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Background This study aimed to analyse data from two different studies (phase II and phase III) regarding the safety and efficacy of treatment with alum formulated glutamic acid decarboxylase GAD65 (GAD-alum) at 30 months after administration to children and adolescents with type 1 diabetes. Methods The phase II trial was a double-blind, randomised placebo-controlled study, including 70 children and adolescents who were followed for 30 months. Participants received a subcutaneous injection of either 20 µg of GAD-alum or placebo at baseline and 1 month later. During a subsequent larger European phase III trial including three treatment arms, participants received two or four subcutaneous injections of either 20 µg of GAD-alum and/or placebo at baseline, 1, 3 and 9 months. The phase III trial was prematurely interrupted at 15 months, but of the 148 Swedish patients, a majority completed the 21 months follow-up, and 45 patients completed the trial at 30 months. Both studies included GAD65 auto-antibodies-positive patients with fasting C-peptide ≥0.10 nmol/l. We have now combined the results of these two trials. Results There were no treatment related adverse events. In patients treated with 2 GAD-alum doses, stimulated C-peptide area under the curve had decreased significantly less (9 m: p 0.2 nmol/L (p

OBJECTIVE GAD formulated in aluminum hydroxide (GAD-alum) has previously been shown to induce preservation of residual insulin secretion in recent-onset type 1 diabetes, but recent phase II and III GAD-alum trials failed to reach primary outcomes. The European phase III study was therefore closed after 15 months, and only a minority of patients completed the 30 months of follow-up. RESEARCH DESIGN AND METHODS This study aimed to characterize cellular and humoral responses in the Swedish patients (n = 148) participating in the phase III trial, receiving four (4D) or two (2D) GAD-alum doses or placebo. Serum GAD65 antibody (GADA) levels, GADA IgG1–4 subclass distribution, cytokine secretion, and proliferative responses in peripheral blood mononuclear cells (PBMCs) were analyzed. RESULTS The GAD65-induced cytokine profile tended to switch toward a predominant Th2-associated profile over time both in the 2D and 4D group. The groups also displayed increased GADA levels and PBMC proliferation compared with placebo, whereas GADA IgG subclass distribution changed in 4D patients. CONCLUSIONS Both 2D and 4D patients displayed GAD65-specifc cellular and humoral effects after GAD-alum treatment, but at different time points and magnitudes. No specific immune markers could be associated with treatment efficacy.

Type 1 diabetes (T1D) patients have numeral and functional defects in peripheral immune cells, but the pre-diabetic period is fairly uncharacterized. Our aim was to analyze expression of immunological markers in T1D high risk children and relate it to clinical/immunological parameters. Children from ABIS (All Babies in Southeast Sweden) with ≥2 diabetes related autoantibodies were considered at high risk. Age-matched controls and new-onset T1D patients were included. Expression of genes related to immune cell function and different arms of the immune system was assessed in peripheral blood mononuclear cells using PCR array. Risk children had lower TNF and CD45, and although there were few differences between the groups, expression of many genes differed when comparing children with regard to residual insulin secretion. Hence, expression of immune related genes seemed related not only to the autoimmune process but rather to residual β-cell function, which was decreased already during the pre-diabetic phase.

Administration of Glutamic Acid Decarboxylase (GAD)65 formulated in aluminium hydroxide preserved insulin secretion in a phase II trial in recent onset Type 1 Diabetes. A subsequent European phase III trial was closed at 15months after failing to reach primary endpoint, but the majority of the Swedish patients completed the 21months follow-up. We studied the frequencies and phenotype of T cells, suppressive capacity of Tregs, GAD65-induced proliferation, and frequencies of T cells with a GAD65-specific TCR in Swedes participating in the trial. Stimulation with GAD65 induced activated T cells and also cells with a suppressive phenotype. Activated GAD65-specific effector T cells were detected by tetramer staining while the frequency of GAD65-specific Treg was not affected by the treatment. Additional doses of GAD-alum increased frequencies of CD25+CD127+, but had no effect on CD25hiCD127lo. Our findings indicate that GAD-alum treatment primarily induced activated T cells. GAD65-specific cells were mainly of activated phenotype.

Background Antibodies to posttranslationally modified insulin (oxPTM-INS-Ab) are a novel biomarker of type 1 diabetes (T1D). Here, we evaluated whether oxPTM-INS-Ab can improve T1D prediction in children with positive standard islet autoantibodies (AAB). Methods We evaluated sensitivity, specificity, accuracy, and risk for progression to T1D associated with oxPTM-INS-Ab and the standard islet AAB that include insulin (IAA), GAD (GADA), and tyrosine phosphatase 2 (IA-2A) in a cohort of islet AAB-positive (AAB+ ) children from the general population (median follow-up 8.8 years). Results oxPTM-INS-Ab was the most sensitive and specific autoantibody biomarker (74% sensitivity, 91% specificity), followed by IA-2A (71% sensitivity, 91% specificity). GADA and IAA showed lower sensitivity (65% and 50%, respectively) and specificity (66% and 68%, respectively). Accuracy (AUC of ROC) of oxPTM-INS-Ab was higher than GADA and IAA (P = 0.003 and P = 0.017, respectively), and similar to IA-2A (P = 0.896). oxPTM-INS-Ab and IA-2A were more effective than IAA for detecting progr-T1D when used as second-line biomarker in GADA+ children. Risk for diabetes was higher (P = 0.03) among multiple AAB+ who were also oxPTM-INS-Ab+ compared with those who were oxPTM-INS-Ab- . Importantly, when replacing IAA with oxPTM-INS-Ab, diabetes risk increased to 100% in children with oxPTM-INS-Ab+ in combination with GADA+ and IA-2A+ , compared with 84.37% in those with IAA+ , GADA+ , and IA-2A+ (P = 0.04). Conclusions Antibodies to oxidized insulin (oxPTM-INS-Ab), compared with IAA which measure autoantibodies to native insulin, improve T1D risk assessment and prediction accuracy in AAB+ children.

Aim To further elucidate the relationship between physical activity and several risk factors for development of diabetes (glucose, C-peptide and obesity) over time. Methods A prospective longitudinal study where physical activity was measured on 199 children from Kalmar and Linkoping at age 8, and the same 107 children from Linkoping again at age 12. Anthropometric data was collected and blood was analyzed for C-peptide and f-glucose. The children in the study were representative for the general Swedish child population, and on an average lean. Results High physical activity was related to lower C-peptide at age 8 and 12. This correlation was especially pronounced in boys, who also were more physically active than girls at both time points. The association seen at 8 years of age was similar at age 12 in most children. Children with higher BMI Z-Score had a higher fasting C-peptide (age 12) but linear regression showed that children with more steps per day were less likely to have a higher fasting C-peptide irrespective of BMI. Longitudinal follow-up showed that a decrease in physical activity increased insulin resistance and beta-cell load. Conclusions Already in young children, physical activity improves insulin sensitivity and decreases the need of C-peptide over time. This seems to become even more pronounced with increasing age when children are followed longitudinally. Low physical activity increases the load on insulin producing beta-cells, might increase the risk for both type 1- and 2 diabetes. Funding Agencies|Swedish Child Diabetes Foundation (Barndiabetesfonden); Novo Nordisk Foundation; Research Council of South-east Sweden (FORSS); Swedish Research Council [K2005-72X-11242-11A]; ALF/County Council of Ostergotland

Type 1 diabetes (T1D) is an autoimmune disease characterized by insulin deficiency, due to immune-mediated destruction of beta cells. Current knowledge regarding the period preceding disease onset comes, to a large extent, from studying risk cohorts based on relatives of T1D-patients, as they have an increased disease risk. Among T1D patients in general, however, few have the disease in their immediate family. It is therefore important to study risk cohorts from the general population as well. An ongoing autoimmune reaction can often be seen in the blood long before disease onset, by detection of autoantibodies directed towards beta cell antigens. By autoantibody screening among participants in the ABIS (All Babies in the South-east of Sweden) cohort, we could identify a group of children from the general population with increased risk for T1D, positive for multiple autoantibodies. They were enrolled in a 2-year prospective follow-up aiming to characterize the prediabetic period and to identify factors indicative of progression/non-progression to T1D. We assessed glucose homeostasis and autoantibody titers over time, and searched for risk-biomarkers by analyzing the expression of immune-related genes (Th1-Th2-Th3) in peripheral blood mononuclear cells (PBMC) from these children, in comparison to healthy children and newly diagnosed T1D patients. In the same groups we also compared serum micro RNA (miRNA) profiles, knowing that miRNA molecules have desirable biomarker properties. We found that two specific autoantibodies, IA2A and ZnT8A, were detected at higher concentrations in risk-individuals who progressed to overt T1D during or after the follow-up period, compared to those who still have not. We also observed disturbed glucose homeostasis long before onset in the progressors, but it was seen among those who remain symptom free as well. Further, we found support for the possible role of insulin resistance as an accelerator of the disease process. For gene expression and serum miRNA, few differences were observed between risk-individuals and healthy children overall. However, for PBMC gene expression and serum miRNA both, there were associations to beta cell function and glucose homeostasis, and for miRNA also to islet autoantibodies. Although specific profiles for prediction of disease onset or identification of risk-individuals could not be found, these results are interesting and deserve to be evaluated further. As part of another sub-study within ABIS, the effects of physical activity on glucose homeostasis were assessed in healthy schoolchildren. The level of physical activity, measured by pedometers, was related to insulin resistance and beta cell-stress, and decreased physical activity was associated with increased insulin resistance and load on the insulin-producing beta cells, already at school-age.

Background There is a need for increased understanding of the pre-diabetic period in individuals with high risk of type 1 diabetes from the general population. Methods High-risk children (n = 21) positive for multiple islet autoantibodies were identified by autoantibody screening within the All Babies in Southeast Sweden study. The children and their parents were enrolled in a 2-year prospective follow-up study aiming to characterize the pre-diabetic period. Blood samples were collected every 6 months for measurement of C-peptide, HbA1c, fasting glucose, and autoantibodies. Human leukocyte antigen-genotype was determined, and oral glucose tolerance test was performed every 12 months. Results Despite positivity for multiple autoantibodies, 9 out of 21 individuals had low-risk human leukocyte antigen-genotypes. Children who progressed to manifest diabetes (progressors, n = 12) had higher levels of IA2A and ZnT8A than children who did not (non-progressors, n = 9). Impaired glucose tolerance and impaired fasting glucose was observed to the same extent in progressors and non-progressors, but HbA1c increased over time in progressors in spite of increased C-peptide. Conclusions Autoantibodies to IA2 and ZnT8 may be useful discriminators for disease progression in at-risk children from the general population. Dysglycemia was observed long before diagnosis, and difficulties in maintaining glucose homeostasis despite increased C-peptide indicate that insulin resistance might be an important accelerator of disease in risk individuals.

Glutamic acid decarboxylase (GAD)65 formulated with aluminium hydroxide (GAD-alum) was effective in preserving insulin secretion in a Phase II clinical trial in children and adolescents with recent-onset type 1 diabetes. In addition, GAD-alum treated patients increased CD4+CD25hi forkhead box protein 3+ (FoxP3+) cell numbers in response to in-vitro GAD65 stimulation. We have carried out a 4-year follow-up study of 59 of the original 70 patients to investigate long-term effects on the frequency and function of regulatory T cells after GAD-alum treatment. Peripheral blood mononuclear cells were stimulated in vitro with GAD65 for 7 days and expression of regulatory T cell markers was measured by flow cytometry. Regulatory T cells (CD4+CD25hiCD127lo) and effector T cells (CD4+CD25–CD127+) were further sorted, expanded and used in suppression assays to assess regulatory T cell function after GAD-alum treatment. GAD-alum-treated patients displayed higher frequencies of in-vitro GAD65-induced CD4+CD25+CD127+ as well as CD4+CD25hiCD127lo and CD4+FoxP3+ cells compared to placebo. Moreover, GAD65 stimulation induced a population of CD4hi cells consisting mainly of CD25+CD127+, which was specific of GAD-alum-treated patients (16 of 25 versus one of 25 in placebo). Assessment of suppressive function in expanded regulatory T cells revealed no difference between GAD-alum- and placebo-treated individuals. Regulatory T cell frequency did not correlate with C-peptide secretion throughout the study. In conclusion, GAD-alum treatment induced both GAD65-reactive CD25+CD127+ and CD25hiCD127lo cells, but no difference in regulatory T cell function 4 years after GAD-alum treatment. Funding Agencies|Swedish Research Council|K2008-55x-20652-01-3|Swedish Child Diabetes Foundation (Barndiabetesfonden)||Medical Research Council of Southeast Sweden||JDRF|1-2008-106|Ile-de-France CODDIM||Inserm Avenir Program

A phase II clinical trial with glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) has shown efficacy in preserving residual insulin secretion in children and adolescents with recent-onset type 1 diabetes (T1D). We have performed a 4-year follow-up study of 59 of the original 70 patients to investigate long-term cellular and humoral immune responses after GAD-alum-treatment. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with GAD(65). Frequencies of naive, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD(65) autoantibody (GADA) levels. We here show that GAD-alum-treated patients display increased memory T-cell frequencies and prompt T-cell activation upon in vitro stimulation with GAD(65), but not with control antigens, compared with placebo subjects. GAD(65)-induced T-cell activation was accompanied by secretion of T helper (Th) 1, Th2 and T regulatory cytokines and by induction of T-cell inhibitory pathways. Moreover, post-treatment serum GADA titres remained persistently increased in the GAD-alum arm, but did not inhibit GAD(65) enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD(65)-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD(65) immunity. Funding Agencies|Swedish Research Council|K2008-55x-20652-01-3|Swedish Child Diabetes Foundation (Barndiabetesfonden)||Medical Research Council of Southeast Sweden||Juvenile Diabetes Research Foundation (JDRF)|1-2008-106|Ile-de-France CODDIM||Inserm Avenir Program

Aims/hypothesis A European Phase III trial of GAD formulated with aluminiumhydroxide (GAD-alum) failed to reach its primary endpoint (preservation of stimulated C-peptide secretion from baseline to 15 months in type 1 diabetes patients), but subgroup analysis showed a clinical effect when participants from Nordic countries were excluded, raising concern as to whether the mass vaccination of the Swedish and Finnish populations with the Pandemrix influenza vaccine could have influenced the study outcomes. In the current study, we aimed to assess whether Pandemrix vaccination affects the specific immune responses induced by GAD-alum and the C-peptide response. Methods In this secondary analysis, we analysed data acquired from the Swedish participants in the Phase III GAD-alum trial who received subcutaneous GAD-alum vaccination (two doses, n = 43; four doses, n = 46) or placebo (n = 48). GAD autoantibodies (GADA) and H1N1 autoantibodies, GAD(65)-induced cytokine secretion and change in fasting and stimulated C-peptide levels from baseline to 15 months were analysed with respect to the relative time between H1N1 vaccination and the first injection of GAD-alum. Results GADA levels at 15 months were associated with the relative time between GAD-alum and Pandemrix administration in participants who received two doses of the GAD-alum vaccine (p = 0.015, r = 0.4). Both in participants treated with two doses and four doses of GAD-alum, GADA levels were higher when the relative time between vaccines was amp;gt;= 210 days (p amp;lt; 0.05). In the group that received two doses of GAD-alum, levels of several GAD(65)-induced cytokines were higher in participants who received the H1N1 vaccination and the first GADalum injection at least 150 days apart, and the change in fasting and stimulated C-peptide at 15 months was associated with the relative time between vaccines. Neither of these effects were observed in individuals who received four doses of GAD-alum. Conclusions/interpretation In individuals who received two doses of GAD-alum, receiving the Pandemrix vaccine closer to the first GAD-alum injection, i.e. amp;lt; 150 days, seemed to affect both GAD(65)-induced immune response and C-peptide preservation. Funding Agencies|JDRF [17-2011-249]; Swedish Research Council [K2008-55x-20,652-01-3]; Swedish Child Diabetes Foundation (Barndiabetesfonden); Medical Research Council of Southeast Sweden; Diamyd Medical