Shilpa Oak's research while affiliated with Baylor College of Medicine and other places

Context: Ketosis-prone diabetes (KPD), defined by presentation with diabetic ketoacidosis (DKA), comprises 4 subgroups based on the presence or absence of islet cell autoantibodies (A(-) or A(+)) and β-cell functional reserve (β(-) or β(+)). Among A(+) KPD, autoantibody epitope reactivity to 65-kDa glutamate decarboxylase (GAD65), defined by monoclonal GAD65Ab(DPD), was associated with greater β-cell functional reserve. In a majority of healthy individuals, GAD65Ab are present in the sera but are masked by anti-idiotypic antibodies; in contrast, overtly GAD65Ab-positive patients with autoimmune type 1 diabetes patients lack these anti-idiotypic antibodies. Objective: Our objective was to determine the presence of masked and overt GAD65Ab(DPD) in relation to β-cell function and genetic risk factors in KPD patients. Design: We investigated the associations of masked and overt GAD65Ab(DPD) with β-cell functional reserve, and their relationship with human leukocyte antigen (HLA) class II haplotypes linked to autoimmune diabetes susceptibility or resistance, in a large KPD cohort. Patients: Adult KPD patients (n = 384) were followed longitudinally in a research clinic. Main outcome measures: β-Cell function, autoantibody status, GAD65Ab epitopes, and HLA class II haplotypes were evaluated. Results: Overall, KPD patients with β-cell functional reserve (β(+) subgroups) showed significantly higher frequency of masked GAD65Ab(DPD) than patients without β-cell functional reserve (β(-) subgroups): 112 of 144 (79%) compared with 59 of 100 (59%), respectively (P = .002). Masked or overt GAD65Ab(DPD) were also more frequent among autoantibody-positive patients with preserved β-cell functional reserve (A(+)β(+) KPD) than those lacking β-cell function (A(+)β(-) KPD): 77% compared with 55% (P = .01). The susceptibility HLA haplotypes DQA1*0301/DQB1*0302 and DQA1*0301/DQB1*0201 were associated with absence of overt or masked GAD65Ab(DPD) (odds Ratios 2.3 and 2.2, respectively). Conclusions: Masked GAD65Ab(DPD) are strongly associated with preserved β-cell functional reserve among patients with KPD. Absence of GAD65Ab(DPD) reactivity is associated with 2 HLA class II susceptibility haplotypes for autoimmune type 1 diabetes.

Previously we reported the presence of anti-idiotypic antibodies (anti-Id)-specific to autoantibodies against GAD65 (GAD65Ab) in healthy individuals while the activity of anti-Id directed to GAD65Ab in type 1 diabetes (T1D) patients was significantly lower. These anti-Id recognize the antigen-binding site of GAD65Ab, thus preventing their binding to GAD65. Here, we characterized the IgG subclass profile of these anti-Id (GAD65Ab specific) and of the associated GAD65Ab themselves. The IgG subclass response of anti-Id in healthy individuals (n = 16) was IgG3-dominated, while in T1D patients (n = 8) IgG1 was the major IgG subclass. The GAD65Ab bound by anti-Id in both healthy individuals (n = 38) and GAD65Ab-negative T1D patients (n = 35) showed a predominant rank order of IgG1 > IgG2 > IgG4 > IgG3. However, the frequency of GAD65Ab of the IgG4 subclass was significantly higher in T1D patients (P < 0.05). We conclude that the IgG subclass profile of anti-Id (GAD65Ab specific) in healthy individuals differs from that in T1D patients. These differences may provide insights into the development of these antibodies.

The previously reported absence of 65-kDa glutamate decarboxylase antibody (GAD65Ab)-specific antiidiotypic antibodies (anti-Id) in type 1 diabetes (T1D) patients at clinical onset could be due to an inability to mount an antibody response to GAD65Ab or a longitudinal decline in anti-Id levels. We investigated anti-Id levels in longitudinal samples obtained from T1D patients (n = 41) (clinical diagnosis - 12 months), and latent autoimmune diabetes in adults (LADA) patients (n = 32) who received alum-formulated human recombinant GAD65 (baseline - 12 months). We also determined anti-Id levels in a small cohort of Type 2 diabetes patients during their development of autoimmune T cell responses. At clinical onset T1D patients presented no or low anti-Id levels. However, 22/41 T1D patients showed ≥50% increase in GAD65Ab-specific anti-Id levels during follow-up; peaking at 3 (n = 1), 6 (n = 10), 9 (n = 10), or 12 (n = 1) months. Increasing anti-Id levels marked patients who experienced a temporary increase in C-peptide levels. Anti-Id levels correlated significantly with glycated hemoglobin and C-peptide levels at 6 and 9 months (P values ranged from <0.001 to <0.05). In LADA patients receiving placebo, anti-Id levels declined in seven of nine patients, whereas four of five patients receiving 20 μg alum-formulated human recombinant GAD65 showed increasing anti-Id levels. Changes in anti-Id and C-peptide levels closely correlated (P < 0.0001). The significant decline in anti-Id levels (P = 0.03) in T2D patients developing T cell autoimmune responses supports our hypothesis that declining anti-Id levels are associated with developing islet autoimmunity. The close association between GAD65Ab-specific anti-Id levels and β-cell function may provide a novel marker for the progression of autoimmune diabetes.

This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-0785

Autoantibodies to the islet-specific zinc transporter isoform 8 (ZnT8) are detected in the majority of type 1 diabetes patients prior to and at clinical diagnosis. The presence of ZnT8Ab after diagnosis has not been investigated. This study analyzed the autoantibody response to ZnT8 in regard to age at onset and disease duration. Two new onset type 1 diabetes patient cohorts with different age distributions at onset (2-17 and 15-34 years of age at onset), a longitudinal subset of the younger type 1 diabetes patient cohort (n = 32), and a cohort of GAD65Ab-positive LADA patients (n = 47) was analyzed for the presence of autoantibodies directed to the two major isoforms, ZnT8-Arginine (ZnT8R) and ZnT8-Tryptophan (ZnT8W). The majority of type 1 diabetes patients tested positive for ZnT8Ab to both isoforms. ZnT8Ab titers were significantly higher in the younger type 1 diabetes patients as compared with the older cohort (ZnT8RAb at a median of 148 and 29 U/ml, respectively, p < 0.001) (ZnT8WAb at a median of 145 and 58 U/ml, respectively, p < 0.01). ZnT8RAb and ZnT8WAb titers were significantly lower in the LADA patients (ZnT8RAb at a median of 14 U/ml, ZnT8WAb at a median of 25 U/ml) as compared with either type 1 diabetes cohorts. In our longitudinal analysis of type 1 diabetes patients after clinical diagnosis, ZnT8Ab levels to both isoforms declined significantly during the initial year of disease (ZnT8RAb from a median of 320-162 U/ml, p = 0.0001; ZnT8WAb from a median of 128-46 U/ml, p = 0.0011). The antibody titers further declined during the following 4 years (p < 0.0001). We conclude that ZnT8Ab presents a useful marker for type 1 diabetes, especially in younger patients at disease diagnosis.

Anti-idiotypic antibodies (anti-Id) to autoantibodies are present in several autoimmune diseases and are hypothesized to have regulatory function. Recently we reported the presence of anti-Id to a major type 1 diabetes-associated autoantibody (GAD65Ab) in sera of healthy individuals. Our current assay for the detection of GAD65Ab-specific anti-Id requires the initial removal of anti-Id from the sera using immobilized monoclonal GAD65Ab, followed by detection of the now exposed GAD65Ab. However, anti-Id in samples that are GAD65Ab-negative cannot be detected in this assay. Furthermore, we cannot distinguish between serum GAD65Ab and the monoclonal GAD65Ab used in the absorption of anti-Id. In this study we evaluated two novel detection assays for GAD65Ab-specific anti-Id. The biotin/streptavidin based absorption assay utilizes the strong interaction of biotin and streptavidin to prevent possible leakage of the immobilized antibody. Moreover, this assay format allows to identify the origin of the detected GAD65Ab. The ECL-based assay allows the direct detection of anti-Id independent of the presence of GAD65Ab. We analyzed new-onset type 1 diabetes patients (n=133) and matched healthy controls (n=178) for the presence of GAD65Ab-specific anti-Id using both new detection assays and the original absorption assay. We found that all three assays can distinguish between the type 1 diabetes cohort and the healthy control samples. The biotin/streptavidin assay allowed us to positively exclude the monoclonal GAD65Ab as the source of the detected GAD65Ab. While the original absorption assay showed the highest sensitivity and specificity, the ECL format showed the highest peak signal-to-noise ratio and excellent linear correlation, making this assay our first choice for quantification of anti-Id.

Administration of exogenous insulin for the treatment of diabetes is often accompanied by the development of insulin antibodies (IA). These antibodies may affect the patient’s requirement for insulin by acting as an insulin binding reservoir. The improvement of insulin purification in the 1970s and the development of human recombinant insulin both reduced the incidence of IA and their binding levels. This study investigates the parameters affecting IA frequency and binding levels in a cohort of type 1 diabetes (T1D) patients. All patients were treated with human recombinant insulin. About half of the patients had received animal insulin prior to the introduction of human recombinant insulin. We tested the IA frequency and binding level for all serum samples. IA were further analyzed for their epitope specificity comparing human and porcine insulin binding. We found that T1D patients who received animal insulin in the past show significantly higher IA binding levels as compared to patients treated exclusively with human recombinant insulin (IA binding level of 0.9 and 0.25 index, respectively, P=0.005). T1D patients who received animal insulin in the past showed a relative bias towards porcine insulin, as compared to T1D patients who were treated with human recombinant insulin exclusively (P<0.0001). We conclude that IA binding level and epitope specificity are biased by treatment with animal insulin. This bias remains for over 20years after animal insulin treatment is terminated. KeywordsType 1 diabetes-Insulin antibodies-Radio ligand binding assay-Antibody epitopes

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells. Here we show that administration of a human monoclonal antibody (b96.11) specific to the 65-kDa isoform of glutamate decarboxylase (GAD65) to prediabetic non-obese diabetic (NOD) mice significantly delays the onset of autoimmune diabetes. We found this effect to be epitope-specific, as only b96.11 showed this therapeutic property, while a GAD65-specific human monoclonal control antibody (b78) derived from the same patient, but specific to a different determinant of GAD65, had no significant effect on the progression of disease. Administration of b96.11 or b78 to NOD mice was accompanied by the generation of anti-idiotypic antibodies. Importantly, the induced anti-idiotypic antibodies were specific for the immunizing antibody and blocked the binding of GAD65 by the respective antibody. These findings suggest a potential role for the internal image of the GAD65 determinant recognized by b96.11 in the anti-idiotypic antibody, supporting an immunomodulatory role for GAD65-specific autoantibodies, as originally postulated by Jerne.

Autoantibodies to glutamate decarboxylase 65 (GAD65Ab) are commonly believed to be a major characteristic for type 1 diabetes (T1D). We investigated the presence of GAD65Ab in healthy individuals (n = 238) and first-degree relatives (FDRs) of T1D patients (n = 27) who tested negative for GAD65Ab in conventional RIAs. Sera were applied to affinity columns coated with GAD65-specific mAbs to absorb anti-idiotypic antibodies (anti-Ids). The absorbed sera were analyzed for binding to GAD65 by RIAs. Both healthy individuals and FDRs present GAD65Ab that are inhibited by anti-Id, masking them in conventional detection methods. The presence of GAD65Ab-specific anti-Ids was confirmed by competitive ELISA. Remarkably, T1D patients (n = 54) and Stiff Person Syndrome patients (n = 8) show a specific lack of anti-Ids to disease-associated GAD65Ab epitopes. Purified anti-Ids from healthy individuals and FDRs inhibited the binding of GAD65Ab from T1D patients to GAD65. We conclude that masked GAD65Ab are present in the healthy population and that a lack of particular anti-Ids, rather than GAD65Ab per se, is a characteristic of T1D. The lack of these inhibitory antibodies may contribute to T cell activation by GAD65Ab.

To determine whether development of insulin requirement in patients with latent autoimmune diabetes in adults (LADA) is accompanied with the emergence of a type 1 diabetes-like autoimmune response. We correlated beta-cell-specific autoimmunity reflected in autoantibodies to the 65-kDa isoform of GAD (GAD65) with insulin requirement. We determined GAD65Ab epitope specificities in type 1 diabetic patients, LADA patients without insulin requirement (nonprogressed), and LADA patients that had developed insulin requirement (progressed). Recognition of a type 1 diabetes-specific GAD65Ab epitope was more pronounced in type 1 diabetic patients than in nonprogressed (P < 0.001) or progressed (P < 0.01) LADA patients, with no significant differences between the two LADA cohorts. These differences were particularly pronounced in samples with GAD65Ab titers <1,000 units/ml, with no differences in epitope specificities in samples with higher GAD65Ab titers. Disease duration (initial diabetes diagnosis until sample collection or development of insulin requirement) in nonprogressed and progressed LADA patients, respectively, was not correlated with epitope specificity, suggesting lack of epitope maturation. This was supported by epitope analyses of longitudinal samples from LADA patients during progression to insulin requirement. First, the GAD65Ab-specific autoimmune reaction in type 1 diabetic patients with low and moderate GAD65Ab titers differs from that in LADA patients, irrespective of insulin requirement. Second, the GAD65Ab-specific autoimmune response in LADA patients does not change after their initial diabetes diagnosis. Finally, LADA patients with high GAD65Ab titers resemble type 1 diabetic patients in their GAD65Ab epitope specificity.