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Distinct Idiotypes of Insulin Autoantibody in Autoimmune Polyendocrine Syndrome Type 2 and Childhood Onset Type 1 Diabetes

Department of University Medicine (D.D., B.F., S.J.H., T.J.W.), Peninsula Medical School, Plymouth PL6 8DH, United Kingdom; Department of Biological Sciences (T.S.G.), University of Plymouth, Plymouth, United Kingdom; and Hospital for Children and Adolescents (M.K.), University of Helsinki, FIN-33014 Helsinki, Finland

Address all correspondence and requests for reprints to: Dr. D. Devendra, The Endocrine Department, Hammersmith Hospital, Du Cane Road, London W12 0HS, United Kingdom. E-mail: ddevendra{at}hhnt nhs.uk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Insulin autoantibodies (IAA) are present in type 1 diabetes (T1D) and other autoimmune diseases. The differences in the IAA epitopes in various clinical diseases have not been evaluated. We used phage display to select phagotopes specific to IAA from a newly diagnosed T1D child (designated FPP) and from an adult-onset T1D subject with autoimmune polyendocrine syndrome type 2 (APS-II). The phagotopes randomly selected were tested as antiidiotope reagents to displace human radiolabeled insulin in the microfiltration radiobinding assay using IAA⁺ sera from T1D subjects and insulin antibody (IA⁺) sera from insulin-treated type 2 diabetes subjects. The DNA of the phagotopes selected from the FPP and APS sera revealed consensus amino acid sequences of GRG and LGKRS, respectively. Phagotope FPP-10 displaced insulin binding in 90% of IAA⁺ subjects but not in the IA⁺ or the APS subject. Phagotope APS-4 was able to displace insulin binding from the APS subject but not in the IAA⁺ or IA⁺ subjects. We have demonstrated antiidiotope reagents able to distinguish childhood-onset T1D-associated IAA⁺ from adult-onset T1D (APS-II-associated IAA⁺) that are different from their specificity for human insulin and from its antiidiotope amino acid sequence.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
IMMUNE MEDIATED TISSUE destruction or dysregulation underpins many common, as well as rare, endocrine disorders such as type 1 diabetes (T1D), Graves’ disease, Hashimoto’s thyroiditis, and Addison’s disease (1). Autoimmune insulitis is associated, from an early stage, with the presence of islet-related autoantibodies (2). Several autoantibodies have been identified, but only insulin autoantibodies (IAA) bind to an antigen specific to the ß-cell. IAA are of particular interest as markers for ß-cell disease and are the first of the islet-related antibodies to appear during early insulitis (3). Insulin-binding antibodies, however, can be isolated from healthy people, as well as from subjects with autoimmune diseases other than T1D, where their implications remain, at present, unclear (4).

The autoimmune polyendocrine syndromes (APS) are clusters of autoimmune endocrine disorders. APS II is defined by the coexistence of autoimmune adrenocortical insufficiency, or serological evidence of adrenalitis, with either autoimmune thyroid disease or T1D (5, 6). Although the prevalence of islet cell antibody (ICA) (7), glutamic acid decarboxylase (GAD), and insulin antibody (IA)-2 (8) have been reported in APS-II subjects, the prevalence of IAA is unknown in these subjects. IAA titers are known to be inversely related with age (3) and therefore are less likely to be present in subjects with APS II, who classically present during the third and fourth decades.

An alternative method for defining the nature of antibody:antigen interactions is the antibody probing of phage-displayed peptide libraries that can reveal both conformational and linear phagotopes that mimic the shape of the immunizing epitopes (9). We have demonstrated that phage display technology can create and sequence a library of phagotopes able to distinguish IAA from IA idiotopes in a subject with insulin autoimmune syndrome (10). Furthermore, using the similar technology, we have demonstrated that insulin-binding idiotypes from newly diagnosed T1D subjects (IAA positive) are distinctively different from insulin-binding idiotypes of insulin-treated individuals (IA) (11). In this current study, we attempt to distinguish the idiotopes of IAA in a newly diagnosed T1D child from IAA detected in an adult-onset T1D subject with APS II, testing whether there was a difference in insulin binding between childhood-onset T1D (acute autoimmunity) and adult-onset T1D (chronic autoimmunity) with a polyendocrine susceptibility.

	Subjects and Methods

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Serum used to select phagotopes using phage display technology

FPP. Serum was obtained within 24 h of insulin initiation from an 8-yr-old Caucasian boy with newly diagnosed T1D. He was GAD (0.046 index) and IA-2 (0.15 index) antibody positive. His human leukocyte antigen genotype was DRB1*0701 DQA1*0201 DQB1*0303; DRB1*0301 DQA1*0501 DQB1*0201.

APS. Serum from a 34-yr-old lady with recent-onset T1D was obtained within 24 h of insulin initiation. She was also suffering from hypothyroidism and Addison’s disease. Thyroid peroxidase antibody and adrenal cortex antibody were positive when measured 4 yr previously. She was also positive for GAD (0.063 index) antibody but not for IA-2 (0.012 index) antibody. Her human leukocyte antigen genotype was DRB1*0301 DQA1*0501 DQB1*0201.

FGP. Serum was obtained from the FPP subject described above 3 months after the initiation of exogenous insulin. The phagotope from this IA-positive subject was used to test whether IA idiotopes were also specific for insulin binding seen in the IAA⁺ childhood-onset T1D subjects or in the IAA⁺ adult-onset T1D subject with APS II.

Serum used to test phagotopes selected

IA⁺ T2D. Serum was obtained from 20 subjects with T2D who had been insulin treated for more than 6 months (mean age, 59.6; SD, ± 11.5 yr). They were all negative for GAD antibody (ICA or IA-2 antibodies were not analyzed). Parietal cell antibody, transglutaminase antibody, thyroid peroxidase antibody and adrenal antibody were all negative in these subjects.

IAA⁺ childhood-onset T1D. Serum was obtained from 10 subjects with newly diagnosed T1D, before insulin injection (mean age, 10.3; SD, ± 2.9 yr). Parietal cell antibody, transglutaminase antibody, thyroid peroxidase antibody, and adrenal antibody were all negative in these subjects.

Controls. Twenty subjects (mean age, 46.5; SD, ± 6.9 yr) were selected as controls. All the subjects were GAD, IAA, parietal cell antibody, transglutaminase antibody, thyroid peroxidase antibody and adrenal antibody negative. The local ethics committee approved the study, and all subjects (or parents as appropriate) gave informed consent.

Microfiltration radiobinding assay of IAA

For the detection of insulin binding in the subjects, we used an assay method described by Yu et al. (12). We tested the insulin-specific binding by trying to displace the IAA with human insulin. The results are expressed as percentage binding. The interassay and intraassay coefficients of variation of this method are 10.3 and 10.5%, respectively.

Measurement of GAD and IA-2 autoantibodies

GAD and IA-2 autoantibodies were measured simultaneously by combined GAD and IA-2 radioassay as previously described (13). The cut-points were set at indexes of 0.032 (mean ± 2 SD, GAD) and 0.049 (mean ± 6 SD, IA-2), the 99th percentile, respectively, of 50 normal controls.

Preparation of affinity-purified antibody

Before phage selection, the sera (a total of 1 ml) FPP, FGP, and APS were subjected to a process of insulin-specific purification as described previously by our group (10).

Phage display random peptide library—biopanning and isolation of phage

The Ph.D-7 random heptapeptide library was purchased from New England Biolabs (Beverly, MA). The library was screened using IgG purified by the insulin-purified antibody from FPP, FGP, and APS, respectively. Single colonies of phage from the same plate were purified after the third round of biopanning and were used for DNA sequencing and tested for reactivity with affinity-purified IAA/IA by capture ELISA as described previously (10).

Displacement of insulin binding by phagotopes

A protocol similar to the microfiltration radiobinding assay (RBA) was used. Instead of using insulin as a specific ligand to displace insulin binding, the phagotope with the highest binding SD score (SDS) from the capture ELISA, and the phagotope with the lowest, were selected to demonstrate any displacement of insulin binding.

Displacement of insulin binding with synthetic peptides derived from sequences of phagotopes

To test the immunospecificity of the sequenced peptides, we employed synthetic custom peptides (Department of Immunohaematology and Blood Transfusion at the Leiden University Medical Center, Leiden, Holland) in the microfiltration RBA to displace insulin binding in sera from childhood-onset T1D (IAA⁺) and insulin-treated T2D (IA⁺), respectively. The heptamer with the sequence LGRGGSK (designated as P1), sequence KRSRLDV (P3), AIHETAT (P2) and IAKAGSK (P4) were employed in displacement studies.

Statistical analysis

A Mann-Whitney U test was used for evaluating the differences of insulin binding displacement observed with the various phagotopes or peptides used in the radiobinding assay.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Insulin binding using RBA with human radiolabeled insulin and human insulin as a displacement ligand in the test sera

The mean percentage of insulin binding in the FPP, FGP, and APS test serum, using the RBA, was 0.53, 0.84, and 0.42%, respectively. With human insulin displacement, the mean percentage of insulin binding of FPP, FGP, and APS fell to 0.04, 0.1, and 0.03%, respectively. The mean and SD of the 20 control subjects were 0.06 and 0.02%, respectively.

Capture ELISA

Of 10 phagotopes from the FPP sera isolated by biopanning with human insulin-purified IgG, seven bound to the test serum with more than 2 SDS: FPP-2, FPP-3, FPP-5, FPP-6, FPP-7, FPP-8, and FPP-10 (Table 1). Furthermore, these positive phagotopes were displaced by insulin (data not shown). In the FGP sera, six phagotopes were considered positive: FGP-1, FGP-2, FGP-5, FGP-6, FGP-7, and FGP-8; and in the APS serum, seven phagotopes were positive: APS-1, APS-4, APS-5, APS-6, APS-7, APS-8, and APS-9. All the reactive phagotopes were displaced by insulin (data not shown). The mean absorbance units of the 20 control subjects were 0.08 U (SD, 0.02).

The positive and negative phagotopes selected from the FPP, FGP, and APS sera were used to demonstrate the displacement of radiolabeled insulin in 20 insulin-treated T2D subjects and 10 T1D subjects. At a dilution of 1:100, FPP-10 was able to significantly (P < 0.01) displace insulin binding in T1D subjects, whereas, FPP-9 did not displace insulin binding for any of them (Fig. 1). FPP-10 and the irrelevant (negative phagotope) FPP-9 were unable to displace insulin binding in the majority of the 20 insulin-treated T2D sera (IA⁺) (Fig. 2). Furthermore, FPP-10 was not able to displace insulin binding in the APS subject (data not shown). FGP-2 and FGP-9 were selected from the FGP sera for insulin displacement studies. These two phagotopes were unable to displace insulin binding in any of the newly diagnosed T1D sera (IAA⁺) (Fig. 1). At a dilution of 1:100, FGP-2 was able to significantly (P < 0.01) displace insulin binding in the majority of insulin-treated (IA⁺) T2D subjects (Fig. 2). In contrast, the irrelevant phagotope FGP-9 was unable to demonstrate any displacement. In addition, FGP-2 was unable to displace insulin binding in the APS subject (data not shown). Phagotope APS-4 (positive phagotope) and APS-10 (negative phagotope) at the dilution of 1:100, selected from the APS subject, was unable to displace any insulin binding in the T1D and T2D subjects (Figs. 1 and 2).

View larger version (25K): [in this window] [in a new window]   FIG. 1. The ability of phagotopes selected from the newly diagnosed childhood-onset T1D subject: FPP-9 and FPP-10; phagotopes selected from the insulin-treated subject (IA+): FGP-2 and FGP-9; and phagotopes selected from the APS-II subject: APS-4 and APS-10, to displace the binding of I125 recombinant human insulin in the sera of 10 T1D-associated IAA+ sera in the microfiltration radiobinding assay.

View larger version (26K): [in this window] [in a new window]   FIG. 2. The ability of phagotopes selected from the newly diagnosed childhood-onset T1D subject: FPP-9 and FPP-10; phagotopes selected from the insulin-treated subject (IA+): FGP-2 and FGP-9; and phagotopes selected from the APS-II subject: APS-4 and APS-10, to displace the binding of I125 recombinant human insulin in the sera of 20 IA+ subjects from insulin-treated type 2 diabetes in the microfiltration radiobinding assay.

The heptamer P1 (LGRGGSK), corresponding to the peptide sequence of FPP-10, was able to significantly displace insulin binding in the microfiltration RBA at a concentration of 20 µmol/liter in six of six childhood-onset T1D sera (see Fig. 3A). In contrast, P2, P3, and P4 were unable to displace insulin binding at a concentration of 20 µmol/liter in the T1D IAA⁺ sera. The heptamer P3 with the sequence KRSRLDV was shown to displace insulin binding in sera from insulin-treated (IA⁺) T2D, whereas no significant displacement of insulin binding in T2D sera could be demonstrated with the irrelevant sequence P2 and P4 or the childhood-onset T1D-associated sequence P1 (see Fig. 3B).

View larger version (31K): [in this window] [in a new window]   FIG. 3. The effect of synthetic peptides P1 (LGRGGSK), P2 (AIHETAT), P3 (KRSRLDV), and P4 (IAKAGSK), at a concentration of 20 µmol/liter, to displace insulin binding in IAA-positive childhood-onset T1D subjects (A) and IA-positive insulin-treated type 2 diabetes subjects (B). The displacement ability of cold insulin is included as a positive control.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In the present study, we evaluate insulin-binding antibodies in a childhood-onset T1D subject and an adult-onset T1D subject with APS-II. The phagotopes selected from the serum of both these subjects were used as an antiidiotope reagent to distinguish insulin binding. We demonstrate that insulin-binding phagotopes selected from the childhood-onset T1D subject was very specific to IAA observed in childhood-onset T1D. In contrast, insulin-binding phagotopes selected from the APS-II subject did not show any specificity for IAA observed in childhood-onset T1D or IA observed in T2D subjects. These observations indicate that IAA in childhood-onset T1D is distinctively different from adult-onset T1D subjects with a polyendocrine susceptibility. In addition, these findings may also suggest a difference between acute rapid-onset autoimmunity observed in childhood-onset T1D and chronic slow-progressing autoimmunity observed in adult-onset T1D with APS-II.

Phagotopes selected from the IAA⁺ childhood-onset T1D subject revealed a consensus region of GRG, which was strikingly different from the APS-II subject, LGKRS. Synthetic peptides derived from the sequences of the phagotopes we isolated were also used as reagents to differentiate insulin binding in a RBA. The IAA-specific sequence from the childhood-onset T1D subject, LGRGGSK, was specific to insulin binding in IAA detected in childhood-onset T1D sera and not to insulin binding observed in insulin-treated type 2 diabetes sera. In contrast, KRSRLDV, a sequence derived from the phagotope isolated from an IA-positive subject (which contained similar motifs to the APS-II subject), was able to displace insulin binding in a group of IA⁺ individuals but not in IAA⁺ childhood-onset T1D subjects. Therefore, we have demonstrated that the combination of phagotopes specific to insulin binding from a IAA- or IA-positive individual and the corresponding synthetic peptide can be used in a RBA to discriminate between these insulin-binding antibodies.

In conclusion, this study provides further evidence that different insulin-binding idiotopes can be detected by phage technology. We have shown that distinct IAA idiotypes and differences in human insulin specificity are observed between adult-onset T1D (chronic autoimmunity) associated with APS-II compared with childhood-onset T1D (acute autoimmunity). Phage display has the potential to create reagents specific to IAA that will be able to distinguish subjects with earlier onset of diabetes from subjects with a delayed onset of T1D (with a polyendocrine susceptibility), which would be a major advance in surrogate-marker technology in T1D prediction and prevention studies.

	Footnotes

 
This work was supported by a grant by the European Commission (CT-983363), Sir Halley Stuart Trust, and Northcott Devon Medical Foundation.

Abbreviations: APS, Autoimmune polyendocrine syndrome(s); GAD, glutamic acid decarboxylase; IA, insulin antibody; IAA, insulin autoantibodies; ICA, islet cell antibody; RBA, radiobinding assay; SDS, SD score; T1D, type 1 diabetes.

Received March 15, 2004.

Accepted June 28, 2004.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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