLA haplotype DQA1*0102 DQB1*0602 confers strong protection from type 1 diabetes, in a dominant fashion (Chapter 7). In our experience, autoantibody-positive relatives with this haplotype have a very low risk of progression to diabetes⁷¹ and usually express only a single autoantibody, namely anti-GAD, although a few also express IAA. Such protection is however not absolute, and approximately 1% of children developing type 1A diabetes⁷² (versus 20% of the general U.S. population) and 3% of adults with type 1 diabetes have DQB1*0602 (DQB1*0602 is usually part of the haplotype DRB1*1501, DQA1*0102, DQB1*0602). Approximately 5% of older individuals developing type 1 diabetes are reported to have the protective HLA allele DQB1*0602⁷³.

In addition to HLA more than 50 loci contribute to risk of type 1 diabetes⁷⁴. Each locus has a small effect but a report by Winkler and coworkers suggests that combining loci can impact prediction⁶⁸. We have followed a set of identical triplets and now all three triplets have progressed to diabetes including the last triplet who was non-diabetic in 1983 (Figure 11.4).

Prediction in the General Population

It is likely that genetic typing will have an even greater impact on assessing diabetes risk in the general population^{25, 68, 75, 76}. Most studies of prediabetic subjects have involved the screening of first-degree relatives of diabetic probands, rather than the general population. However, less than 10% of new cases of type 1 diabetes have an affected relative, so the general population will need to be screened eventually if an effective intervention is to have a major impact. Screening the general population is likely to be more difficult than screening relatives. Bayes' theorem states that a screening test will have a lower positive predictive value in the general population than in a selected group with a higher prevalence of disease, such as first-degree relatives. One approach toward solving this problem is to screen the general population with markers of genetic susceptibility first, followed by autoantibody testing of susceptible individuals. For example, among the general Denver population 2.4% of individuals express both DR3 and DR4 (with associated DQ2 and DQ8). Of this subgroup, it is predicted that approximately 6% will develop type 1 diabetes, similar to the risk among first-degree relatives.

Unexpectedly, studies performed in Florida suggest that ICA have a predictive value in the general population similar to that in relatives⁷⁷. In contrast, studies in England suggest that ICA will have a lower positive predictive value in the general population. The prevalence of ICAs of 20 or more JDF units was only two to three times higher in siblings than in the general population, compared with a 13 times greater risk of diabetes in the siblings⁷⁸. Many population studies have been studied (Table 11.1). The difference between the above two studies probably relates to differences in the ICA assays, with primarily individuals with higher levels of ICA (that associated with multiple biochemical autoantibodies) followed in the Gainesville general population studies. With analysis of biochemical autoantibodies it appears that even general population individuals expressing multiple anti-islet autoantibodies are at very high risk of progressing to diabetes similar to first degree relatives.⁷⁹ Those expressing single autoantibody (of insulin, GAD, IA-2 or ZnT8 autoantibodies) are at low risk for progression. This very likely results from the high specificity of expression of multiple autoantibodies if the islet autoantibody assays are set at the 99^th percentile. Assuming independent “false positives” one can use the binomial theorem to calculate the probability of expressing two or more of the four autoantibodies by chance, and this probability is very low (.05%, specificity .9995; Binomial theorem Pn(k)=n!/(n-k)! times p^k*(1-p)^n-k where p=probability of antibodies positive in given population, n= number of trials, and k=number of antibodies positive). With four biochemical autoantibodies to measure n=4, K=2,3,and 4 for >=2 autoantibodies, and for control population with assays set with 99% specificity, p=.01. We believe it is important to utilize assays with high specificity as well as >=2 autoantibodies in evaluation of individuals without diabetes or individuals clinically having type 2 diabetes with attempts to diagnose LADA (Latent Autoimmune Diabetes of Adults). For instance if assay specificity is 95% with four assays approximately 20% normals will express a single autoantibody but only 0.25% would express >=2 autoantibodies. As shown in DASP (Diabetes Autoantibody Standardization Program of the Immunology of Diabetes Society and the CDC) the different assays vary as do the different laboratories^{80, 81}. In particular many laboratories have great difficulty measuring insulin autoantibodies⁸², while most laboratories have high sensitivity/specificity GAD65 autoantibody assays and IA-2 assays. The difference appears to relate to the separation of signals between normal control samples and patients with Type 1 diabetes, with relatively separation for approximately ½ of patients positive for insulin autoantibodies. A new electrochemiluminescent assay has the potential to improve standardization of insulin autoantibodies⁸¹.

Figure 11.4: Development of autoantibodies and loss of first phase insulin secretion in identical triplets of a patient with type 1A diabetes.

Several studies have now been initiated where children are followed from birth for the development of anti-islet autoantibodies. The three studies with the longest follow-up are the BabyDiab study from Germany, the DAISY study from Denver Colorado, and major studies in Finland^{39, 83-86}. The BabyDiab study evaluates offspring of patients with diabetes while the DAISY study has prospectively followed both first degree relatives (offspring and siblings) as well as children stratified by HLA type from the general population. All three studies are providing generally concordant results. Anti-islet autoimmunity frequently develops in the two years of life, but can develop at any age²⁶. Often insulin autoantibodies appear first, but GAD65 and less often IA-2 and ZnT8 autoantibodies can also be the first to develop^{11, 87}. Several years can elapse between the appearance of insulin and GAD65 autoantibodies in young children before IA-2 and ZnT8 autoantibodies develop. The presence of multiple anti-islet autoantibodies in young children portends a high risk of progression to diabetes for both relatives and the general population^{31, 36}. High titer autoantibodies and a broad immune response (multiple different molecules, multiple epitopes of given autoantigens, multiple immunoglobulin subclasses) are associated with higher risk as is high affinity anti-insulin autoantibodies¹¹. Of note the insulin autoantibodies that first occur in young children are already of high affinity when first detected⁸⁷. Lower affinity insulin autoantibodies are more often transient or not associated with expression of autoantibodies reacting with multiple islet antigens, and thus of lower risk (Figure 11.5). IA-2beta autoantibodies enhances risk, with almost all patients who have IA-2 beta also having IA-2 autoatnibodies⁸⁸.

Figure 11.5 Affinity of insulin autoantibodies of children in the BABY-DIAB study, with high affinity autoantibodies associated with development of multiple islet autoantibodies and progression to diabetes.

It is likely that there is no age at which a genetically susceptible individual has no increased risk of converting to anti-islet autoantibody positivity⁸⁹, but progression to diabetes is usually less rapid in older individuals. Figure 11.4 represents updated follow up of a set of non-diabetic monozygotic triplets of a patient with type 1 diabetes, where a second triplet progressed to diabetes at age 21, and at 42 years of age the remaining non-diabetic triplet developed anti-islet autoantibodies and has now progressed to diabetes at age 57. A recent publication updates our studies of monozygotic twins with very high concordance for anti-islet autoimmunity (>60%) given long-term follow up of initially discordant identical twins⁹⁰ In addition the age of onset of the proband twin and the HLA DR/DQ genotype of the twins contribute to differences in long term risk (higher cumulative risk the younger the age of onset of the proband and higher risk with the DR3/-DQ2/8 genotype).

Figure 11.6

Prediction/Diagnosis in Adults

Both patients with gestational diabetes (Figure 11.7) and adults with a diagnosis of type 2 diabetes (Figure 11.6)⁹¹ have a significant risk of having type 1A diabetes^{92, 93}. Between 5 and 10% of patients with a diagnosis of gestational diabetes express anti-islet autoantibodies and the great majority progress to type 1A diabetes. In a similar manner, the diagnosis of type 1 diabetes is associated with expression of islet autoantibodies⁹⁴ in adults thought initially to have type 2 diabetes^{95, 96, 96}. Assays for GAD65 autoantibodies are the most useful for type 1A diabetes in both patients with gestational or adult onset type 1 diabetes^97-99 and the term LADA (Latent Autoimmunity of Adults) has been applied to latter this group. ¹⁰⁰ In addition to autoantibody assays specialized assays for detection of T cells reacting with islet autoantigens may identify a subset of patients with a clinical diagnosis of Type 2 diabetes who have anti-islet autoimmunity.^{101, 102} At present such T cell assays are performed in relatively few laboratories without workshop standardization.

Figure 11.7 Progression to insulin dependent diabetes of individuals with gestational diabetes (diabetes developing during pregnancy) analyzed relative to the number of anti-islet autoantibodies.

At What Stage in Life is Beta Cell Autoimmunity Initiated, What Is the Initial Target Antigen, and in What Sequence Do Different Autoantibodies Appear?

Studies in first-degree relatives have detected evidence of beta cell autoimmunity many years before clinical presentation with overt hyperglycemia^{27, 28, 103}. However, the existence of young infants with type 1A diabetes indicates that beta cell destruction may also occur rapidly in some individuals. A plausible hypothesis is that beta cell autoimmunity can begin early in life, with variation in the age of onset of overt diabetes explained by differences in the rate of beta cell destruction from one individual to another, but there is also extreme variation in the age which autoantibodies first develop. Thus both the timing of appearance of islet autoantibodies and the rate of progression once autoantibodies are detected are associated with age of onset²⁵. The first autoantibody to appear is usually insulin autoantibodies, followed by GAD65 autoantibodies (Figure 11.8) but this is a generalization, as is the observation that IA-2 and ZnT8 autoantibodies follow after several years. Any of the autoantibodies can be the first or the only autoantibody expressed. There is a misconception that the levels of insulin autoantibodies are determined by the age of the individual. Though levels of insulin autoantibodies are much higher in children developing type 1 diabetes with onset less than age 5¹⁰⁴, when prediabetic children are followed from birth the levels are not related to age but the rate of progression to diabetes²⁵. Figure 11.9 illustrates a child in the DAISY study followed from birth with 13 years of autoantibody positivity before progression to diabetes. Such long prodromes are associated with either none or low levels of insulin autoantibodies (bottom line) even at one year of age.

Figure 11.8 Cumulative development of islet autoantibodies in the BabyDiab study 2009).

In support of the hypothesis that the timing of diabetes onset is influenced by factors such as insulin resistance, a major peak in the incidence of type 1A diabetes occurs during early adolescence in nearly all populations studied¹⁰⁵. This increased incidence during adolescence is probably due to the insulin resistance associated with puberty, with individuals requiring more insulin than can be produced given beta cell destruction.

Figure 11.9 Child with long prodrome preceding diabetes with typical lack of insulin autoantibodies throughout follow up

After the age of about 15 years, measuring the incidence of type 1A diabetes is complicated by misclassification of some patients as type II diabetics. Nevertheless, it has been estimated that at least 37% of type 1 diabetes occurs after the age of 19 years and 15% after the age of 30 years¹⁰⁶. Adult autoimmune diabetes differs relatively little genetically from pediatric diabetes¹⁰⁷. It has been suggested that there is an excess of type 2 diabetes in parents of children with type 1A diabetes, but a report of the analysis of parents in the Barts-Windsor study indicates that the majority of diabetic parents have type 1A diabetes, and the prevalence of type 2 diabetes is not higher compared to the general population¹⁰⁸.

Table 11.1

Country	Age Range	Number Screened	Prevalence of ICA	Cutoff (JDF Units)	Incidence of Type 1 Diabetes (a)	References
Japan	7-18	473	0.4%	--	2.0	^109, 110
Germany	6-21	4287	1.05%	>/=5	7.0	^111, 112
			0.4%	>/=20
France	6-17	8363	1.79%	>/=4.5	7.1	^113, 114
			0.2%	>/=24
Netherlands	5-19	4806	0.42%	>/=.06	11.0	^115, 116
Spain	14-17	2291	0.35%	>/=5	11.3	^117, 118
New Zealand	5-7	20000	1.2%	>/=10	12.8	^119, 120
USA
Florida	5-18	9696	0.59%	>/=10	13.7(b)	^77
Washington State	12-18	3992	1.7%	>/=1		^121
Pennsylvan						^122
Australia	4-18	1900	0.68%	>/=20	14.5	^123, 124
England	9-13	2925	2.8%	>/=4	15.6	^78, 125
			0.8%	>/=20
Sweden	0-14	420	3%	>/=5	23.6	^126, 127
Finland	3-18	1212	4.1%	>/=3	35.3	^128, 129

(a) Per 100,000 per year in the 0-14 age group

(b) Incidence in Pennslyvania

Table 11.1. The Prevalence of Islet Cell Antibodies (ICA) among Schoolchildren in the General Population of Different Countries Compared with Incidence of Type 1 Diabetes.

New cases of type 1A diabetes presenting in adult life tend to have a longer duration of symptoms before diagnosis and higher C peptide levels remaining at diagnosis compared with those presenting in childhood¹²⁹, suggesting a slower rate of beta cell destruction. "Slow onset" type 1A diabetes, presenting in late adult life, may be diagnosed initially as type II diabetes. Tuomi and coworkers studied 102 adults who were treated for type II diabetes, of whom one third had low stimulated C peptide levels and two thirds had normal levels¹³⁰. Of the group with low C peptide, 76% were positive for anti-GAD (similar to the frequency among children with newly diagnosed type 1A diabetes). In comparison, only 12% of this group with normal C peptide was positive for anti-GAD.

In many studies, it is unknown how long autoantibodies were present in these individuals before they were discovered. However, studies by Pilcher and Elliott in New Zealand suggest that ICAs develop early in childhood and are usually preceded by IAA¹³¹. In the studies 666 first-degree relatives, aged less than 20 years and initially ICA negative (<10 JDF units), were followed longitudinally. Those under 5 years of age were retested annually, with older subjects retested every 3 to 5 years. Sixteen (2.4%) became ICA positive during follow-up, with seroconversion occurring at an average age of 3.2 years, ranging from 1.6 to 7.1 years. All except the two oldest seroconverters were IAA positive at the time of seroconversion and the appearance of IAA was documented to precede ICA in 6 (46%) of 13 seroconverters who could be retrospectively checked for IAA.

Ziegler and coworkers in Germany studied a cohort of infants of mothers with type 1A or gestational diabetes^{132, 133}. Data from the study also indicate that autoantibodies may appear very early in life, with IAAs appearing first¹¹. A high frequency of both IAAs and ICAs was found in cord blood samples. This was attributable to transplacental passage of maternal IgG and to falsely positive IAA results (that may be obtained in up to one half of cord blood samples from normal pregnancies)¹³⁴. However, by 9 months of age all became ICA negative and only 3 of 90 infants remained IAA positive. By 2 years of age, one of these infants became IAA negative, two remained IAA positive, and another child became persistently IAA positive for the first time. It is not possible to absolutely distinguish in the first year of life transplacental anti-islet autoantibodies from autoantibodies produced by the infant but the presence of IgG1-insulin autoantibodies was associated with autoantibody persistence and risk of type 1A diabetes¹³³. Measurement of cord blood or mothers serum at birth allows distinction, with extremely high levels of the autoantibodies usually associated with autoantibodies in the infant that persist for up to 12 months. Transplacental anti-insulin autoantibodies are usually of the IgG4 subclass in contrast the dominance of IgG1 spontaneous autoantibodies¹³⁵. A rising level of autoantibodies is almost always associated with spontaneous autoantibodies.

Both insulin and GAD have been suggested as candidates for the initiating antigen but another (unidentified) autoantigen could be responsible^{136, 137}. Neither IAA nor anti-GAD is universally present among patients with newly diagnosed type 1A diabetes^{104, 138-140, 140-143}. Of note a small subset of children followed from birth in the DAISY study have expressed multiple islet autoantibodies that were lost prior to the onset of diabetes. It is clear that although insulin autoantibodies usually appear first a significant percentage of children followed from birth initially express GAD65 autoantibodies, while IA-2 and ZnT8 autoantibodies are usually the last to develop. With improved assays for insulin autoantibodies⁸¹, in the DAISY study insulin autoantibodies are almost always first or present with other autoantibodies and >95% of those progressing to diabetes at some point express insulin autoantibodies (DAISY study, unpublished.)

Several observations provide circumstantial evidence for insulin as an initiating autoantigen. Insulin is the only antigen unique to the beta cell and it is present on the cell surface^{144, 145}. In contrast, GAD is also present in alpha cells¹⁴⁶, which are not destroyed¹⁵, and insulitis disappears when all insulin-containing cells have been destroyed¹⁴⁷. IAAs occur with greater frequency and at higher levels in younger newly diagnosed patients^148-150 and the level of IAA correlate with the rate²⁵ of beta cell destruction in prediabetic individuals¹⁵¹. When 29 patients were studied, their IAAs recognized the same epitope¹⁵². Williams and coworkers improved the assay for insulin autoantibodies with the development of a microassay^{153, 154}. This assay utilizes protein A or protein A/protein G for autoantibody precipitation and thus avoids the false autoantibody positives for cord or hemolyzed blood associated with the polyethylene glycol based assays. We have modified the Williams assay such that it is performed in 96-well micro titer plates utilizing membrane filtration and direct b-counting in the micro titer plate¹⁵⁵. With this assay not only do prediabetic individuals express readily detectable anti-insulin autoantibodies but also NOD mice express high levels of the autoantibody¹⁵⁵. Of note administration of a dominant peptide of insulin (insulin B chain peptide B:9-23) induces autoantibodies to insulin, even in Balb/c normal mice that react with intact insulin and are not absorbed by the immunizing peptide¹⁵⁶. This autoantibody induction is MHC restricted, suggesting that with the MHC of either Balb/c or NOD mice, T cell activation by an insulin peptide activates already sensitized B-lymphocytes recognizing insulin¹⁵⁶. In addition in the NOD mouse model an insulin 1 gene knockout almost completely prevents the development of diabetes, while an insulin 2 gene knockout (the mouse insulin expressed within the thymus) accelerates the development of diabetes¹⁵⁷, and NOD mice with both native insulin genes knocked out (with mutated insulin transgene) do not develop diabetes¹⁵⁸. Insulin peptide B:9-23 is a dominant NOD target recognized in a specific MHC class II register^{159, 160}

Are there measurable abnormalities that precede development of islet autoantibodies.

With the existence of many children, both relatives and general population children, followed from birth to the development of diabetes and high throughput, proteomic, metabolomic^{161, 161-163}, messenger RNA assays, as well as improving T cell assays a number of investigators have reported abnormalities that may precede development of islet autoantibodies. This includes reported abnormalities of lipids, metabolites such as glutamate reported from studies in Finland, evaluation of serum effects on messenger RNA expression arrays from Wisconsin, and T cell assays evaluating LADA patients in the state of Washington^{101, 102, 164} At present with single reports with identification of different metabolites and lack of standardization and utilization of such assays by multiple groups there is not a clear consensus that specific abnormalities precede the development of islet autoantibodies. We personally doubt there will be “non-genetically” determined abnormalities preceding the appearance of islet autoantibodies. Given type 1A diabetes as a T cell mediated autoimmune disorder an analogy would be to search for metabolomic abnormalities preceding anti-polio antibodies induced by polio vaccination.

Hampe and coworkers have reported the presence of anti-idiotypic antibodies that mask the presence of GAD65 autoantibodies in normal controls, with the additional report that such anti-idiotypic antibodies are not present in patients with diabetes, and that individuals progressing to diabetes lose anti-iditiotypic antibodies¹⁶⁵. In that the detection of anti-idiotypic antibodies relies upon the use of columns with human monoclonal anti-GAD autoantibodies, We are not convinced as to the actual presence of anti-idiotypic antibodies and this is an area being further studied¹⁶⁵ and a recent report correlated changed of anti-idiotypic antibodies with c-peptide changes^{166, 167}.

Are Environmental Factors Important, Either in the Initiation of Beta Cell Autoimmunity or in Modulating the Process Once It Has Begun?

The diabetes susceptibility genes identified so far are present in a high proportion of the general population, indicating that they are not sufficient to cause the disease. Furthermore, the concordance rate observed in HLA-identical siblings is lower than that in identical twins^{35, 168} and there are more than 50 loci discovered^{68, 74, 169}. As type 1A diabetes is associated with other autoimmune diseases^{170, 171}, it is known that at least some of the important genes will not be specific to type 1A diabetes but also involved in the susceptibility to other^{172, 173} autoimmune diseases¹⁷⁴ such as celiac disease¹⁷⁵ and rheumatoid arthritis¹⁷⁶.

Despite the importance of genetic factors, the concordance rate is less than 100% in identical twins^{148-150, 177} and the rapidly increasing incidence of Type 1 diabetes in Western societies¹⁷⁸ implies a critical role for non-genetic random re-arrangement of T cell receptor or immunoglobulin genes during the differentiation of T and B lymphocytes¹⁷⁹, somatic mutation, and or environmental factors. Epidemiological studies with validated case ascertainment support a role for environmental factors. A rising incidence of type 1A diabetes over time, too rapid to be explained by an increase in the frequency of diabetogenic genes in the population, has been reported in multiple countries^180-189. In addition, increased rates have been observed in migrant populations compared with their countries of origin¹⁹⁰, though the change in migrant populations is relatively small¹⁹¹.

An environmental factor could be involved either by initiating autoimmunity or by altering its progression, once established. There is a seasonal variation in the onset of type 1A diabetes^{123, 126, 180, 181, 192}, with more cases in the winter months, associated with viral infections. It is likely that such viral infections alter the process late in its course by increasing insulin requirements, thereby advancing the time of diabetes onset. In humans, the best evidence for the involvement of the specific viral agent in the initiation of autoimmunity comes from the observation that patients with congenital rubella have an increased prevalence of type 1A diabetes^{193, 194}, as well as other autoimmune diseases¹⁹⁵. Of note anti-islet autoantibodies are relatively uncommon¹⁹⁶. Coxsackievirus B infection (enterovirus^{197, 198}) has also been suggested as an environmental trigger¹⁹⁹, although a large Swedish study found no significant difference in the levels of IgM antibodies against Coxsackievirus B types 1-5 between newly diagnosed diabetic children and population controls²⁰⁰. There is likely to be a long lag time between a triggering infection and the onset of diabetes (perhaps short time between infection and anti-islet autoantibody induction[weeks])but IgG levels were not measured in this study. It has been suggested that an infectious agent might activate autoimmunity by molecular mimicry, whereby the immune response to a viral antigen, such as the P2-C protein of Coxsackievirus B4²⁰¹ or the rubella capsid²⁰², might cross-react with a beta cell antigen. Some evidence against a significant role for an infectious agent comes from Japan. Despite wide variation in the incidence rates for infectious diseases in different parts of the country, the incidence of type 1A diabetes is uniformly low²⁰³ with the additional occurrence of an unusual fulminant form of diabetes²⁰⁴. Approximately 20% of Japanese type 1 patients have the fulminant form²⁰⁵. There are several case reports from Japan of induction fulminant diabetes by medications inducing drug induced hypersensitivity syndrome (DIHS)^{206, 207}. Given recent greater understanding of the pathogenesis of DIHS this suggests that profound T cell activation can induce beta cell destruction^{208, 209}.

At present studies of potential environmental factors triggering the development of anti-islet autoantibodies in children followed from birth have not led to the identification of major precipitating factors, and contradictory results are a problem²¹⁰. Studies from Finland have implicated both enterovirus infection and ingestion of bovine milk formula^{86, 211, 212}. In contrast studies from DAISY and BabyDiab have failed to associate these factors with the initiation of autoimmunity^213-215. A report of rotavirus infection ²¹⁶ associated with development of anti-islet autoantibodies from the Australian Baby study has not been replicated with studies from Finland²¹⁷. It is likely that environmental factors contributing to the initiation of anti-islet autoimmunity are relatively ubiquitous and multiple factors may contribute. This is readily demonstrated in animal models where injection of poly-IC into multiple rat strains with the HLA haplotype RT1-U develops either insulitis and/or diabetes. Poly-IC (inosinic cytodylic acid) is a mimic of viral RNA and a potent inducer of interferon-a through Toll receptors of the innate immune system^{218, 219}. A number of viruses (including KRV: Kilham rat virus) induce diabetes in a Lewis strain as well as BB-DR (diabetes resistant) rats.²²⁰ The rat strains that develop diabetes share the same high risk alleles in the major histocompatibility complex. Induction islet autoimmunity in these models follows activation of innate immunity and can be blocked by a single injection of dexamethasone²²¹.

In oral communications Dotta and coworkers have reported the finding of enterovirus immunoreactivity within islets that have died with new onset diabetes. The number of pancreases analyzed is small (approximately 6) but ½ of patients had enteroviral antigen within islets,. Apparently an enterovirus sequenced was a laboratory related sequence. The presence of viruses in a subset of pancreases of patients with type 1 diabetes is an active area of investigation.

In addition, in animal models of type 1 diabetes, exposure to infectious agents may suppress rather than trigger autoimmunity. Diabetes-prone BB rat housed in "viral antibody free" conditions have a higher frequency of diabetes than those housed in less stringent "specific pathogen free" conditions²²². In contrast NOD mice with a specific mutation blocking toll receptor signaling have autoimmune diabetes dependent upon specific gut flora²²³. Specific infection with mouse hepatitis virus is associated with reduced incidence of diabetes in the NOD mouse²²⁴, and infection with lymphocytic choriomeningitis virus is associated with reduced incidence in both the NOD mouse and BB rat^{225, 226}. However, in at least one instance infection is implicated as a trigger. Kilham's rat virus, a parvovirus, has been shown to trigger beta cell autoimmunity and diabetes in the diabetes-resistant BB rat²²⁷. It appears that the Kilham virus acts similar to poly-IC rather than by infecting islet cells²²⁸. Other reports of viruses triggering diabetes in animals may involve direct viral damage of beta cells without an autoimmune mechanism²²⁹ or bystander stimulation^{230, 231}.

In humans, associations with infant feeding have been reported. Several population-based case-control studies found a protective effect associated with breast-feeding^232-234 and an increased risk associated with the early introduction of supplemental feeding^{232, 233, 235}. A Danish study, which eliminated several possible sources of bias by using the records of postnatal health visitors, found no evidence to support an associated with the total duration of breast feeding²³⁶, but did not examine the timing of introduction of supplemental feeding. Both BabyDiab and DAISY found no evidence for infant bovine milk ingestion²¹³ but a pilot study of the elimination of bovine milk from infant formula was associated with a reduction of development of cytoplasmic ICA, with little reduction of GAD65 autoantibodies²¹².

The reported associations with infant feeding are weak (odds ratios ~1.5)²³⁷, but this may be consistent with the effect of a common exposure acting only on genetically susceptible individuals. Several mechanisms could explain the associations with infant feeding. Breast-feeding could protect the infant from an infection. The increased caloric intake and weight gain associated with artificial feeding might cause increased insulin secretion and presentation of beta cell autoantigens²³⁸. Karjalainen and coworkers have suggested that intact cow's milk peptides might cross the immature gut, initiating an immune response to cross-reacts with a beta cell surface antigen(146). Higher levels of antibodies against several components of cow's milk have been reported in children with newly diagnosed type 1A diabetes than in controls^239-243. T cell proliferation in response to the ABBOS peptide was higher in newly diagnosed patients than in controls²⁴⁴. These results could not be reproduced by Atkinson and coworkers(153).They found no difference between newly diagnosed patients and controls for either anti-BSA antibodies or T cell proliferation to ABBOS. In animal studies, the addition of cow's milk to the diet of the BB rat at the time of weaning increases the incidence of diabetes^{245, 246}, but there are conflicting reports on the effect of cow's milk in the diet of the NOD^{247, 248}. Of interest there is insulin in both human milk and bovine milk and a reported induction of anti-bovine insulin antibodies with bovine milk ingestion²⁴⁹ as well as an initial clinical trial of removing insulin from milk ²⁵⁰.

Ziegler and coworkers and Norris and coworkers (figure 11.10) have reported in JAMA an association with the induction of anti-islet autoantibodies with the introduction of cereals/gluten prior to three months of age^{251, 252}. In the report by Norris and coworkers, there was a biphasic association with autoantibodies, namely increased risk with introduction before 3 months and after 6 months of age. Decreased omega-3-fatty acid consumption has been reported to be associated with increased risk in the DAISY study^{253, 254}. The National Institutes of Health have established a major international consortium termed TEDDY (The Environmental Determinants of Type 1 Diabetes in the Young) that will follow infants from birth for the development of anti-islet autoimmunity and diabetes⁷⁵. No association with development of islet autoantibodies with Vitamin D intake was found in the DAISY study²⁵⁵.

Figure 11.10: Life table analysis of expression of anti-islet autoantibodies for infants followed from birth relative to the age of introduction of cereal/gluten.

Once Autoimmunity Is Initiated, Are Beta Cells Destroyed in a Progressive, Linear Fashion or Are There Spontaneous Remissions?

The long-term follow-up of high-titer ICA-positive relatives in the Joslin family study with serial IVGTTs suggested a model in which beta cell destruction is a progressive, linear process^{256, 256-258} . The rate of beta cell destruction may vary widely from one individual to another. It may be so slow in some individuals that such overt diabetes does not develop during the person's lifetime. Such individuals might appear to have decreased but stable beta cell function when assessed with serial IVGTTs over many years. A correlation between serial FPIR levels and the number of years to diabetes in ICA-positive relatives²⁵⁹, and the addition of IAA as a risk marker²⁶⁰, led to the development of a "dual parameter" linear regression model¹⁵¹. In this model the time remaining before the onset of diabetes is predicted by a combination of the FPIR (reflecting the remaining beta cell mass) and the IAA level (reflecting the rate of loss of beta cells). Recent analysis of children followed from birth indicates age at appearance of islet autoantibodies and mean levels of insulin autoantibodies predict approximate age of diabetes onset²⁵.

Other studies, including relatives with lower titer ICA, have suggested that progression to diabetes may be less predictable^261-263. They have suggested an alternative model, in which the rate of beta cell destruction may fluctuate, with spontaneous remission in some individuals. This model is similar to that suggested for autoimmune thyroiditis²⁶⁴. Reports of transient ICA in relatives^{261, 265-267}, in discordant identical twins(169), in the general population^{113, 127, 268, 269}, and in patients initially treated for type II diabetes²⁶³ lend support to this hypothesis. Most, but not all^{261, 267-269} of these transient ICAs have been reported in subjects with initially low titers, raising the possibility that the phenomenon may frequently be explained by variability in the ICA assay²⁷⁰. Some of these studies did not report the levels of ICA in terms of endpoint titer or JDF units^{263, 265, 266}. However, several confirmed the change in ICA status by retesting this sera from all time points for a given subjects in one assay^{127, 261, 263, 266}. Even with the utilization of “biochemical” autoantibody assays there is some conflicting data concerning the persistence of anti-islet autoantibodies, but a number of prospective studies suggest that individuals with readily detectable levels of islet autoantibodies, high risk HLA genotypes of multiple autoantibodies, have extremely persistent autoantibodies^271-273. It is likely that even with “biochemical” autoantibody assays, a subset of autoantibody positivity is not associated with islet autoimmunity and in particular individuals expressing a single islet autoantibody. For instance we have recently found that for the rare individual with transient ICA512 autoantibodies, the autoantibodies do not recognize multiple epitopes of the molecules in sharp contrast to the persistent autoantibodies associated with diabetes risk and in prospective studies screening general population a significant percentage of low titer anti-islet autoantibodies are either not confirmed or not persistent²⁷⁴. Studies measuring multiple anti-islet autoantibodies “suggest” that all family members with multiple islet autoantibodies are destined to develop autoimmune diabetes²⁷⁵. The latter statement is likely to be perhaps too strong as rarely someone expressing multiple anti-islet autoantibodies loses expression of all autoantibodies without having developed diabetes, though some prospectively followed children who lose expression of autoantibodies have gone on to Type diabetes. It is important not to count cytoplasmic ICA as a distinct autoantibody as it may represent GAD, IA-2 or ZnT8⁷ autoantibodies as on e counts numbers of “biochemical” autoantibodies to identify extreme risk.

Figure 11.11 Progression of autoantibody positive first degree relatives to diabetes, subdivided by the number of biochemical autoantibodies expressed.

Low-titer ICAs carry a reduced risk of type 1A diabetes, compared with high titer^{27, 28}, and it may be that fluctuating levels do not signify a disease process, especially unaccompanied by other autoantibodies. Non-persistent CAs may represent a nonspecific immunological response associated with infection, as Helmke and coworkers reported the transient appearance of ICAs in normal schoolchildren during mumps infection²⁶⁸. The development of more reproducible assays for antibodies against biochemically defined autoantigens may improve the assessment of diabetes risk. For relatives who express more than one autoantibody, levels of IAA⁵⁹, anti-GAD, and anti-ICA512 can be remarkably persistent over time^{11, 271, 276}.

Carel and coworkers reported that ICA-negative siblings of diabetic children have significantly lower mean FPIR than controls²⁷⁷. They suggested that autoimmune beta cell destruction may have been initiated in some of these siblings, but spontaneously remitted. However, other autoantibodies may have been present in some of the ICA-negative siblings in this study. At most, only 94% of children with newly diagnosed type 1A diabetes are ICA positive, using a very sensitive assay²⁷⁸ but the addition of other autoantibodies may reduce the autoantibody-negative proportion. In addition, although the diet was standardized for the 3 days prior to the IVGTT, there may be differences over the longer term in the diets of children with and without a diabetic sibling that could have affected the IVGTT results.

Experiments with streptozotocin-treated baboons indicate that beta cell function (assessed by IVGTT) correlates with beta cell mass²⁷⁹. In humans, however, the IVGTT has several limitations. The results of beta cell function tests may vary with insulin sensitivity according to age^{277, 280}, physical fitness²⁸¹, body mass index^{280, 282}, pubertal status^{282, 282, 283, 283, 283}, and physical or psychological stress. Different protocols for the IVGTT give different results²⁸⁴ but a standard protocol has been developed to allow meta-analyses²⁸⁵.

Figure 11.12 Intravenous glucose tolerance insulin levels of antibody positive markedly obese female progressing to diabetes with fasting insulin typically above 25 (illustration 2 times fasting insulin) and sum 1+3 minute declining progressively, until there is no first phase secretion at onset of diabetes.

The interpretation of serial FPIR data is complicated by within-subject variability in the measurement. Figures 11.12 and11.13 illustrates the course of the FPIR over time for two ICA-positive relatives. One has a clearly linear loss of secretion (Fig. 11.12). In contrast, the other relative (Fig. 11.13) has what appears to be a stepwise loss of secretion, with remissions and periods of recovery. However, this second pattern could result

Figure 11.13 Marked variation in loss of first phase insulin secretion on intravenous glucose tolerance testing during progressing to diabetes (years prior to diabetes from right to left).

from wide within-subject measurement variation, superimposed on an underlying loss of secretion. In the large DPT-1 study loss of first phase insulin secretion was strongly associated with diabetes risk⁵⁷ as it was in the combined ICARUS dataset⁵⁸ and with large datasets considerable variability exists in the patterns of loss of first phase insulin secretion prior to diabetes. A significant percentage of prediabetic individuals have retained first phase secretion within one year of diabetes, with many of these appearing to have high fasting insulin and potentially severe insulin resistance. Recent data in the youngest children expressing anti-islet autoantibodies indicates that at the time of detection of autoantibodies many of those children already have severely depressed FPIR, though a higher risk for diabetes is also in that study associated with an average earlier onset of diabetes⁵⁶ (Figure 11.14).

Figure 11.14 Early loss IVGTT response in ICA+ infants that have not (Panel B) or have (Panel C) progressed to diabetes.

Several centers have performed duplicate IVGTTs, spaced 1 or more weeks apart, on healthy volunteers. The median within-subject coefficients of variation (CV) reported for FPIR varied from 4% to 36%^{280, 286, 287} but the CV was as high as 56% for some subjects. There are conflicting reports about whether calculating the area under the curve for insulin release improves reproducibility, compared with a sum of the 1- and 3-minute insulin levels^{280, 286, 287}. McCulloch and coworkers studied 18 normal post pubertal subjects on two occasions separated by an average of 1 year²⁶¹. They used an IVGTT protocol with frequent sampling and a bolus of tolbutamide at 20 minutes, allowing the calculation of the insulin sensitivity index. The within-subject CV for the incremental area under the insulin curve (above fasting levels) from 0 to 10 minutes was 11.3%. Adjusting for the insulin sensitivity index resulted in a similar CV (10.5%). However, it is likely that adjusting for insulin sensitivity may improve reproducibility in pubertal subjects and those followed over a longer period. The subjects also underwent arginine stimulation tests (both at physiological plasma glucose concentration and hyperglycemic clamp). The reproducibility of the response to arginine was similar to that of the response to glucose, indicating little advantage gained by the use of arginine.

Rayman and coworkers achieved highly reproducible results using retrograde venous cannulation and arterialization of the hand from which the samples are drawn²⁸⁶, but recent studies found no improvement with these techniques^{288, 289}. Other maneuvers that have been evaluated to improve reproducibility include placement of the intravenous line 1 hour before the test to minimize the effects of stress hormones. Allen and coworkers tested the hypothesis that catecholamine release, associated with anxiety about the test, may inhibit insulin release but found no correlation between FPIR and plasma catecholamine levels or standardized anxiety scores²⁸⁰. However, a significant "first test effect" was observed. Among 11 normal subjects who underwent two tests, 9 had a higher FPIR on the second test²⁸⁰. One subject who fainted during the first test had a very low FPIR (2 mU/L) that became normal on a subsequent occasion.

Though loss of first phase insulin secretion is a sensitive marker of beta cell abnormalities and aids in the prediction of diabetes it is a labor intensive test. At present in the DAISY study we follow autoantibody positive individuals with measurement of HbA₁c with fingerstick blood sampling. For the great majority of children progressing to diabetes HbA₁c begins to rise one to several years prior to the onset of diabetes in the normal range²⁹⁰. When HbA₁c approaches the upper limit or exceeds the upper limit of normal we perform a formal oral glucose tolerance test to confirm the diagnosis of diabetes. In addition fasting glucose, glucose at 120 minutes on oral glucose tolerance testing, and C-peptide on oral glucose tolerance testing can all be used to stage progression to diabetes of islet autoantibody positive individuals24673}. HbA₁c >6.5% has an approximate 25% sensitivity to identify early diabetes in prospective studies with high specificity²⁹¹.

The FPIR is a measure of beta cell function, but a measure of beta cell mass might be more useful. In the future, new ways of assessing the beta cells may be developed, including high-performance glycated hemoglobin assays and imaging with nuclear magnetic resonance (MRI) or isotopic scans. Leech and coworkers measured the HbA₁c by high-performance liquid chromatography and healthy teenagers who were also tested for ICA²⁹². The mean HbA₁c was slightly, but significantly, higher in the ICA-positive subjects, suggesting that elevation of the HbA₁c within the normal range may be an early marker of loss of beta cell function. Signore and coworkers reported preliminary data suggesting that scans following the injection of²⁹³ I-labeled interleukin-2 (IL-2) may be useful in the detection of beta cell autoimmunity²⁹⁴. A significant accumulation of tracer was detected in the pancreatic region of four newly diagnosed and two prediabetic subjects, compared with four controls. MRI has been used to detect pancreatic graft rejection^295-297. With refinements in MRI techniques it may be possible in the future to detect insulitis or to approximate beta cell mass^{298, 299}. In the NOD mouse model with its extensive insulitis several methods for detecting insulitis using iron particles have been developed. One apparently measures vascular leakage and the other specific binding of autoantigenic T cells to their cognate receptor. Studies to apply such techniques to man are underway, but the amount of insulitis given current analysis of pancreas from patients of the nPOD study indicate that insulitis can be be minimal³⁰⁰. In addition several groups are attempting to develop technology for non-invasive imaging of beta cell mass with at present controversial results³⁰¹.

There is little information on changes in islet histology before the onset of overt diabetes in humans. However, Sutherland and coworkers studied patients with longstanding type 1A diabetes who received pancreatic transplants from their non-diabetic identical twins¹⁴⁷. Of these, three received either delayed immunosuppression or no immunosuppression after the transplants and all three became insulin dependent again within 5 to 12 weeks. Serial biopsies of the graft revealed T cell infiltration with selective beta cell destruction, unlike graft rejection. Rather, these histological findings were consistent with reactivation of beta cell autoimmunity. Of note, the insulitis disappeared after the insulin-containing cells were destroyed.

Foulis and coworkers studied the pancreatic histology of 119 patients with type 1 diabetes who had died of ketoacidosis, of whom 60 had died within 1 year of diagnosis¹⁵. More recent studies from Japan indicate that the prescience of anti-islet autoantibodies correlate with insulitis on biopsy. They found that insulitis is a patchy process, in which islets with or without insulitis tend to be grouped separately in different lobules. At diagnosis, the beta cells in some islets were destroyed while other islets were left untouched, but most islets were insulin deficient after a 1-year duration of diabetes. Hanafusa and coworkers performed pancreatic biopsy on seven Japanese adults, 2-4 months after the diagnosis of type 1 diabetes. The islets were atrophic, with reduced or absent insulin staining, but surprisingly there was hardly any lymphocytic infiltration³⁰². A form of diabetes characterized by marked hyperglycemia yet with normal HbA₁c suggests extremely rapid development of disease and because there was a lack of anti-islet autoantibodies was originally thought to be a form of type 1B(non-immune mediated) diabetes. Subsequent studies from Japan have revealed the presence of HLA alleles associated with type 1A diabetes in such patients, and with histology showing lymphocytes throughout the pancreas this disorder may represent extremely fulminant type 1A diabetes^{303, 304}. Such patients are very rare in Caucasian populations³⁰⁵. The nPOD study headed by Mark Atkinson promises to supply essential information concerning the pathology of Type 1A diabetes and “prediabetes” (Figure 11.15). The whole pancreas is obtained from organ donors with diabetes and non-diabetic cadaveric donors are also being screened for expression of multiple biochemical islet autoantibodies to detect individuals who had been at risk of progession. We estimate that 1/300 cadaveric donors expresses multiple islet autoantibodies³⁰⁶. One can already view on the nPOD web site histology of patients with long-term diabetes using ones office computer www.jdrfnpod.org)³⁰⁷. Most patients with long-term Type 1A diabetes have essentially no beta cells within their islets, but the small subset with beta cells have the lobular destructive pattern described by Foulis in new onset patients. Lobules of the pancreas have islets where all express insulin, while adjvacent regions where only islets lacking beta cells are present (pseudoatrophic islets)³⁰⁸. It is likely that this lobular pattern of destruction underlies the slow development of Type 1 diabetes.

Figure 11.15 Pancreatic section from nPOD website showing marked atrophy of acinar pancreas in area where all beta cells within islets are destroyed (right panel) versus lack of atrophy on left of section where 100% of islets have extant beta cells. Inset illustrates vitiligo on legs of patients with similar lobular loss of target cells, in this case melanocytes. Suggest that Type 1A diabetes develops as vitiligo of the pancreas with immune mediated lobular killing of beta cells.

The diagnosis of diabetes is to some extent an arbitrary point in the natural history of the autoimmune disease. The timing of overt diabetes may be influenced by factors that increase insulin requirements, such as infections, obesity and puberty. Recent studies indicate that insulin secretion in normal individuals and individuals at risk for type 1 diabetes who do not progress to diabetes usually increases with age¹⁰. This is likely related to increasing insulin resistance with age, and thus when an individual remains with stable insulin secretion (e.g. C-peptide secretion on mixed meal tolerance test) or with decreasing insulin secretion, marked functional abnormalities of beta cell function are present. Wilkins and coworkers have hypothesized (Accelerator Hypothesis)³⁰⁹ that type 1 and type 2 diabetes are essentially the same disorder with three factors “accelerating” loss of beta cells in type 1 diabetes (high rate of beta cell apoptosis; insulin resistance; beta cell autoimmunity)³¹⁰. Given the increasing understanding of the genetics of type 1A diabetes and ability to predict the disease with immunologic assays, both of which are lacking in type 2 diabetes, we believe it is more likely that insulin resistance may impact development of type 1 diabetes simply by influencing the timing of hyperglycemia in the presence of limited insulin secretion. Such insulin resistance may relate to reports relating BMI and higher energy intake prior to type 1 diabetes^{311, 312}. Consistent with this, insulin resistance in analysis of the ENDIT study influenced progression only in those with low insulin secretion³¹³. Experiments with streptozotocin-treated baboons suggest that the FPIR may be undetectable even when about 20%-50% of beta cell mass remains²⁷⁹, although pancreatic insulin content was close to zero at this point. Autoimmune beta cell destruction continues after the diagnosis of diabetes. A temporary remission from insulin dependency may occur in up to 27% of patients, soon after diagnosis³¹⁴, and is attributed to the beta cell rest caused by insulin treatment and slowing of loss of C-peptide is associated with intensive insulin therapy in the DCCT trial¹⁰. Younger age at onset, male sex, high-titer ICA, severe ketoacidosis at diagnosis, and a short duration of symptoms prior to diagnosis are associated with a more rapid loss of C peptide secretion³¹⁵. In one study a more rapid loss was seen in patients heterozygous for DR3 and DR4³¹⁶, although another study came to the opposite conclusion³¹⁴, and another found no significant DR associations³¹⁵. It is likely that insulin resistant individuals present earlier with overt diabetes for any given loss of beta cell mass and increasing fasting glucose prior to onset of diabetes has been reported, a trend toward increasing fasting insulin is also found, and most important impaired glucose tolerance on oral glucose tolerance testing¹⁰ frequently precedes overt diabetes ²⁵⁹.

Studies by Butler and coworkers provide evidence for retention of islet beta cells decades after the onset of diabetes in a subset of patients (Figure 11.1). The amount of beta cells retained is reported to usually be less than 1% of normal and there is continuing evidence of apoptosis, though evidence for beta cell proliferation was not detected²³. There are interesting exceptions with a small subset (<2%) of patients with type 1A diabetes retaining lobules with beta cells for decades⁹. There is a report of spontaneous remission to type 1 diabetes with loss of GAD65 autoantibodies and follow-up till five years³¹⁷. This is an extremely rare occurrence for individuals presenting with overt diabetes and long-term follow-up will be of interest.

Conclusions

We propose a model in which immunological abnormalities appear early in life in many genetically susceptible individuals but in which anti-islet autoimmunity can remain discordant for years for both children and adults for genetically identical individuals. The presence of autoimmunity is most easily marked by the appearance high affinity anti-islet autoantibodies. Anti-insulin autoantibodies when first present in children followed from birth are already of high affinity. Environmental triggers activating innate immunity and T cells may rapidly (weeks) activate autoimmunity^{206, 207, 221}. Alternatively autoimmunity may be aborted by environmental factors (e.g., various infections). In a subset of individuals, these abnormalities may be transient and are not associated with beta cell destruction, though once multiple autoantibodies are present on more than one occasion almost all individuals progress to diabetes, even though the specific autoantibodies positive varies over years. Once beta cell destruction is initiated, marked by the appearance of multiple immunological abnormalities and later by measurable loss of FPIR and progressive loss of glucose tolerance. Anti-islet autoantibodies can be present for decades without loss of insulin secretion in some individuals, but over time most individuals expressing multiple biochemical anti-islet autoantibodies progress to overt diabetes. Genetic and environmental factors may modulate the process, affecting the rate of beta cell destruction. There is not a clear pattern in the variation with some autoantibodies rising in level while others are falling with a time course often measured in years. Levels of only insulin autoantibodies correlate with rate of progression to diabetes²⁵ and since these levels can vary over time, the rate of destruction of beta cells likely varies. The loss of beta cells may be so slow in some individuals that overt diabetes does not occur during the person's lifetime. The presence of more than one of these autoantibodies (GAD65, ICA512, ZnT8, or insulin), combined with FPIR or glucose tolerance measurements and typing for HLA-DQ alleles, allows the identification of a subset of relatives with sufficiently high-risk for type 1A diabetes to carry out preventive trials.

The hypothesis that type 1A diabetes developed in a chronic and predictable manner forms the basis for prevention trials. It is equally important to identify those autoantibody-positive relatives who are unlikely to progress to diabetes (e.g. great majority of those with a single autoantibody). Knowledge about the natural history of the prediabetic period may also facilitate the diagnosis of type 1A diabetes. Such information becomes clinically relevant in genetic counseling, the diagnosis of diabetes in adults (type 1A versus type II), and in children with an unusual clinical course.

Figure 11.16 Lack of progression to diabetes of ICA positive relatives with the protective HLA genotype DQB1*0602.

Evaluation of each of the above circumstances will usually include genetic, immunological, and metabolic information. The presence of the HLA allele DQA1*0102/DQB1*0602 is so rare (approximately 1%) among children with type 1A diabetes^{318, 319} that an alternative disorder (non Type 1A) should be considered as the cause of diabetes if it’s haplotype is present(Figure 11.16). Lack of all biochemical autoantibodies at onset in children should also stimulate search for unusual forms of diabetes³²⁰. Other diseases that can present as insulin-requiring diabetes in childhood include Kir6.2 mutations (sulfonylurea therapy may suffice) for “neonatal” diabetes³²¹ Wolfram's syndrome (DIDMOAD-diabetes insipidus, diabetes mellitus, optic atrophy, and nerve deafness)³²², mitochondrial mutations (often associated with nerve deafness³²³), and MODY³²⁴ (maturity onset diabetes of youth, that may be caused by mutations in the glucokinase and HLA genes)(204). The DQB1*0602 protective haplotype is likely to be present in these disorders at the normal population frequency of approximately 20%.

In adults, the presence of specific autoantibodies in a diabetic individual is probably the best available evidence of type 1A diabetes. At an epidemiological level, the diagnosis of type 1A diabetes by presence of ketoacidosis, insulin requirement, and young age of onset is useful, but for an individual may be of little value^{91, 325}. In the past, determination of ICA was the only immunological test available. In most series high titers of ICA are present in less than 70% of new-onset patients with classic type 1A diabetes³⁶. The use of four recombinant antigen based assays (anti-insulin, anti-ZnT8, anti-GAD, and anti-IA2) appears to detect more than 90% of individuals with type 1A diabetes. Two or more of these antibodies are present in approximately 80% of patients. As such assays become more utilized, the detection of slow-onset type 1A diabetes among adults (LADA: Latent Autoimmune Diabetes of Adults) should be enhanced. Such patients will likely compose more than 10% of adults developing diabetes as has now been confirmed in multiple studies^{95, 96, 326}. Whether this will lead to the immediate institution of insulin therapy rather than oral hypoglycemic agents requires clinical trials of the potential risks and benefits of such therapy.

Of 63 children with transient hyperglycemia we have evaluated, most (89%) remain non-diabetic³²⁷. All of the few with ICAs or IAAs became permanently diabetic within 18 months. It is likely that the measurement of antibodies against multiple biochemically defined antigens will also improve diagnosis of this group. The IVGTT is the diagnostic test choice in this group. Among children with resolved transient hyperglycemia, none of those with normal FPIR (greater than the 5th percentile) has progressed to diabetes, though with increasing insulin resistance in the population there are bound to be exceptions, and we usually subtract 2*fasting insulin from the 1+3 minute insulin following intravenous glucose as a measure of FPIR. Of those with low FPIR, a subset normalizes upon repeat testing, but those remaining abnormal have all become overtly diabetic within 1 year.

An individual contemplating the donation of a kidney to a diabetic relative should have an OGTT to rule out the presence of overt diabetes, an IVGTT by ICARUS criteria, and determination of autoantibodies with assays that have high specificity and sensitivity (e.g., the combination of anti-GAD, anti-insulin, anti-ZnT8 and anti-ICA512). Most of these assays are available now in commercial laboratories.

Finally, the most important reason for detecting individuals at risk for type 1A diabetes is the potential for preventive therapy. It is likely that with more detailed analysis and improvements in the assays available, the prediction of type 1A diabetes will improve further. Sosenko and coworkers have developed a series of combined risk scores for autoantibody positive individuals^{53, 54}. It will be very important to begin such prediction in the general population, as the majority of new cases have no affected relative. The large ENDIT trial demonstrated the feasibility of international trials based upon screening for cytoplasmic islet cell autoantibodies, though nicotinamide did not influence progression to diabetes³²⁸. The large DPT-1 trial (parenteral and oral insulin therapy trials) is now complete and this study with more than 90,000 first degree relatives screened for islet autoantibodies provides a wealth of information. Parenteral and oral insulin therapy did not slow progression to diabetes though a subset of individuals entering the oral trial with high levels of insulin autoantibodies may have had a slowing of progression to diabetes and TrialNet is planning a study to confirm or refute this observation^{329, 329}. Initial analysis of more than 70,000 relatives tested for GAD65 and ICA512 autoantibodies confirms the predominant influence of presence of multiple autoantibodies for prediction⁴³. One half of cytoplasmic ICA positive relatives did not express any of the biochemical autoantibodies, and just as important the cytoplasmic ICA assay failed to detect GAD65 or ICA512 autoantibodies in approximately 2% of the relatives⁴³. The presence of biochemical anti-islet autoantibodies at the initial screening was associated with high risk for eventual eligibility for the DPT trial (e.g. low first phase insulin secretion). The presence of a single biochemical autoantibody and abnormal glucose tolerance will likely identify an additional high-risk group. Presence of ICA adds to risk engendered by biochemical autoantibodies but only if levels of biochemical autoantibodies are not considered.

A very large NIH effort is underway to prevent type 1A diabetes, including TrialNet, the Immune Tolerance Network, Autoimmunity Centers of Excellence and Autoimmunity Prevention Centers. TrialNet will evaluate multiple therapies in new onset patients (aimed at preserving C-peptide secretion) and selective studies of diabetes prevention (1-800-Halt-DM1). This organization is encouraging proposals from the diabetes and immunology community, whether or not individuals are members of the core set of Centers. In a similar manner the Immune Tolerance Network is seeking protocols, and has a web site, (www.immunetolerance.org) to receive concept proposals. The Immune Tolerance Network will support core laboratories and trials and its funding is available to international studies. A major deficiency in our current efforts to prevent type 1A diabetes is a lack of proven quantitative assays of the antigen specific T cells that destroy islet B-cells. In animal models such as the NOD mouse using tetramers reacting with CD8 T lymphocytes it now appears possible to predict diabetes using T cell assays ^330-332 and recently tetramers for CD4 T cells have been developed with “register fixing”¹⁵⁹. Further basic work in parallel with clinical trials will hopefully take advantage of the international effort to prevent type 1 diabetes.

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