K. Savola

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]

Enterovirus antibody levels during the first two years of life in prediabetic autoantibody-positive children

Aims/hypothesis. We evaluated the role of enterovirus infections in the pathogenesis of Type I (insulin-dependent) diabetes mellitus by monitoring enterovirus antibody levels in prediabetic children who turned positive for diabetes-associated autoantibodies in a prospective birth cohort study. Methods. Serial serum samples taken during prospective observation starting at birth were analysed for IgG and IgA class antibodies against enterovirus antigens including purified coxsackievirus B4, echovirus 11, poliovirus 1 and a synthetic enterovirus peptide antigen using enzyme immunoassay. Maternal samples taken at the end of the third month of pregnancy were also studied. Analyses were done from 21 childen who developed autoantibodies and from 104 autoantibody-negative control children who were matched for the time of birth, gender and HLA susceptibility alleles. For comparison, adenovirus antibodies were also analysed from all samples collected. Results. IgG class enterovirus antibody levels were high in maternal samples and in cord blood in both case and control children. After birth the IgG levels decreased reaching a nadir at the age of 6 months. No IgA class antibodies were detected at birth but started to emerge postnatally. Antibody levels did not differ between the autoantibody positive and the control children during the first 6 months of life. From 6 months to 24 months of age, the autoantibody positive children had higher IgG and IgA levels against coxsackievirus B4, echovirus 11 and the synthetic enterovirus peptide antigens than control children but poliovirus 1 and adenovirus antibodies were closely similar in the two groups. The difference between children with autoantibodies and control children was predominantly seen among boys and among those with the HLA-DQB1*0302/x genotype. Conclusions/interpretation. Our data show that children who seroconverted for diabetes-associated autoantibodies develop stronger humoral immune responses to coxsackievirus B4, echovirus 11 and a synthetic enterovirus peptide antigen than children who remained negative for autoantibodies. Poliovirus antibodies induced by uniform vaccinations did not differ between the prediabetic and control children suggesting that the regulation of antibody responses to enteroviruses is not disturbed. Accordingly, the results imply a stronger enterovirus exposure in prediabetic children supporting the role of enteroviruses in the pathogenesis of Type I diabetes. [Diabetologia (2001) 44: 818–823]

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.

Disease-associated autoantibodies and HLA-DQB1 genotypes in children with newly diagnosed insulin-dependent diabetes mellitus (IDDM)

The possible relation between HLA‐DQ genotypes and both frequencies and levels of autoantibodies associated with IDDM was assessed by examining HLA‐DQB1 alleles and antibodies to islet cells (ICA), insulin (IAA), glutamic acid decarboxylase (GADA) and the protein tyrosine phosphatase‐related IA‐2 molecule (IA‐2A) in 631 newly diagnosed diabetic children under the age of 15 years. ICA were found in 530 children (84.0%), while close to half of the subjects (n = 307; 48.7%) tested positive for IAA. GADA were detected in 461 index cases (73.1%), with a higher frequency in those older than 10 years (78.9% versus 69.2% in the younger ones; P = 0.006). More than 85% of the children (n = 541; 85.7%) tested positive for IA‐2A. Altogether there were only 11 children (1.7%) who had no detectable autoantibodies at diagnosis. There were no differences in the prevalence of ICA or GADA between four groups formed according to their HLA‐DQB1 genotype (DQB1*0302/02, *0302/X (X = other than *02), *02/Y (Y = other than *0302) and other DQB1 genotypes). The children with the *0302/X genotype had a higher frequency of IA‐2A and IAA than those carrying the *02/Y genotype (93.8% versus 67.3%, P < 0.001; and 49.0% versus 33.6%, P = 0.002, respectively). The children with the *02/Y genotype had the highest GADA levels (median 36.2 relative units (RU) versus 14.9 RU in those with *0302/X; P = 0.005). Serum levels of IA‐2A and IAA were increased among subjects carrying the *0302/X genotype (median 76.1 RU versus 1.6 RU, P = 0.001; and 50 nU/ml versus 36 nU/ml, P = 0.004) compared with those positive for *02/Y. Only three out of 11 subjects homozygous for *02 (27.3%) tested positive for IA‐2A, and they had particularly low IA‐2A (median 0.23 RU versus 47.6 RU in the other subjects; P < 0.001). The distribution of HLA‐DQB1 genotypes among autoantibody‐negative children was similar to that in the other patients. These results show that DQB1*0302, the most important single IDDM susceptibility allele, is associated with a strong antibody response to IA‐2 and insulin, while GAD‐specific humoral autoimmunity is linked to the *02 allele, in common with a series of other autoimmune diseases as well as IDDM. We suggest that IA‐2A may represent β cell‐specific autoimmunity, while GADA may represent a propensity to general autoimmunity.

Cow's milk consumption, disease-associated autoantibodies and type 1 diabetes mellitus: a follow-up study in siblings of diabetic children. Childhood Diabetes in Finland Study Group.

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.

Staging of Preclinical Type 1 Diabetes in Siblings of Affected Children

Objectives. To assess whether it is clinically relevant to classify siblings of children with recent-onset type 1 diabetes mellitus (T1DM) into various stages of preclinical diabetes, and to compare the risk of developing clinical disease and the time to diagnosis between these stages. Study Design. From a total of 801 families taking part in the Childhood Diabetes in Finland Study, 758 initially unaffected siblings were graded into four stages of preclinical T1DM based on the number of disease-associated autoantibodies detectable close to the time of diagnosis in the index case: no (no antibodies), early (one antibody specificity), advanced (two antibodies), and late prediabetes (more than three antibodies). Another classification system, used with 712 siblings, was based on a combination of the number of antibodies and the first-phase insulin response (FPIR) to intravenous glucose: no (no antibodies), early (one antibody specificity, normal FPIR), advanced (two or more antibodies, normal FPIR), and late prediabetes (one or more antibodies, reduced FPIR). Results. Six out of 661 siblings who initially presented no signs of prediabetes (0.9%; 95% confidence interval [CI], 0.3%–2.0%) progressed to clinical T1DM. Based on the first set of criteria, 3 out of 49 individuals (6.1%; CI, 1.3%–16.9%; odds ratio [OR], 7.1; CI, 1.7–29.4) from the early prediabetes category, 3 out of 13 with advanced prediabetes (23.1%; CI, 5.0%–53.8%; OR, 32.8; CI, 7.2–150), and 23 out of 35 with late prediabetes (65.7%; CI, 47.8%–80.9%; OR, 209; CI, 72.2–607) presented with clinical signs of T1DM. According to the second set of criteria 1 out of 15 siblings with early prediabetes (6.7%; CI, 0.2%–32.0%; OR, 7.8; CI, 0.9–69.1), 6 out of 23 with advanced prediabetes (26.1%; CI, 10.2%–48.4%; OR, 38.5; CI, 11.3–132), and 12 out of 13 with late prediabetes (92.3%; CI, 64.0%–99.8%; OR, 1310; CI, 146–11 737) presented with clinical signs of T1DM. The time to diagnosis was significantly shorter in those with late prediabetes initially than in those with no signs of prediabetes. Conclusions. Our observations indicate that it is possible to grade siblings of children with newly diagnosed T1DM into categories with significant differences in the subsequent risk of clinical T1DM and time to diagnosis. Such a classification will become clinically relevant as soon as effective measures are available for preventing or delaying the manifestation of overt T1DM. autoantibodies, classification, prospective, first-phase insulin response.

Natural course of preclinical type 1 diabetes in siblings of affected children

Aim: To define the dynamics of preclinical type 1 diabetes in siblings of affected children and to characterize the siblings experiencing a progressive process. Methods: From 801 families taking part in the “Childhood Diabetes in Finland” (DiMe) Study, 715 initially unaffected siblings were graded into four stages of preclinical type 1 diabetes based on the initial number of disease‐associated autoantibodies detectable close to the time of diagnosis of the index case, while another classification system covering 641 of the siblings was based on a combination of the initial number of antibodies and the first‐phase insulin response (FPIR) to intravenous glucose. Results: Based on the first classification, there was a total of 95 siblings with initial signs of prediabetes, out of whom 34 (36%) progressed, 26 (27%) remained stable and 35 (37%) regressed during prospective observation for a median of 3.6 y (range 0.01–9.8 y). The siblings who progressed were younger, had a higher initial number of detectable autoantibodies, higher initial levels of various antibodies, with the exception of insulin autoantibodies, lower FPIR and a retarded glucose elimination rate in the first intravenous glucose tolerance test as compared with those that regressed. According to the second classification there were 41 siblings with initial signs of prediabetes, among whom 23 (56%) progressed, 14 (34%) remained stable and 4 (10%) regressed during the observation period.