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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.

Previous studies have indicated phenotypical differences in glutamic acid decarboxylase 65 autoantibodies (GADA) found in type 1 diabetes (T1D) patients, individuals at risk of developing T1D and stiff-person syndrome (SPS) patients. In a Phase II trial using aluminium-formulated GAD65 (GAD-alum) as an immunomodulator in T1D, several patients responded with high GADA titres after treatment, raising concerns as to whether GAD-alum could induce GADA with SPS-associated phenotypes. This study aimed to analyse GADA levels, immunoglobulin (Ig)G1–4 subclass frequencies, b78- and b96·11-defined epitope distribution and GAD65 enzyme activity in sera from four cohorts with very high GADA titres: T1D patients (n = 7), GAD-alum-treated T1D patients (n = 9), T1D high-risk individuals (n = 6) and SPS patients (n = 12). SPS patients showed significantly higher GADA levels and inhibited the in-vitro GAD65 enzyme activity more strongly compared to the other groups. A higher binding frequency to the b78-defined epitope was found in the SPS group compared to T1D and GAD-alum individuals, whereas no differences were detected for the b96·11-defined epitope. GADA IgG1–4 subclass levels did not differ between the groups, but SPS patients had higher IgG2 and lower IgG4 distribution more frequently. In conclusion, the in-vitro GADA phenotypes from SPS patients differed from the T1D- and high-risk groups, and GAD-alum treatment did not induce SPS-associated phenotypes. However, occasional overlap between the groups exists, and caution is indicated when drawing conclusions to health or disease status. The title of the article in manuscript form was "Characteristics of GAD65 autoantibodies (GADA) in high titer individuals".

Serotonin (5-HT) applied at 1, 3, and 10 microM into the striatum of halothane-anesthetized rats by in vivo microdialysis enhanced dopamine (DA) outflow up to 173, 283, and 584% of baseline values, respectively. The 5-HT effect was partially reduced by 1 or 10 microM GR 125,487, a 5-HT4 antagonist, and by 100 microM DAU 6285, a 5-HT3/4 antagonist, whereas the 5-HT1/2/6 antagonist methiothepin (50 microM) was ineffective. In the presence of tetrodotoxin the effect of 1 microM 5-HT was not affected by 5-HT4 antagonists. In addition, tetrodotoxin abolished the increase in DA release induced by the 5-HT4 agonist (S)- zacopride (100 microM). In striatal synaptosomes, 1 and 10 microM 5-HT increased the outflow of newly synthesized [3H]DA up to 163 and 635% of control values, respectively. The 5-HT4 agonists BIMU 8 and (S)-zacopride (1 and 10 microM) failed to modify [3H]DA outflow, whereas 5- methoxytryptamine (5-MeOT) at 10 microM increased it (62%). In prelabeled [3H]DA synaptosomes, 1 microM 5-HT, but not (S)-zacopride (1 and 10 microM), increased [3H]DA outflow. DAU 6285 (10 microM) failed to modify the enhancement of newly synthesized [3H]DA outflow induced by 5-MeOT or 5-HT (1 microM), whereas the effect of 5-HT was reduced to the same extent by the DA reuptake inhibitor nomifensine (1 microM) alone or in the presence of DAU 6285. These results show that striatal 5-HT4 receptors are involved in the 5-HT-induced enhancement of striatal DA release in vivo and that they are not located on striatal DA terminals.

In vivo experiments carried out in halothane-anaesthetized cats implanted with push-pull cannulae demonstrated that glutamate (GLU) released from corticostriatal fibers triggers the release of dopamine (DA), even in the absence of activity in nigral DA cells. As shown in vitro, using rat striatal slices or synaptosomes or in vivo in the cat, both NMDA and AMPA receptors subtypes are involved in the GLU-induced release of DA. Beside this direct regulation, GLU also exert several indirect facilitatory and inhibitory controls on DA release, particularly through cholinergic and GABAergic striatal neurons. Indeed, as shown by numerous authors, the GLU-evoked release of DA is markedly reduced in the presence of tetrodotoxin, bicuculline or atropine or by previous kainate- or ibotenate-induced lesion of striatum. Differences in the presynaptic regulation of DA release in striosomal and matrix compartments have also been found with NMDA and acetylcholine. The effect of acetylcholine was of shorter duration in the matrix than in the striosomal-enriched areas. Two opposite indirect regulations of DA release could be demonstrated: one is facilitatory and involves nicotinic receptors, the other is inhibitory, involves muscarinic receptors and mediated, at least in the matrix by dynorphin containing neurons. The NMDA-evoked responses are of larger amplitude and more sensitive to tetrodotoxin in the matrix than in the striosomes. In conclusion, GLU released from corticostriatal fibers, is able to control the release of DA from terminals of nigrostriatal neurons through direct facilitatory mechanisms (NMDA and AMPA receptors), but also through indirect facilitatory and inhibitory local circuits involving cholinergic and GABAergic neurons.

31p.; International audience; The Avionics Full Duplex Switched Ethernet (AFDX) backbone constitutes one of the major technological breakthroughs in modern avionic architectures. This network is based on routing Ethernet frames through isolated data tunnels referred to as Virtual Links (VL). VLs can be thought of as multicast trees, each serving for data transmission between one and only one end of the network to several others. Multiple VLs are deployed for exchanging data between avionic systems with a reserved amount of bandwidth. In this paper, we propose different methods to define VL characteristics and to route VLs in the network while minimizing the maximum utilization rate of the links. The proposed methods provide the basis for a more efficient design of the VLs, and have to be completed later on by the verification of the worst-case network latencies. The industrial applicability is shown on experimental results and on a representative benchmark.

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

Type 1 diabetes (T1D) is a serious autoimmune disease which increases worldwide and affects children at a young age, but there still is no cure available. Clinical intervention trials in recent onset T1D patients are therefore very important, since even a modest preservation of β-cell function has proven to reduce end-organ complications. Glutamic acid decarboxylase 65 (GAD65) is one of the major antigens in T1D, to which autoantibodies (GADA) are formed. Immunomodulation with aluminum-formulated GAD65 (GAD-alum) has been considered both in the prevention and intervention of T1D. In a phase II trial using GADalum we showed clinical benefits in C-peptide preservation, but unfortunately a following larger European phase III trial failed to reach primary end-point. The general aim of this thesis was to study the characteristics and phenotypes of GADA following immunomodulation with GAD-alum in T1D patients during a phase II and III trial. In the phase II trial, a transient increase of the GADA IgG3 and IgG4 subclasses, and a decrease in IgG1 was detected as part of the treatment-induced GADA levels after 2 GADalum doses, a result interpreted to be T helper (Th) 2-associated. This Th2-associated immune response was also observed, in parallel to increased GADA levels, during the following phase III trial including a larger group of patients. However, enhanced Th2-like IgG subclass distribution, reflected as increased IgG4 frequency, was in contrast only observed in the group treated with 4 doses of GAD-alum. In addition, the GADA fold-change was associated with in vitro GAD65-stimulated cytokine secretion, but only in patients receiving 2 GAD-alum doses. Furthermore, a 4-year follow-up of the phase II trial showed that the effect of GAD-alum treatment was long-lasting as GADA titers remained elevated. Even though the phase III trial did not reach primary end-point, and was closed after 15 months, preservation of β-cell function was observed in the small sub-group of Swedish patients receiving 2 GAD-alum doses that completed the 30 months trial-period. During the trials, concerns were raised whether the elevated GADA titers might induce Stiff person syndrome (SPS), a disease affecting the nervous system, but in vitro analysis of GADA phenotypes showed that the GAD65-enzyme activity and GADA epitope distribution differed from that detected in SPS patients. Continued research to clarify how immunomodulation with autoantigens affects immune responses and also to identify which patients are suitable for treatment, is crucial for optimizing future T1D intervention- and prevention trials.

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

We have previously shown that two injections of glutamic acid decarboxylase formulated in alum (GAD-alum) preserved residual insulin secretion in children and adolescents with recent onset type 1 diabetes (T1D), and was accompanied by increased GAD autoantibody (GADA) titers. The aim of this study was to investigate whether GAD-alum treatment affected the GADA epitope pattern.Serum samples from patients treated with GAD-alum (n = 33) or placebo (n = 27), at baseline, 1, 3, 9, and 15 months after the initial injection, were tested for their binding capacity to specific GADA epitopes in an epitope-specific radioligand binding assay with six recombinant Fab (rFab) (b96.11, DPA, DPD, MICA3, b78, and N-GAD(65) mAb).No significant differences in variability of binding to any of the tested rFab were observed from baseline to 15 months. There was a sustained low binding of GADA to the b78- and N-GAD(65) mAb-defined epitopes, often recognized by GADA in patients with stiff person syndrome (SPS) and seldom in T1D patients. However, binding of GADA to the T1D-associated b96.11-defined epitope increased between baseline and 3 months in GAD-alum (-8.1%, min -72.4%, max 39.6%) compared to placebo patients (1.5%, min -28.3%, max 28.6%) (p = 0.02). Subsequently, the b96.11-defined epitope recognition returned to levels similar to that observed at baseline.GAD-alum injections did not affect binding of GADA to SPS-related epitopes, further supporting the safety of the treatment. There were no changes in GADA epitope specificity to the T1D-related epitopes, except for a temporarily increased binding to one of the tested epitopes.

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/

CD4+CD25hi T cells are thought to be crucial for the maintenance of immunological tolerance to self antigens. In this study, we investigated the frequencies of these cells in the early stage of type 1 diabetes, as well as in a setting of possible pre-diabetic autoimmunity. Hence, the expression of FOXP3, CTLA-4, and CD27 in CD4+ CD25hi T cells was analyzed using flow cytometry in 14 patients with recent onset type 1 diabetes, in 9 at-risk individuals, and 9 healthy individuals with no known risk for type 1 diabetes. Our results show there were no differences in the frequency of CD4+CD25hi cells between groups. However, compared to controls, recent-onset type 1 diabetic patients had higher expression of FOXP3, CTLA-4, and CD27 in CD4+ CD25hi cells from peripheral blood. The median fluorescence intensity of FOXP3 was significantly higher in CD4+CD25hi cells from patients with type 1 diabetes than from controls. Furthermore, a positive correlation between the frequency of FOXP3+ cells and the median fluorescence intensity of FOXP3 was observed among patients with type 1 diabetes. These data suggest that the frequency of CD4+CD25hi FOXP3+ T cells in the periphery is not decreased but rather increased at onset of type 1 diabetes. Thus, functional deficiencies rather than reduced numbers of CD4+CD25hi cells could contribute to the development of type 1 diabetes.

We have previously shown that two injections of 20 mu g GAD-alum to recent onset type 1 diabetic children induced GADA levels in parallel to preservation of insulin secretion. Here we investigated if boosted GADA induced changes in IgG1, 2, 3 and 4 subclass distributions or affected GAD(65) enzyme activity. We further studied the specific effect of GAD-alum through analyses of IA-2A, tetanus toxoid and total IgE antibodies. Serum from children receiving GAD alum or placebo was collected pre-treatment and after 3, 9, 15 and 21 months. At 3 months a reduced percentage of IgG1 and increased IgG3/IgG4 were detected in GAD-alum treated. Further, IA-2A, IgE and tetanus toxoid antibodies, as well as GAD(65) enzyme activity, were unaffected confirming the specific effect of treatment. In the GAD-alum group, higher pretreatment GADA were associated to more pronounced C-peptide preservation. The induced IgG3/IgG4 and reduced IgG1 suggest a Th2 deviation of the immune response. Original Publication: Mikael Chéramy, Camilla Skoglund, Ingela Johansson, Johnny Ludvigsson, Christiane S Hampe and Rosaura Casas, GAD-alum treatment in patients with type 1 diabetes and the subsequent effect on GADA IgG subclass distribution, GAD(65) enzyme activity and humoral response, 2010, CLINICAL IMMUNOLOGY, (137), 1, 31-40. http://dx.doi.org/10.1016/j.clim.2010.06.001 Copyright: Elsevier Science B.V., Amsterdam http://www.elsevier.com/

It has been suggested that some anesthetic agents could exert their hypnotic/anesthetic effects by selectively blocking receptors involved in the central excitatory neurotransmission mediated by glutamate. In the present study, we analyzed whether riluzole (54274 RP), a novel compound that inhibits both the release and some postsynaptic effects of glutamate in some brain structures, has anesthetic properties in rats. For this purpose, we investigated whether 1) riluzole administered intraperitoneally (ip) at doses ranging from 2.5 to 45 mg/kg induces loss of righting reflex (LRR); 2) riluzole (2.5 and 5 mg/kg) prolongs sleep-times induced by either ketamine (30 or 80 mg/kg ip) or thiopental (25 or 35 mg/kg ip); 3) a 5-mg/kg subanesthetic riluzole dose affects the minimum alveolar concentration of halothane (MACh). Onset of drug action was defined as the period of time from the ip injection to LRR. Sleep-time was considered the period of time from LRR to restoration of righting reflex. Riluzole at doses greater than 15 mg/kg was able to induce LRR (riluzole dose for which LRR was achieved in 50% of the rats [ED50 = 25.6 mg/kg]). A positive correlation was found between the dose of riluzole and sleep-time (r = 0.92, P less than 0.001). A 5-mg/kg (but not 2.5-mg/kg) riluzole dose significantly prolonged sleep-times induced by both ketamine (30 and 80 mg/kg) and thiopental (25 but not 35 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine can be released in the substantia nigra for the dendrites of nigrostriatal dopaminergic neurones, to be involved there in the self-regulation of the dopaminergic cells, to control the release of neurotransmitters from nigral afferent fibres and to influence the activity of nigral non-dopaminergic cells.

Acetylcholinesterase (AChE), which is known to inactivate acetylcholine (ACh), is present in great abundance in the substantia nigra, although ACh levels and choline acetylase activity in this region are relatively low1. Nigral dopaminergic cell bodies and their dendrites also contain AChE2–4. The functional significance of this enzyme in nigro-striatal dopaminergic neurones has been questioned1,4,5. Earlier studies demonstrated an evoked release of AChE from unidentified central neurones into cerebrospinal fluid (CSF) in cats6, rabbits7,8 and dogs9. Later experiments have provided indirect evidence that the substantia nigra may contribute to a substantial amount of AChE detected: a unilateral nigral lesion in rabbits reduced AChE levels in the CSF, whereas electrical stimulation of the substantia nigra induced the opposite effect10. To investigate the possible release of AChE from dopaminergic dendrites and terminals we measured the in vivo release of this enzyme from the substantiae nigrae and caudate nuclei of cats implanted with four push–pull cannulae and compared it with that of dopamine (DA). DA is released from dendrites in the substantia nigra11,12 as well as nerve terminals in the caudate nucleus. Spontaneous AChE release was observed in the substantia nigra and in the caudate nucleus. Moreover, the application of potassium (30 mM) in one substantia nigra increased the local release of AChE. This was accompanied by remote changes in the enzyme release from the other three structures which differed from that seen for DA. The different patterns of responses observed for AChE and DA suggest that AChE may also originate from other neurones in both the substantia nigra and the caudate nucleus.

USING cats implanted with push–pull cannulae, we have previously demonstrated a reciprocal regulation of the activity of the two nigro-striatal dopaminergic pathways. Asymmetric fluctuations in the spontaneous release of dopamine(DA) were simultaneously seen in the two caudate nuclei (CN)1. As revealed by the changes in DA release from nerve terminals, the unilateral nigral application of DA1 or of drugs enhancing the dendritic release of DA, such as amphetamine or benztropine (2), reduced the activity of ipsilateral dopaminergic neurones and induced an opposite effect in the contralateral side. Conversely, the unilateral nigral blockade of dopaminergic transmission by local application of neuroleptics enhanced the release of DA in the ipsilateral CN and decreased the transmitter release in the contralateral structure2. Opposite changes in the release of DA were also seen in the two CN under unilateral delivery of sensory stimuli3 or during unilateral electrical stimulation of the cerebellar dentate nucleus4. These latter effects were associated with asymmetric changes in the dendritic release of DA in the two substantia nigrae (SN) which were in an opposite direction to those seen in the two CN (an increased release of DA from nerve terminals corresponded to a decreased release of the transmitter from dendrites and vice versa). These experiments indicated that DA which is released from dendrites in one SN regulates the activity of the ipsilateral dopaminergic neurones; they also suggested that it contributes to the control of the contralateral dopaminergic neurones by influencing the dendritic release of DA in the contralateral SN. This hypothesis was confirmed in the present study by measuring the changes in DA release from nerve terminals and dendrites of the two pathways under pharmacological blockade or facilitation of dopaminergic transmission in one SN.