Timing of onset of autoimmunity is a prerequisite for unmasking triggers and pathogenesis of type 1 diabetes. We followed 4,590 consecutive newborns with 8 or 3% HLA-DQB1 conferred risk for type 1 diabetes at 3-, 6-, or 12-month intervals up to 5.5 years of age. Islet cell autoantibodies (ICAs) and, in the 137 children with ICAs, insulin autoantibodies (IAAs), GAD65 autoantibodies (GADAs), and IA-2 protein autoantibodies (IA2As) were measured. Children with high genetic risk developed ICAs more often than those with moderate risk (log-rank P ‫؍‬ 0.0015); 85 and 91% remained ICA negative by 5 years of age, respectively. The time of appearance of biochemical autoantibodies was then compared with the appearance of ICAs. IAAs and GADAs emerged usually before ICAs (means ؊1.8 and ؊1.5 months, respectively) and IA-2As after ICAs (mean 2.0 months). Ninety-five percent of all IAAs, GADAs, and IA-2As seroconversions occurred in a cluster (؊12 to 8 months) around the ICA seroconversion. We conclude that diabetes-associated autoantibodies emerged in children with predisposing HLA-DQB1 alleles after 3 months of age at a constant tempo, determined by the genetic risk level, usually in the order of IAA, GADA, ICA, and IA-2A. Seroconversion to multiple autoantibody positivity usually occurred tightly clustered in time. Diabetes 51:646 -651, 2002

SUMMARYWe observed 42 initially non-diabetic siblings of affected children to characterize the humoral immune response to the 65 kDa isoform of glutamic acid decarboxylase (GAD65) in preclinical type I diabetes. During the observation period with a mean duration of 9·6 years 21 siblings progressed to type I diabetes. The humoral immune response to GAD65 was observed initially as a simultaneous response to the middle (M) and carboxy (C)-terminal regions of the GAD65 molecule in most cases, and if the response was restricted initially to the middle region, it spread rapidly to the C-terminal domain and in a few cases later to the amino (N)-terminal domain. There was some heterogeneity in the GAD65 isotype response, but it was composed mainly of antibodies of immunoglobulin (Ig) G1 subclass. Responses of IgG2-, IgG4-, IgM-and IgA-GAD65Ab were observed frequently, whereas IgE-and IgG3-GAD65Ab responses were seen more rarely. Initially, the non-progressors tended more often to have IgG2-and IgG4-GAD65Ab than the progressors. As a sign of a dynamic process a significant isotype spreading was seen for IgG2-GAD65Ab ( P < 0·05) and close to significant for IgM ( P = 0·06) among progressors and for IgM-GAD65Ab ( P < 0·05) among non-progressors during the observation period. This study failed to identify any GAD65 epitope-or isotype-specific antibody reactivity that could be used as a marker for progression to disease, as such progression was not associated with any specific changes in reactivity over time. Our findings indicate that epitope-and isotype-specific GAD65 antibodies are hardly capable of separating progressors from non-progressors among GAD65Ab-positive first-degree relatives of children with type I diabetes.

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SUMMARYTo evaluate whether pregnancy has any effect on insulin antibody levels and to test the concordance between a conventional radioimmunoassay and a new microassay for the detection of insulin antibodies, insulin antibodies were analysed in 104 mothers in early pregnancy and at delivery and in their newborn infants. Thirty-eight of the mothers had type 1 diabetes. The concordance between the assays was high in the samples taken in early pregnancy (95%), but substantially lower in the samples taken at delivery (40%) and in the cord blood samples (68%). A considerable proportion of the mothers at delivery, especially the unaffected mothers (71%), and the newborn infants of the unaffected mothers (32%) were positive for insulin antibodies in the conventional assay but not in the microassay. Insulin antibody levels increased in the mothers, significantly so in the unaffected mothers (P , 0´001), during pregnancy in the conventional assay, whereas in the microassay they decreased significantly (P , 0´01) in affected mothers and remained negative in the unaffected mothers. Since immune complexes are precipitated with protein A specific for IgG in the microassay and with polyethylene glycol lacking specificity for immunoglobulins in the conventional assay, our data indicate that insulin antibody levels decrease on average during pregnancy and that the increasing non-IgG anti-insulin activity observed in the conventional assay is induced by pregnancy and is present in both the maternal and the foetal circulation.

SummaryWe studied whether serum interferon (IFN)-γ γ γ γ or interleukin (IL)-10 levels and their corresponding functional polymorphic genotypes are associated with partial remission of type 1 diabetes (T1D). A multi-centre study was undertaken in patients with newly diagnosed T1D and matched controls. T1D patients were followed for 3 months and characterized for remission status. Partial clinical remission was defined as a daily insulin dose ≤ ≤ ≤ ≤ 0·38 units/kg/ 24 h with an HbA1c ≤ ≤ ≤ ≤ 7·5%. Thirty-three patients and 32 controls were phenotyped for serum concentrations of IFN-γ γ γ γ and IL-10 and genotyped for functional polymorphisms of the IFN-γ γ γ γ and IL-10 genes. Sixteen of 25 informative patients (63%) remitted. Serum IFN-γ γ γ γ concentrations were significantly decreased in remitters but increased in non-remitters compared to controls, and did not change over time in any group. IFN-γ γ γ γ genotypes corresponded with serum levels in controls and non-remitters, but not in remitters who displayed the lowest serum IFN-γ γ γ γ levels despite more often carrying high-producing IFN-γ γ γ γ genotypes. Neither the frequency of IL-10 genotypes nor serum IL-10 concentration differed between patients and controls. The combination of high-producing IFN-γ γ γ γ genotype together with low serum IFN-γ γ γ γ concentration at the time of diagnosis provided a strong positive predictive value for remission. Serum IFN-γ γ γ γ concentrations predicted by genotype and observed serum levels were discordant in remitters, suggestive of regulation overruling genetic predisposition. Although high-producing genotypes were less frequent in remitters, they were predictive of remission in combination with low serum IFN-γ γ γ γ levels. These data imply that remission is partially immunemediated and involves regulation of IFN-γ γ γ γ transcription.

Objective: The pattern of the humoral immunity to disease-associated autoantigens may reflect the severity of the autoimmune disease process. The purpose of this study was to delineate the maturation of the humoral immunity to one of the main autoantigens in type 1 diabetes (T1D), glutamic acid decarboxylase (GAD65). Design and methods: Serum samples were obtained for the detection of epitope-and isotype-specific antibodies sequentially with short intervals from 36 young children with HLA-conferred genetic susceptibility to T1D starting from the first appearance of GAD65Ab. During prospective observation, ten children developed T1D. Antibodies were analyzed using biotinylated anti-human immunoglobulin (Ig) antibodies and chimeric GAD molecules in radio-binding assays. Results: The immune response to GAD65 started as reactivity to the middle region and spread rapidly to the C-terminal region. IgG1 antibodies dominated among the isotypes from the first appearance of GAD65Ab, while other IgG subclasses were observed to a lesser extent. IgG4 antibodies emerged clearly as the last IgG subclass. A broad initial response comprising three to four IgG subclasses and the lack of an emerging IgG4 response during follow-up was associated with increased risk for progression to clinical diabetes (P!0.05). Conclusions:The humoral response to GAD65 epitope clusters is relatively uniform in young children, whereas there is conspicuous individual variation in IgG subclass responses except for IgG1. A narrow initial IgG subclass response to GAD65 and the emergence of IgG4 antibodies were characteristic of those who remained non-diabetic over the first few years of GAD65 autoimmunity.European Journal of Endocrinology 155 633-642

To test for the existence of a reproductive cost, we manipulated brood sizes (‐2 and +2 nestlings) over 6 years in a northern population of Willow Tits Parus montanus breeding in natural holes. Possible effects were sought in subsequent survival and fecundity of the parents. Parents given extra chicks made more feeding visits than did parents with reduced and control broods. However, this was not reflected in differences in parental body‐weight between groups at the end of the nestling period. Brood size manipulation did not significantly affect female or male survival. In 4 out of 6 years, there was a weak and nonsignificant effect on male survival, consistent with a cost of reproduction. Female and male fecundity in the year following the experiment was not affected by the manipulations. Thus, the data do not give evidence of an intragenerational cost of reproduction in the Willow Tit. Parents appeared unwilling to increase their breeding effort to a level which jeopardized their own survival or future breeding success. It is possible that, because of the time constraints in northern latitudes, females work under their capacity and lay smaller clutches than would otherwise be most profitable. Thus, no costs to the parents would be expected as a consequence of manipulations. These results suggest that the current reproductive rate is not maintained by reproductive cost in the Willow Tit. However, the results do not rule out the possibility that selection has operated outside the current range of reproductive rates during evolutionary history of the species.

SummaryThe natural history of preclinical diabetes is partly characterized, but there is still limited information on the dynamics of the immune response to β β β β -cell autoantigens during the course of preclinical disease. The aim of this work was to assess the maturation of the humoral immune response to the protein tyrosine phosphatase(PTP)-related proteins (IA-2 and IA-2β β β β ) in preclinical type I diabetes (TID). Forty-five children participating in the Finnish Type I Diabetes Prediction and Prevention (DIPP) Study who had seroconverted to IA-2 antibody positivity were analysed. Specific radiobinding assays were used to determine IA-2/IA-2β β β β epitope-specific antibodies (the juxtamembrane (JM) region of IA-2, PTP-like domain and β β β β PTP-like domain) and isotype-specific IA-2 antibodies. Individual areas under the curve (AUC) over the observation period were calculated for total IA-2 antibodies, each isotype and specific epitope responses. The children who progressed to TID tended to have an initial IA-2 JM epitope response more frequently ( P = = = = 0·06), and this response was more often dominant during the observation period ( P < < < < 0·05). The children who did not progress to TID had IgE-IA-2 more frequently (70%; versus progressors 27%; P < < < < 0·05), and had higher integrated titres of IgE-IA-2 antibodies ( P < < < < 0·05). The occurrence of IgE-IA-2 antibodies was protective even when combined with positivity for IA-2 JM antibodies ( P = = = = 0·002). IgE-IA-2 antibody reactivity may be a marker of a regulatory immune response providing protection against or delaying progression to TID among IA-2 antibody-positive young children with HLA-conferred disease susceptibility.