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Article, This article contributes the knowledge of Finnish aphyllophoroid funga with nationally or regionally new species, and records of rare species. Ceriporia bresadolae, Clavaria tenuipes and Renatobasidium notabile are presented as new aphyllophoroid species to Finland. Ceriporia bresadolae and R. notabile are globally rare species. The records of Ceriporia aurantiocarnescens, Crustomyces subabruptus, Sistotrema autumnale, Trechispora elongata, and Trechispora silvae-ryae are the second in Finland. New records (or localities) are provided for 33 species with no more than 10 records in Finland. In addition, 76 records of aphyllophoroid species are reported as new to some subzones of the boreal vegetation zone in Finland. Notes on substrata and habitats of each record are given, and the ecology and distribution of some species are discussed., published version, peerReviewed

Background & Aims Feeding during the first months of life might affect risk for celiac disease. Individuals with celiac disease or type 1 diabetes have been reported to have high titers of antibodies against cow's milk proteins. Avoidance of cow's milk−based formula for infants with genetic susceptibility for type 1 diabetes reduced the cumulative incidence of diabetes-associated autoantibodies. We performed a randomized controlled trial in the same population to study whether weaning to an extensively hydrolyzed formula reduced the risk of celiac disease autoimmunity or celiac disease. Methods We performed a double-blind controlled trial of 230 infants with HLA-defined predisposition to type 1 diabetes and at least 1 family member with type 1 diabetes. The infants were randomly assigned to groups fed a casein hydrolysate formula (n = 113) or a conventional formula (control, n = 117) whenever breast milk was not available during the first 6−8 months of life. Serum samples were collected over a median time period of 10 years and analyzed for antibodies to tissue transglutaminase (anti-TG2A) using a radiobinding assay, to endomysium using an immunofluorescence assay, and antibodies to a deamidated gliadine peptide using an immunofluorometry assay. Duodenal biopsies were collected if levels of anti-TG2A exceeded 20 relative units. Cow's milk antibodies were measured during the first 2 years of life. Results Of the 189 participants analyzed for anti-TG2A, 25 (13.2%) tested positive. Of the 230 study participants observed, 10 (4.3%) were diagnosed with celiac disease. We did not find any significant differences at the cumulative incidence of anti-TG2A positivity (hazard ratio, 1.14; 95% confidence interval, 0.51−2.54) or celiac disease (hazard ratio, 4.13; 95% confidence interval, 0.81−21.02) between the casein hydrolysate and cow's milk groups. Children who developed celiac disease had increased titers of cow's milk antibodies before the appearance of anti-TG2A or celiac disease. Conclusions In a randomized controlled trial of 230 infants with genetic risk factors for celiac disease, we did not find evidence that weaning to a diet of extensively hydrolyzed formula compared with cow's milk−based formula would decrease the risk for celiac disease later in life. Increased titers of cow's milk antibody before anti-TG2A and celiac disease indicates that subjects with celiac disease might have increased intestinal permeability in early life. ClinicalTrials.gov Number: NCT00570102.

Antibodies to glutamate decarboxylase (GAD65Ab) may persist, and their titres even increase after the clinical onset of type 1 diabetes. To characterize this phenomenon in detail, we analysed sequentially antibodies to GAD65 epitope clusters in a radio-binding assay in patients with type 1 diabetes. Serum samples were taken at diagnosis and 2, 5 and 10 years later from 50 young patients who had tested positive for GAD65Ab at least once during observation. The levels of GAD65Ab peaked in 21 patients after diagnosis. Antibodies to the middle region of GAD65 (GAD65-M-Ab, 88%) were more common at diagnosis than antibodies to the C-terminal (GAD65-C-Ab, 68%, P

This prospective case-control study aimed at evaluating the time course of serum concentrations of soluble adhesion molecules; intercellular adhesion molecule-1 and L-selectin in siblings with signs of pre-clinical Type 1 diabetes in order to relate these concentrations to autoantibody status and to assess whether these markers could discriminate between those siblings who progressed to clinical diabetes and those who remained non-diabetic. Serum levels of soluble adhesion molecules were measured with enzyme-linked immunosorbent assays in autoantibody-positive initially healthy siblings of diabetic children who progressed to clinical disease during the observation period of 10 years and in sex- and age-matched autoantibody-positive siblings who have remained unaffected. The intraindividual and interindividual variability in the concentrations of soluble adhesion molecules was conspicuous both among the progressors and non-progressors. Integrated concentrations (area-under-the curve) of intercellular adhesion molecule-1 over a period of 6 to 48 months before the diagnosis was higher in the progressors (p=0.035), the difference being most evident 18 to 24 months before diagnosis (p=0.015). The integrated concentrations of soluble L-selectin were similar in progressors and non-progressors over the total pre-clinical period. There were no differences in the integrated concentrations of soluble adhesion molecules in relation to the initial or maximal number of autoantibodies detected during the follow-up. These observations suggest that the process of destructive insulitis could be initiated approximately 4 years before the manifestation of clinical diabetes, being most active about 1.5 years before diagnosis. Peripheral concentrations of soluble intercellular adhesion molecule-1 or L-selectin are not helpful in the identification of those prediabetic subjects who will progress to clinical disease over the next 10 years, since there is substantial overlapping in these concentrations between progressors and non-progressors.

Aims/hypothesis. To assess the role of HLA-defined genetic diabetes susceptibility in the appearance of signs of beta-cell autoimmunity in a series of children derived from the general population. Methods. Tests for five HLA DQB1 alleles and four diabetes-associated autoantibodies were carried out on 1,584 older sibs of infants with an increased HLA-defined genetic risk of Type 1 diabetes. The DQB1 genotypes were classified into those conferring high (*02/0302), moderate (*0302/x; where x indicates *0302 or a non-defined allele), low (*0301/0302, *02/0301, *02/x, *0302/0602, *0302/0603; where x indicates *02 or a non-defined allele) or decreased risk (other genotypes). Results. Both islet cell antibodies (ICA) and GAD65 antibodies (GADA) were more frequent among the sibs with the high-risk genotype than among those with a low or decreased risk. Insulin autoantibodies and IA-2 antibodies (IA-2A) were more prevalent in the high-risk than low-risk sibs. Sibs with moderate-risk genotypes tested positive for ICA, GADA and IA-2A more often than sibs with genotypes conferring decreased risk. Autoantibody titres were also dependent on the genetic risk with high risk sibs having the highest values. Sibs carrying high-risk or moderate-risk genotypes tested positive for multiple antibodies (≥2) more often than did the sibs with low or decreased genetic risk. Conclusions/interpretation. The data show that HLA-defined susceptibility to Type 1 diabetes has an effect on both the quality and quantity of humoral beta-cell autoimmunity in unaffected children derived from the general population.

Background Islet cell antibodies (ICA) represent a heterogenous group of autoantibodies to diabetes-associated antigens, including glutamic acid decarboxylase (GAD) and the IA-2 protein. The objectives of the present study were to compare the prevalence of autoantibodies to known biochemically characterized autoantigens between ICA-positive non-diabetic parents and siblings of children with type 1 diabetes and to evaluate how such antibodies explain ICA reactivity. Methods The presence and levels of GAD antibodies (GADA), IA-2 antibodies (IA-2A) and insulin autoantibodies (IAA) were analyzed in the sera of 184 ICA-positive first-degree relatives (79 parents and 105 siblings). Results The prevalences of GADA (61.9% in siblings vs 32.9% in parents), IA-2A (55.2% vs 15.2%) and IAA (41.0% vs 0%) were increased among ICA-positive siblings relative to ICA-positive parents (p

Background Cord blood samples were collected from 1002 consecutive births at Turku University Hospital to study the prevalence and fate of type 1 diabetes-associated autoantibodies in newborn infants of unaffected mothers. Methods The samples were analysed for cytoplasmic islet cell antibodies (ICA), autoantibodies to the 65 kD isoform of glutamic acid decarboxylase (GADA), autoantibodies to the protein tyrosine phosphatase related IA-2 antigen (IA-2A), insulin autoantibodies (IAA) and HLA DQB1 genotypes. Results ICA were detected in 27 cord blood samples (2.7%), with a median of 6 (range 4–34) JDF units. GADA were found to be positive (≥6.6 RU) in six samples (0.6%), with a median of 66 (range 19–125) RU. IA–2A (≥0.43 RU) were observed in three samples (0.3%), with a median of 1.3 (range 0.8–57) RU, while only one cord blood sample (0.1%) tested positive for IAA (≥1.56 nU/ml) with a value of 5.4 RU. Maternal or gestational age, sex and birth weight of the infant were not related to antibody prevalence or titres. Altogether there were 29 infants with antibody positivity in their cord blood (2.9%). Five of these (0.5%) tested positive for two antibodies (ICA and GADA), and one was positive for all four antibodies measured. All nine infants from whom follow-up samples were available became antibody negative by the age of 15 months, and in all but one case inverse seroconversion occurred by the age of 9 months. Conclusions Around 3% of infants of non-diabetic mothers in Finland have diabetes-associated autoantibodies at birth, and these antibodies disappear at the latest by the age of 15 months. Copyright © 2001 John Wiley & Sons, Ltd.

To study the dynamics of disease-associated humoral immune responses, we analyzed autoantibodies to the IA-2 protein (IA-2A), glutamic acid decarboxylase (GADA), and insulin (IAA) and also islet cell antibodies (ICA) in a population-based, prospective, representative series of 710 siblings (

OBJECTIVE: To assess whether there are any differences in genetic, autoimmune, or clinical features between type 1 diabetes presenting in childhood and that diagnosed later. RESEARCH DESIGN AND METHODS: We studied 352 individuals (252 children and adolescents or =20 years of age) manifesting clinical signs of type 1 diabetes over a period of 7.5 years at a university hospital in northern Finland with a primary catchment area population of approximately 300,000. The patients were analyzed for susceptible and protective HLA-DQB1 alleles (*02, *0302, *0301, *0602, *0603, and *0604), islet cell antibodies (ICA), insulin autoantibodies, and antibodies to GAD and IA-2 (IA-2A). Their clinical symptoms and signs were recorded at diagnosis. RESULTS: The adult patients carried the high-risk DQB1*02/0302 genotype less frequently than the children and more often had protective genotypes. They also had a decreased frequency of all 4 single autoantibody specificities and of multiple (> or =3) autoantibodies. The proportion of patients testing negative for all autoantibodies was lower among the children than among the adults. IA-2A were associated with the DQB1*0302/x genotype in both the children and adults, and the same held true for ICA among the adults. The adults were characterized by a higher proportion of males, a longer duration of symptoms, and a lower frequency of infections during the preceding 3 months. In addition, they had a higher relative body weight on admission and milder signs of metabolic decompensation (higher pH, base excess, and bicarbonate concentrations) and a lower glycated hemoglobin level at diagnosis than the children. CONCLUSIONS: Clinical manifestation of type 1 diabetes before the age of 20 years is associated with a strong HLA-defined genetic disease susceptibility, an intensive humoral immune response to various beta-cell antigens, a higher frequency of preceding infections, and a shorter duration of symptoms and more severe metabolic decompensation at diagnosis. Taken together, these observations suggest that the age at clinical onset of type 1 diabetes is determined by the intensity of the beta-cell-destructive process, which is modulated by both genetic and environmental factors.

Previous studies suggest that enterovirus infections may initiate and accelerate beta-cell damage years before the clinical manifestation of type 1 diabetes. We have now analyzed the role of enterovirus infections in the initiation of autoimmunity in children who have tested positive for diabetes-associated autoantibodies in a prospective study starting at birth (the Finnish Diabetes Prediction and Prevention Study). The frequency of enterovirus infections was studied using both serology and testing for the presence of enterovirus RNA in the sera of 21 children who developed and retained autoantibodies and in 104 control subjects chosen from the same study cohort and matched for the time of birth, sex, and HLA alleles determining genetic diabetes susceptibility. Sample intervals were taken as basic units of follow-up, to which the observed number of infections was adjusted. Enterovirus infections were detected in 26% of sample intervals in the case subjects and in 18% of the sample intervals in the control children (P = 0.03). A temporal relationship between enterovirus infections and the induction of autoimmunity was found; enterovirus infections were detected in 57% of the case subjects during a 6-month follow-up period preceding the first appearance of autoantibodies compared with 31% of the matched control children in the same age-group (odds ratio 3.7, 95% CI 1.2-11.4). The frequency of adenovirus infections did not differ between the patient and control groups. Our data imply that enterovirus infections are associated with the development of beta-cell autoimmunity and provide evidence for the role of enteroviruses in the initiation of beta-cell destruction.

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.

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.

Aims/hypothesis. To study temporal changes in positivity for autoantibodies associated with Type I (insulin-dependent) diabetes mellitus and the relations between these antibodies, HLA-DQB1-risk markers and first-phase insulin response (FPIR) in non-diabetic schoolchildren.¶Methods. The stability of the antibody status over 2 years was assessed in 104 schoolchildren initially positive for islet cell antibodies (ICA) or antibodies to the 65 000 Mr isoform of the glutamic acid decarboxylase (GADA) or both and in 104 antibody-negative control children matched for sex, age and place of residence. All children were also studied for their first-phase insulin response and HLA-DQB1 alleles on the second occasion.¶Results. On the second occasion 3 of the 98 initially ICA-positive children, 3/13 of those positive for antibodies to the IA-2 protein (IA-2A), 1/17 GADA-positive and 2/7 of those positive for insulin autoantibodies (IAA) tested negative for these antibodies. Children with IA-2A, GADA, IAA and multiple ( ≥ 2) antibodies had significantly lower first-phase insulin responses than the control children. In contrast, these responses did not differ between subjects with and without specific HLA-DQB1-risk alleles or genotypes. Of the six subjects with a considerably reduced first-phase insulin response three had multiple antibodies on both occasions but none of them had a DQB1 genotype conferring increased diabetes risk. Two subjects progressed to Type I diabetes within 3.4 years of follow-up, both of them having multiple antibodies and a considerably reduced first-phase insulin response but neither of them having a DQB1-risk genotype.¶Conclusions/interpretation. Positivity for diabetes-associated autoantibodies is a relatively stable phenomenon in unaffected schoolchildren, although conversion to seronegativity can occur occasionally. Our observations also indicate that DQB1 alleles associated with decreased susceptibility to Type I diabetes do not protect from impaired beta-cell function or from progression to overt disease in initially unaffected schoolchildren. [Diabetologia (2000) 43: 457–464]

To evaluate the emergence of diabetes-associated autoantibodies in young children and to assess whether such antibodies can be used as surrogate markers of type 1 diabetes in young subjects at increased genetic risk, we studied 180 initially unaffected siblings (92 boys and 88 girls) of children with newly diagnosed type 1 diabetes. All siblings were younger than 6 yr of age at the initial sampling, and they were monitored for the emergence of islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamate decarboxylase antibodies (GADA), and IA-2 antibodies (IA-2A) up to the age of 6 yr and for progression to clinical type 1 diabetes up to the age of 10 yr. All 160 siblings with DNA samples available were typed for susceptible (DQB1*02 and *0302) and protective (DQB1*0301 and *0602-03) HLA DQB1 alleles. Twenty-two siblings (12.2%) tested positive for ICA in their first antibody-positive sample before the age of 6 yr, 13 (7.2%) tested positive for IAA, 15 (8.3%) tested positive for GADA, and 14 (7.8%) tested positive for IA-2A. There were 16 siblings (8.9%) who had 1 detectable autoantibody, 5 (2.8%) had 2, and 12 (6.7%) had 3 or more. In the group of 82 siblings with increased human leukocyte antigen-defined genetic susceptibility [DQB1*02/*0302, *0302/x (x = other than *02 or a protective allele), *02/y (y = other than *0302 or a protective allele)], 18 (22.0%) tested positive for ICA in their first antibody-positive sample, 10 (12.2%) tested positive for IAA, 14 (17.1%) tested positive for GADA, and 12 (14.6%) tested positive for IA-2A. One antibody was detectable in 6 siblings (7.3%), 2 were detectable in 5 (6.1%), and 3 or more were detectable in 12 (14.6%). Fifteen siblings (18.3%) presented with clinical type 1 diabetes before the age of 10 yr. All of the progressors showed increased human leukocyte antigen-defined genetic susceptibility. Thirteen of those 15 siblings, who presented with clinical type 1 diabetes before the age of 10 yr, had at least 2 antibodies detectable before the age of 6 yr (disease sensitivity, 87%; 95% confidence interval, 60-98%). Thirteen of the 17 siblings who tested positive for 2 or more autoantibodies before the age of 6 yr developed type 1 diabetes before the age of 10 yr (positive predictive value, 76%; 95% confidence interval, 50-93%). These observations suggest that disease-associated autoantibodies can well be used as surrogate markers of clinical type 1 diabetes in primary prevention trials targeting young subjects with increased genetic disease susceptibility.

The relationships between genetic markers and disease-associated autoantibodies were studied in an unselected population of 701 siblings of children with type 1 diabetes, and the predictive characteristics of these markers over a period of 9 years were determined. Increased prevalences of all the antibodies were closely associated with HLA identity to the index case, the DR4 and DQB1*0302 alleles, and the DR3/4 phenotype and the DQB1*02/0302 genotype. Antibodies to GAD (GADA) were also associated with the DR3 and DQB1*02 alleles. Siblings carrying the protective DR2 and DQB1*0602-3 alleles were characterized by lower frequencies of islet cell antibodies (ICA), antibodies to IA-2 (IA-2A), and GADA. Higher levels of ICA were related to HLA identity, the DR4 and DQB1*0302 alleles, and the susceptible DQB1 genotypes, while no significant differences were observed in the levels of IA-2A, GADA, or insulin autoantibodies among siblings with different HLA risk markers. The DR2 or DQB1*0602-3 alleles were not related to the levels of any antibody specificity. A combination of the genetic markers and autoantibodies increased the positive predictive values of all autoantibodies substantially, which may have clinical implications when evaluating the risk of developing type 1 diabetes at the individual level or when recruiting high-risk individuals for intervention trials. However, because such combinations also resulted in reduced sensitivity, autoantibodies alone rather than in combination with genetic markers are recommended as the first-line screening in siblings. Finally, not all siblings with a broad humoral autoimmune response or high-risk genetic markers present with type 1 diabetes, while some with a low genetic risk and weak initial signs of humoral autoimmunity may progress to disease.

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.

Background: the role of enteroviruses has been implicated in the etiology of insulin-dependent diabetes mellitus (IDDM). A possible connection between glutamate decarboxylase (GAD) autoimmunity and enterovirus infections in IDDM has been suggested to be based on a homology region between GAD and the non-structural protein 2C of coxsackievirus B4 (CVB4). Objectives: the aims of the study were to measure the occurrence of cellular immunity to GAD and CVB4 in Finnish patients with newly diagnosed IDDM, and to study the relation between these two responses. T-cell responses to GAD and CVB4 were analyzed in relation to HLA DQB1 risk alleles for IDDM and antibodies to GAD and CVB4. Study design: T-cell and antibody responses to GAD65 and purified CVB4 were measured in patients with newly diagnosed IDDM and in healthy children. The purified CVB4 did not contain the non-structural protein 2C thus lacking the reported homology region with GAD. Results: high proliferative responses of PBMC to both GAD and CVB4 were more frequent in IDDM patients than in the control children (40 vs. 16%, 27 vs. 10%; P=0.03 and 0.04, respectively; Fisher’s exact test), when the cut-off for positivity was three multiples of the median SI in the healthy children. Median SI to GAD was higher in the patients with IDDM than in the control subjects (3.10 vs. 1.55; P=0.03, Mann–Whitney U-test). T-cell responses to GAD and CVB4 showed a positive correlation in the patients (r=0.62, P=0.001), but not in the control children (r=0.23; P=0.38). Conclusions: enhanced T-cell responsiveness to CVB4 in patients with newly diagnosed IDDM support the involvement of enteroviral infections in the development of IDDM. The observed correlation between T-cell reactivity to GAD and CVB4, lacking the crossreactive protein 2C, in patients with IDDM suggests that CBV4 reactivity is associated with GAD autoimmunity in IDDM but does not reflect immunization to GAD.

We analyzed 747 children, younger than 15 yr of age, with newly diagnosed diabetes, for antibodies to glutamic acid decarboxylase (GADA), the IA-2 protein (IA-2A), insulin (IAA), and islet cells, to evaluate the influence of positivity for GADA, IA-2A, IAA, or multiple (> or = 3) autoantibodies at diagnosis, on the clinical presentation and natural course of the disease over the first 2 yr and to characterize autoantibody-negative patients. At diagnosis, 73.2% of the children tested positive for GADA, 85.7% for IA-2A, 54.2% for IAA, and 72.6% for multiple autoantibodies. Only 17 subjects (2.3%) had no detectable autoantibodies. The patients testing positive for multiple autoantibodies were younger than the remaining children (P < 0.001). A similar age difference was seen when comparing IAA-positive and -negative patients (P < 0.001). There was no significant difference between the GADA-positive and -negative subjects in the degree of metabolic decompensation at diagnosis, whereas those testing positive for IA-2A had reduced serum C-peptide concentrations (P = 0.003), and those positive for IAA had lower glycated hemoglobin values. The patients with no detectable autoantibodies had higher serum C-peptide levels (P = 0.007) at diagnosis than did the other subjects. The children initially positive for IA-2A had decreased serum C-peptide concentrations at 24 months (P = 0.045), and their daily insulin dose was higher at 18 (P = 0.005) and 24 months (P < 0.001). The patients who tested positive for multiple autoantibodies at diagnosis had decreased serum C-peptide levels (P < 0.001) and higher insulin doses (P = 0.005) at 12, 18, and 24 months. A lower proportion of them were also in clinical remission at 12 and 18 months (P = 0.01). Autoantibody-negative subjects needed less exogenous insulin at 6 and 18 (P = 0.01) and at 24 months (P < 0.001) than the other subjects, and a higher proportion of them were in clinical remission at 18 months (P < 0.001). We conclude that positivity for multiple diabetes-related autoantibodies is associated with accelerated beta-cell destruction and an increased requirement for exogenous insulin over the second year of clinical disease, indicating that multiple autoantibodies reflect an aggressive progression to total beta-cell destruction. Patients testing negative for diabetes-associated autoantibodies at diagnosis seem to have a milder degree of beta-cell destruction, but their metabolic decompensation is similar to that seen in other affected children, suggesting that they do represent classical type 1 diabetes.

SUMMARY 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

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]

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 (> or =3 glasses daily) was associated with more frequent emergence of Type 1 DM-associated autoantibodies than low consumption (

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.

The current focus of the screening for individuals at risk for type 1 diabetes has moved from first-degree relatives to the general population. However, there is a shortage of data on the predictive utility of various risk markers in the background population in various countries, and predictive strategies for the general population remains open. We studied the frequency of diabetes-associated autoantibodies in a series of 3,652 unaffected Finnish school-children, and determined the relationships between autoantibodies and HLA-DQB1 risk markers. In addition, all subjects were observed for progression to type 1 diabetes. The reported frequencies of autoantibodies in the general population have varied widely. In the present study, the frequencies of ICA (islet cell antibodies), GADA (GAD antibodies), IA-2A (antibodies to IA-2 protein), and IAA (insulin autoantibodies) were 2.8, 0.5, 0.6, and 0.9%, respectively. Multiple antibodies (i.e., two or more) were detected in 21 children (0.6%), and 9 children (0.25%) had three or four antibody specificities in their initial blood sample. The present cut-off limits for antibody-positivity (determined as the 99th percentile in >370 healthy control subjects) are close to the 99.5th percentile for IA-2A and GADA and the 99.0th percentile for IAA in the present series of 3,652 children. The use of different approaches for the definition of cut-off limits (1,2) resulted in somewhat higher frequencies of autoantibodies, although this did not improve the diagnostic sensitivity of any autoantibody. These data illustrate the difficulties in defining borderline positivity …

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. Conclusion: These data show that almost half of the siblings with signs of prediabetes at the time of diagnosis of the index case progressed further in their preclinical disease process during prospective observation. Young age, a strong humoral immune response to β-cell antigens and reduced insulin secretory capacity appeared to be characteristic of those with a progressive process. Advanced and late prediabetes seem to represent a point of no return, as regression from these stages to no prediabetes was extremely rare.

We set out to study the association between human leukocyte antigen-defined genetic disease susceptibility and the stage of preclinical type 1 diabetes and whether genetic predisposition affects the natural course of preclinical diabetes in initially nondiabetic siblings of affected children. A total of 701 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: no prediabetes (no antibodies), early (one antibody specificity), advanced (two antibodies), and late prediabetes (three or more antibodies). Another classification system covering 659 siblings was based on a combination of the initial number of antibodies and the first-phase insulin response (FPIR) to iv glucose: no prediabetes (no antibodies), early (one antibody specificity, normal FPIR), advanced (two or more antibodies, normal FPIR), and late prediabetes (at least one antibody, reduced FPIR). Genetic susceptibility to type 1 diabetes was defined by human leukocyte antigen identity and DR and DQ genotypes. There was a higher proportion of siblings with late prediabetes initially among those with strong genetic disease susceptibility than among those with decreased genetic predisposition (16.7% vs. 0.5%; P < 0.001 for DQB1 genotypes according to the first classification), whereas there was a higher proportion of siblings with no signs of prediabetes among those with genotypes conferring decreased risk (91.2% vs. 70.4% among those with high-risk DQB1 genotypes; P < 0.001 according to the first classification). Autoantibodies alone were more sensitive in the prediction of future diabetes in siblings than when combined with genetic susceptibility. Genetic susceptibility played a role in whether the initial prediabetic stage progressed (progression in 29.6% of the high-risk siblings compared with 6.6% of the siblings with DQB1 genotypes conferring decreased risk; P < 0.001 according to the first classification) and whether overt type 1 diabetes became manifest or not. Genetic susceptibility has an impact on both the initiation and progression of the autoimmune process leading to clinical diabetes in siblings of affected children.

Background: Type 1 diabetes mellitus results from destruction of the pancreatic insulin-producing beta cells by a chronic autoimmune process. Methods are needed for the detection of circulating autoantibodies to glutamic acid decarboxylase (GAD65), a major marker of this process. Methods: Streptavidin-coated microtiter plates were incubated with biotinylated GAD65, and after incubation with serum samples from patients with type 1 diabetes mellitus and control individuals, europium-labeled GAD65 was added. After washing steps, the delayed fluorescence was measured in duplicate in a fluorometer. Samples collected from 100 patients with newly diagnosed type 1 diabetes mellitus and 100 healthy controls were measured by the new assay and by a radiobinding assay. Results: The detection limit of the new assay was 1.49 WHO units/mL, the calibration curve was linear to 4 140 WHO units/mL, and no hook effect was observed up to 41 400 WHO units/mL. The intraassay CV was 2.1–6.3% over the calibration range. For patient serum samples, the intraassay, interassay, and total CVs were 5.4–7.0%, 9.8–13%, and 12–14%, respectively. Compared with conventional radioimmunologic methods, the analytical range was broader and the analysis time required to perform the measurements was shorter. At a cutoff with 99% specificity, the new assay and the radiobinding assay were positive in 71 and 67 patients, respectively. Conclusions: The new assay provides a rapid and sensitive nonradioactive method applicable for large-scale screening for beta-cell autoimmunity. It has a broad linear analytical range, is easy to perform and automate, and has sensitivity and specificity comparable to those for the conventional radioisotope assay.

The aim of this study was to evaluate the frequency and predictive value of diabetes-associated autoantibodies, such as islet cell antibodies (ICA) and autoantibodies to insulin (IAA), GAD65 (GADA), and the IA-2 molecule (IA-2A) in genetically susceptible children from the general population during the first 2 yr of life. Of 12,170 newborn infants, 1,005 with increased genetic risk of type 1 diabetes (high risk, human leukocyte antigen DQB1*02/*0302; moderate risk, DQB1*0302/x, where x = other than *02, *0301, or *0602) were monitored for ICA, IAA, GADA, and IA-2A at 3- to 6-month intervals from birth up to a minimum age of 2 yr. In addition, all 15 genetically susceptible children from the general population who had participated in regular immunological follow-up and developed clinical type 1 diabetes by the end of April 2000 were analyzed for the development of autoantibodies. Among 1,005 children, 63 (6.3%) tested positive for at least one autoantibody, 31 for ICA (3.1%), 48 for IAA (4.8%), 23 for GADA (2.3%), and 13 for IA-2A (1.3%) at least once by the age of 2 yr. Both ICA and IAA identified 95% [95% confidence interval (CI), 77.2-99.9%] of those who tested persistently positive for multiple (or = 2) antibodies at the age of 2 yr, GADA identified 86% (CI, 65.1-97.1%), and IA-2A identified 55% (CI, 32.2-75.6%). Close to half of the antibody-positive children (29 of 63) reverted back to antibody negativity. Autoantibodies disappeared more often among those who tested positive for IAA than among those who tested positive for other autoantibodies (Por = 0.021). Among the 15 children who developed type 1 diabetes, the disease sensitivity of ICA was 80% (CI, 51.9-95.7%), that of IAA was 93% (CI, 68.0-99.8%), that of GADA was 60% (CI, 32.3-83.7%), and that of IA-2A was 40% (CI, 16.3-67.7%). These results suggest that IAA are characterized by high sensitivity, early appearance, and high frequency of transient antibody positivity, whereas ICA detected with a thoroughly standardized assay appear to be more specific for the screening of beta-cell autoimmunity in young children with increased genetic susceptibility to type 1 diabetes in the Finnish population, which has the highest incidence of type 1 diabetes in the world.

Summary We studied the pattern of type 1 diabetes-associated autoantibodies during pregnancy and the transplacental transfer of these autoantibodies to the fetal circulation and searched for possible signs of prenatal induction of β-cell autoimmunity in newborn infants. The population comprised 208 mothers and their newborn infants. Seventy-four of the mothers (36%) had type 1 diabetes and 134 (64%) of the infants had an affected father or sibling. Blood samples were obtained from the mother at the end of the first trimester and at delivery, and from the cord blood of the newborn infant. Close to 40% of the mothers with type 1 diabetes had antibodies to islet cells (ICA), 55% to glutamic acid decarboxylase (GADA) and 54% to the IA-2 protein (IA-2A) in early pregnancy, whereas the corresponding frequencies in the nonaffected mothers were 5·2%, 5·2% and 3·0%. No significant changes could be seen in autoantibody levels during pregnancy, and there was a close correlation between the two maternal samples. One third of the infants of mothers with type 1 diabetes tested positive for ICA, 50% for GADA and 51% for IA-2A. Six percent of the infants of nondiabetic mothers had ICA, 2·2% GADA and none had IA-2A. None of the infants of the antibody negative mothers had antibodies in their cord blood. These observations indicate that the immunomodulatory effect of pregnancy on signs of β-cell autoimmunity is weak, but if diabetes-associated autoantibodies are present in the mother, most of them are transferred to the fetal circulation. Our data do not provide any support for fetal induction of β-cell autoimmunity.

To study the dynamics of disease-associated humoral immune responses, we analyzed autoantibodies to the IA-2 protein (IA-2A), glutamic acid decarboxylase (GADA), and insulin (IAA) and also islet cell antibodies (ICA) in a population-based, prospective, representative series of 710 siblings (20 years of age) of children with type 1 diabetes. Positivity for single autoantibodies was observed in 8-13% of these siblings during an average follow-up of 4 years. The overall incidence rates per 1,000 years (number of cases/person-years in parentheses) for positive seroconversion of IA-2A were nine (19/2,123), followed by six (12/2,049) for GADA, 19 (40/2,111) for IAA, and 16 (31/1965) for ICA. Positive seroconversions seemed to be associated with a young age of the sibling, HLA DR3/DR4 heterozygosity, HLA identity, and a high initial number of detectable autoantibodies. The overall incidence rates per 1,000 years (number of cases/person-years in parentheses) for inverse seroconversion of IA-2A were 76 (12/157), followed by 42 (10/237) for GADA, 460 (32/70) for IAA, and 27 (9/331) for ICA. No consistent risk factor for inverse seroconversions was present, although seroconversions were most frequent in siblings with older age, male sex, HLA phenotypes other than DR3/DR4, a small family size, and no other autoantibodies detectable at seroconversion. Altogether, these observations indicate that beta-cell autoimmunity may be induced at any age in childhood and adolescence. HLA-conferred genetic disease susceptibility is a strong determinant of persistent beta-cell autoimmunity, but environmental factors may also contribute to such autoimmunity.

Little is known about the timing of the etiological events and the preclinical process of type 1 diabetes during the first years of life in the general population. In this population-based prospective birth cohort study, the appearance of diabetes-associated autoantibodies as a sign of beta-cell autoimmunity and the development of type 1 diabetes were monitored from birth. Of 25,983 newborn infants, 2,448 genetically susceptible children were monitored for islet cell antibodies (ICA) at 3- to 6-month intervals. If an infant seroconverted to ICA positivity, all his/her samples were also analyzed for insulin autoantibodies (IAA), antibodies to the 65-kDa isoform of glutamic acid decarboxylase, and antibodies to the protein tyrosine phosphatase-related IA-2 molecule. Fifteen children of those who carried the high-risk genotype (2.7%) and 23 of those who carried the moderate-risk genotype (1.2%; P = 0.019) tested positive for ICA at least once. Among those who showed positivity for at least 2 antibodies during the observation period (25 of 38), IAA appeared as the first or among the first antibodies in 22 children (88%) and emerged earlier than the other antibodies (P < 0.019 or less). The first autoantibodies appeared in the majority of the children in the fall and winter (30 of 38 vs. 8 of 38 in the spring and summer, P < 0.001). These observations suggest that young children in the general population with a strong human-leukocyte-antigen-DQ-defined genetic risk of type 1 diabetes show signs of beta-cell autoimmunity proportionally more often than those with a moderate genetic risk. IAA emerge as the first detectable antibody more commonly than any other antibody specificity, implying that insulin may be the primary antigen in most cases of human type 1 diabetes associated with the DR4-DQB1*0302 haplotype. The seasonal variation in the emergence of the first signs of beta-cell autoimmunity suggests that infectious agents may play a role in the induction of such autoimmunity.

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]

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, P0.001). 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, P0.01) and glutamic acid decarboxylase (OR 15.4, P0.05), 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.

To evaluate the emergence of diabetes-associated autoantibodies in young children and to assess whether such antibodies can be used as surrogate markers of type 1 diabetes in young subjects at increased genetic risk, we studied 180 initially unaffected siblings (92 boys and 88 girls) of children with newly diagnosed type 1 diabetes. All siblings were younger than 6 yr of age at the initial sampling, and they were monitored for the emergence of islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamate decarboxylase antibodies (GADA), and IA-2 antibodies (IA-2A) up to the age of 6 yr and for progression to clinical type 1 diabetes up to the age of 10 yr. All 160 siblings with DNA samples available were typed for susceptible (DQB1*02 and *0302) and protective (DQB1*0301 and *0602-03) HLA DQB1 alleles. Twenty-two siblings (12.2%) tested positive for ICA in their first antibody-positive sample before the age of 6 yr, 13 (7.2%) tested positive for IAA, 15 (8.3%) tested positive for GADA, and 14 (7.8%) tested positive for IA-2A. There were 16 siblings (8.9%) who had 1 detectable autoantibody, 5 (2.8%) had 2, and 12 (6.7%) had 3 or more. In the group of 82 siblings with increased human leukocyte antigen-defined genetic susceptibility [DQB1*02/*0302, *0302/x (x = other than *02 or a protective allele), *02/y (y = other than *0302 or a protective allele)], 18 (22.0%) tested positive for ICA in their first antibody-positive sample, 10 (12.2%) tested positive for IAA, 14 (17.1%) tested positive for GADA, and 12 (14.6%) tested positive for IA-2A. One antibody was detectable in 6 siblings (7.3%), 2 were detectable in 5 (6.1%), and 3 or more were detectable in 12 (14.6%). Fifteen siblings (18.3%) presented with clinical type 1 diabetes before the age of 10 yr. All of the progressors showed increased human leukocyte antigen-defined genetic susceptibility. Thirteen of those 15 siblings, who presented with clinical type 1 diabetes before the age of 10 yr, had at least 2 antibodies detectable before the age of 6 yr (disease sensitivity, 87%; 95% confidence interval, 60-98%). Thirteen of the 17 siblings who tested positive for 2 or more autoantibodies before the age of 6 yr developed type 1 diabetes before the age of 10 yr (positive predictive value, 76%; 95% confidence interval, 50-93%). These observations suggest that disease-associated autoantibodies can well be used as surrogate markers of clinical type 1 diabetes in primary prevention trials targeting young subjects with increased genetic disease susceptibility.

Glutamic acid decarboxylase antibodies are present in some patients with therapy-resistant epilepsy. The authors measured glutamic acid decarboxylase antibodies in an unselected population of 114 children with different types of epilepsy. Three children with temporal lobe epilepsy and six children with various other types of epilepsy had intractable epilepsy. None of the children tested positive for glutamic acid decarboxylase antibodies. The study suggests that glutamic acid decarboxylase antibody testing cannot be recommended in unselected cases of childhood epilepsy.

OBJECTIVE: To study the characteristics of type 1 diabetes in very young children. RESEARCH DESIGN AND METHODS: Clinical outcome, islet cell antibodies (ICA), insulin autoantibodies (IAA), antibodies against GAD (GADA), IA-2 antibodies (IA-2A), and HLA-DQB1-defined genetic risk were analyzed in 35 children diagnosed with type 1 diabetes before 2 years of age and compared with those in 146 children who were diagnosed between 2.0 and 4.9 years of age and with those in 620 children diagnosed between 5.0 and 14.9 years of age. RESULTS: The youngest age-group had severer metabolic decompensation at clinical onset, and their serum C-peptide levels, compared with those of older children, were lower at the time of diagnosis and during the first 2 years after the diagnosis. The levels of ICA and IAA were highest in children < 2 years of age, but there were no differences in GADA levels among the three age-groups. The youngest age-group had the lowest IA-2A levels. The HLA DQB1*02/*0302 genotype associated with strong genetic susceptibility was more frequent in children diagnosed < 5 years of age, whereas the proportion of children carrying a genotype, which includes protective alleles, was higher among those diagnosed at > or = 5 years of age. CONCLUSIONS: The clinical presentation of type 1 diabetes at a very young age is associated with severe metabolic decompensation, poorly preserved residual beta-cell function, strong humoral autoimmunity against islet cells and insulin, and strong HLA-defined disease susceptibility.

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.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).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.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.

We analyzed 747 children, younger than 15 yr of age, with newly diagnosed diabetes, for antibodies to glutamic acid decarboxylase (GADA), the IA-2 protein (IA-2A), insulin (IAA), and islet cells, to evaluate the influence of positivity for GADA, IA-2A, IAA, or multiple (or = 3) autoantibodies at diagnosis, on the clinical presentation and natural course of the disease over the first 2 yr and to characterize autoantibody-negative patients. At diagnosis, 73.2% of the children tested positive for GADA, 85.7% for IA-2A, 54.2% for IAA, and 72.6% for multiple autoantibodies. Only 17 subjects (2.3%) had no detectable autoantibodies. The patients testing positive for multiple autoantibodies were younger than the remaining children (P0.001). A similar age difference was seen when comparing IAA-positive and -negative patients (P0.001). There was no significant difference between the GADA-positive and -negative subjects in the degree of metabolic decompensation at diagnosis, whereas those testing positive for IA-2A had reduced serum C-peptide concentrations (P = 0.003), and those positive for IAA had lower glycated hemoglobin values. The patients with no detectable autoantibodies had higher serum C-peptide levels (P = 0.007) at diagnosis than did the other subjects. The children initially positive for IA-2A had decreased serum C-peptide concentrations at 24 months (P = 0.045), and their daily insulin dose was higher at 18 (P = 0.005) and 24 months (P0.001). The patients who tested positive for multiple autoantibodies at diagnosis had decreased serum C-peptide levels (P0.001) and higher insulin doses (P = 0.005) at 12, 18, and 24 months. A lower proportion of them were also in clinical remission at 12 and 18 months (P = 0.01). Autoantibody-negative subjects needed less exogenous insulin at 6 and 18 (P = 0.01) and at 24 months (P0.001) than the other subjects, and a higher proportion of them were in clinical remission at 18 months (P0.001). We conclude that positivity for multiple diabetes-related autoantibodies is associated with accelerated beta-cell destruction and an increased requirement for exogenous insulin over the second year of clinical disease, indicating that multiple autoantibodies reflect an aggressive progression to total beta-cell destruction. Patients testing negative for diabetes-associated autoantibodies at diagnosis seem to have a milder degree of beta-cell destruction, but their metabolic decompensation is similar to that seen in other affected children, suggesting that they do represent classical type 1 diabetes.

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 (or =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.

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%, p0.001). 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.

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.