Medical care from childhood to adulthood in type 1 and type 2 diabetes.

Diabetes mellitus comprises a heterogeneous group of diseases that have in common the development of macro- and microvascular complications. It is now possible to identify subjects at high risk of Type 1 or Type 2 diabetes, especially in the patient's family members. Preventive interventions are quickly becoming available, and can help delay the onset of the disease and thereby reduce complications in these subjects. Furthermore the correct etiological diagnosis of diabetes is fundamental in providing the best treatment for the patient. Maturity-onset diabetes of the young (MODY) syndrome should be suspected in cases of a subtle onset of diabetes and autosomal dominant inheritance. Mitochondrial DNA mutations should be considered when a diabetic patient also suffers from deafness or if there is a family history of this combination in the mother side of the family. Atypical diabetes has to be identified by the physician to avoid mistakes when the patient enters the non-insulin-dependent phase. In the case of Wolfram's syndrome a gene analysis for each family member should be performed to identify heterozygote subjects. Recently, many discoveries in genetics help us better understand the pathogenesis of the diseases and diagnose the monogenic form of diabetes more easily. If all family members are followed in the same center, clues from the family history are readily available for differential diagnosis and preventive interventions can be established more effectively.

Autoantibodies and human leucocyte antigen class II in first-degree family members of Mexican-American type 1 diabetic patients.

As part of a genetic study of type 1 diabetes in Mexican-Americans, 360 first-degree relatives of 108 type 1 diabetic probands were studied. Islet cell antibody (ICA), insulin autoantibody, glutamic acid decarboxylase (GAD(65)), and protein tyrosine phosphatase autoantibodies were measured and human leucocyte antigen (HLA) class II alleles DRB1 and DQB1 genotyping was performed. ICA was positive in 37% of the probands and 5.8% of the relatives. A subgroup of 26 probands (12 ICA+, 14 ICA-) was tested for GAD(65) and was found positive. 4/14 ICA+ first-degree relatives were GAD(65) positive. Four relatives, positive for two antibodies, subsequently developed type 1 diabetes. Life-Table analysis of first-degree relatives with autoantibodies indicated an 80% disease-free survival at 3.5 yr. HLA-DRB1 was found to be associated with the presence of ICA in both probands and relatives, whereas HLA-DPB1 was associated with autoantibody in relatives of type 1 diabetic probands. These results suggest that autoimmunity occurs in type 1 diabetes families of Mexican descent in similar frequencies to that of non-Hispanic, Caucasian families. The presence of autoantibodies appears to be regulated in part by HLA class II genes, even in the absence of overt diabetes.

Islet cell autoimmunity in youth onset diabetes mellitus in Northern India.

We characterised a consecutive cohort of 132 youth onset diabetic individuals (age at onset<30 years, mean duration of disease 5.5+/-6.0 years) from North India, by serological determination of the determination of the islet cell autoantibodies, GAD(65) and IA2, and clinically for coexisting autoimmune thyroid disease, malnutrition and pancreatic calcification. Five types of diabetes were delineated: Type 1 (37%), ketosis resistant (32%), Type 2 (13%), fibrocalculous pancreatopathy (11%) and autoimmune polyglandular syndrome (7%). C-peptide response to glucagon was assessed in a representative subset of 50 patients with Type 1, ketosis resistant, and autoimmune polyglandular syndrome. A total of 22.4% of Type 1 and 30% of autoimmune polyglandular syndrome subjects showed both GAD(65) plus IA-2 autoantibody positivity, significantly more than the 4.7% positivity shown by the ketosis resistant type. However, GAD(65) antibody positivity alone was seen in 38% of ketosis resistant subjects which was significantly more than the 14.2 and 10% positivity seen in Type 1 and autoimmune polyglandular groups, respectively. The fibrocalculous pancreatopathy group showed GAD(65) plus IA-2 autoantibody positivity in 14.2% and GAD(65) autoantibody alone positivity in 7.1%. 26 and 60%, respectively, of the Type 1 and autoimmune polyglandular syndrome groups had thyroid microsomal autoantibody positivity. Type 1 showed significantly less C-peptide response to glucagon when compared to the ketosis resistant and autoimmune polyglandular syndrome groups. The controls and Type 2 diabetic individuals tested negative for islet cell autoimmunity markers. These findings demonstrate a role of islet cell autoimmunity in the pathogenesis of four out of the five clinical types of youth onset diabetes seen in North India.

Diabetes vaccines: a future to be realized.

Immune-mediated (type 1) diabetes mellitus (IMD) is an autoimmune disease resulting from the chronic destruction of pancreatic islet cells by autoreactive T lymphocytes. Although there has been much advancement in diabetes management, targeting the precise etiology of the disease process has remained elusive. Recent progress in the understanding of the immunopathogenesis of IMD, however, has led to new intervention strategies, especially antigen-based therapies given as altered peptide ligands (APLs) or as vaccines. Instead of using immunosuppressive agents to suppress an already dysfunctional immune system, antigen specific vaccines or even non-antigen specific immunostimulants present a unique opportunity to boost regulatory function and thereby regain tolerance to self. We discuss here the pathogenesis of IMD as it relates to therapeutic possibilities, review various intervention strategies that have been successful in rodent models, and then present recent progress in human trials of diabetes intervention and prevention through vaccine prototypes.

Current cases in which epitope mimicry is considered as a component cause of autoimmune disease: immune-mediated (type 1) diabetes.

Autoimmune diseases result from a combination of genetic, immunologic, hormonal, and environmental factors. Infectious agents may induce the breakdown of immunological tolerance and the appearance of autoreactivity. However, the specific relationship between infection and autoimmunity is still unclear. One of the mechanisms responsible could be molecular mimicry between the infectious agent and self. The concept of molecular mimicry is a viable hypothesis in the investigation of the etiology, pathogenesis, treatment, and prevention of autoimmune disorders. Immune-mediated (type 1) diabetes in humans and in non-obese diabetic (NOD) mice is polygenic and characterized by autoimmune destruction of insulin-producing pancreatic beta cells in islets of Langerhans. In NOD mice, a T-helper 1 (Th1)-based autoimmune response arises spontaneously against glutamate decarboxylase (GAD) concurrently with the onset of insulitis. Subsequently. this Th1-type autoreactivity spreads intra- and intermolecularly to other beta cell autoantigens, suggesting that a Th1-type response is responsible for the progression of the disease, whereas Th2 responses when experimentally induced are protective. In humans, a homology between GAD and the P2-C protein of Coxsackie B make a cause-and-effect molecular mimicry an attractive hypothesis. Evidence to support the concept of molecular mimicry in diabetes is reviewed.

The human leukocyte antigen HLA DRB3*020/DQA1*0501 haplotype is associated with Graves' disease in African Americans.

Information on genetic susceptibility to Graves' disease in African Americans is limited. We studied DRB1, DQB1, DRB3 subtypes, DQA1*0501, DQA1*0201, and CTLA-4 polymorphisms in 49 African American patients with adult onset Graves' disease and 47 racially-matched controls using PCR-based sequence-specific priming methods. There were no significant differences in DRB1 or DQB1 allelic frequencies or CTLA-4 polymorphisms between patients and controls. However, we found that the frequency of DRB3 was significantly increased in the patients (75.5% vs. 57.4%, P = 0.006, X2 = 3.52), especially for the DRB3*0202 subtype (53.1% vs. 23.4, P = 0.003, X2 = 8.91). In this one respect, the finding was in concordance with our previous observations in Caucasian patients with adult-onset Graves' disease. In addition, whereas the frequency of DQA1*0501 was increased (P = 0.018, X2 = 5.63) in our patients, the haplotype of DRB3/DQA1*0501, or DRB3*0202/DQA1*0501 was found to be more strongly associated (P = 0.008, X2 = 7.0; P = 0.0008, X2 = 11.34, respectively). These data suggest that DRB3*0202, particularly when found with DQA1*0501 in a haplotype is a susceptible gene(s) for Graves' disease in adult African Americans. Considering these data with those in Caucasian patients, our results would suggest that the primary Graves susceptible locus is likely DRB3 and not DRB1.

Autoimmunity and diabetes.

The face of immune-mediated (type 1) diabetes is changing. No longer considered a disease confined to childhood, the incidence rate in Western countries is clearly rising and affecting younger children. Such a secular trend can only be explained on the basis of increased contacts with adverse environmental factors acting on a background of complex genetics. Multiple defects in immunological tolerance to "self' predispose to immune-mediated (type 1) diabetes. Initiation of immune responses involves the cytokine rich natural killer T cells. Such cells appear deficient in both humans and the rodent models of the disease. Furthermore, the regulatory abilities of T cells in general seem to be compromised. Effector mechanisms probably are dominated by cell-mediated beta cell destruction through apoptosis induction. Surprisingly, the essential antigen-presenting cells in the autoimmune processes involved appear to be B lymphocytes. The improved understanding of the beta cell autoantigens involved has led to better disease prediction. The long prodromal phase now readily identifiable through autoantibodies is spawning hopes of disease prevention, notably through antigen-based interventions or diabetes "vaccines."

HLA-DRB1*08, DRB1*03/DRB3*0101, and DRB3*0202 are susceptibility genes for Graves' disease in North American Caucasians, whereas DRB1*07 is protective.

Graves' disease is known to be HLA-D associated; however, the primary loci involved remain unclear. We examined HLA genotypes of DRB1 and DQB1 plus DRB3 subtypes using PCR-based sequence-specific priming in two groups of North American (Gainesville, FL; and Toronto, Canada) Caucasian patients with Graves' disease. We stratified patients into those with either early age at onset (<20 yr; 13.1 +/- 4.8 yr; n = 30) and later age at onset of disease (38.8 +/- 9.7 yr; n = 62) and compared the results to 192 normal controls. As expected, we found that DRB1*03 was associated with Graves' disease, but at a higher odds ratios for early-onset than later-onset patients (3.7 vs. 2.2). The frequency of DRB1*08 was also increased in both groups of patients, but significantly so only in patients with early-onset Graves' (P = 0.001; chi2 = 10.8). DRB3 was highly associated with Graves' in both groups of patients (P = 0.009; chi2 = 6.83 and P = 0.0015; chi2 = 10.1, respectively); however, the subtypes of DRB3 revealed differential susceptibilities. Whereas the frequencies of both DRB3*0101 and DRB3*0202 were increased over the entire cohort, that of DRB3*0301 was not. Significant P values were found for DRB3*0101 in patients with early-onset and for DRB3*0202 in patients with later onset of Graves' disease. When the haplotypes of DRB1*03-DRB3 of all subtypes were removed for analysis (all DRB1*03 positive also had DRB3*0101), the frequency of DRB3*0202 remained significantly higher in the patients with later onset of Graves' disease than in controls (P = 0.0043; chi2 = 8.13), but DRB3 was no longer positively associated with the early-onset group. In addition, we found that DRB1*07 was negatively associated with both groups of patients (P = 0.024; chi2 = 5.10 and P = 0.0085; chi2 = 6.93). These data suggest that DRB3*0202 is more likely to be the primary susceptible locus than DRB1*03 for patients with later onset of Graves' disease.

Association of diabetes mellitus and chronic hepatitis C virus infection.

While patients with liver disease are known to have a higher prevalence of glucose intolerance, preliminary studies suggest that hepatitis C virus (HCV) infection may be an additional risk factor for the development of diabetes mellitus. To further study the correlation of HCV infection and diabetes, we performed a retrospective analysis of 1,117 patients with chronic viral hepatitis and analyzed whether age, sex, race, hepatitis B virus (HBV) infection, HCV infection, and cirrhosis were independently associated with diabetes. In addition, a case-control study was conducted to determine the seroprevalence of HCV infection in a cohort of 594 diabetics and 377 clinic patients assessed for thyroid disease. In the former study after the exclusion of patients with conditions predisposing to hyperglycemia, diabetes was observed in 21% of HCV-infected patients compared with 12% of HBV-infected subjects (P =.0004). Multivariate analysis revealed that HCV infection (P =.02) and age (P =.01) were independent predictors of diabetes. In the diabetes cohort, 4.2% of patients were found to be infected with HCV compared with 1.6% of control patients (P =.02). HCV genotype 2a was observed in 29% of HCV-RNA-positive diabetic patients versus 3% of local HCV-infected controls (P <.005). In conclusion, the data suggest a relatively strong association between HCV infection and diabetes, because diabetics have an increased frequency of HCV infection, particularly with genotype 2a. Furthermore, it is possible that HCV infection may serve as an additional risk factor for the development of diabetes, beyond that attributable to chronic liver disease alone.

The gene responsible for autoimmune polyglandular syndrome type 1 maps to chromosome 21q22.3 in US patients.

Autoimmune polyglandular syndrome type 1 [APS-1] comprises multiple organ-specific autoimmunities such as acquired hypoparathyroidism and autoimmune Addison's disease, and a predisposition to certain infections such as chronic mucocutaneous candidiasis. An APS-1 candidate gene was assigned to chromosome 21q22.3 by linkage analyses in patients with APS-1 from Finland. To examine the influence of ethnic and geographic differences on the location of the candidate gene locus, we studied 24 US patients with APS-1 by microsatellite marker typing, using five microsatellite markers, D21S49, PFKL, D21S171, D21S1903 and CD18, selected from chromosome 21q22.3. By allelic association analyses, the frequencies of allele number 5 for D21S171 and allele number 8 for D21S1903 were significantly higher in the 24 patients with APS-1 than in 33 controls (33/48 vs. 31/66, P = 0.0207, X2 = 5.35; 12/48 vs. 7/66, P = 0.0418, X2 = 4.15 respectively). The frequency of homozygosity for allele number 5 of D21S171 was also significantly higher in the patients than in controls, 15/24 vs. 9/33 (P = 0.0078, X2 = 7.07). Maximum lod scores detected for the five markers in nine families (containing 15 of the patients with APS-1) were: 2.384 for D21S49, 3.144 for PFKL, 3.506 for D21S171, 4.329 for D21S1903, and 1.130 for CD18. These results confirm the linkage of the candidate APS-1 gene to 21q22.3 in US APS-1 patients, and suggest that the candidate gene is located near the D21S1903 marker. The demonstration of the location of the APS-1 candidate gene to 21q22.3 in an out-bred heterogeneous patient population should promote the physical mapping of the responsible gene.

The relationship between humoral and cellular immunity to IA-2 in IDDM.

Autoantibodies to the neuroendocrine protein insulinoma-associated protein 2 (IA-2), a member of the tyrosine phosphatase family, have been observed in individuals with or at increased risk for IDDM. Because this disease is thought to result from a T-cell-mediated autoimmune destruction of the insulin-producing pancreatic beta-cells, we analyzed humoral and cellular immune reactivity to this autoantigen to further define its role in the pathogenesis of IDDM. Peripheral blood mononuclear cells (PBMC) from individuals with newly diagnosed IDDM or at varying levels of risk for the disease were stimulated in vitro with the entire 42-kDa internal domain of IA-2 (amino acids 603-979), a series of control antigens (glutathionine-S-transferase, tetanus toxoid, Candida albicans, mumps, bovine serum albumin), and a mitogen (phytohemagglutinin). The frequency and mean stimulation index of PBMC proliferation against IA-2 was significantly higher in newly diagnosed IDDM subjects (14 of 33 [42%]; 3.8+/-4.5 at 10 microg/ml) and autoantibody-positive relatives at increased risk for IDDM (6 of 9 [66%]; 3.9+/-3.2) compared with autoantibody-negative relatives (1 of 15 [7%]; 1.8+/-1.0) or healthy control subjects (1 of 12 [8%]; 1.5+/-1.0). The frequencies of cellular immune reactivities to all other antigens were remarkably similar between each subject group. Sera from 58% of the newly diagnosed IDDM patients tested were IA-2 autoantibody positive. Despite investigations suggesting an inverse association between humoral and cellular immune reactivities against islet-cell-associated autoantigens, no such relationship was observed (rs=0.18, P=0.39) with respect to IA-2. These studies support the autoantigenic nature of IA-2 in IDDM and suggest the inclusion of cellular immune responses as an adjunct marker for the disease.

Insulin-dependent diabetes mellitus (IDDM) is associated with CTLA4 polymorphisms in multiple ethnic groups.

Linkage disequilibrium (association) analysis was used to evaluate a candidate region near the CTLA4/CD28 genes using a multi-ethnic collection of families with one or more children affected by IDDM. In the data set unique to this study (Spanish, French, Mexican-American, Chinese and Korean), the transmission/disequilibrium test (TDT) revealed a highly significant deviation for transmission of alleles at the (AT)n microsatellite marker in the 3' untranslated region (P = 0.002) and the A/G polymorphism in the first exon (P = 0.00002) of the CTLA4 gene. The overall evidence for transmission deviation of the CTLA4 A/G alleles is also highly significant (P = 0.00005) in the combined data set (669 multiplex and 357 simplex families) from this study and a previous report on families from USA, Italy, UK, Spain and Sardinia. Significant heterogeneity was observed in these data sets. The British, Sardinian and Chinese data sets did not show any deviation for the A/G polymorphism, while the Caucasian-American data set showed a weak transmission deviation. Strong deviation for transmission was seen in the three Mediterranean-European populations (Italian, Spanish and French) (P = 10(-5)), the Mexican-American population (P = 0.002) and the Korean population (P = 0.03). These results suggest that a true IDDM susceptibility locus (designated IDDM12) is located near CTLA4.

Autoantigens in insulin-dependent diabetes mellitus: molecular cloning and characterization of human IA-2 beta.

In this study, we describe the isolation, expression, and characterization of a new member of the transmembrane protein tyrosine phosphatase family from human brain, designated IA-2 beta. The 3853-bp cDNA encodes 986 amino acids with a molecular mass of 108,044 daltons (a predicted pI value of 5.8). The intracellular domain of human IA-2 beta is 74% identical to human IA-2. Northern blot analysis showed that IA-2 beta cDNA recognized two transcripts (approximately 5.0 kb and 4.0 kb) in four of five human insulinomas, one glucagonoma, and in normal human brain, pituitary, and pancreas, but not in a variety of other normal tissues. Rabbit antiserum, raised against the intracellular domain of IA-2 beta, reacted with pancreatic islets. Treatment of in vitro-translated full-length IA-2 beta protein with trypsin converted it into a 37-kD fragment. Using recombinant human IA-2 beta, we developed a radioimmunoprecipitation assay to measure autoantibodies in the sera of patients with insulin-dependent diabetes mellitus (IDDM). Seventy-six new-onset IDDM patients were tested. Thirty-seven percent (28 of 76) of the IDDM sera-but less than 1% of the control sera (1 of 174)-reacted with IA-2 beta. The same IDDM sera tested for autoantibodies to IA-2 and glutamic acid decarboxylase (GAD65) showed that 64% (49 of 76) and 57% (43 of 76), respectively, were positive. All but two of the IA-2 beta autoantibody-positive sera also reacted with IA-2, supporting the close sequence similarity between the two molecules. Combination of any two markers, such as IA-2 beta and IA-2, or IA-2 beta and GAD65, or IA-2 and GAD65, revealed that 67%, 74%, and 87% of IDDM sera were positive for autoantibodies, respectively. Blocking of IDDM sera with recombinant IA-2, IA-2 beta, or GAD65 resulted in marked inhibition of reactivity of IDDM sera with pancreatic islet sections as measured by islet cell autoantibody immunofluorescence. This result suggests that these three autoantigens are the major targets of islet-cell autoantibody reactivity.

Immunization therapies in the prevention of diabetes.

Insulin-dependent diabetes (IDD), being an autoimmune disease, offers several opportunities for immunological interventions that may result either in the reduction of disease severity or in delaying diabetes onset. Among the various experimental preventative approaches, parenteral immunization with islet-specific autoantigens appears to be practically simpler and promising. We have previously shown that immunization with insulin, insulin B chain and B chain epitope (p9-23), but not insulin A chain, in incomplete Freund's adjuvant (IFA) and in alum (with B chain) delayed/prevented diabetes onset in NOD mice. Here we demonstrate the protective efficacy of affinity purified GAD65 in IFA. While both insulin B chain and GAD65 significantly delayed the onset of diabetes (P=0.001), a recently described tyrosine phosphatase (IA-2) antigen did not (P=0.38). Interestingly, B chain immunization reduced the incidence of cyclophosphamide (CY)-accelerated diabetes by about 50-55%. We also provide further evidence that B chain, upon increased adsorption to alum, could improve on its protective capacity in NOD mice.

Insulin-dependent diabetes mellitus: the hypothesis of molecular mimicry between islet cell antigens and microorganisms.

Insulin-dependent diabetes mellitus (IDDM) in humans and the non-obese diabetic mouse is a polygenic disease, resulting from an autoimmune destruction of the insulin-secreting pancreatic beta cells. At least in NOD mice, the process is mediated through a T helper 1-cell-mediated cytotoxicity pathway. Although there is much circumstantial evidence to suggest that IDDM is environmentally induced, recent studies support the possibility that the inductive event involves cross-reactive immune responses to antigenic epitopes acting as molecular mimics between microbial proteins and autoantigens expressed by pancreatic insulin-secreting beta cells. The following article reviews the evidence for this concept.