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Autoimmunity to Islet Proteins in Children Diagnosed with New-Onset Diabetes

Departments of Medicine (B.M.B.-W., J.P.P.) and Pediatrics (C.P.), University of Washington, Seattle, Washington 98195; Department of Medicine (B.M.B.-W., J.P.P.), Department of Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108; Benaroya Research Institute at Virginia Mason (C.J.G.); and Children’s Hospital and Regional Medical Center (C.P.), Seattle, Washington 98105

Address all correspondence and requests for reprints to: Barbara Brooks-Worrell, Ph.D., 1660 South Columbian Way (111), DVA Puget Sound Health Care System, Seattle, Washington 98108. E-mail: bbrooks{at}u.washington.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Autoantibody and T cells reacting with islet proteins have been demonstrated in patients with type 1 diabetes. In recent years an increasing number of children have been clinically classified with type 2 (not ketosis prone, evidence of insulin resistance, presence of acanthosis nigricans, and obesity) or indeterminant diabetes (admixture of clinical features of types 1 and 2). In this study, we compared the islet cell autoantibody and T-cell responses to islet proteins in type 2 (n = 19) and indeterminant (n = 16) children (<18 yr of age) to classic type 1 (n = 37) diabetic patients. We observed that 37 of 37 type 1 diabetic children demonstrated autoantibody and/or T-cell reactivity to islet proteins. Fourteen of the 19 type 2 patients were positive for islet cell autoantibodies, and six of 14 were positive for T-cell responses to islet proteins. For the indeterminant patients, 11 of 16 of the patients were positive for autoantibodies, and six of 16 patients were positive for T-cell responses to islet proteins. These results demonstrate that autoimmunity to islet proteins is present in a high percentage of children classified as indeterminant or type 2 diabetes. Moreover, the presence of obesity or acanthosis nigracans does not reliably distinguish children with or without islet cell autoimmunity.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
CHILDREN DIAGNOSED WITH diabetes are very commonly presumed to have autoimmune type 1 diabetes. In clinical practice, physicians use their assessment of phenotypic signs and symptoms to determine whether a child has type 1 or type 2 diabetes. These include factors such as age at onset, apparent abruptness of onset of hyperglycemic symptoms, presence of diabetic ketoacidosis, degree of obesity, presence of acanthosis nigricans, weight loss, and the perceived need for insulin replacement. Health care providers and researchers (1, 2, 3, 4, 5, 6) have been reporting an increased prevalence of type 2 diabetes among children largely based on these clinical phenotypes. However, an alternative hypothesis is that many of these children still have type 1 diabetes, a disease caused by immune-mediated destruction of the pancreatic ß-cell but that the phenotype is changing with the marked increase in childhood obesity.

Type 1 diabetes is characterized by T-cell and autoantibody responses to islet proteins (7, 8, 9), whereas type 2 diabetes is not autoimmune (10). Our laboratory has been interested in studying the autoantibodies and T-cell autoreactivity to human islet proteins in type 1 diabetes patients. We previously demonstrated that patients diagnosed with classic type 1 diabetes between 7 and 35 yr of age have T cells responsive to numerous islet proteins within 1 yr of diagnosis, whereas normal controls do not (8, 11, 12). Moreover, classic (autoantibody negative) adult type 2 diabetes patients do not demonstrate T-cell reactivity to islet proteins (11). However, there is a subgroup of adult patients clinically classified as type 2 diabetes who are positive for autoantibodies to islet proteins. We previously demonstrated that these patients commonly also have T-cell responses to islet proteins (11).

In this study, we asked whether children clinically classified as having type 2 (not ketosis prone, evidence of insulin resistance, presence of acanthosis nigricans, and obesity, n = 19) or indeterminant diabetes (admixture of types 1 and 2 clinical features, n = 16) had autoantibodies to islet proteins. We also examined these patients for T cells reactive to islet proteins and compared their autoantibody and T cell response with those of classic type 1 diabetes patients (ketosis prone, insulin requiring, not obese, n = 37). Moreover, we asked whether the presence of obesity, urinary ketones, ketoacidosis, or acanthosis nigracans would distinguish children with or without islet cell autoimmunity.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Experimental subjects

Peripheral blood was collected from subjects within 1 month of diagnosis. Assays for autoantibodies, human leukocyte antigen (HLA), and T-cell assays were performed on the sample. These studies were approved by the Institutional Review Boards at Children’s Hospital and Regional Medical Center and the University of Washington. The first 72 newly diagnosed subjects who agreed to participate in the study were clinically classified by their treating physician as type 1, type 2, or indeterminant. All nonobese (<85th percentile for age and gender) subjects presenting with diabetic ketoacidosis (DKA) were classified as having classic type 1 diabetes (n = 37). Type 2 subjects (n = 19) demonstrated obesity and/or acanthosis nigricans and were negative for DKA. Indeterminant subjects (n = 16) had characteristics that overlapped both types 1 and 2 diabetes. The clinical characteristics, demographic information, and ethnicity of these subjects are shown in Tables 1 and 2.

Cryostat sections (4 µm thick) of human, blood group O pancreas were adhered to dichromic acid-washed slides coated with poly-L-lysine hydrobromide. Test sera, preabsorbed with rat liver powder (Sigma Chemical Co., St. Louis, MO), at dilutions of 1:4 and 1:16, were applied to slides and the slides incubated overnight in a covered moist chamber at 4 C. After rinsing with PBS, the appropriate dilution of goat antihuman IgG conjugated with FITC was applied and incubated overnight at room temperature in a moist chamber. The slides were rinsed with PBS and a coverslip applied using DABCO mounting media (Sigma). Slides were coded, randomly arranged, and read in a masked fashion using a episcopic fluorescent microscope (Nikon Instruments Inc., Melville, NY). Each section was scored before breaking the code. Our laboratory has participated in a total of eight International Diabetes Society Workshops and the International Diabetes Society (IDS)-sponsored proficiency programs for ICA with an average sensitivity of 80% and a specificity of 100% each time. In the IDS-sponsored Combined Antibody Workshop, our ICA assay had a specificity of 98% and a sensitivity of 76%. Our ICA assay has been validated in a serum exchange with the Diabetes Prevention Trial-Type 1 ICA core laboratory. In this exchange, the sensitivity of our assay was 85% with a specificity of 100%. The lower detection limit for our ICA assay is 1 Juvenile Diabetes Foundation (JDF) unit and values above the 99th percentile established from approximately 4000 normal school children (4 JDF units) were considered positive.

Glutamic acid decarboxylase 65-kDa autoantibody (GAD65Ab) assay

The determination of GAD65Ab levels was performed at the Immunoassay Core of the Diabetes Endocrinology Research Center, University of Washington. GAD65Abs were measured in a radiobinding immunoassay on coded serum samples as described (13). The levels of GAD65Ab were expressed as a relative index (GAD65 index) using one positive serum (JDF world standard for ICA) and three negative standard sera from healthy subjects. GAD65 index was calculated and a positive was considered at 0.085 or less, which is the 99th percentile based on 200 normal controls. The positive and negative controls are run in duplicate in each assay. The performance of the assays is monitored by a set of quality controls and participation in external laboratory proficiency comparison. The laboratory has participated in the Diabetes Antibody Standardization Program (DASP). In the 2001 evaluation, the laboratory scored 76% sensitivity and 94% specificity (14). In the 2002 evaluation, the laboratory scored 84% sensitivity and 96% specificity in the DASP program.

Insulinoma associated protein-2 autoantibody (IA-2) assay

Autoantibodies to IA-2 were measured under identical conditions as described for GAD65Ab (13). The plasmid containing the cDNA coding for the cytoplasmic portion of IA-2 was kindly donated by Dr. G. Eisenbarth (Barbara Davis Research Center, Denver, CO). The same JDF standard serum and control sera as in the GAD65 Ab assay were used to calculate the IA-2 Ab index for each sample. An IA-2 index of 0.017 or more, the 99th percentile based on 200 normal controls, is taken as the cut-off for positivity. The performance of the assays is monitored by a set of quality controls and participation in external laboratory proficiency comparison. The laboratory has participated in the DASP. In the 2001 evaluation, the laboratory scored 54% sensitivity and 96% specificity (14). In the 2002 evaluation, the laboratory scored 62% sensitivity and 89% specificity in the DASP program.

Insulin autoantibody (IAA) assay

The IAA assay is performed as previously described (11, 12). The threshold for positivity was also the 99th percentile of values in normal controls. IAAs were evaluated before the start of insulin therapy. We participated in the International Diabetes Society Workshops- and IDS-sponsored workshops and proficiency programs for IAA with a sensitivity of 100% and specificity of 100%.

Cellular immunoblotting

Cellular immunoblotting was performed as previously described for islets (8, 11, 12, 15) and nonislet proteins (16, 17, 18). Human pancreata were obtained by University of Washington transplant surgeons, and pancreatic islets were isolated within 48 h, post mortem, at the Islet Satellite of the Tissue Culture Core of the Diabetes Endocrinology Research Center (University of Washington, Seattle, WA). Islet cells and tetanus toxoid were subjected to preparative 10% SDS-PAGE (19). After electrophoresis, the gels were electroblotted onto nitrocellulose (Bio-Rad Laboratories, Richmond, CA) at 30 mA overnight, nitrocellulose particles prepared (20), and the nitrocellulose particles used to stimulate peripheral blood mononuclear cell (PBMCs) in vitro.

A stimulation index (SI) for each molecular weight section was calculated as follows:

Control wells contained nitrocellulose particles without antigen. Positive proliferation was considered to be an SI greater than 2.0, which corresponds to greater than the mean + 3 SD of control values (8). Antigen doses and specificity of PBMC responses of type 1 diabetes patients to the islet protein preparations and known islet autoantigens using cellular immunoblotting have been previously described (8). PBMC responses to tetanus toxoid were used as a control antigen along with the PBMC responses to mitogens, which were included to test for viability of the cultures. PBMC responses of type 1 diabetes patients, autoantibody positive type 2 diabetes patients, autoantibody-negative type 2 diabetes patients, and controls to tetanus toxoid have been shown to be similar (11). Based on results in more than 60 controls and 60 patients with type 1 diabetes, cellular immunoblotting is considered positive when more than three blot sections have an SI greater than 2.0 (12).

HLA typing

DR typing of subjects was performed by the Molecular and Genetics Core of the Diabetes Endocrinology Research Center at the University of Washington.

Briefly, DNA was isolated from peripheral blood leukocytes by an automatic extractor (model 340 A, Applied Biosystems, Foster City, CA) and the HLA class II typing was performed by an oligoblot hybridization method (21). DRB1 was amplified by the PCR and labeled by incorporation of digoxigenin-labeled dUTP during the PCR. High-resolution HLA typing was achieved by the hybridization of the labeled PCR products to nylon membranes containing allele-specific oligo-probes selected from various functional DRB loci. Positive reactions were visualized by a chemiluminescent detection method (Roche Diagnostics, Indianapolis, IN).

Statistics

Autoantibodies and T-cell responses to islet proteins were tested in a blinded fashion. Clinical classification was determined before knowledge of T-cell or autoantibody status. Unpaired t tests and Fisher’s exact test were performed to determine significance among groups.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Children clinically classified with type 1 diabetes (n = 37)

Weight loss was reported in 81% of type 1 subjects, 78% had urinary ketones, with 19% having acidosis as well. Clinical characteristics are summarized in Table 1.

Thirty-six of the 37 children classified with clinical type 1 diabetes were positive for two or more autoantibodies. One child was autoantibody negative at diagnosis. Four of the 37 patients (11%) were positive for only two autoantibodies (three positive for GAD/IA-2 and one positive for ICA/GAD), 17 of 37 (46%) were positive for only three autoantibodies (11 for ICA/GAD/IA-2 and six for ICA/IAA/IA-2) and 15 of 37 (41%) were positive for all four autoantibodies tested (ICA/IAA/GAD/IA-2). Autoantibody results are summarized in Table 3 and Fig. 1.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Summary of autoantibody and T-cell responses for all subjects.

View larger version (16K): [in this window] [in a new window]   FIG. 2. T-cell responses to islet proteins, using cellular immunoblotting, of children classified with type 1 diabetes, n = 37; type 2 diabetes, n = 19; and indeterminant diabetes, n = 16. Shaded symbols indicate individuals positive for at least one autoantibody.

Children clinically classified with type 2 diabetes (n = 19)

Eighteen of the 19 type 2 patients were obese (>85th percentile) and all 19 patients demonstrated the presence of acanthosis nigricans. Although none had DKA, 26% of the patients had urinary ketones, and 32% demonstrated weight loss (Table 1).

Autoantibody status is summarized in Table 3 and Fig. 1. Five of the 19 patients (26%) classified with type 2 diabetes were negative for autoantibodies. Of the 14 patients (74%) positive for autoantibodies, four patients (21%) were positive for only one autoantibody [one patient was ICA positive only (6 JDF units), one patient was IAA only, two patients were GAD only], six patients were positive for only two autoantibodies (one patient was IAA/GAD, four patients were positive for ICA/IA-2, one patient was ICA/GAD), two patients were positive for only three autoantibodies (one patient ICA/GAD/IA-2 and one patient ICA/IAA/IA-2), and two patients (11%) were positive for all four autoantibodies tested.

Four of the five patients that were autoantibody negative were also negative for T-cell responses (Fig. 2). The other autoantibody-negative patient responded to six blot sections. This subject was Hispanic, was obese, had acanthosis nigracans, and presented with urinary ketones. Of the 14 patients that were positive for autoantibodies, five were also positive for T-cell reactivity. Of the five patients that were positive for T-cell responses, one was positive for GAD autoantibodies only, one for ICA/GAD, one for ICA/GAD/IA-2, and two were positive for all four autoantibodies. The other nine patients who were negative for T-cell reactivity, four patients were positive for ICA/IA-2, one was positive for ICA/IAA/IA-2, one was positive for ICA (6 JDF units) only, two were GAD positive only, and one was IAA positive only.

Children clinically classified with indeterminant diabetes (n = 16)

Of the indeterminant patients, 31% were obese, 56% had urinary ketones, 19% had acanthosis nigracans, 31% had weight loss, and 19% presented in DKA (Table 1).

Five of the 16 patients (31%) classified with indeterminant diabetes were negative for autoantibodies (Table 3 and Fig. 1). Of the 11 patients positive for autoantibodies, two were positive for only one autoantibody (one was positive for ICA, one for IA-2), three patients were positive for only two autoantibodies (ICA/IA-2), and six were positive for only three autoantibodies (four for ICA/GAD/IA-2, one for IAA/GAD/IA-2, and one for ICA/IAA/GAD).

Four of the five autoantibody-negative patients were also negative for T-cell responses (Fig. 2). The autoantibodynegative patient who was positive for T-cell responses, responding to four blot sections, was a lean Asian with urinary ketones. Of the 11 patients who were positive for autoantibodies, five were also positive for T-cell responses to islet proteins. Of these five, one was positive for ICA only, one for ICA/IAA/GAD, and three were positive for ICA/GAD/IA-2. None of the three patients that were positive for ICA/IA-2 demonstrated T-cell reactivity to islet proteins.

Comparisons among type 1, indeterminant, and type 2 diabetes patients

Significant differences were observed between clinically classified type 1 and type 2 diabetes children for the number of autoantibodies positive (P < 0.0001) and the number of blot sections recognized by their T cells (P < 0.005). Differences were also observed for the number of autoantibodies positive (P < 0.001) between type 1 and indeterminant diabetes patients and for the number of blot sections positive (P < 0.05). No significant differences were observed between type 2 and indeterminant patients in autoantibody or T-cell reactivity. Autoantibody and T-cell reactivity to islet proteins are summarized in Table 3 for the three groups of patients.

DR typing was available on 28 type 1 patients, 15 type 2 patients, and 11 indeterminant patients. Eighty-nine percent (25 of 28) of the type 1 patients had diabetes-associated HLA (DR3 and/or DR4). Of these 25, 24 were autoantibody positive. Of the type 2 patients, 67% (10 of 15) were positive for DR3 and/or DR4 and eight of the 10 were autoantibody positive. For the indeterminant patients, 82% (9 of 11) were found to have DR3 and/or DR4, and six of these nine were autoantibody positive. The frequency of DR3 and/or DR4 in the three groups was not significantly different, but this may be due to the relatively small number of subjects in each group. The DR typing and autoantibody data are summarized in Table 4.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In this study, we observed that all but one of the children classified with classic type 1 diabetes (n = 37) were positive for autoantibodies to islet proteins, whereas 74% of the type 2 and 69% of the indeterminant patients were positive for islet cell autoantibodies. In our previous studies of adults with phenotypic type 2 diabetes (11, 25), we observed a much lower frequency of autoantibodies to islet proteins. Thus, the high percentage of children classified with type 2 or indeterminant diabetes who were positive for autoantibodies was an unexpected result. However, based on the sensitivity and specificity of the autoantibody assays we employed (see Subjects and Methods), we are confident in the results we observed. Because positivity for autoantibodies has been set at the 99th percentile of normal controls, we would expect only 1% of the apparent autoantibodies observed to be false positives. We also investigated whether the presence of autoimmunity was evident in these patients by investigating T-cell reactivity and HLA of the patients. The T-cell assay that we used, cellular immunoblotting, recently performed well at distinguishing controls from recently diagnosed type 1 diabetes patients in a blinded study funded by National Institutes of Health (data not published).

A high percentage of type 2 and indeterminant patients demonstrated autoimmunity as determined by T-cell reactivity to islet proteins. The diabetes susceptibility alleles HLA DR3 and/or DR4 exist in approximately 44% of the general population. In this study, we found these susceptibility alleles in 89% (25 of 28) of type 1 patients, 67% (10 of 15) of type 2 patients, and 82% (9 of 11) of indeterminant patients available for typing. This high percentage of subjects with diabetes-associated HLA in the type 2 and indeterminant diabetes groups further supports the autoantibody and T-cell results that a large number of patients identified by physicians as type 2 diabetes in fact demonstrate autoimmunity. Therefore, taken together the autoantibody, T-cell, and HLA status of these patients support the conclusion that many patients classified as either type 2 or indeterminant may have autoimmune diabetes. Clinical characteristics did not clearly distinguish type 1 vs. type 2 or indeterminant diabetes.

Our results report a higher percentage of patients classified with type 2 or indeterminant diabetes demonstrating autoreactivity to islet proteins than those reported by others (5, 6, 26); however, this appears to most likely be due to different patient populations studied. In our study 26% of our type 2 patients were Caucasian, 21% Hispanic, 11% Asian, 11% African-American, and 5% other. Whereas patients in the studies by Libman et al. (6) and Katz et al. (26) were primarily African-American (67 and 51.7%, respectively), and in a study by Hathout et al. (5), the type 2 patient population studied was 62.9% Hispanic. The percentage of patients with autoimmunity appears to be strongly influenced by race; thus, varying patient populations may significantly vary the percentages of patients positive for autoreactivity. However, the important issue is the relatively high percentage of patients diagnosed with type 2 or indeterminant diabetes that are positive for islet cell autoimmunity in all these studies.

Another interesting observation concerning autoantibodies involved a cohort of seven children, three diagnosed with indeterminant diabetes and four with type 2 diabetes. All seven of these children were positive for ICA/IA-2 but did not demonstrate T-cell reactivity to islet proteins. Five of the seven children were obese and demonstrated the presence of acanthosis nigricans. Future studies may help us identify whether children with ICA and IA-2 alone, or possibly the absence of GAD autoantibodies in children, represent a subgroup of patients with potentially unique autoimmune characteristics. Seissler et al. (27) observed that the presence of IA-2 autoantibodies, restricted to the IgG4 subclass, may in fact be associated with protection from type 1 diabetes. Further studies will help to confirm whether the patient population in our study has a similar antibody restriction.

In this study, we also observed T-cell responses to islet proteins in children with type 2 and indeterminant diabetes. Taken together, the results of our study and others demonstrate that autoreactivity to islet proteins, whether autoantibodies or T cells or both, is more prevalent than expected in children classified with type 2 or indeterminant diabetes. Moreover, these studies also demonstrate that classic autoantibody-negative type 2 diabetes may be less common than believed in patients with diabetes diagnosed during childhood. We also found that diabetes-associated HLA (DR3 and/or DR4) occurred more commonly in the autoantibody-positive patients in each of the three groups. The presence of diabetes-associated HLA, however, is also present in some of the autoantibody-negative patients. Thus, having diabetes-associated DR alleles is not a strong marker of type 1 diabetes or of autoimmunity.

Katz et al. (26) observed that children with new-onset type 2 diabetes could be distinguished from those with type 1 diabetes with a combination of age, race, body mass index, acanthosis nigracans, c-peptide, and urine ketones. Hardin et al. (28) observed that type 2 diabetes in youth could be distinguished from type 1 diabetes by age, ethnicity, obesity, presence of acanthosis nigricans, serum pH, Tanner stage, and family history. In our study we compared obesity, presence of acanthosis nigricans, ethnicity, weight loss, diabetic ketoacidosis, and urine ketones. We observed that none of the clinical characteristics, with the possible exception of urine ketones, segregated those patients with islet autoimmunity from those patients who did not have islet autoimmunity. The presence of ketosis (as did Katz et al.) significantly correlated (P < 0.05) with the presence of autoimmunity to islet proteins, but the absence of ketones did not indicate the absence of autoimmunity to islet proteins. Two of the nonobese patients (one type 2 and one indeterminant) demonstrated acanthosis nigricans. Both of these patients were positive for autoantibody, T cells, urinary ketones, and weight loss. One of the two patients presented in DKA. Thus, it would be of interest to investigate what level of insulin resistance may be present in these two patients.

In adults, most patients with phenotypic type 2 diabetes are autoantibody and T cell negative. However, this does not appear to be the case in children. Our unexpected results demonstrate that autoimmunity to islet proteins consistent with type 1 diabetes is present in a high percentage of children clinically classified as type 2 or indeterminant diabetes. In agreement with the adult autoantibody-positive type 2 patients, clinical phenotypic characteristics typically associated with a diagnosis of type 2 diabetes do not reliably distinguish autoimmune vs. nonautoimmune children (25). These results suggest that the reported increase in clinical type 2 diabetes may actually represent an increase in obese children with type 1 diabetes and that classic type 2 diabetes (negative for autoantibodies and T-cell autoreactivity to islet proteins) is less common. We are currently investigating whether the clinical course is different in children who are autoantibody and T cell positive or negative for both or cases in which autoantibodies and T-cell results are different. Such a study requires a much larger number of patients followed up prospectively and is currently underway. Currently, there is a 5-yr multicenter study (SEARCH) funded by the Centers for Disease Control and Prevention and the National Institute of Diabetes and Digestive and Kidney Diseases investigating how types 1 and 2 diabetes differ in children by age and race/ethnicity. With more studies in the future incorporating autoantibody and T-cell assays to study diabetes in children, the questions concerning the presence of autoreactivity in these patients can be answered.

One of the extreme difficulties, as demonstrated by the results in this study, is the identification of autoantibody-negative type 2 children. Thus, a much larger study will be needed to identify a large number of autoantibody-negative subjects. The results of this current study stress the need for more studies to investigate the presence vs. the absence of autoimmune markers in children with diabetes, even if the clinical features strongly suggest a diagnosis of type 2 diabetes. Our results also demonstrate that children with diabetes should be tested for the presence of HLA, autoantibodies, and T cells reactive with islet proteins, regardless of their clinical presentation. Those children with islet autoimmunity should probably not be classified as type 2 diabetes patients.

	Acknowledgments

 
We sincerely thank Rick Mauseth, M.D., for providing some of the diabetes patients. Also, we thank Connaught Laboratories for supplying the tetanus toxoid. We also thank Dr. G. Eisenbarth (Barbara Davis Research Center, Denver, CO) for supplying the plasmid containing the cDNA coding for the cytoplasmic portion of IA-2.

	Footnotes

 
This work was supported in part by the Medical Research Service of the Department of Veterans Affairs through a VA Merit Review grant and by the following grants from the NIH: MO1 RR00037-41, P01 DK53004, and P30 DK17047.

Abbreviations: DKA, Diabetic ketoacidosis; GAD65Ab, glutamic acid decarboxylase 65-kDa autoantibody; HLA, human leukocyte antigen; IA-2, insulinoma associated protein-2 autoantibody; IAA, insulin autoantibody; ICA, islet cell antibody; PBMC, peripheral blood mononuclear cell; SI, stimulation index.

Received August 4, 2003.

Accepted February 4, 2004.

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