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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1719
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 2 462-467
Copyright © 2007 by The Endocrine Society

Association of Amino-Terminal-Specific Antiglutamate Decarboxylase (GAD65) Autoantibodies with ß-Cell Functional Reserve and a Milder Clinical Phenotype in Patients with GAD65 Antibodies and Ketosis-Prone Diabetes Mellitus

Christiane S. Hampe, Ramaswami Nalini, Mario R. Maldonado, Tyler R. Hall, Gilberto Garza, Dinakar Iyer and Ashok Balasubramanyam

Robert H. Williams Laboratory (C.S.H., T.R.H.), Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98195; Translational Metabolism Unit (R.N., M.R.M., D.I., A.B.), Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, and Endocrine Service (R.N., G.G., A.B.), Ben Taub General Hospital, Houston, Texas 77030; and Bristol-Myers-Squibb, Co. (M.R.M.), Princeton, New Jersey 08540

Address all correspondence and requests for reprints to: Christiane S. Hampe, Ph.D., Department of Medicine, University of Washington, Seattle, Washington 98195. E-mail: champe{at}u.washington.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: We previously characterized patients presenting with diabetic ketoacidosis prospectively into four subgroups of ketosis-prone diabetes mellitus (KPDM), based on the presence or absence of ß-cell autoimmunity (A+ or A–) and ß-cell functional reserve (B+ or B–). The A+B– KPDM subgroup comprises patients with classic, autoimmune type 1 diabetes, whereas the A+B+ KPDM subgroup has only partial ß-cell loss and a distinct clinical phenotype.

Objective: We hypothesized that epitope specificity of autoantibodies directed against the 65-kDa isoform of glutamate decarboxylase (GAD65) reflects differences in ß-cell destruction.

Design: Sera of sequential GAD65Ab-positive KPDM patients admitted for diabetic ketoacidosis (n = 36) were analyzed for their epitope recognition using five GAD65-specific recombinant Fab and their ability to inhibit GAD65 enzymatic activity. All patients were followed longitudinally to assess ß-cell functional reserve and insulin dependence.

Results: Binding to an amino-terminal epitope defined by monoclonal antibody DPD correlated positively with fasting serum C-peptide levels at baseline (P = 0.0008) and after 1 yr (P = 0.007). Binding to the DPD-defined epitope also correlated positively with area under the curve for C-peptide after glucagon stimulation (P = 0.007) and with homeostasis model assessment percent B at 1 yr (P = 0.03). Binding to the DPD-defined epitope was significantly stronger in A+B+ than in A+B– patients (P = 0.001). Sera of 16 patients (44%) significantly inhibited GAD65 enzymatic activity, but this did not correlate with ß-cell function.

Conclusion: DPD-defined epitope specificity is correlated directly with preserved ß-cell functional reserve in GAD65Ab-positive patients and is associated with the milder clinical phenotype of A+B+ KPDM.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
DIABETIC KETOACIDOSIS (DKA) is thought to be due to virtually complete lack of insulin secretion by pancreatic ß-cells or, rarely, overwhelming insulin resistance (1). DKA has traditionally been viewed as a complication of type 1 diabetes (T1D), but several recent studies have shown that patients presenting with DKA in multiethnic, urban settings are heterogeneous in regard to the type of diabetes (2, 3, 4, 5, 6) (for review, see Ref. 7). We prospectively characterized these heterogeneous forms of ketosis-prone diabetes mellitus (KPDM) into four subgroups, based on the presence or absence of autoantibodies (A+ or A–) and ß-cell functional reserve (B+ or B–), measured longitudinally after the index episode of DKA (8). Two subgroups of KPDM patients show signs of ß-cell-specific autoimmunity, reflected in circulating autoantibodies against ß-cell antigens (9, 10, 11). One of these subgroups (A+B– KPDM) comprises patients with classic T1D (early age of onset of diabetes, tendency to leanness, complete absence of ß-cell functional reserve, total dependence on exogenous insulin), whereas the other (A+B+ KPDM) comprises patients with a distinctly different phenotype (older age of onset, tendency to overweight/obesity, partially preserved ß-cell functional reserve, and insulin dependence or independence) (8). Other investigators have described patients with phenotypes resembling A+B+ KPDM and classified them as latent autoimmune diabetes in adults (LADA) (12), type 1.5 diabetes (12, 13, 14, 15), or slowly progressive type 1 (insulin-dependent) diabetes mellitus (for review, see Ref. 16).

In addition to their baseline phenotypic differences, A+B– and A+B+ KPDM patients also follow distinctly different clinical courses after the index episode of DKA. In longitudinal follow-up over more than 2 yr in a dedicated research clinic, we observed that A+B– KPDM patients never recover ß-cell function and therefore remain completely insulin dependent, whereas a third to half of the patients classified as A+B+ maintain well-preserved ß-cell function and remain insulin independent, whereas the others experience gradual deterioration of ß-cell function and become insulin dependent (17).

There is ongoing debate as to the key factors that differentiate the autoimmune processes that lead on the one hand to rapid, complete destruction of ß-cells in classic, autoimmune T1D and on the other hand to the slower, later-onset, and variably progressive forms of ß-cell destruction in A+B+ KPDM, LADA, or type 1.5 diabetes (for review, see Ref. 18). Identification of such factors might not only provide clinically useful or prognostic markers but also identify molecular mechanisms of autoimmune ß-cell destruction underlying the pathophysiology of these distinct syndromes. We hypothesized that these two subgroups of KPDM would differ in the characteristics of their ß-cell-specific autoimmunity.

Autoantibodies directed to the 65-kDa isoform of glutamate decarboxylase (GAD65), insulin, and a protein tyrosine phosphatase-like islet cell antigen (IA-2) predict the disease (19, 20). Whereas insulin autoantibodies and IA-2Ab are negatively associated with age at onset, autoantibodies directed against GAD65 (GAD65Ab) are directly associated with age at onset (21). In our previous work, we found that epitope specificity of GAD65Ab is a better indicator of the degree of underlying ß-cell destruction and the associated clinical effects than GAD65Ab titers alone (22, 23). Therefore, we analyzed the epitope specificity of GAD65Ab as a reflection of ß-cell autoimmunity in A+B– and A+B+ KPDM patients.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The protocol was approved by the Institutional Review Boards for Human Studies of Baylor College of Medicine and the Harris County Hospital District, Houston, Texas. Informed consent was obtained from all patients. We analyzed the sera of adult, GAD65Ab-positive patients (n = 36) who were admitted sequentially for DKA to Ben Taub General Hospital and followed up thereafter as outpatients in a dedicated research clinic.

DKA was defined by the presence of all of the following: anion gap 15 or greater, blood pH less than 7.30, serum bicarbonate 17 mmol/liter or less, serum glucose greater than 200 mg/dl, serum ketones 5.2 mmol/liter or greater, or urine ketones moderate to large, as described previously (8).

ß-Cell secretory capacity was measured at the time of the initial presentation with DKA (within 1 wk after resolution of ketoacidosis) and again after 12 months of follow-up by the following tests: fasting serum C-peptide concentration and C-peptide response to glucagon (8). (The methods for performing these tests as well as receiver-operator curve analysis to establish the C-peptide cutoffs that distinguish B– from B+ status have been previously established and published by us (8). Briefly, ß-cell functional reserve was defined as preserved (B+) if the peak C-peptide response to glucagon was at least 1.5 ng/dl (0.5 nmol/liter) or fasting C-peptide concentration was at least 1 ng/dl (0.33 nmol/liter). ß-Cell functional reserve was defined as absent (B–) if the glucagon-stimulated or fasting C-peptide concentrations did not meet these criteria. Receiver operator characteristic analysis of these levels were used to determine these cut-off values [area under the curve (AUC) value = 0.97776 for fasting C-peptide, 0.96751 for peak C-peptide response to glucagon, and 0.96089 for C-peptide to glucose ratio].

GAD65Ab in the patients’ sera was measured as described below. The upper limit of the normal range for the autoantibody level was established independently for each ethnic group (8). Patients were classified as GAD65Ab positive if the GAD65Ab level exceeded the ethnic-specific 99th percentile.

Patients who were GAD65Ab positive were classified as A+B+ if the fasting serum C-peptide concentration was at least 1 ng/ml (0.33 nmol/liter) or the peak serum C peptide response to glucagon was at least 1.5 ng/ml (0.5 nmol/liter) (receiver operator characteristic AUC for peak serum C-peptide concentration after glucagon stimulation = 0.96751). Patients were classified as A+B– if the fasting serum C-peptide concentration and peak serum C-peptide concentration after glucagon stimulation were less than these cut-off values. These definitions follow the criteria for patient definition as described previously (17).

Long-term insulin dependence was assessed in all patients who were initially B+ by the following clinical protocol. All patients were placed on twice daily isophane insulin at the time of hospital discharge. If the self-monitored blood glucose values before each meal and at bedtime during a 2-wk period attained American Diabetes Association-defined goals for fasting and/or bedtime plasma glucose levels, the insulin dose was reduced by 50% and the patient was reassessed 1 wk later. If the mean blood glucose values remained at American Diabetes Association goals at two consecutive clinic visits made 2–4 wk apart, insulin was discontinued and the patient was monitored closely. If blood glucose values increased, but ketosis did not develop, the patients were placed on oral hypoglycemic agents. Conversely, if the patient developed ketosis on decreasing insulin dosage, the insulin regimen was intensified and no further attempts were made to discontinue insulin in such patients.

Monoclonal recombinant antigen binding fragment of the antibody (rFab) used in this study

Monoclonal antibodies DPA, DPC, and DPD were isolated from a patient with T1D (24); they recognize epitopes located at amino acids 483–585, 195–412, and 96–173, respectively (25, 26). Monoclonal antibodies b96.11 and b78 were derived from a patient with autoimmune polyendocrine syndrome type 1 (27) and recognize epitopes located at amino acid residues 308–365 and 451–585, respectively (26, 27). All monoclonal antibodies used in this study are specific to GAD65 and do not recognize the larger isoform of GAD, namely GAD67. rFab of these antibodies were generated and expressed as previously described (28).

Radioligand binding assay

Recombinant [35S]GAD65 was produced in an in vitro-coupled transcription/translation system with SP6 RNA polymerase and nuclease-treated rabbit reticulocyte lysate (Promega, Madison, WI) as described previously (29). The in vitro-translated [35S]antigen was kept at –70 C and used within 2 wk. Binding of rFab to radiolabeled antigen was determined as described previously (23), using protein G Sepharose (Zymed Laboratories, Carlton Court, CA) as the precipitating agent.

Competition studies of rFab

The capacity of the rFab to inhibit GAD65 binding by human serum GAD65Ab was tested in a competitive RBA using protein A Sepharose (Zymed Laboratories) as described (23). The rFab were added at the optimal concentration (0.7–1 µg/ml) as determined in competition assays using the intact monoclonal antibody as a competitor. This rFab concentration was confirmed as optimal in titration experiments showing that an increase of 100% or a decrease by 50% in the rFab concentration did not yield a significant change in the competition with human sera. The background competition for each rFab was established in competition experiments with normal control sera. The background was subtracted before calculation of percent inhibition. The cutoff for specific competition was determined as more than 10% by using as a negative control rFab D1.3 (a kind gift from Dr. J. Foote, Arrowsmith Technologies, Seattle, WA), specific to an irrelevant target, hen-egg lysozyme, at 5 µg/ml.

Binding of GAD65Ab to GAD65 in the presence of rFab was expressed as follows:

Formula

GAD65 enzymatic activity assay

Glutamate decarboxylase activity was measured by the 14CO2-trapping method described previously (30). Briefly, recombinant human GAD65 (a kind gift from Zymogenetics, Seattle, WA) (100 ng) was incubated with or without the indicated amounts of serum for 1 h at room temperature. The enzymatic reaction was initiated by the addition of 0.56 mM L-glutamate and 0.018 µCi 14C-glutamate (Amersham Life Science Inc., Arlington Heights, IL) and allowed to continue for 2 h at 37 C. During incubation, the released 14CO2 was captured on filter paper (Kontes, Vineland, NJ) soaked in 50 µl 1 M NaOH. After the incubation, the absorbed radioactivity was determined in a scintillation counter (Beckman, Fullerton, CA). The results are presented as follows: percent inhibition = (counts per minute in the presence of serum/counts per minute in the absence of serum) x 100.

Statistical analysis

All samples were analyzed in triplicate determinations. The mean intraassay coefficient of variation was 5% (13–0.04%). Our assay for GAD65Ab showed good sensitivity (80%) and high specificity (91%) in the 2005 Diabetes Antibody Standardization Program Workshop. Descriptive statistics (mean, SD) were used to characterize the A+B– and A+B+ groups. Comparisons between the A+B+ and A+B– groups were analyzed using the nonparametric Mann Whitney U test. Fisher’s exact test was used to assess the differences in categorical variables (insulin dependence) between the A+B+ and the A+B– groups. Significance was defined by P < 0.05 and a trend by P < 0.1. The nonparametric Spearman test was used to analyze correlations.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The demographics, ß-cell functional characteristics, insulin sensitivity, and glycemic control of the 36 GAD65Ab-positive KPDM patients at baseline and after 12 months of follow-up are summarized in Table 1Go. Twenty-four patients were classified as A+B– and 12 patients as A+B+. Consistent with our initial report of the phenotypes of KPDM (8) and our recent validation of the AB classification scheme (17), the A+B+ patients had a significantly higher body mass index than the A+B– patients (P = 0.03) and a trend toward later age of onset of diabetes. The initial hemoglobin A1c (HbA1c) level was not significantly different between the two groups. Glycemic control after 12 months of treatment in the research clinic was significantly better in the A+B+ group, compared with the A+B– group (P < 0.01). In addition, insulin sensitivity at 12 months assessed by homeostasis model assessment using C-peptide measurements (HOMA-2 IR) (31) was significantly lower in the A+B+ group than the A+B– group (P < 0.001) (Table 1Go) consistent with the metabolic phenotype of these patients.


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  		TABLE 1. Demographics, ß-cell function, insulin sensitivity, and glycemic control in GAD65Ab-positive KPDM patients

 
All A+B– patients remained completely insulin dependent, whereas seven of the 12 A+B+ patients required varying amounts of insulin with or without oral hypoglycemic agents for more than 1 yr after the index episode of DKA (P < 0.01). Of these seven A+B+ patients, three were lost to follow-up after 12 months, so attempts to wean off insulin were not completed. Five A+B+ patients remained insulin independent for 2–6 yr after the DKA episode.

Epitope analysis of GAD65Ab-positive KPDM patients

We tested every serum sample for its ability to compete with rFabs b78, DPA, b96.11, DPC, and DPD for binding to GAD65 (Fig. 1AGo). Overall we observed a wide range of binding to the different epitopes. We found no correlation of epitope specificity to age at onset, duration, gender, human leukocyte antigen, race, or GAD65Ab titer (data not shown). When comparing epitope specificity between the two subgroups, we found that binding to GAD65 in the presence of rFab DPD was significantly lower in A+B+ patients than in A+B– patients (median binding 69%, compared with 88%, P = 0.001).


Figure 1
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  		FIG. 1. A and B, GAD65Ab characteristics. A, Binding of serum samples obtained from A+B– (black symbols) and A+B+ (white symbols) KPDM patients to GAD65 in the presence of rFab b78 (square), DPA (circle), b96.11 (triangle), DPC (diamond), and DPD (inverted triangle). Binding to GAD65 in the absence of rFab is defined as 100%. Median binding is indicated. B, Ability of serum samples to inhibit GAD65 enzymatic activity. Enzymatic activity is reported as a percentage, with activity in the absence of serum defined as 100%.

 
Relationship of specific epitope recognition to ß-cell function

We tested whether the humoral immune response to GAD65 reflected in the GAD65Ab epitope specificities correlated with any clinical or biochemical parameters relevant to the KPDM phenotype. We found a significant positive correlation between binding to the DPD-defined amino-terminal conformational epitope and measures of ß-cell functional reserve. Binding to the DPD-defined amino-terminal epitope was correlated positively with the fasting serum C-peptide level both at baseline (R = 0.56, P = 0.0008) and after 12 months (R = 0.54, P = 0.007) (Fig. 2AGo). There was also a trend toward a positive correlation with HOMA%B (31) at baseline (R = 0.27; P ≤ 0.1), which reached statistical significance after 12 months (R = 0.4, P = 0.03) (Fig. 2BGo). Binding to the DPD-defined epitope also correlated with area under the curve for C-peptide after glucagon stimulation (R = 0.53, P = 0.007) (Fig. 2CGo).


Figure 2
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  		FIG. 2. A–C, Correlation between binding to the DPD-defined amino-terminal epitope and ß-cell function. Binding to the DPD-defined amino-terminal epitope is correlated with measurements of ß-cell functional reserve at the time of presentation with the index episode of DKA (white squares) and after 12 months of follow-up (black squares). A, Correlation with fasting serum C-peptide (FCP) concentration. B, Correlation with HOMA%B. C, Correlation with AUC for C-peptide after glucagon stimulation (GST CP-AUC). Binding of serum to GAD65 in the presence of rFab DPD is presented as a percentage, with binding to GAD65 in the absence of rFab defined as 100%. Linear regression curves with R2 and P values are indicated.

 
Correlation between GAD65Ab epitope binding and ß-cell functional reserve is not due to differences in disease duration

It is established that fasting and stimulated C-peptide levels are inversely correlated with the duration of diabetes. Whereas differences in disease duration between the A+B– and A+B+ groups were not statistically significant (Table 1Go), the patients in the A+B+ group showed a trend toward a shorter disease duration. To eliminate the confounding effect of duration of diabetes, we compared binding to GAD65 in the presence of rFab DPD in patients in both groups who presented with DKA at the initial diagnosis of diabetes (n = 7 in both groups). We observed that the binding to GAD65 in the presence of rFab DPD among the new-onset patients in the A+B+ group was significantly lower than among those in the A+B– group (65%, SD 12, compared with 81%, SD 10, P = 0.025; data not shown). Moreover, we found no relationship between binding to the DPD-defined epitope and duration of disease (data not shown).

Inhibition of GAD65 enzymatic activity

To assess whether the association of GAD65Ab epitope specificity with ß-cell functional reserve might be due to differences in glutamate decarboxylase activity in the presence of these antibodies, we tested the ability of the individual sera to inhibit GAD65 enzymatic activity. Sera of 16 of the 36 patients (44%) did in fact inhibit GAD65 enzymatic activity significantly, with the degree of inhibition ranging from 68 to 28% (Fig. 1BGo), a surprising and novel finding because inhibition of GAD65 enzymatic activity has not previously been noted in patients with GAD65Ab-positive T1D, as it has in patients with stiff person syndrome (SPS) (31, 32). However, there was no relationship between inhibition of enzymatic activity, GAD65Ab titer, specific GAD65Ab epitope, ß-cell function, or any other clinical or biochemical parameters (data not shown). Hence, the correlation of DPD epitope specificity with preservation of ß-cell functional reserve could not be ascribed to altered GAD65 enzymatic activity in patients with DPD-specific GAD65Ab.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
KPDM is a heterogeneous condition in which some patients have evidence of ß-cell-specific autoimmunity (for review, see Ref. 7). These patients can be differentiated into those with no ß-cell reserve (A+B–) and those with residual ß-cell reserve (A+B+). The two autoimmune subgroups have distinct clinical phenotypes. In this study we tested the hypothesis that characteristics of ß-cell autoimmunity differ in these two subgroups of KDPM. We analyzed the autoimmune response reflected in GAD65Ab epitope specificity in all A+ patients in our KPDM cohort to determine possible correlations with ß-cell function. Our analysis of other T1D-associated autoantibodies such as IAA and IA-2Ab was impractical because of the insulin treatment of some patients and low frequency of autoantibodies (6% of the patients were IA-2Ab positive), respectively. We analyzed the GAD65Ab present in these patients’ sera both for their ability to recognize five different epitopes and inhibit GAD65 enzymatic function. The results were tested for possible correlations between GAD65Ab epitope specificities and ß-cell function as well as several distinctive clinical characteristics of A+B–, compared with A+B+ patients. Whereas the difference in GAD65Ab titers between the two groups was only weakly significant, we observed a strong positive correlation between binding to an amino-terminal epitope defined by monoclonal antibody DPD and ß-cell functional reserve, i.e. possession of DPD-specific GAD65Ab was associated with sustained, preserved ß-cell functional reserve. On bivariate analysis of the two autoimmune KPDM subgroups, it was also associated strongly with the A+B+ phenotype. No correlation between disease duration and presence of DPD-defined GAD65Ab was observed. The positive association between DPD-defined epitope specificity and the A+B+ phenotype persisted in the subset of new-onset patients with DKA. Thus, it is possible that DPD-defined epitope specificity of GAD65Ab is a characteristic of more slowly progressive ß-cell destruction, resulting clinically in a milder, often insulin-independent course after an episode of DKA. It would be very useful to investigate, in a prospective manner, whether DPD-defined epitope specificity can be used to predict ß-cell functional reserve and a more benign clinical course in a larger group of autoimmune KPDM patients.

The presence of GAD65Ab specific to amino-terminal epitopes was previously reported in patients with type 1.5 diabetes (32) and in Japanese slowly progressive type 1 (insulin-dependent) diabetes mellitus patients (33). Thus, it appears that autoantibodies specific to the amino-terminal region of GAD65 occur with high frequency in patients with autoimmune forms of diabetes who have a later onset or more benign clinical course, even if they present with DKA.

Another interesting and unique feature of this cohort of KPDM patients is that the sera of 16 of the 36 patients (44%) inhibited GAD65 enzymatic activity. Inhibition of GAD65 enzymatic activity is a feature that has been described heretofore almost exclusively in patients with SPS (34, 35). Inhibition of GAD65 enzymatic activity characteristically is absent in patients with autoimmune T1D (35). We did not observe any correlation between ß-cell function or other clinical features and the ability of the patient’s serum to inhibit GAD65 enzymatic activity. However, the relatively small sample size and lack of detailed neurological and neurophysiological analyses of the patients could have obscured such a correlation. In a recent study we found that GAD65 enzyme inhibition by GAD65Ab in patients with SPS correlated with binding to a C-terminal b78-defined epitope (36). In the present study, we did not observe binding to the b78-defined epitope, either by sera that inhibited GAD65 enzymatic activity or sera that did not, suggesting that inhibition of enzymatic activity can be caused by GAD65Ab of different epitope specificities. The differences in the underlying autoimmune responses in SPS and T1D patients are also reflected in the T cell epitope specificities detected in these patients (37, 38). We are currently performing detailed neurological and neurophysiological investigations of GAD65Ab-positive KPDM patients with and without enzyme inhibiting serum activities to determine whether some KPDM patients have a coexisting neurological syndrome linked to this activity.

Previous reports have shown that GAD65Ab levels remain stable for years after diagnosis of T1D (39), and our previous work has shown that GAD65Ab epitope specificity remains stable once the disease is established (22, 40). However, we cannot exclude the possibility that the epitope specificities may change over time. This possibility may be especially relevant in the A+B+ patients, in whom autoimmune destruction of the pancreatic ß-cells could be a slowly progressive, ongoing process.

There is considerable interest in the field of autoimmune diabetes in determining the different pathogenic mechanisms underlying clinically distinct patterns of ß-cell loss. The quest for such mechanisms requires both precise phenotyping of the clinical syndromes as well as identification of the responsible molecular factors in different autoimmune pathways. We previously described differences in GAD65Ab epitope specificity between patients with LADA and those with classic autoimmune T1D, which might explain the long latency in the development of clinical diabetes in the former syndrome (for review, see Ref. 18). Recently further differences in autoantibody pattern and T cell reactivity have been reported to distinguish the pathogenesis of LADA and autoimmune T1D (18). DPD- and other amino-terminal GAD65 epitope-defined autoantibodies may be additional molecular factors that characterize and potentially underlie the pathogenesis of other distinct syndromes of autoimmune ß-cell destruction. Careful delineation of these factors could have important implications for the classification and immunomodulatory therapy of autoimmune diabetes.


   Footnotes
 
This work was supported by an American Diabetes Association Career Development Award (to C.S.H.), a Juvenile Diabetes Foundation Award (to M.R.M.), a Juvenile Diabetes Foundation Career Development Award, the Caroline Wiess Law Award, and RO1-HL73696 (to A.B.).

Disclosure Statement: M.R.M is currently an employee of Bristol-Myers Squibb, Co., which supports his activities related to the collaborative study of ketosis-prone diabetes at Baylor College of Medicine and Ben Taub General Hospital (Houston, TX) and the University of Washington (Seattle, WA).

First Published Online November 7, 2006

Abbreviations: AUC, Area under the curve; DKA, diabetic ketoacidosis; GAD65, 65-kDa isoform of glutamate decarboxylase; HbA1c, hemoglobin A1c; HOMA%B, homeostasis model assessment of ß-cell function; HOMA2-IR, homeostasis model assessment using C-peptide measurements; IA-2, islet cell antigen; KPDM, ketosis-prone diabetes mellitus; LADA, latent autoimmune diabetes in adults; rFab, recombinant antigen binding fragment of the antibody; SPS, stiff person syndrome; T1D, type 1 diabetes.

Received August 9, 2006.

Accepted October 27, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Foster DW, McGarry JD 1983 The metabolic derangements and treatment of diabetic ketoacidosis. N Engl J Med 309:159–169[Medline]
  2. Banerji MA, Chaiken RL, Huey H, Tuomi T, Norin AJ, Mackay IR, Rowley MJ, Zimmet PZ, Lebovitz HE 1994 GAD antibody negative NIDDM in adult black subjects with diabetic ketoacidosis and increased frequency of human leukocyte antigen DR3 and DR4. Flatbush diabetes. Diabetes 43:741–745[Abstract]
  3. Balasubramanyam A, Zern JW, Hyman DJ, Pavlik V 1999 New profiles of diabetic ketoacidosis: type 1 vs type 2 diabetes and the effect of ethnicity. Arch Intern Med 159:2317–2322[Abstract/Free Full Text]
  4. Umpierrez GE, Woo W, Hagopian WA, Isaacs SD, Palmer JP, Gaur LK, Nepom GT, Clark WS, Mixon PS, Kitabchi AE 1999 Immunogenetic analysis suggests different pathogenesis for obese and lean African-Americans with diabetic ketoacidosis. Diabetes Care 22:1517–1523[Abstract/Free Full Text]
  5. Otiniano ME, Balasubramanyam A, Maldonado M 2005 Presence of the metabolic syndrome distinguishes patients with ketosis-prone diabetes who have a type 2 diabetic phenotype. J Diabetes Complications 19:313–318[CrossRef][Medline]
  6. Sapru A, Gitelman SE, Bhatia S, Dubin RF, Newman TB, Flori H 2005 Prevalence and characteristics of type 2 diabetes mellitus in 9–18 year-old children with diabetic ketoacidosis. J Pediatr Endocrinol Metab 18:865–872[Medline]
  7. Umpierrez GE, Smiley D, Kitabchi AE 2006 Narrative review: ketosis-prone type 2 diabetes mellitus. Ann Intern Med 144:350–357[Abstract/Free Full Text]
  8. Maldonado M, Hampe CS, Gaur LK, D’Amico S, Iyer D, Hammerle LP, Bolgiano D, Rodriguez L, Rajan A, Lernmark A, Balasubramanyam A 2003 Ketosis-prone diabetes: dissection of a heterogeneous syndrome using an immunogenetic and ß-cell functional classification, prospective analysis, and clinical outcomes. J Clin Endocrinol Metab 88:5090–5098[Abstract/Free Full Text]
  9. Franke B, Galloway TS, Wilkin TJ 2005 Developments in the prediction of type 1 diabetes mellitus, with special reference to insulin autoantibodies. Diabetes Metab Res Rev 21:395–415[CrossRef][Medline]
  10. Falorni A, Brozzetti A 2005 Diabetes-related antibodies in adult diabetic patients. Best Pract Res Clin Endocrinol Metab 19:119–133[CrossRef][Medline]
  11. Schmidt KD, Valeri C, Leslie RD 2005 Autoantibodies in type 1 diabetes. Clin Chim Acta 354:35–40[CrossRef][Medline]
  12. Tuomi T, Groop LC, Zimmet PZ, Rowley MJ, Knowles W, Mackay IR 1993 Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with a non-insulin-dependent onset of disease. Diabetes 42:359–362[Abstract]
  13. Pietropaolo M, Barinas-Mitchell E, Pietropaolo SL, Kuller LH, Trucco M 2000 Evidence of islet cell autoimmunity in elderly patients with type 2 diabetes. Diabetes 49:32–38[Abstract]
  14. Groop LC, Bottazzo GF, Doniac D 1986 Islet cell antibodies identify latent type 1 diabetes in patients aged 35–75 years at diagnosis. Diabetes 35:237–241[Abstract]
  15. Naik RG, Palmer JP 2003 Latent autoimmune diabetes in adults (LADA). Rev Endocr Metab Disord 4:233–241[CrossRef][Medline]
  16. Kobayashi T 1994 Subtype of insulin-dependent diabetes mellitus (IDDM) in Japan: slowly progressive IDDM—the clinical characteristics and pathogenesis of the syndrome. Diabetes Res Clin Pract 24(Suppl):S95–S99
  17. Balasubramanyam A, Garza G, Rodriguez L, Hampe C, Gaur L, Lernmark A, Maldonado M 2006 Accuracy and predictive value of classification schemes for ketosis-prone diabetes. Diabetes Care 29:2575–2579[Abstract/Free Full Text]
  18. Palmer JP, Hampe CS, Chiu H, Goel A, Brooks-Worrell BM 2005 Is latent autoimmune diabetes in adults distinct from type 1 diabetes or just type 1 diabetes at an older age? Diabetes 54(Suppl 2):S62–S67
  19. Bingley PJ, Bonifacio E, Williams AJK, Genovese S, Bottazzo GF, Gale EAM 1997 Prediction of IDDM in the general population: strategies based on combinations of autoantibody markers. Diabetes 46:1701–1710[Abstract]
  20. Verge CF, Stenger D, Bonifacio E, Colman PG, Pilcher C, Bingley PJ, Eisenbarth GS 1998 Combined use of autoantibodies (IA-2) autoantibody, GAD autoantibody, insulin autoantibody, cytoplasmic islet cell antibodies in type 1 diabetes: Combinatorial Islet Autoantibody Workshop. Diabetes 47:1857–1866[Abstract]
  21. Graham J, Hagopian WA, Kockum I, Li LS, Sanjeevi CB, Lowe RM, Schaefer JB, Zarghami M, Day HL, Landin-Olsson M, Palmer JP, Janer-Villanueva M, Hood L, Sundkvist G, Lernmark A, Breslow N, Dahlquist G, Blohme G 2002 Genetic effects on age-dependent onset and islet cell autoantibody markers in type 1 diabetes. Diabetes 51:1346–1355[Abstract/Free Full Text]
  22. Schlosser M, Banga JP, Madec AM, Binder KA, Strebelow M, Rjasanowski I, Wassmuth R, Gilliam LK, Luo D, Hampe CS 2005 Dynamic changes of GAD65 autoantibody epitope specificities in individuals at risk of developing type 1 diabetes. Diabetologia 48:922–930[CrossRef][Medline]
  23. Padoa CJ, Banga JP, Madec AM, Ziegler M, Schlosser M, Ortqvist E, Kockum I, Palmer J, Rolandsson O, Binder KA, Foote J, Luo D, Hampe CS 2003 Recombinant Fabs of human monoclonal antibodies specific to the middle epitope of GAD65 inhibit type 1 diabetes-specific GAD65Abs. Diabetes 52:2689–2695[Abstract/Free Full Text]
  24. Madec AM, Rousset F, Ho S, Robert F, Thivolet C, Orgiazzi J, Lebecque S 1996 Four IgG anti-islet human monoclonal antibodies isolated from a type 1 diabetes patient recognize distinct epitopes of glutamic acid decarboxylase 65 and are somatically mutated. J Immunol 156:3541–3549[Abstract]
  25. Jaume JC, Parry SL, Madec AM, Sonderstrup G, Baekkeskov S 2002 Suppressive effect of glutamic acid decarboxylase 65-specific autoimmune B lymphocytes on processing of T cell determinants located within the antibody epitope. J Immunol 169:665–672[Abstract/Free Full Text]
  26. Schwartz HL, Chandonia JM, Kash SF, Kanaani J, Tunnell E, Domingo A, Cohen FE, Banga JP, Madec AM, Richter W, Baekkeskov S 1999 High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase. J Mol Biol 287:983–999[CrossRef][Medline]
  27. Tremble J, Morgenthaler NG, Vlug A, Powers AC, Christie MR, Scherbaum WA, Banga JP 1997 Human ß cells secreting immunoglobulin G to glutamic acid decarboxylase-65 from a nondiabetic patient with multiple autoantibodies and Graves’ disease: a comparison with those present in type 1 diabetes. J Clin Endocrinol Metab 82:2664–2670[Abstract/Free Full Text]
  28. Carter P, Kelley RF, Rodrigues ML, Snedecor B, Covarrubias M, Velligan MD, Wong WL, Rowland AM, Kotts CE, Carver ME, Yang M, Bourell JH, Shepard M, Henne D 1992 High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Biotechnology (NY) 10:163–167
  29. Grubin C, Daniels T, Karlsen AE, Boel E, Hagopian WA, Lernmark Å 1992 The cDNA-directed, in vitro-synthesized nascent peptide of glutamic acid decarboxylase (GAD2) is the autoantigen in insulin-dependent diabetes. Clin Res 40:299A
  30. Hampe CS, Hammerle LP, Falorni A, Robertson J, Lernmark A 2001 Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding. FEBS Lett 488:185–189[CrossRef][Medline]
  31. Wallace TM, Levy JC, Matthews DR 2004 Use and abuse of HOMA modeling. Diabetes Care 27:1487–1495[Abstract/Free Full Text]
  32. Hampe CS, Kockum I, Landin-Olsson M, Torn C, Ortqvist E, Persson B, Rolandsson O, Palmer J, Lernmark A 2002 GAD65 antibody epitope patterns of type 1.5 diabetic patients are consistent with slow-onset autoimmune diabetes. Diabetes Care 25:1481–1482[Free Full Text]
  33. Kobayashi T, Tanaka S, Okubo M, Nakanishi K, Murase T, Lernmark A 2003 Unique epitopes of glutamic acid decarboxylase autoantibodies in slowly progressive type 1 diabetes. J Clin Endocrinol Metab 88:4768–4775[Abstract/Free Full Text]
  34. Dinkel K, Meinck H, Jury KM, Karges W, Richter W 1998 Inhibition of {gamma}-aminobutyric acid synthesis by glutamic acid decarboxylase autoantibodies in stiff-man syndrome. Ann Neurol 44:194–201[CrossRef][Medline]
  35. Bjork E, Velloso LA, Kampe O, Karlsson FA 1994 GAD autoantibodies in IDDM, stiff-man syndrome, and autoimmune polyendocrine syndrome type I recognize different epitopes. Diabetes 43:161–165[Abstract]
  36. Raju R, Foote J, Banga JP, Hall TR, Padoa CJ, Dalakas MC, Ortqvist E, Hampe CS 2005 Analysis of GAD65 autoantibodies in stiff-person syndrome patients. J Immunol 175:7755–7762[Abstract/Free Full Text]
  37. Lohmann T, Hawa M, Leslie RD, Lane R, Picard J, Londei M 2000 Immune reactivity to glutamic acid decarboxylase 65 in stiffman syndrome and type 1 diabetes mellitus. Lancet 356:31–35[CrossRef][Medline]
  38. Lohmann T, Londei M, Hawa M, Leslie RD 2003 Humoral and cellular autoimmune responses in stiff person syndrome. Ann NY Acad Sci 998:215–222[Abstract/Free Full Text]
  39. Rowley MJ, Mackay IR, Chen QY, Knowles WJ, Zimmet PZ 1992 Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus. Diabetes 41:548–551[Abstract]
  40. Hampe CS, Hammerle LP, Bekris L, Ortqvist E, Persson B, Lernmark A 2002 Stable GAD65 autoantibody epitope patterns in type 1 diabetes children five years after onset. J Autoimmun 18:49–53[CrossRef][Medline]



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