Search

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.

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.

Type 1 diabetes (T1D) is caused by a deficiency of insulin as a result of an autoimmune destruction of the pancreatic ² -cells. A possibility to preserve remaining ² -cells in children with newly diagnosed T1D is of great importance since sustained ² -cell function is recognized to result in reduced end-organ complications. Glutamic acid decarboxylase 65 (GAD65) is one of the major antigens targeted by self-reactive T cells in T1D, and immunomodulation with GAD65 formulated in aluminum (GAD-alum) has been considered both in prevention and treatment of T1D. Results from a Phase II trial have shown clinical effect of subcutaneous injections with GAD-alum, this was unfortunately not fully confirmed in the following larger Phase III trial which therefore was closed after 15 months. The general aim of this thesis was to study the immunomodulatory effect of GAD-alum-treatment in children with T1D participating in the Phase II and Phase III trials. We hypothesized that treatment with GAD-alum contributes to the preservation of residual insulin secretion through deviation of the GAD65-specific immune response from a destructive to a protective process, accompanied by a shift from T helper (Th) 1 towards a predominant Th2 profile. In the Phase II trial, GAD-alum-treated patients responded with an early GAD65-specific Th2 skewed cytokine secretion, with highest IL-5 and IL-13 secretion in clinical responders. Also, the CCR4/CCR5 ratio indicating balance between Th2/Tc2 and Th1/Tc1 responses, increased in treated patients. The recall response to GAD65 was characterized by a wide range of cytokines, but the relative contribution of each cytokine suggests a shift towards a more pronounced Th2-associated profile over time. Induction of a CD4+ cell subset upon GAD65-stimulation 4 years after treatment, suggesting clonal expansion of the memory T-cell compartment upon antigen re-challenge, was seen in parallel to a persistent GAD65-specific cytokine response. Finally, even if the phase III trial failed to reach the primary endpoint at 15 months, a subgroup analysis showed that the treatment had an effect on preservation of residual insulin secretion, but the effect was not seen until after 30 months. Taken together, these results suggest that GAD-alum treatment might exert its effect through induction of an early Th2 skewed immune response which tends to deviate away from a destructive Th1/Tc1 response upon GAD65 re-challenge, and generation of GAD65-specific memory T cells that produce cytokines and exert effector responses which may be important for regulating GAD65 immunity. Continued research to better understand how immunomodulation with autoantigen modifies T-cell responses and also which patients are suitable for treatment, is crucial for optimizing future intervention trials using ² -cell antigens.

Type 1 diabetes results from autoimmune destruction of insulin producing pancreatic β-cells. We have shown that treatment with alum-formulated glutamic acid decarboxylase 65 (GAD-alum) preserved residual insulin secretion and induced antigen-specific responses in children with recent onset type 1 diabetes. The aim of this study was to further investigate the immunomodulatory effect of GAD-alum, focusing on CD4(+)CD25(high) cells and their association to cytokine secretion. Samples obtained 21 and 30months after the initial injection of GAD-alum or placebo were included in the present study. GAD(65)-stimulation enhanced the percentage of CD4(+)CD25(high)FOXP3(+) cells, but reduced the percentage of CD4(+)CD25(+) cells, in samples from the GAD-alum treated group. Further, the GAD(65)-induced secretion of IL-5, -10, and -13 correlated with the expression of CD4(+)CD25(high)FOXP3(+) cells, but inversely with CD4(+)CD25(+) cells. These new data suggest that GAD-alum treatment induced GAD(65)-specific T cells with regulatory features.

Background The 65-kD isoform of glutamic acid decarboxylase (GAD) is a major autoantigen in patients with type 1 diabetes mellitus. This trial assessed the ability of alum-formulated GAD (GAD-alum) to reverse recent-onset type 1 diabetes in patients 10 to 18 years of age. Methods We randomly assigned 70 patients with type 1 diabetes who had fasting C-peptide levels above 0.1 nmol per liter (0.3 ng per milliliter) and GAD autoantibodies, recruited within 18 months after receiving the diagnosis of diabetes, to receive subcutaneous injections of 20 μg of GAD-alum (35 patients) or placebo (alum alone, 35 patients) on study days 1 and 30. At day 1 and months 3, 9, 15, 21, and 30, patients underwent a mixed-meal tolerance test to stimulate residual insulin secretion (measured as the C-peptide level). The effect of GAD-alum on the immune system was also studied. Results Insulin secretion gradually decreased in both study groups. The study treatment had no significant effect on change in fasting C-peptide level after 15 months (the primary end point). Fasting C-peptide levels declined from baseline levels significantly less over 30 months in the GAD-alum group than in the placebo group (−0.21 vs. −0.27 nmol per liter [−0.62 vs. −0.81 ng per milliliter], P = 0.045), as did stimulated secretion measured as the area under the curve (−0.72 vs. −1.02 nmol per liter per 2 hours [−2.20 vs. −3.08 ng per milliliter per 2 hours], P = 0.04). No protective effect was seen in patients treated 6 months or more after receiving the diagnosis. Adverse events appeared to be mild and similar in frequency between the two groups. The GAD-alum treatment induced a GAD-specific immune response. Conclusions GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent-onset type 1 diabetes, although it did not change the insulin requirement. (ClinicalTrials.gov number, NCT00435981.)

1. Several studies suggest an association between venous thromboembolism and the use of antipsychotic drugs, especially clozapine, but the biological mechanisms are unknown. It has been suggested that antipsychotic drugs enhance aggregation of platelets and thereby increase the risk of venous thrombosis. The purpose of the present study was to examine the effects of clozapine and its main metabolite, N-desmethyl clozapine, as well as olanzapine, risperidone and haloperidol, on platelet adhesion and aggregation and on plasma coagulation in vitro. 2. Blood was collected from healthy subjects free of medication. Platelet adhesion to different protein surfaces and aggregation were measured in microplates. The coagulation methods of activated partial thromboplastin time (APTT) and prothrombin time were performed in platelet-poor plasma. 3. Clozapine was the only compound that increased platelet adhesion and aggregation and shortened APTT. The effect appeared at therapeutic concentrations and was significant but weak. 4. This weak effect of clozapine on haemostasis may explain, in part, the association of this compound and venous thromboembolism.

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.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.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 0.037; 15 m: p 0.032; 21 m: p 0.003 and 30 m: p 0.004), and a larger proportion of these patients were also able to achieve a peak stimulated C-peptide0.2 nmol/L (p 0.05), as compared with placebo.Treatment with two doses of GAD-alum in children and adolescents with recent-onset type 1 diabetes shows no adverse events and preserves residual insulin secretion.

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

The aim of this study was to investigate the safety and efficacy of alum formulated glutamic acid decarboxylase GAD(65) (GAD-alum) treatment of children and adolescents with type 1 diabetes after 4 years of follow-up. Seventy children and adolescents aged 10-18 years with recent onset type 1 diabetes participated in a phase II, double-blind, randomised placebo-controlled clinical trial. Patients identified as possible participants attended one of eight clinics in Sweden to receive information about the study and for an eligibility check, including a medical history. Participants were randomised to one of the two treatment groups and received either a subcutaneous injection of 20 mu g of GAD-alum or placebo at baseline and 1 month later. The study was blinded to participants and investigators until month 30. The study was unblinded at 15 months to the sponsor and statistician in order to evaluate the data. At follow-up after 30 months there was a significant preservation of residual insulin secretion, as measured by C-peptide, in the group receiving GAD-alum compared with placebo. This was particularly evident in patients with andlt; 6 months disease duration at baseline. There were no treatment-related serious adverse events. We have now followed these patients for 4 years. Overall, 59 patients, 29 who had been treated with GAD-alum and 30 who had received placebo, gave their informed consent. One patient in each treatment group experienced an episode of keto-acidosis between months 30 and 48. There were no treatment-related adverse events. The primary efficacy endpoint was the change in fasting C-peptide concentration from baseline to 15 months after the prime injection for all participants per protocol set. In the GAD-alum group fasting C-peptide was 0.332 +/- 0.032 nmol/l at day 1 and 0.215 +/- 0.031 nmol/l at month 15. The corresponding figures for the placebo group were 0.354 +/- 0.039 and 0.184 +/- 0.033 nmol/l, respectively. The decline in fasting C-peptide levels between day 1 and month 1, was smaller in the GAD-alum group than the placebo group. The difference between the treatment groups was not statistically significant. In those patients who were treated within 6 months of diabetes diagnosis, fasting C-peptide had decreased significantly less in the GAD-alum group than in the placebo-treated group after 4 years. Four years after treatment with GAD-alum, children and adolescents with recent-onset type 1 diabetes continue to show no adverse events and possibly to show clinically relevant preservation of C-peptide. ClinicalTrials.gov NCT00435981 The study was funded by The Swedish Research Council K2008-55X-20652-01-3, Barndiabetesfonden (The Swedish Child Diabetes Foundation), the Research Council of Southeast Sweden, and an unrestricted grant from Diamyd Medical AB. The original publication is available at www.springerlink.com:Johnny Ludvigsson, Maria Hjorth, Mikael Chéramy, Stina Axelsson, Mikael Pihl, G Forsander, N -O Nilsson, B-O Samuelsson, T Wood, J Aman, E Ortqvist and Rosaura Casas, Extended evaluation of the safety and efficacy of GAD treatment of children and adolescents with recent-onset type 1 diabetes: a randomised controlled trial, 2011, DIABETOLOGIA, (54), 3, 634-640.http://dx.doi.org/10.1007/s00125-010-1988-1Copyright: Springer Science Business Mediahttp://www.springerlink.com/

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD(65) or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-gamma and TNF-alpha) and chemokines (IP-10, MCP-1, MIP-1alpha, MIP-1beta and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-beta mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-gamma and MCP-1, and mRNA expression of FOXP3 and TGF-beta, was detected after cryopreservation. Stimulation with GAD(65) induced higher levels of IL-6, IFN-gamma, TNF-alpha and MIP-1alpha, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.

Although the role of the T cell-mediated autoimmune reaction in type 1 diabetes (T1D) is conclusive, studies including data from human circulating CD4+ and CD8+ lymphocytes subsets during the disease onset and posterior development are scarce. Further, chemokines and chemokine receptors are key players in the migration of pathogenic T cells into the islets of NOD mice developing T1D, but few studies have investigated these markers in human T1D patients. We studied the expression of T helper 1 (Th1) and Th2 associated chemokine receptors, and the two isoforms of CD45 leukocyte antigen on CD4+ and CD8+ lymphocytes from T1D and healthy children, as well as the secretion of chemokines in cell supernatants in peripheral blood mononuclear cells. Our results showed increased expression of CCR7 and CD45RA, and reduced CD45RO on CD8+ cells among recent-onset T1D patients. The percentages of CD4+ cells expressing CXC chemokine receptor 3 (CXCR3), CXCR6 and CCR5, and the secretion of interferon-γ-induced protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1a and MIP-1β was lower among diabetics. Low expression of Th1-associated receptors and secretion of chemokines, together with an increased amount of CD8+ cells expressing CD45RA and CCR7 in T1D patients therefore might represent suboptimal Th function in T1D, leading to impaired T cytotoxic (Tc) responses or alternatively reflect a selective recruitment of Th1 cells into the pancreas. The definitive version is available at www.blackwell-synergy.com: M. Hedman, M. Faresjö, S. Axelsson, J. Ludvigsson and R. Casas, Impaired CD4+ and CD8+ T cell phenotype and reduced chemokine secretion in recent-onset type 1 diabetic children, 2008, Clinical and Experimental Immunology, (153), 3, 360-368.http://dx.doi.org/10.1111/j.1365-2249.2008.03720.x. Copyright: Blackwell Publishing www.blackwell-synergy.com

Introduction Plusieurs essais cliniques de vaccination a l’aide d’antigenes des cellules beta tels que l’insuline et la GAD se sont reveles decevants chez les patients diabetiques de type 1. Il est donc important de comprendre les effets immunologiques induits par ces traitements afin d’ameliorer les essais cliniques ulterieurs. Patients et methodes Nous avons participe a l’analyse des reponses des anticorps et des lymphocytes T diriges contre l’insuline et la GAD apres traitement par ces deux antigenes dans le cadre de deux protocoles cliniques: un protocole australien de vaccination par insuline intra-nasale, sans adjuvant, administree chez des patients diabetiques adultes non encore traites par insuline; et une vaccination sous-cutanee avec la GAD en presence de l’adjuvant alun chez des enfants diabetiques de type 1 suedois. Resultats Le traitement par insuline intra-nasale n’a pas retarde la perte de fonction beta-cellulaire residuelle ni le passage a l’insulinotherapie chez les patients traites par rapport au groupe placebo. Toutefois, les analyses immunologiques ont revele une induction efficace de la tolerance immunitaire vis-a-vis de l’insuline, au niveau de reponses des lymphocytes T ainsi que des reponses anticorpales. Le traitement par GAD-alun en sous-cutane chez les enfants diabetiques suedois a ete associe a une meilleur preservation de la fonction beta-cellulaire pour les patients traites precocement. Le traitement a induit aussi une augmentation des titres des anticorps anti-GAD associes a des reponses T productrices des cytokines de type Th1 (IFN-γ, TNF-α), Th2 (IL-5, IL-13) et T regulatrices (IL-10). Conclusion Ces resultats suggerent que la formulation antigenique (sans ou avec adjuvant) et la voie de vaccination induisent des effets immunologiques differents. La presence de biomarqueurs de tolerance immunitaire ne s’associe pas a des benefices cliniques significatifs, suggerant la necessite d’intervenir plus precocement ou d’associer plusieurs antigenes pour obtenir un effet de tolerance plus complet.

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