Antibodies to GAD65 and peripheral nerve function in the DCCT

Journal of Neuroimmunology 185 (2007) 182 – 189 www.elsevier.com/locate/jneuroim

Antibodies to GAD65 and peripheral nerve function in the DCCT Robert D. Hoeldtke a,⁎, Christiane S. Hampe c , Lynn M. Bekris c , Gerald Hobbs b , Kimberly D. Bryner a , Ake Lernmark c The DCCT Research Group b

a Department of Medicine, West Virginia University, Morgantown WV 26506–9159, United States Department of Community Medicine and Statistics, West Virginia University, Morgantown WV, United States c Department of Medicine, University of Washington, Seattle WA, United States

Received 1 November 2006; received in revised form 19 January 2007; accepted 23 January 2007

Abstract Antibodies to the smaller isoform of glutamic acid decarboxylase (GAD65Ab) have been linked to the presence of neuropathy in Type 1 diabetes in several small studies. We attempted to confirm this association by measuring GAD65Ab, GAD65Ab epitopes and IA-2Ab in 511 patients who participated in the Diabetes Control and Complications Trial (DCCT). We also tested for correlations between these autoantibodies and C-peptide and glycemic control. We only included patients for whom serum was available from the first 4 years of their illness. The presence or absence of neuropathy was determined by electrophysiological studies, autonomic testing and clinical evaluation at baseline and 5 years into the trial or at close out. Samples from controls (patients without neuropathy at 5 years) were selected for patients who had similar C-peptide responses to a standardized meal at baseline. The GAD65Ab index correlated with HgbA1c only in the adult participants and only at baseline. The adults initially in poor control (upper tertile for glycemia) had higher GAD65Ab and lower C-peptides. The GAD65Ab index was not significantly different in patients with confirmed clinical neuropathy at 5 years versus controls matched for C-peptide (.248 ± .03 versus .278 ± .03). Epitope analysis, based on the blocking of conformational epitopes by recombinant Fab, revealed that the binding to multiple epitopes was decreased in the patients with neuropathy. © 2007 Elsevier B.V. All rights reserved. Keywords: Autoantibodies; GAD65Ab; Neuropathy

Analysis of the data gathered in the Diabetes Control and Complications Trial (DCCT) has revealed that hyperglycemia is a major factor but not the only determinant for tissue damage in patients with diabetes (Nathan, 1996). There are indications, for example, that autoimmune mechanisms distinct from those directed at the beta cell promote the development of neuropathy. Lymphocytic infiltrates have been observed in the sympathetic ganglia of patients with diabetic autonomic neuropathy (Duchen et al., 1980) and a variety of antibodies to the autonomic and somatosensory nervous system have been reported in patients with neuropathy (Rabinowe et al., 1990; Vinik et al., 1995). A possible association between beta-cell specific autoimmunity in diabetes and neuropathy was indicated in an earlier study by the presence of increased levels of autoantibodies to glutamic acid decarboxylase (GAD) in patients with diabetic ⁎ Corresponding author. Tel.: +1 304 293 4125; fax: +1 304 293 2544. E-mail address: [email protected] (R.D. Hoeldtke). 0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.01.009

neuropathy (Kaufman et al., 1992). We confirmed an association between the presence of autoantibodies to the smaller isoform of GAD (GAD65Ab) and decreased peripheral nerve function in a small cohort (n = 37) of patients with early Type 1 diabetes (Hoeldtke et al., 2000). GAD65Ab are also reported in patients with neurological diseases, such as cerebellar ataxia (Honnorat et al., 1995; Saiz et al., 1997), drug-refractory epilepsy (Giometto et al., 1998; Peltola et al., 2000), palatal myoclonus (Nemni et al., 1994), and the stiff person syndrome (Murinson, 2004; Raju et al., 2005). Patients with this condition suffer from muscle rigidity, muscle spasms, and an increased sensitivity to stimuli. This disease is often accompanied with other autoimmune diseases, such as Type 1 diabetes (Saiz et al., 1997; Murinson, 2004; Raju et al., 2005). A possible pathophysiological role for the high GAD65Ab titers in stiff person syndrome has been suggested, as these inhibit the formation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) a characteristic not observed in patients with

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Type 1 diabetes (Bjork et al., 1994). Moreover, we found that the capacity of the sera obtained from patients with stiff person syndrome to inhibit the enzymatic activity of GAD correlated with binding to a C-terminal epitope (Raju et al., 2005). Based on this possible connection of GAD65Ab and neurological disorders it was of interest to determine whether the binding of GAD65Ab epitopes differed in Type 1 diabetes patients with and without neuropathy. The second purpose of this study was to test for possible indirect effects of GAD65Ab on peripheral nerves of patients with diabetes. It seemed possible that GAD65Ab titers might reflect ongoing beta-cell damage that might affect peripheral nerves indirectly through elevated blood glucose levels. We therefore postulated that GAD65Ab might indicate beta-cell destruction and subsequent increased glycemia, which in turn causes deterioration in peripheral nerve function. On the basis of this reasoning we tested for possible correlations between beta-cell destruction (reflected in C-peptide secretion, GAD65Ab and epitopes), glycemia and peripheral nerve function in participants in the DCCT. 1. Materials and methods 1.1. Patients The patients who developed neuropathy during the DCCT have been previously described (Diabetes Control and Complications Trial (DCCT) Research Group, 1995; Diabetes Control and Complications Trial Research Group, 1998b). The present analysis included a subset of patients with neuropathy (n = 285) who entered the trial and provided serum during the first 4 years of their illness and had two complete peripheral nerve assessments, one at baseline and a second (between 4 and 5 years into the study) at the time of their five year evaluation or close out. DCCT participants without neuropathy (n = 230) served as controls and were matched to the neuropathy patients as described below. 1.2. Samples The NIDDK-saved serum samples from participants of the DCCT are now generally available to scientists who have raised questions about the study subsequent to its completion. Samples have been stored at − 70 °C at the University of Minnesota. The peripheral nerve function data (Diabetes Control and Complications Trial (DCCT) Research Group, 1995; Diabetes Control and Complications Trial Research Group, 1998b) and C-peptide data (Diabetes Control and Complications Trial Research Group, 1998a) have been published. This information and all clinical and demographic studies are archived at the Biostatistics Center at George Washington University (Cowie and Kenny, 2001).

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remainder. All samples were obtained in patients with a duration of diabetes of less than 4 years. 1.4. Autoantibody radioligand binding assay (RBA) 1.4.1. GAD65Ab GAD65Ab were measured in serum samples coded before the analysis in a RBA (Grubin et al., 1994). Briefly, recombinant 35S-GAD65 was produced in an in vitro coupled transcription and translation system with SP6 RNA polymerase and nuclease treated rabbit reticulocyte lysate (Promega, Madison, WI, U.S.A.). The in vitro translated 35S-GAD65 was kept at − 70 °C and used within 2 weeks of preparation. Free 35S-GAD65 was separated from the antibody-bound tracer by precipitation with Protein A Sepharose (Zymed Laboratories, Carlton Court, CA). The immunoprecipitated radioactivity was counted on a Wallac Microbeta Liquid Scintillation Counter (Perkin Elmer Life and Analytical Sciences, Inc, Boston, MA). All samples were analyzed in triplicate determinations and the intra-assay average coefficient of variation was 15%. The levels of GAD65Ab were expressed as a relative GAD65Ab index using one positive serum (JDF World Standard for ICA) and three negative standard sera from healthy subjects, as previously described (Grubin et al., 1994; Falorni et al., 1995). Serum samples were first analyzed at a final serum dilution of 1:25. If the GAD65Ab index exceeded 1, the sample was further diluted until the GAD65Ab index was b1. The upper level of normal was evaluated as mean ± 3SD sera from healthy individuals included in each assay. The interassay coefficient of variation was 14.7%, as evaluated in the positive standard serum in 53 consecutive assays. In the First and Second International GAD Autoantibody Workshops, our GAD65Ab assay showed 100% and 82% sensitivity and 100% and 96% specificity, respectively. 1.4.2. IA-2Ab Antibodies to the islet cell antigen, IA-2, were measured under identical conditions as described for GAD65Ab (Rabin et al., 1994). The plasmid containing the cDNA for the cytoplasmic portion of islet antigen 512 was kindly donated by Dr. G. Eisenbarth, Barbara Davis Research Center, Denver, Colorado. The same JDF standard serum and control sera as in the GAD65Ab assay were used to correct inter-assay coefficient of variation by calculating an IA-2Ab index for each sample. The inter-assay coefficient of variation for the positive standard serum was 19.0% as determined in 38 consecutive assays. The GAD65Ab index was called positive if it was greater than the 98th percentile for healthy controls (.043). The IA2-Ab index was called positive if it was greater than the 98th percentile for healthy controls (.0067). 1.5. Recombinant Fab (rFab) used in this study

1.3. Biochemical measurements Autoantibodies were measured in baseline samples in 52% of the patients and in samples gathered 1 year later in the

Monoclonal antibodies N-GAD65 mAb recognized epitopes representing amino acids 4–22 (Binder et al., 2004; Padoa et al., 2003; Hampe et al., 2001). Monoclonal antibodies DPA, DPC,

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and DPD were isolated from a patient with Type 1 diabetes (Madec et al., 1996; Jaume et al., 2002) and recognized epitopes located at amino acids 483–585, 195–412, and 96–173, respectively (Schwartz et al., 1999). Monoclonal antibody b96.11 was isolated from a patient with Autoimmune Polyendocrine Syndrome Type 1 (APS-1) (Schwartz et al., 1999), and recognized an epitope located at amino acid residues 308–365 (Schwartz et al., 1999; Tremble et al., 1997). rFab of these monoclonal antibodies were cloned as described (Padoa et al., 2003). 1.6. Expression and purification of the rFab rFab were expressed in Escherichia coli 25F2 cells as previously described (Carter et al., 1992). Briefly, bacteria containing the recombinant plasmids encoding the respective rFab were grown for 16 h at 30 °C in complete MOPS medium. Cells were then subcultured and grown in the absence of phosphate at 30 °C for 4 h. The rFab were isolated from the bacteria as described previously (Carter et al., 1992). The rFab were purified by two subsequent affinity chromatography steps on Ni-NTA Agarose (Qiagen Inc., Valencia, CA) and Protein G Sepharose (PGS) (Zymed Laboratories, Carlton Court, CA). Fractions were examined by immunoblot for the presence of rFab and by RBA for GAD65 binding. Active fractions were pooled and the protein concentration was determined. The yield of functional purified rFab was ∼ 0.5–1 mg/l bacterial culture. 1.7. Epitope-specific RBA (ES-RBA) The capacity of the rFab to inhibit GAD65 binding by human serum GAD65Ab was tested in a competitive RBA using Protein A Sepharose (PAS) (Zymed Laboratories) as the precipitating agent. Fab lack the CH2 domain of the Fc region and do not bind Protein A. The rFab were added at the optimal concentration as determined in competition assays using the intact mAb as a competitor. Binding of GAD65Ab to GAD65 in the presence of rFab was expressed as follows: cpm of ½S 35 GAD65 bound in the presence of rFab  100: cpm of ½S 35 GAD65 bound in the absence of rFab All samples were analyzed in triplicate determinations and the intra-assay average coefficient of variation was 4% with the highest value of 15 and the lowest being 0.04%. 1.8. Peripheral nerve function Nerve conduction evaluations were performed at baseline, at 5 years and at the end of the study. Nerve conduction evaluations included the dominant median (motor and sensory), peroneal motor and sural nerves as previously described (Diabetes Control and Complications Trial (DCCT) Research Group, 1995). Compound muscle action potentials amplitudes, F wave latencies, median and sural sensory nerve action potentials and sensory nerve conduction velocities were also determined.

Autonomic function was evaluated by measuring the beat to beat variation with deep breathing, the Valsalva ratio, and the orthostatic change in blood pressure as previously described (Diabetes Control and Complications Trial Research Group, 1998a,b). 1.9. Categorization of patients A history and physical examination was performed by a neurologist on all participants at the initiation of the study and 5 years later. A close-out evaluation during the fourth year of the study substituted for the five year evaluation in some patients. Patients were categorized into three types of neuropathy: clinical, subclinical, and confirmed clinical. Clinical neuropathy was diagnosed if patients had at least two of the following: decreased peripheral sensation, decreased or absent deep tendon reflexes or symptoms indicative of diabetic polyneuropathy. Electrophysiological studies and autonomic testing were also performed. Somatosensory nerve dysfunction was diagnosed if the patients had at least one abnormality among amplitude, conduction velocity or F wave latency in at least two anatomically distinct nerves (Diabetes Control and Complications Trial (DCCT) Research Group, 1995). Autonomic dysfunction was diagnosed if the patient had an R–R interval variation of less than 15, or an R–R interval variation between 15 and 19.9 and a Valsalva ratio less than 1.5, or orthostatic hypotension confirmed by low catecholamines (Diabetes Control and Complications Trial Research Group, 1998b). If patients with clinical neuropathy had either somatosensory dysfunction or autonomic dysfunction they were categorized as having confirmed clinical neuropathy (Diabetes Control and Complications Trial (DCCT) Research Group, 1995). If the objective tests were abnormal but the neurological evaluation was normal patients were categorized as having subclinical neuropathy. If the neurological evaluation was abnormal but the electrophysiological and autonomic tests were normal, they were categorized as clinical neuropathy. 1.10. Assessment of symptoms The neurologists who evaluated the patients made a judgment as to the presence or absence of symptoms of diabetic neuropathy (Diabetes Control and Complications Trial (DCCT) Research Group, 1995). We compared patients with symptoms diagnosed to patients who were symptom-free or experiencing complaints thought unrelated to diabetic neuropathy by the neurologist. All patients were also given a questionnaire which listed a number of somatosensory symptoms each of which was rated as present or absent. Our analysis focused on sensory symptoms (dysesthesias or paresthesias, hypersensitivity to touch, burning pain and numbness). We included cramps, the most common symptom. 1.11. Experimental design The analysis followed the case–control format. We will describe the approach for patients with confirmed clinical

R.D. Hoeldtke et al. / Journal of Neuroimmunology 185 (2007) 182–189 Table 1 GAD65Ab, C-peptide and glycemic control at baseline in adults HgbA1c (%) C-peptide (pmol/ml) GAD65Ab index GAD65Ab tertile

8.54 ± .13 .176 ± .012 .045 ± .003 Lowest

8.55 ± .13 .185 ± .011 .212 ± .006 Middle

9.00 ± .14⁎ .173 ± .011 .660 ± .018 Highest

⁎Different from the other tertiles, p b .01.

neuropathy at 5 years but identical separate analyses were performed for patients with subclinical neuropathy and clinical neuropathy. We based our analysis on the assumption that the autoantibodies would correlate with the C-peptide response to a meal challenge at baseline (Hoeldtke et al., 2000). Confirmed clinical neuropathy patients were ranked according to their C-peptide levels and each matched with a DCCT participant without neuropathy with a similar C-peptide, age and duration of diabetes. The process was repeated for every patient in each category of neuropathy. On the basis of this information we were provided samples from 67 patients with confirmed clinical neuropathy, 185 patients with subclinical neuropathy, 33 patients with clinical neuropathy and 230 control subjects. There were less controls than patients with neuropathy since some of the controls were suitable matches for more than one of the patients with neuropathy. For the patients with confirmed clinical neuropathy we also performed case–control matching on the basis of cumulative glycemia (the average HgbA1c during the first 5 years of the trial) using the same format described above. Treatment category (standard or experimental) had a large effect on cumulative glycemia. Therefore, for this analysis all matched controls belonged to the same treatment category as the patients to whom they were matched. For example, we first identified the patient with confirmed clinical neuropathy on standard therapy with the worst HgbA1c and then found controls in this treatment category with a similar mean HgbA1c (within 0.2%) and chose a match with the most similar age and duration. Then we found the confirmed clinical neuropathy patient on standard therapy with the second worse mean HgbA1c and proceeded according to the format just described. After matches had been found for all the patients on standard therapy, we performed the same analysis on the patients who developed confirmed clinical neuropathy on intensive therapy. 1.12. C-peptide analysis Comparison of C-peptide secretion in patients with and without neuropathy in the present study was not possible since each control was matched according to C-peptide levels. Despite this, stimulated C-peptide data were available on a broader cohort of DCCT controls (patients without neuropathy) who entered the trial within 4 years of diagnosis (Diabetes Control and Complications Trial Research Group, 1998a) and this information was provided to us from the Biostatistics Center at George Washington University (Cowie and Kenny, 2001). This made it possible to compare stimulated C-peptide in this large

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group of controls (n = 349) to the neuropathy patients in this study. 1.13. Statistical analysis Patients with neuropathy were compared to matched controls using analysis of variance and logistic regression analysis (Winer, 1979). Statistical principles in experimental design. In: Ed 2. New York, McGraw-Hill, pp. 514–603). In a few instances we have specified when nonparametric comparisons were employed. 2. Results 2.1. Autoantibodies and glycemia At baseline, 81% of the patients were positive for GAD65Ab and 53% were positive for IA-2Ab. Forty-five percent of patients were positive for both antibodies and 12% were negative for both. Patients positive for GAD65Ab were older (mean age 28.9 ± .51) than those positive for IA-2Ab (23.1 ± 1.1) ( p b .001). Neither GAD65Ab nor IA-2Ab correlated with baseline glycemia in the whole cohort but this analysis was obscured by the presence of decreased GAD65Ab ( p b .005) but increased mean HgbA1c ( p b .001) in the adolescent (less than 21 years old) patients. There was a weak (r = .13) but significant ( p b .005) association between GAD65Ab and baseline HgbA1c in the adult participants. Stratification of the adult patients according to their GAD65Ab tertiles confirmed that those in the highest tertile had increased HgbA1c at baseline ( p b .01) (Table 1) but this was not seen in subsequent evaluations during the trial. A similar analysis of adolescent patients showed no relation between GAD65Ab and HgbA1c at baseline or subsequently. Stratification of patients according to their IA-2Ab tertiles revealed no relationship with glycemia in adolescents or adults. Neither GAD65Ab (Table 1) nor IA-2Ab correlated with C-peptide. The adults in poor control (the upper tertile for glycemia) at baseline, however, had higher GAD65Ab and lower C-peptides (Table 2). This relationship was not seen for the adolescent patients. 2.2. Autoantibodies and peripheral nerve function at 5 years Neither GAD65Ab nor IA-2Ab were increased in any of the categories of neuropathy at the five year time point when

Table 2 Autoantibodies and C-peptide in adults with poor versus good control at baseline GAD65Ab index IA-2Ab index C-peptide (pmol/ml) HgbA1c (%) Glycemic tertile

.264 ± .02 .142 ± .02 .231 ± .01 7.01 ± .04 Lowest

⁎Different from the other tertiles, p b .01. ⁎⁎p b .001.

.291 ± .02 .150 ± .02 .178 ± .01 8.45 ± .04 Middle

.351 ± .03⁎ .160 ± .02 .131 ± .01⁎⁎ 10.5 ± .09 Highest

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Table 3 Autoantibodies, C-peptide and cumulative glycemia in patients with or without neuropathy at 5 years n 64 Confirmed clinicalneuropathy

GAD65Ab index IA-2Ab index Mean HgbA1c (%) a C-peptide (pmol/ml) Clinical neuropathy 33 GAD65Ab index IA-2Ab index Mean HgbA1c (%) a C-peptide (pmol/ml) Subclinical 183 GAD65Ab index neuropathy IA-2Ab index Mean HgbA1c (%) a C-peptide (pmol/ml)

Patients

C-peptide matchedcontrols

.247 ± .035 .140 ± .03 8.66 ± .22 .153 ± .06 .232 ± .04 .222 ± .05 8.21 ± .23 .144 ± .02 .309 ± .02 .136 ± .017 8.76 ± .12 .175 ± .01

.278 ± .03 .172 ± .03 7.75 ± .15⁎ .155 ± .015 .270 ± .04 .160 ± .04 8.18 ± .22 .144 ± .02 .291 ± .02 .156 ± .02 7.96 ± .10⁎ .172 ± .01

⁎Different from patients, p b .001. a Mean HgbA1c from entry through year 5.

developed confirmed clinical neuropathy at 5 years showed less competition by any of the rFab than did sera of C-peptide matched controls and glycemia matched controls (Table 4). A similar trend was seen in the patients with subclinical neuropathy for competition by rFab DPA, DPC, DPD and NGAD65Ab mAb, however did not approach significance. 2.4. C-peptide and neuropathy Patients with confirmed clinical neuropathy at 5 years had a stimulated C-peptide of .153 ± .015 pmol/ml at baseline, significantly less than controls (.184 ± .007, p b 01). Those with clinical neuropathy at 5 years had a stimulated C-peptide at baseline of .144 ± .02 pmol/ml, significantly lower than controls ( p b .001) (Table 5). C-peptide secretion was normal at baseline in those with subclinical neuropathy. 2.5. Analysis of symptoms

patients were compared to C-peptide matched controls (Table 3). The mean HgbA1c during the first 5 years of the trial was increased in those with subclinical neuropathy ( p b .001) and confirmed clinical neuropathy ( p b .001). Patients with autonomic nerve dysfunction 5 years into the trial did not have increased autoantibodies during the first 4 years of their illness. 2.3. Epitopes detected using rFab All samples taken at baseline were analyzed for their conformational epitopes using five GAD65-specific rFab. None of the epitopes detected by ES-RBA correlated with the age of the patients, duration of diabetes or glycemic control. C-peptide correlated positively with binding to the N-GAD65 mAb defined epitope ( p b .01, r = .16) but showed no relationship with the other epitopes (Table 4). Baseline sera of patients who Table 4 Autoantibodies in patients with confirmed neuropathy at 5 years Patients with Controls matched Controls matched neuropathy for glycemia for C-peptide n GAD65Ab index Epitope DPA (% inhibition) Epitope b96.11 Epitope DPC Epitope DPD Epitope N-GAD65 mAb IA2-Ab index C-peptide (pmol/ml) Mean HgbA1c (%) Duration at entry (months) Age at entry (years)

67 .248 ± .04 4.33 ± .88

67 .335 ± .04 10.7 ± 1.6⁎⁎⁎⁎

64 .278 ± .03 9.58 ± 1.7⁎⁎⁎

11.5 ± 2.0 8.5 ± 1.7 4.37 ± 1.2 6.03 ± 1.2 .152 ± .03 .153 ± .02 8.63 ± .21 28.4 ± 1.3

17.8 ± 2.2⁎ 14.9 ± 2.3⁎⁎ 11.7 ± 1.9⁎⁎⁎⁎ 12.5 ± 1.6⁎⁎⁎⁎ .192 ± .03 .169 ± .02 8.59 ± .21 25.6 ± 1.1

18.6 ± 2.4⁎⁎ 14.8 ± 2.3⁎ 9.14 ± 1.8⁎⁎ 11.8 ± 1.7⁎⁎⁎ .172 ± .03 .155 ± .02 7.75 ± .15⁎⁎⁎ 30.3 ± 1.3

28.6 ± .93

26.6 ± .92

27.3 ± .80

⁎Different from patients with neuropathy, p b .05. ⁎⁎p b .025. ⁎⁎⁎p b .01. ⁎⁎⁎⁎p b .001.

At baseline only 25 patients were judged to have symptoms attributable to diabetic neuropathy by the examining neurologists. Their mean GAD65Ab titer was increased (0.370 ± .06) when compared to the mean titer for asymptomatic patients with (0.27 ± .03) and without neuropathy (.29 ± .02). This difference was significant by the Kruskal–Wallis rank sums test (p b .05). The competition by rFab DPC with GAD65Ab in the symptomatic patients (16.7 ± 3.2) was significantly stronger than that in the asymptomatic neuropathy patients (9.72 ± 1.5), or the asymptomatic patients without neuropathy (12.8 ± .99) ( p b .05). Analysis of questionnaire data, which provided an independent assessment of symptoms, revealed that sensory symptoms or cramps were very common at baseline, occurring in 121 patients. Twenty-five percent of patients with neuropathy and 24% of patients without neuropathy had positive questionnaires for sensory symptoms or cramps. The GAD65Ab index and competition by rFab DPC were increased in the patients with symptoms (Table 6).

Table 5 Stimulated C-peptide at baseline in patients with and without neuropathy at 5 years Controls (n = 349) C-peptide(pmol/ 0.184 ± .007 ml) Age at entry 27.0 ± .44 28.8 ± .54 Duration at eligibility (months) Average HgbA1c 7.89 ± .07 from baseline to year 5 (%)

Confirmed clinical Clinical neuropathy neuropathy (n = 67) (n = 33)

Subclinical neuropathy (n = 183)

.153 ± .015⁎

.144 ± .02⁎⁎ .175 ± .01

28.7 ± .81 26.8 ± .62

27.3 ± 1.0 25.2 ± 1.9

27.0 ± .52 25.8 ± .79

8.81 ± .20⁎⁎

8.19 ± .23

8.76 ± .12⁎⁎

The number of controls is greater in this table than in the other tables because we included controls in which autoantibodies were not measured. ⁎Different from controls, p b .01. ⁎⁎Different from controls, p b .001.

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Table 6 Autoantibodies and GAD65Ab epitopes detected by recombinant Fab at baseline

n GAD65Ab index Epitope DPA (% inhibition) Epitope b96.11 Epitope DPC Epitope DPD Epitope N-GAD65 mAb IA2-Ab index HgbA1c at baseline

No neuropathy

No neuropathy

No neuropathy

Neuropathy

Neuropathy

No symptoms

No symptoms

Symptoms

No symptoms

Symptoms

All patients

Group matched for glycemia

All patients

All patients

All patients

285 .275 ± .02 7.99 ± .65 14.6 ± 1.0 11.3 ± .95 7.10 ± .68 8.76 ± .68 .154 ± .01 8.60 ± 1.0

223 .279 ± .02 8.04 ± .73 14.4 ± 1.1 11.1 ± 1.1 7.19 ± .75 8.51 ± .75 .164 ± .02 9.12 ± 1.0

89 .337 ± .03 8.72 ± 1.2 17.3 ± 2.1 16.6 ± 2.0⁎ 9.72 ± 1.4 11.9 ± 1.3⁎ .183 ± .03 9.21 ± .17

95 .260 ± .03 7.28 ± 1.2 14.0 ± 2.0 10.6 ± 1.7 6.41 ± 1.2 7.31 ± 1.1 .122 ± .02 9.21 ± .19

32 .333 ± .05⁎⁎ 8.57 ± 2.0 20.7 ± 3.7 14.1 ± 3.4⁎⁎⁎ 5.53 ± 1.8 8.31 ± 1.9 .118 ± .04 9.31 ± .35

The neuropathy group included patients with clinical neuropathy, confirmed clinical neuropathy and subclinical neuropathy. The presence of symptoms was determined from questionnaires given at baseline. ⁎Different from patients without neuropathy and without symptoms, p b .01. ⁎⁎Patients with symptoms were different from those without symptoms by the Kruskal–Wallis rank sums test, p b .05. ⁎⁎⁎Patients with symptoms were different from those without symptoms by ANOVA when data from patients with and without neuropathy were pooled, p b .01.

Only one of the sensory symptoms, namely presence of dysesthesias or paresthesias (one item on the questionnaire) was associated with increased GAD65Ab titers. Twenty-nine patients with dysesthesias or paresthesias at baseline had higher titers of GAD65Ab (.371 ± .06) than glycemia matched asymptomatic patients with and without neuropathy (Table 7). Moreover, competition was stronger in the patients with dysesthesias for rFab b96.11, DPC, DPD and N-GAD65 mAb (Table 7). The neurologists’ assessment of symptoms and the results of questionnaires were also examined from the five year or closeout evaluations. Ten percent of patients were assessed to have symptoms of diabetic polyneuropathy by the neurologists. Nineteen percent of patients had positive questionnaires. No correlation was observed between either measure of symptoms and GAD65Ab.

Table 7 Autoantibodies and GAD65Ab epitopes detected by recombinant Fab at baseline in patients with dysesthesias or paresthesias

n GAD65Ab index Epitope DPA (% inhibition) Epitope b96.11 Epitope DPC Epitope DPD Epitope N-GAD65 mAb IA2-Ab index HgbA1c at baseline

No neuropathy

Neuropathy

No symptoms

No symptoms

278 .276 ± .02 8.01 ± .67 14.7 ± 1.0 11.2 ± .96 7.25 ± .70 8.86 ± .69 .155 ± .01 8.67 ± .09

83 .240 ± .03 7.39 ± 1.28 13.5 ± 2.1 10.4 ± 1.9 6.34 ± 1.3 6.97 ± 1.2 .129 ± .03 8.77 ± .16

Patients with dysesthesias 29 .371 ± .06⁎ 6.90 ± 1.4 21.1 ± 3.5⁎⁎⁎ 20.8 ± 3.8⁎⁎ 11.4 ± 2.5⁎⁎⁎ 14.4 ± 2.6⁎⁎ .171 ± .05 8.72 ± .28

Patients were group matched for glycemia. The presence of symptoms was determined from questionnaires given at baseline. Patients without dysesthesias who had other sensory symptoms or cramps were excluded from the analysis. ⁎Patients with dysesthesias were different from the other two groups by ANOVA, p b .05. ⁎⁎p b .01. ⁎⁎⁎Different from the asymptomatic patients by the Wilcoxon rank sums test, p b .05.

3. Discussion The major finding in this study is that while patients with confirmed clinical neuropathy at 5 years had normal GAD65Ab at baseline, their beta-cell specific autoimmune response was distinct as reflected in their GAD65Ab epitope binding characteristics. This observation will be discussed after we have reviewed a) our efforts to relate beta-cell specific autoimmunity and beta-cell function b) data relating beta-cell function to neuropathy. Our analysis of baseline data revealed that the adults in poor glycemic control had increased GAD65Ab titers and low C-peptide levels. GAD65Ab and glycemia in the adult patients could both be elevated due to increased beta-cell destruction (Petersen et al., 1994). We were unable, however, to document a correlation between GAD65Ab and C-peptide. The correlation between GAD65Ab and glycemia was present only transiently, presumably because it was reversed by the large improvements in glycemia, which occurred in those treated intensively. This study also made it possible to assess the correlation of C-peptide secretion in early diabetes with the subsequent development of neuropathy. Patients with clinical neuropathy and confirmed clinical neuropathy at 5 years had decreased stimulated C-peptide at baseline. Those with confirmed clinical neuropathy also had worse glycemia than the controls, which is the most likely explanation of the presence of neuropathy in those with decreased C-peptide. This analysis included a large group of controls (n = 349) (Diabetes Control and Complications Trial Research Group, 1998a,b), not only the smaller subset who were included in the present study of autoantibodies. This result supports previous studies indicating that the loss of beta-cell function in early Type 1 diabetes has widespread manifestations. Steffes et al. have similarly reported that patients with decreased C-peptide secretion in early diabetes have an increased prevalence of retinopathy and nephropathy subsequently (Steffes et al., 2003). Kaufman et al. originally reported an association between GAD65Ab and neuropathy in Type 1 diabetes and suggested

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that these antibodies affect peripheral nerves (Kaufman et al., 1992). Earlier, we confirmed a negative association between GAD65Ab and peripheral nerve function in a small group (n = 37) of patients with recently diagnosed Type 1 diabetes. It remained unclear whether this association was causative, or whether the peripheral nerve function was impaired by increased glycemia, caused by rapid beta-cell destruction (indicated by the higher GAD65Ab titers) (Hoeldtke et al., 1997; Hoeldtke et al., 2000). Other studies of patients with longstanding diabetes did not, however, observe any relation between GAD65Ab and neuropathy (Zanone et al., 1994; Roll et al., 1995). We have questioned the latter reports since many of the GAD65Ab analyses were performed on patients with long-standing disease who might have had high titers of antibodies initially, but not years later when the antibodies were measured in patients with overt neuropathy. The present study resolves this controversy. The antibodies were measured early in the disease (within 4 years of diagnosis) and peripheral nerve function was determined 5 years later. The case–control design made it possible to control for both C-peptide and cumulative glycemia. In this format we could determine that the GAD65Ab were clearly not elevated in those with confirmed clinical neuropathy. Moreover, sera from affected patients failed to inhibit the activity of GAD65 in vitro (data not shown), a characteristic often found for GAD65Ab in stiff person syndrome (Dinkel et al., 1998). Thus there was no evidence of a direct neurotoxic effect of GAD65Ab on peripheral nerves. While the GAD65Ab titer did not differ between patients with and without neuropathy, we found that the beta-cell specific autoimmunity reflected in GAD65Ab epitope specificity present in sera of patients that developed confirmed clinical neuropathy within 5 years differed significantly from patients that remained without neuropathy. GAD65Ab in patients with confirmed clinical neuropathy recognized fewer epitopes and binding to all the epitopes we studied was decreased. Thus patients with diabetic neuropathy may have a distinct beta-cell specific autoimmune response. It is possible, for example that the GAD65Ab in the neuropathy patients were interacting with one or more novel epitopes not included in this analysis. Finally, the prevalence of sensory symptoms and cramps early in the DCCT is noteworthy. At the baseline evaluation, sensory symptoms or cramps were reported on the DCCT questionnaires in 25% of patients with neuropathy and 24% of patients without neuropathy. We investigated the possibility that GAD65Ab might affect the central processing of sensory input from the periphery and thereby exacerbate sensory symptoms. Gamma-aminobutyric acid (GABA), the product of GAD, modulates sensory input in the spinal cord, brain stem, and thalamus (Castro-Lopes et al., 1995; Potes et al., 2006) and a disruption of GABA formation could, in theory, exacerbate sensory symptoms or cause cramps, as suggested for patients with the stiff person syndrome (Levy et al., 1999). While the GAD65Ab did not inhibit the enzymatic activity of GAD65, we found that patients with symptoms of diabetic neuropathy, as determined by a neurologist, had increased GAD65Ab levels and increased recognition of the middle epitope defined by rFab DPC. A similar pattern was seen for patients who had sensory

symptoms or cramps on the DCCT questionnaires at baseline. Our finding that GAD65Ab levels were elevated in the symptomatic patients, but not in those with neuropathy, suggests that the autoimmune response, reflected in GAD65Ab, may be acting centrally to enhance the impact of sensory input from the periphery. Any effect of the autoantibodies on sensory symptoms, however, was only temporary in the DCCT. Dysesthesias and paresthesias correlated with GAD65Ab at baseline but this was not confirmed at the five year (or close out) evaluations presumably because the antibody titers had decreased over time. In summary, GAD65Ab titers correlated with poor glycemic control in adult but not adolescent patients in the DCCTat baseline. IA-2Ab did not correlate with glycemia in either age group. Patients with clinical neuropathy and confirmed clinical neuropathy at 5 years into the trial had decreased stimulated C-peptide at baseline. When diabetic patients with and without neuropathy were matched for C-peptide and cumulative glycemia, however, we found no evidence of increased GAD65Ab in patients with neuropathy. Analysis of conformational epitopes revealed that the sera of patients with confirmed clinical neuropathy showed decreased recognition of multiple epitopes. These results indicate the underlying beta-cell specific autoimmune response may be different in patients who develop neuropathy. References Binder, K.A., Banga, J.P., Madec, A.M., Ortqvist, E., Luo, D., Hampe, C.S., 2004. Epitope analysis of GAD65Ab using fusion proteins and rFab. J. Immunol. Methods 295, 101–109. Bjork, E., Velloso, L.A., Kampe, O., Karlsson, F.A., 1994. GAD autoantibodies in IDDM, stiff-man syndrome, and autoimmune polyendocrine syndrome type 1 recognize different epitopes. Diabetes 43, 61–165. Carter, P., Kelley, R.F., Rodrigues, M.L., Snedecor, B., Covarrubias, M., Velligan, M.D., Wong, W.L., Rowland, A.M., Kotts, C.E., Carver, M.E., 1992. High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Biotechnology (NY) 10, 163–167. Castro-Lopes, J.M., Malcangio, M., Pan, B.H., Bowery, N.G., 1995. Complex changes of GABAA and GABAB receptor binding in the spinal cord dorsal horn following peripheral inflammation or neurectomy. Brain Res. 679, 289–297. Cowie, C.C., Kenny, D., 2001. Diabetes Control and Complications Trial Data Archives. Biostatistics Center at George Washington University, Washington DC. (provided by NIDDK and the Diabetes Control and Complications Trial Research Group). Diabetes Control and Complications Trial (DCCT) Research Group, 1995. Effect of intensive diabetes treatment on nerve conduction in the Diabetes Control and Complications Trial. Ann. Neurol. 38, 869–880. Diabetes Control and Complications Trial Research Group, 1998a. Effect of intensive therapy on residual beta-cell function in patients with type 1 diabetes in the diabetes control and complications trial. A randomized, controlled trial. Ann. Intern. Med. 128, 517–523. Diabetes Control and Complications Trial Research Group, 1998b. The effect of intensive diabetes therapy on measures of autonomic nervous system function in the Diabetes Control and Complications Trial (DCCT). Diabetologia 41, 416–423. Dinkel, K., Meinck, H.M., Jury, K.M., 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. Duchen, L.W., Anjorin, A., Watkins, P.J., 1980. Pathology of autonomic neuropathy in diabetes mellitus. Ann. Intern. Med. 92, 301–303. Falorni, A., Ortqvist, E., Persson, B., Lernmark, A., 1995. Radioimmunoassays for glutamic acid decarboxylase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human ligands. J. Immunol. Methods 186, 89–99.

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