Petri Kulmala

Prediction of insulin-dependent diabetes mellitus in siblings of children with diabetes. A population-based study. The Childhood Diabetes in Finland Study Group.

An unselected population of 755 siblings of children with insulin-dependent diabetes mellitus (IDDM) was studied to evaluate the predictive characteristics of islet cell antibodies (ICA), antibodies to the IA-2 protein (IA-2A), antibodies to the 65-kD isoform of glutamic acid decarboxylase (GADA), insulin autoantibodies (IAA), and combinations of these markers. We also evaluated whether the histochemical ICA test could be replaced by the combined detection of other markers. 32 siblings progressed to IDDM within 7.7 yr of the initial sample taken at or close to the diagnosis of the index case (median follow-up, 9.1 yr). The positive predictive values of ICA, IA-2A, GADA, and IAA were 43, 55, 42, and 29%, and their sensitivities 81, 69, 69, and 25%, respectively. In contrast to the other three antibody specificities, GADA levels were not related to the risk for IDDM. The risk for IDDM in siblings with four, three, two, one, or no antibodies was 40, 70, 25, 2, and 0.8%, respectively. Combined screening for IA-2A and GADA identified 70% of all ICA-positive siblings, and all of the ICA-positive progressors were also positive for at least one of the three other markers. The sensitivity of the combined analysis of IA-2A and GADA was 81%, and the positive predictive value was 41%. In conclusion, combined screening for IA-2A and GADA may replace the ICA assay, giving comparable sensitivity, specificity, and positive predictive value. Accurate assessment of the risk for IDDM in siblings is complicated, as not even all those with four antibody specificities contract the disease, and some with only one or no antibodies initially will progress to IDDM.

Autoantibodies to glutamic acid decarboxylase in patients with therapy-resistant epilepsy

Background: Autoantibodies to glutamic acid decarboxylase (GAD-A) are present in type 1 diabetes and stiff man syndrome (SMS), and have also been reported in cerebellar ataxia. Epilepsy was present in 4 of 19 patients with SMS and GAD-A, implying that epilepsy sometimes is associated with anti-GAD autoimmunity. Methods: The authors investigated the prevalence of GAD-A in patients with therapy-resistant localization-related epilepsy (n = 51) and generalized epilepsy (n = 49) by a radiobinding assay. The positive samples were confirmed by immunohistochemistry and immunoblotting of recombinant human GAD65. Results: GAD-A were found in eight patients with localization-related epilepsy, whereas none of the patients with generalized epilepsy, other neurologic disorders (n = 38), or the control subjects (n = 48) had GAD-A. Two patients had high levels of GAD-A, similar to SMS, whereas six patients had significantly lower titers, characteristic of type 1 diabetes. The two patients with high levels of GAD-A had GAD-A both in serum and CSF by immunohistochemistry and immunoblotting. Both of them had longstanding therapy-resistant temporal lobe epilepsy but did not have diabetes. One had a history of autoimmune disease, whereas the other had serologic evidence of multiple autoantibodies without any clinical signs of autoimmune disease. Conclusions: GAD autoimmunity may be associated with refractory localization-related epilepsy.

IA-2 antibodies - a sensitive marker of IDDM with clinical onset in childhood and adolescence

Summary To study the relationship of IA-2 antibodies (IA-2A) to other autoantibodies and genetic risk markers in insulin-dependent diabetes mellitus (IDDM), 758 children and adolescents younger than 15 years of age (mean age 8.4 years) with newly diagnosed diabetes were analysed for IA-2A, GAD antibodies (GADA) and insulin autoantibodies (IAA) with radiobinding assays, for islet cell antibodies (ICA) with immunofluorescence and for HLA DR alleles by serology. IA-2A were detected in 85.9 % of cases with no association with gender or age. An overwhelming majority of the patients (71.3 %) tested positive for three or more antibodies, and 90.7 % for at least two. Fifty-four subjects (7.1 %) had one antibody detectable, whereas only 2.1 % of the patients tested negative for all four. A higher proportion of patients was positive for IA-2A and/or GADA than for ICA alone (95.5 vs 84.2 %, p < 0.001). The prevalence and level of IA-2A were increased in cases carrying HLA DR4/non-DR3 compared with other DR combinations. The results indicate that almost all patients with newly diagnosed childhood IDDM can be identified by screening with these four autoantibodies. The combination of IA-2A and/or GADA had a higher sensitivity for IDDM than ICA alone. The close association between IA-2A and HLA DR4, the strongest single allele predisposing to IDDM, suggests that IA-2A may be a more specific marker of beta-cell destruction than GADA, which have been shown to associate with the DR3 allele and thyroid autoimmunity. [Diabetologia (1998) 41: 424–429]

Enterovirus RNA in serum is a risk factor for beta‐cell autoimmunity and clinical type 1 diabetes: A prospective study

Recent prospective studies have documented serologically an increased frequency of enterovirus infections in prediabetic children, indicating that these infections may initiate and accelerate the beta‐cell damaging process several years before the clinical manifestation of type 1 diabetes. The aim of the present study was to establish whether these serological findings would be supported by the detection of enterovirus RNA in a unique prospective series of sera collected from prediabetic children 0–10 years before the manifestation of clinical type 1 diabetes. Reverse transcription followed by polymerase chain reaction employing highly conserved primers among enteroviruses were used to amplify enteroviral sequences. Viral RNA was found in 22% (11/49) of follow‐up samples from prediabetic children but in only 2% (2/105) of those from controls (OR 14.9, P < 0.001). Persisting RNA positivity was not observed in any of these children. The presence of enterovirus RNA was associated with concomitant increases in the levels of autoantibodies against islet cells (OR 21.7, P < 0.01) and glutamic acid decarboxylase (OR 15.4, P < 0.05), but not in the levels of antibodies against insulin or the tyrosine phosphatase‐like IA‐2 protein. In contrast to the prediabetic children, those with newly diagnosed type 1 diabetes were negative for enterovirus RNA. The results thus complement previous serological data, suggesting that enterovirus infections are an important risk factor underlying type 1 diabetes and associated with the induction of beta‐cell autoimmunity even years before symptoms appear. J. Med. Virol. 61:214–220, 2000.

Autoantibodies associated with Type I diabetes mellitus persist after diagnosis in children

Summary To study the persistence of Type I (insulin-dependent) diabetes mellitus associated autoantibodies and their relation to genetic risk markers and clinical characteristics of the disease after clinical manifestation, serum samples were obtained from 90 children and adolescents at diagnosis and 2, 5 and 10 years later. The samples were analysed for islet cell antibodies (ICA) by immunofluorescence and for antibodies to glutamic acid decarboxylase (GADA), intracellular portion of the protein tyrosine phosphatase related IA-2 antigen (IA-2A) and insulin autoantibodies by specific radiobinding assays. Of the subjects tested 79 % were positive for IA-2A at diagnosis, 62 % for GADA, 81 % for ICA and 28 % for insulin autoantibodies, but the prevalence of IA-2A, GADA and ICA decreased substantially as a function of increasing duration of the disease (p < 0.05 or less), their levels following the same pattern (p < 0.001 for all three autoantibodies). Two thirds of the subjects still tested positive for at least one autoantibody specificity after the first 10 years of the disease and 42 % had two or three antibodies detectable. An increase over the initial antibody concentrations after the diagnosis was seen more often for GADA than for ICA (p < 0.001) or IA-2A (p < 0.05). In conclusion, autoantibodies associated with Type I diabetes appear to persist longer than expected after manifestation of the clinical disease, possibly due to small scale continuous beta-cell regeneration after diagnosis or to structural and/or functional mimicry between exogenous proteins and beta-cell antigens or both. [Diabetologia (1998) 41: 1293–1297]

Several different enterovirus serotypes can be associated with prediabetic autoimmune episodes and onset of overt IDDM

In a prospective multicentre study described previously on prediabetic events in siblings of index cases with insulin‐dependent diabetes mellitus, 31 children developed clinical diabetes during the observation period and 51 children seroconverted for islet cell antibodies or insulin autoantibodies. By using nonserotype specific EIA and RIA, it has shown recently that enterovirus infections in both groups were frequently associated with increases of islet cell antibody and/or insulin autoantibody titres. Serum specimens sequentially collected from 12 children during the prediabetic period were still available and were then tested for serotype‐specific neutralizing antibodies. Plaque‐neutralization assays were carried out for coxsackievirus A9, coxsackievirus B types 1 to 6, and echovirus types 1 and 11. An unequivocal monotypic increase in neutralizing antibodies was observed on seven occasions in six children, on one occasion with coxsackievirus A9, one with coxsackievirus B1, two with coxsackievirus B2, two with coxsackievirus B3, and one with coxsackievirus B5. In four patients, the infection was associated temporally with increases in the levels of islet cell antibodies, insulin autoantibodies and/or antibodies to glutamic acid decarboxylase, and in three other patients, it coincided with the clinical onset of insulin‐dependent diabetes mellitus. These results suggest that the association of enterovirus infections with insulin‐dependent diabetes mellitus is not restricted to serotype 4 of coxsackie B viruses suspected previously, but that several different serotypes might play a role in the pathogenesis of the disease. J. Med. Virol. 56:74–78, 1998.

Prediction of Type 1 Diabetes in the General Population

OBJECTIVE To evaluate the utility of GAD antibodies (GADAs) and islet antigen-2 antibodies (IA-2As) in prediction of type 1 diabetes over 27 years in the general population and to assess the 6-year rates of seroconversion. RESEARCH DESIGN AND METHODS A total of 3,475 nondiabetic subjects aged 3–18 years were sampled in 1980, and 2,375 subjects (68.3%) were resampled in 1986. All subjects were observed for development of diabetes to the end of 2007. GADAs and IA-2As were analyzed in all samples obtained in 1980 and 1986. RESULTS A total of 34 individuals (1.0%; 9 developed diabetes) initially had GADAs and 22 (0.6%; 9 developed diabetes) IA-2As. Seven subjects (0.2%) tested positive for both autoantibodies. The positive seroconversion rate over 6 years was 0.4% for GADAs and 0.2% for IA-2As, while the inverse seroconversion rates were 33 and 57%, respectively. Eighteen subjects (0.5%) developed type 1 diabetes after a median pre-diabetic period of 8.6 years (range 0.9–20.3). Initial positivity for GADAs and/or IA-2As had a sensitivity of 61% (95% CI 36–83) for type 1 diabetes. Combined positivity for GADAs and IA-2As had both a specificity and a positive predictive value of 100% (95% CI 59–100). CONCLUSIONS One-time screening for GADAs and IA-2As in the general childhood population in Finland would identify ∼60% of those individuals who will develop type 1 diabetes over the next 27 years, and those subjects who have both autoantibodies carry an extremely high risk for diabetes. Both positive and inverse seroconversions do occur over time reflecting a dynamic process of β-cell autoimmunity.

Cow's milk consumption, disease-associated autoantibodies and Type 1 diabetes mellitus: A follow-up study in siblings of diabetic children

Evidence from case–control studies for the diabetogenicity of introduction of cow’s milk‐based formulas at early age in infancy is inconclusive. We followed siblings of children with Type 1 diabetes mellitus (Type 1 DM) to investigate a possible relationship between cow’s milk consumption during infancy or later in childhood and the emergence of diabetes‐associated autoantibodies and progression to clinical Type 1 DM. A cohort of 725 initially unaffected 0 to 25‐year‐old siblings of 801 index children with Type 1 DM diagnosed in 1986–1989 participated in the study (82 % of those invited). The siblings were observed for Type 1 DM associated autoantibodies at intervals of 3–12 months for 4 years, starting from the diagnosis of Type 1 DM in the index child. The follow‐up for Type 1 DM started at the same time and ended on 31 October 1995. The combined prevalence of Type 1 DM associated autoantibodies (islet cell antibodies (ICA), insulin autoantibodies (IAA), GAD autoantibodies (GADA), and/or antibodies to the insulinoma associated cDNA2 protein (IA‐2A)) was 13.6 % (95/697) at the beginning of the study. Of the initially seronegative siblings, 7.5 % (45/602) converted to antibody positivity during 4 years, and of all siblings 4.6 % (33/725) developed Type 1 DM during the total follow‐up time. The age at introduction of supplementary milk feeding was not significantly related to seroconversion to positivity for Type 1 DM associated autoantibodies or to the development of Type 1 DM in the siblings. When adjusted for age, sex, infant feeding patterns, and maternal age and education, high milk consumption in childhood (≥3 glasses daily) was associated with more frequent emergence of Type 1 DM‐associated autoantibodies than low consumption (<3 glasses daily) (adjusted odds ratio 3.97, 95 % confidence interval 1.3–11.7, p = 0.01). There was a non‐significant association between high milk consumption and progression to clinical Type 1 DM (adjusted hazard ratio 2.75, 95 % confidence interval 0.9–8.4, p = 0.07). To conclude, this study suggests that high consumption of cow’s milk during childhood may be associated both with seroconversion to positivity for diabetes‐associated autoantibodies and progression to clinical Type 1 DM among siblings of children with diabetes.

Antibody cross‐reactivity induced by the homologous regions in glutamic acid decarboxylase (GAD65) and 2C protein of coxsackievirus B4

GAD65 contains an amino acid sequence which is highly homologous with a sequence in the 2C protein of coxsackievirus B4 (CBV4‐2C). In the present study the possibility that this region could contain an epitope capable of inducing immunological cross‐reactivity between CBV4‐2C and GAD65 was evaluated. Six out of seven rabbit sera, which were raised against seven different synthetic peptides carrying various modifications of the homology sequence, showed cross‐reactivity between 2C, GAD65 and GAD67 derived peptides in ELISA. There was substantial cross‐reactivity between 2C and GAD65 peptides, but not between 2C and GAD67 peptides. The most cross‐reactive peptides were those corresponding to the 2C sequences FIEWLKVKILPEVKEK and KILPEVKEKHEFLSRL. When the binding of the four 2C peptide‐specific sera to the GAD65 protein was analysed in immunoprecipitation, two sera were found to be cross‐reactive (anti‐FIEWLKVKILPEVKEK and anti‐WLKVKILPEVKEKHEF). One of these (anti‐WLKVKILPEVKEKHEF) reacted also with coxsackie B virus (CBV)‐infected cells. Antibodies against this epitope were induced during enterovirus (including CBV) infections in initially healthy children who later progressed to clinical insulin‐dependent diabetes mellitus (IDDM). Antibody responses were frequent also in constantly GAD65 antibody‐negative non‐diabetic children, and antibody levels did not differ between newly diagnosed IDDM patients and matched control subjects. Blocking experiments confirmed that the observed reactivity of both rabbit and human antibodies was immunologically specific. The results suggest that the epitope is antigenically highly similar in 2C and GAD65, and that peptide immunization induces antibodies which cross‐react with these molecules. However, the significance of this phenomenon in the pathogenesis of IDDM remains to be confirmed, as the peptide antibody levels were similar in patients with recent‐onset IDDM and in control subjects.

HLA-DQB1-defined genetic susceptibility, beta cell autoimmunity, and metabolic characteristics in familial and nonfamilial insulin-dependent diabetes mellitus. Childhood Diabetes in Finland (DiMe) Study Group.

Familial aggregation of insulin-dependent diabetes mellitus (IDDM) is a common phenomenon, but the reasons behind it are poorly understood. To investigate whether there is heterogeneity between familial and nonfamilial forms of IDDM we compared genetic, immunological, and clinical characteristics of diabetic children with and without an affected first-degree relative in a population-based series of Finnish children with IDDM. The frequencies of HLA-DQB1 genotypes known to be associated with high (DQB1*0302/0201) or moderate (*0302/x) IDDM risk in the Finnish population were increased, while the proportions of DQB1 genotypes associated with low or decreased risk for IDDM were reduced in the 121 familial cases as compared with the 574 nonfamilial cases (32.7 vs. 21.3%, 41.3 vs. 35.9%, 18.3 vs. 31.4%, and 7.7 vs. 11.4%, respectively; P = 0.002). The frequencies and serum concentrations of islet cell antibodies, insulin autoantibodies, and antibodies to the 65-kD isoform of glutamic acid decarboxylase were similar at diagnosis in the familial and nonfamilial cases. The 31 first-affected cases in the multiple case families were younger at diagnosis than the nonfamilial cases (6.9 vs. 8.5 yr; P < 0.05). The 90 second-affected familial cases had less severe metabolic decompensation at diagnosis than either the first-affected familial or nonfamilial cases. In conclusion, familial aggregation of IDDM in Finland is at least partly explained by a higher frequency of IDDM susceptibility genes in families with multiple affected individuals. The lack of differences in autoantibody levels between the familial and nonfamilial cases indicates homogeneity rather than heterogeneity in the pathogenetic process of beta cell destruction.