Absolute risk for type 1 diabetes mellitus according to DR/DQ genotypes
DR 3/4, DQ 0201/0302 DR 4/4, DQ 0300/0302 DR 3/3, DQ 0201/0201
DR0403 or DQ0602
As indicated, non-HLA genes are also associated with T1D. For example, the IDDM2 locus has been mapped to a variable number of tandem repeats located upstream of the insulin gene. Disease association studies in case control and family cohorts show that the number of tandem repeats is associated with T1D risk, with shorter repeats conferring higher risk and longer repeats conferring lower risk. Another non-HLA gene associated with T1D is CTLA-4 (cytotoxic T lymphocyte associated-4) . First identified in a family study of T1D, this gene encodes a molecule that plays an important role in the regulation of T-cell function and immune responses. Other specific genes finding some degree of recent support include PTPN22 and SUMO4, each of which is believed to provide influence to immune responsiveness [16,17]. As our understanding of the function of susceptibility and resistant genes grows, we will continue to gain new insights into the relationship between genetic risk and the autoimmunity that culminates in T1D.
As indicated, T1D is an autoimmune disease that culminates in destruction of the pancreatic beta cells, characterized histologically by insulitis and associated islet cell damage. The autoimmunity in T1D is specific to the insulin-producing beta cells. The specific mechanisms responsible for inducing the autoimmunity in T1D have yet to be elucidated, however. Many different theories have been promulgated to explain this induction, including molecular mimicry (sharing of antigenic properties) (ie, amino acid sequences between beta cells and possible environmental agents leading to the generation of an immune response), alteration of self-antigens, defective major histocompatibility complex expression, breakdown in central tolerance (ie, failure to establish immunity to self-antigens in early life), deleterious trafficking of dendritic cells from beta cells to pancreatic lymph nodes, and defects in peripheral tolerance (ie, aberrant T-cell activation). The role for the cellular immune response in T1D has been remarkably controversial. Because nearly all studies performed to date have involved characterization away from the site of injury (ie, performed on cells from peripheral blood and not the pancreatic islets), we have elected to avoid their review in this article but reference other articles dedicated to such descriptions [18-20].
Regardless of the proposed causes of the autoimmunity, biopsy is the only true means of directly demonstrating beta cell injury. Because in most settings pancreatic biopsies have not been considered ethically feasible (because of reasons of safety), autoimmunity in T1D typically has been identified by the presence of circulating autoantibodies to islet cell antigens, which in addition to their presence at the time of diagnosis often can be detected long before the disease becomes clinically evident. Islet cell autoantibodies (ICAs), autoantibodies to glutamic acid decarboxylase (GAD65A), insulin autoantibodies (lAAs), and autoantibodies directed at a transmembrane tyrosine phosphatase (ICA512A) are the most prevalent and best characterized, but the potential for other autoanti-body/autoantigen combinations remains. It is critical to note that autoantibodies have no known etiologic role in diabetes and—simply put—are believed to represent the ''smoke of the fire'' in the pancreas and not the fire itself. Recent studies in animal models of T1D purporting a crucial role for B lymphocytes in disease development may allow for a previously unappreciated role for autoantibodies in presentation of self-antigens to the cytotoxic T cells responsible for beta cell destruction.
TID-associated autoantibodies are present in 70% to 80% of patients newly diagnosed with the disease. In contrast, 0.5% of the general population and 3% to 4% of relatives of patients who have T1D are autoantibody positive . Although autoantibodies are surrogate measures for beta cell autoimmunity, autoantibody titer and the absolute number of autoantibodies (eg, one, two) are independent predictors of T1D risk. Specifically, when present in combination, at higher titers, at a younger age, or with the high-risk HLA genes, autoantibodies allow for a more accurate prediction of T1D risk. ICA titers of > 40 Juvenile Diabetes Foundation units carry a 60% to 70% risk of developing diabetes over 5 to 7 years . When present at a young age, ICAs denote a much greater risk than when present in older subjects. When present in the first few years of life, the 10-year risk of developing diabetes is nearly 90%, whereas a 40-year-old individual who is ICA positive has a 30% 10-year risk. After 10 years of overt diabetes, less than 5% of patients have detectable ICA. Although IAAs may be the first autoantibodies to appear in the development of T1D, they are not by themselves strong predictors of developing T1D. When present in combination with other autoantibodies, however, the risk for T1D increases significantly. In the large, NIH-funded Diabetes Prevention Trial-Type 1, the 5-year risk of T1D was 20% to 25% for subjects with one autoantibody, 50% to 60% for subjects with two autoantibodies, nearly 70% for persons with three autoantibodies, and almost 80% for persons with four autoantibodies .
Another peculiar aspect of IAAs is that they must be measured within 1 week of the start of exogenous insulin therapy, because insulin antibodies in response to exogenously injected insulin also are detected and are indistinguishable from IAAs. GAD65A, like ICAs, are observed in 60% to 70% of patients who have new-onset T1D. Unlike ICA, GAD65A often persist for many years after diagnosis in patients with T1D and may be more useful in diagnosing latent autoimmune disease of adults than ICAs . IA-2 has an extracellular, trans-
membrane, and cytoplasmic domain, and autoantibodies to several forms of IA-2 have been observed in persons who have T1D. Because more than one laboratory independently identified this molecule, it also has been referred to in the literature as the ICA512 autoantigen. The IA-2 antigen, like GAD, is expressed in many tissues, including brain, pituitary, and pancreas .
In summary, the identification and description of autoantibodies in T1D has allowed us to gain remarkable insight into the natural history of this disease. In combination with a growing understanding of genetic susceptibility, we are currently able to predict accurately which patients will develop T1D . As efforts continue to help researchers understand the etioimmunopathogenesis of T1D, many questions still remain as to the role for cellular immunity and issues related to which specific environmental triggers induce or regulate the auto-immunity related to T1D.
T1D results from the interaction of genes, the environment, and the immune system. The presence of disparate geographic prevalence, rising worldwide incidence, and 50% discordance rate in identical twins provides evidence that environmental agents are operative . Because the islet-specific autoantibodies frequently can be detected within the first few years of life [27-29], it seems that triggering environmental encounters may occur early in development. Because there is invariably a latent period between the appearance of T1D-associated autoantibodies and onset of disease, additional environmental factors—probably interacting with genetic factors—also seem to modulate the rate of development of the disease .
Early nutrition and infection have been the most frequently implicated early environmental influences . There is, however, no direct evidence to date that either nutrition or infection plays a major role in causation, albeit one example is often cited as providing such evidence [32,33]. Prenatal rubella infection is associated with beta cell autoimmunity in up to 70% and diabetes in up to 40% of infected children [32,34-38]. Postnatal infection is not associated with increased risk, however. The introduction of universal rubella vaccination has virtually abolished this disease and the occurrence of this form of diabetes, which proves that in some cases T1D can be prevented by modification of environmental factors.
A relationship between beta cell autoimmunity and exposure to enteroviral infections in utero also has been proposed [33,39,40]. Studies from Finland and Sweden suggested that maternal enterovirus infection may increase the likelihood of subsequent T1D development in offspring [33,39]. Higher levels of antibodies to procapsid enterovirus antigens were found in the pregnant sera of mothers of children who developed diabetes. The presence of antibodies against entero-viruses in people with autoimmunity does not prove a causal relationship, however. It should be noted that the number of women exposed to enteroviral infection during pregnancy is decreasing and that infection in early childhood has become less common . Islet-related autoantibodies also have been detected after mumps, measles, chickenpox, and rotavirus infections [42-44]. These considerations do not exclude arguments based on changing antigenicity of foods or viruses or timing of exposure to them. Persons with autoimmunity also may be more prone to enteroviral infection, may have a stronger humoral response to infection because of their particular HLA genotype, or may be in a nonspecific hyperimmune state marked by elevation of antibody levels to various exogenous antigens . With this background, it is clear that well-planned prospective studies in larger populations are essential. The Environmental Determinants of Type 1 Diabetes in Youth study is one such example that was formed recently and at an international level will document exposure to various infectious and other environmental agents throughout pregnancy and early infancy.
In terms of noninfectious influences, the association between a potential protective effect of breastfeeding and early exposure to cow's milk on the incidence of autoimmunity and T1D remains controversial [45-50]. Gerstein's extensive meta-analysis demonstrated a weak but statistically significant association (odds ratio approximately 1.5) between T1D and a shortened period of breastfeeding and cow's milk exposure before 3 to 4 months of age . In the biobreeding rat and nonobese diabetic mouse models of T1D, diet plays an important role in the development of the disease, yet which dietary components are important remains unclear. Among them is casein, a major protein fraction of cow's milk. Feeding semi-purified diets with simple sugars replacing complex carbohydrates and hydrolyzed casein as the protein source routinely retards the development of diabetes in rodents [52,53]. Another component is bovine serum albumin. Kar-jalainen and colleagues showed that antibodies to bovine serum albumin, which are immunologically distinct from human serum albumin, were present in 100% of Finnish children who have new-onset T1D but were absent in controls . Structural similarities between bovine serum albumin and an islet protein (ICA69) were proposed as an appealing pathogenic concept of molecular mimicry, by which the early introduction of cow's milk would allow absorption of the intact protein before gut maturation, thus immunizing an infant and directing an immune response to the islets through its ICA69 mimic . There is strong evidence to counter each argument put forth to advance the cow's milk hypothesis, however . No association between early exposure to cow's milk and beta cell autoimmunity in young siblings and offspring of patients who have diabetes has been shown in several other studies. Increased practice of breastfeeding in developed countries is inconsistent with a rising incidence of childhood diabetes . We and others were unable to show any link between the presence of antibodies to bovine serum albumin and T1D [47,56]. Finally, ICA69 has been found in several other organs beside pancreatic cells, and cross-reactivity of these antibodies with bovine serum albumin has not been confirmed.
The ingestion of nutrient-containing elements of plants such as soy and wheat also seem to have an effect on the development of diabetes, at least as defined in studies of nonobese diabetic mice . In humans, two recent studies—the Diabetes Autoimmunity Study in the Young and the German study of offspring of T1D parents—provided evidence that susceptibility to T1D is associated with the timing of exposure to cereal and gluten [58,59]. In the Diabetes Autoimmunity Study in the Young, initial exposure to cereal between birth and 3 months of age and after 7 months of age imparted risk of autoimmunity. In the German study of offspring of T1D parents, Zeigler and colleagues  demonstrated an increased risk for autoimmunity in infants initially exposed to gluten before 3 months of age and found no increased risk in infants initially exposed to gluten after 6 months of age. Although both studies provided interesting findings, their conclusions are in some ways contradictory and demonstrate the need for larger collaborative investigations to determine appropriately how early dietary exposures affect risk for autoimmunity.
The highest incidence of T1D worldwide occurs in Finland (currently approximately 50 cases per 100,000/year). Sun exposure in northern Finland is limited and low serum concentrations of vitamin D in Finland are common. Hypponen and colleagues  have suggested that ensuring adequate vitamin D supplementation for infants could help to reverse the increasing incidence of T1D. It has been proposed that vitamin D compounds may act as selective im-munosuppressants, as illustrated by their ability to either prevent or markedly suppress development of autoimmune disease in animal models of T1D . Vitamin D has been shown to stimulate transforming growth factor beta-1 and interleukin-4, which may suppress inflammatory T cell (Th1) activity .
Toxic doses of nitrosamine compounds also can cause diabetes because of the generation of free radicals [63,64]. The effect of dietary nitrate, nitrite, or nitrosamine exposure on human T1D risk is less clear [65,66]. Several perinatal risk factors for childhood diabetes are also associated with the development of T1D . The effect of maternal-child blood group incompatibility is fairly strong (both ABO and Rh factor with ABO > Rh) and must be explored further. Other perinatal factors that confer increased risk include pre-eclampsia, neonatal respiratory distress, neonatal infections, caesarian section, birth weight, gestational age, birth order, and maternal age [68-72]. It is important to determine whether these factors really contribute and how they may act or be confounded by other unknown risk factors. Rodent studies also suggested that administration of diphtheria-tetanus-pertussis vaccine at 2 months of age increases the incidence of diabetes compared with the incidence in unvaccinated individuals or in individuals vaccinated at birth. In prospective studies, however, no association has been demonstrated between early childhood immunizations and beta cell autoimmunity [73,74].
Finally, researchers have argued that the rising incidence of T1D could be accounted for by protective factors in the environment that have been lost . In support of this theory, the rise in the rates of asthma and allergy has been parallel to that of T1D. The hygiene hypothesis proposes that exposure to infective agents in early childhood is necessary for maturation of the neonatal immune response. In the absence of such exposure, the model predicts a failure of early immune regulation that may permit, depending on genetic susceptibility, the development of autoimmunity (Th1) or allergic (Th2) disease [76,77]. This is consistent with the fact that the nonobese diabetic mouse is less likely to develop diabetes in the presence of pinworms and other infections, yet specific evidence for the hygiene hypothesis in human T1D is minimal .
The list of suggested environmental triggers and regulators of disease in T1D remains considerable. Only through the continued effort of large, prospective, multicenter screening programs will we be able to determine which environmental and genetic factors are most responsible for the development of this disease.
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