Regulatory T cell phenotype and function 4 years after GAD-alum treatment in children with type 1 diabetes.

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(+) CD25(hi) forkhead box protein 3(+) (FoxP3(+)) cell numbers in response to in-vitro GAD(65) 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(+) CD25(hi) CD127(lo)) 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 GAD(65) -induced CD4(+) CD25(+) CD127(+) as well as CD4(+) CD25(hi) CD127(lo) and CD4(+) FoxP3(+) cells compared to placebo. Moreover, GAD(65) stimulation induced a population of CD4(hi) 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 GAD(65) -reactive CD25(+) CD127(+) and CD25(hi) CD127(lo) cells, but no difference in regulatory T cell function 4 years after GAD-alum treatment.

Extended evaluation of the safety and efficacy of GAD treatment of children and adolescents with recent-onset type 1 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: 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. METHODS: 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 µg 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 <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. RESULTS: 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. CONCLUSION/INTERPRETATION: 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. TRIAL REGISTRATION: ClinicalTrials.gov NCT00435981 FUNDING: 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.

Regulatory T cell-associated activity in photopheresis-induced immune tolerance in recent onset type 1 diabetes children.

Extracorporeal photochemotherapy (ECP) has demonstrated immunological effects. The proposed cytotoxic lymphocyte antigen 4 (CTLA-4) involvement, together with forkhead box P3 (FoxP3) and transforming growth factor (TGF)-beta are associated with regulatory T cell activity. The aim of the study was to evaluate the regulatory T cell-associated effect of ECP in recent onset type 1 diabetic (T1D) children. Children (n = 20) with T1D received photopheresis 8-methoxypsoralen + ECP or placebo + shampheresis. Peripheral blood mononuclear cells (PBMC) collected pretreatment (day 1) and post-treatment (day 90) were stimulated with phytohaemagglutinin (PHA) and T1D-associated glutamic acid decarboxylase 65 (GAD(65)) peptide a.a. 247-279. CTLA-4, sCTLA-4, FoxP3 and TGF-beta mRNA transcription was quantified. Photopheresis-treated individuals' relative mRNA expression was generally maintained during the course of the study. Placebo individuals increased in spontaneous CTLA-4 mRNA (P < 0.05) but decreased in expression after stimulation with GAD(65)-peptide (P < 0.05) and PHA (P < 0.05). Spontaneous TGF-beta (P < 0.05) increased whereas PHA- (P < 0.01) and GAD(65)-peptide (P < 0.01)-induced TGF-beta expression decreased in the placebo group, whereas it was maintained in the treated group. Without intervention, expression of CTLA-4 and TGF-beta, stimulated with PHA and GAD(65) peptide, decreased with time, with a parallel reduction of GAD(65)-peptide and PHA-stimulated TGF-beta expression. These parameters were counteracted by ECP. In conclusion, our results indicate that ECP maintains regulatory T cell-associated activity in recent-onset T1D.

Mortality and treatment side-effects during long-term intensified conventional insulin treatment in the Stockholm Diabetes Intervention Study.

Altogether, 102 patients were randomized to intensified conventional treatment (ICT) (n = 48) or standard treatment (ST) (n = 54). After 7.5 years, 89 patients remained, and it was shown that microangiopathy was retarded by the lower blood glucose concentrations seen in the patients in the ICT group. HbA1c was reduced from (means +/- SE) 9.5 +/- 0.2% to 7.1 +/- 0.1% in the ICT group and from 9.4 +/- 0.2% to 8.5 +/- 0.1% in the ST group (P < 0.001). Of the patients, 4 in the ICT group and 3 in the ST group died. Mortality was predicted by albuminuria, the amplitude of the sural nerve action potential, and the test of arm blood flow during contraction of the contralateral hand (sympathetic nerve function) at baseline (P < 0.05). Weight increased by 4.4 +/- 1.1 kg in the ICT group and 1.8 +/- 0.7 kg in the ST group (P = 0.05). Atherosclerosis, measured with digital pulse plethysmography, was approximately the same in the groups at baseline and after five years. In each group, 3 patients had myocardial infarctions, and 2 from each group had ketoacidosis once. There was a mean of 1.1 episodes per patient and per year of serious hypoglycemia in the ICT group and 0.4 episodes per patient and per year in the ST group. No adverse incidents or accidents were observed in either group, and there were no differences between the groups with regard to cognitive function measured with a battery of tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and determination of specificity of monoclonal IgG anti-mouse-blastocyst antibodies.

Five monoclonal anti-mouse-blastocyst IgG antibodies were raised by intrasplenic immunization of three mice with adhesive-stage mouse blastocysts. Each mouse received a total of 60-70 blastocysts which were either nitrocellulose-immobilized or living but irradiated. Tests for pre-implantation stage-specificity showed that the antibodies differed in specificity. None were specific for surface epitopes. One antibody recognized epitopes only on blastocysts. Other antibodies were able to discriminate between unfertilized and fertilized oocytes, uncompacted and compacted morulae, or delayed and adhesive blastocysts. By applying reduced SDS-PAGE and Western blotting to blastocysts the blastocyst-specific antibody was seen to be bound to a peptide of M(r) 34.

Complications in IDDM are caused by elevated blood glucose level: the Stockholm Diabetes Intervention Study (SDIS) at 10-year follow up.

Blood glucose values close to normal reduce the microvascular complications of insulin-dependent diabetes mellitus. The Stockholm study of this effect continued after the initial 7.5-year period in order to see what happened when intensively treated patients were left to control their own treatment while treatment was intensified in the control group. Forty-three patients with insulin-dependent diabetes randomised to intensified conventional treatment (ICT) and 48 patients randomised to standard treatment (ST) were followed-up for 10 years. Vascular complications, treatment side-effects and well-being were studied. Risk factors for complications were sought. HbA1c (normal range 3.9-5.7%) was reduced from 9.5 +/- 1.4% (mean +/- SD) in the ICT group and 9.4 +/- 1.2% in the ST group to a mean (during 10 years) of 7.2 +/- 0.6% and 8.3 +/- 1.0%, respectively (p < 0.001). Serious retinopathy (63 vs 33%, p = 0.003), nephropathy (26 vs 7%, p = 0.012) and symptoms of neuropathy (32 vs 14%, p = 0.041) were more common in the ST group after 10 years. HbA1c and age were the only risk factors for complications. Self-reported well-being increased to a greater degree and severe hypoglycaemia was more common in the ICT group. Cognitive function after 10 years was similar in both treatment groups, and was not related to the number of severe hypoglycaemic episodes. Intensified insulin treatment leads to reduced long-term complications and increased well-being without causing undue side-effects.