Glucose evaluation trial for remission (GETREM) in type 1 diabetes: a European multicentre study.

OBJECTIVE: Strict metabolic control during the 1st year of type 1 diabetes is thought to be a key factor for achieving clinical remission. The aims of this study were two-fold: (i) to evaluate the frequency and duration of spontaneous remission (defined according to the parameters issued by the International Diabetic Immunotherapy Group (IDIG)) in a European population of consecutive recent onset type 1 diabetes patients (aged 5-35 years), followed-up for a period of 36 months with a common protocol of intensive insulin therapy and without adjunct immune-intervention; and (ii) to identify the predictive factors for clinical remission. RESEARCH DESIGN AND METHOD: A total of 189 consecutive patients with newly diagnosed type 1 diabetes according to ADA criteria were recruited in participating centres (Belgium, Czech Republic, Estonia, France, Germany, Hungary, Italy, Poland, Romania, Sweden and Turkey) and followed-up for a period of up to 36 months. In all patients, intensive insulin therapy was implemented consisting of three or four injections of regular insulin daily with NPH insulin at bedtime. Adjustment of insulin dose was made according to a common protocol. Various clinical characteristics (age, gender, severity of presentation, etc.), history (presence of diabetic siblings in the family, etc.) and integrated parameters of metabolic control (HbA(1c), blood glucose, the total insulin dose at hospital discharge adjusted for body weight) were collected. RESULTS: Twenty-two patients (11.6%) experienced remission. The median duration of remission was 9.6 months and the range was 31 months. There was a wide variation among centres. Logistic regression analysis focused on the centre as the main variable in achieving remission. CONCLUSION: Remission was shown to be very heterogeneous between centres depending on 'other factors' such as patient care and family awareness of the disease rather than on 'measurable factors' such as sex, age, HbA(1c) and severity of presentation at diagnosis. Using intensive insulin therapy and optimisation of metabolic control, remission occurred in nearly one out of eight patients.

Cytokine secretion patterns in twins discordant for Type I diabetes.

AIMS/HYPOTHESIS: The search for T-cell reactions that are associated with disease in Type I (insulin-dependent) diabetes mellitus is severely hampered because control groups cannot be matched for relevant immune response genes. We therefore compared T-cell responses between identical twins discordant for Type I diabetes. METHODS: Pairs of monozygotic twins (n = 17) discordant for Type I diabetes were studied. Cultures were set up from whole blood immediately after sampling and cells were challenged with human recombinant hsp60, with the mitogen phytohaemagglutinin or with the staphylococcal superantigen. Supernatants were removed after 48 or 96 h and analysed for T-helper1 type cytokines interferon-gamma, TNFalpha and T-helper2 type cytokines IL-4, IL-10 by sandwich-ELISA. RESULTS: The height of the T-helper1 type cytokine response to hsp60, phytohaemagglutinin or staphylococcal enterotoxin B did not show disease association, i. e. it was similar between discordant twins. In contrast, the production of T-helper2 type cytokines differed between discordant twins. The IL-10 response to hsp60 was higher in twins at low disease risk (islet cell antibody-negative) than in their diabetic cotwins (p < 0.01), as was the IL-4 response to phytohaemagglutinin (p < 0.05). No difference was seen in the cytokine response between islet cell antibody-positive twins and their diabetic cotwins. CONCLUSIONS/INTERPRETATION: The data indicate an association between T-helper2 type cytokine secretion patterns and disease or disease risk.

The Deutsche Nicotinamide Intervention Study: an attempt to prevent type 1 diabetes. DENIS Group.

On the basis of the positive outcome of animal experiments, several large placebo-controlled trials are underway and aiming for the first time at the prevention of an immune-mediated disease, type 1 diabetes. The first of these trials, The Deutsche Nicotinamide Intervention Study (DENIS), evaluated the clinical efficacy of high doses of nicotinamide in children at high risk for IDDM. Nicotinamide has been shown to protect beta-cells from inflammatory insults and to improve residual beta-cell function in patients after onset of IDDM. Individuals at high risk for developing IDDM within 3 years were identified by screening the siblings (age 3-12 years) of patients with IDDM for the presence of high titer (> or =20 Juvenile Diabetes Foundation [JDF] U) islet cell antibodies. Probands (n = 55) were randomized into placebo and nicotinamide (slow release, 1.2 g x m(-2) x day(-1)) receiving groups and followed prospectively in a controlled clinical trial using a sequential design. Rates of diabetes onset were similar in both groups throughout the observation period (maximum 3.8 years, median 2.1 years). This sequential design provides a 10% probability of a type II error against a reduction of the cumulative diabetes incidence at 3 years from 30 to 6% by nicotinamide. The trial was terminated when the second sequential interim analysis after the eleventh case of diabetes showed that the trial had failed to detect a reduction of the cumulative diabetes incidence at 3 years from 30 to 6% (P = 0.97). The group receiving nicotinamide exhibited decreased first-phase insulin secretion in response to intravenous glucose (P = 0.03). No other side effects were observed. We conclude that in this subgroup of diabetes-prone individuals at very high risk and with an assumed rapid disease progression, nicotinamide treatment did not cause a major decrease or delay of diabetes development. However, the data do not exclude the possibility of a less strong, but potentially meaningful, risk reduction in this cohort, or a major clinical effect of nicotinamide in individuals with less risk of progression to IDDM than studied here.

Activated platelets in subjects at increased risk of IDDM. DENIS Study Group. Deutsche Nikotinamid Interventionsstudie.

Activated platelets respond to activated leukocytes and endothelial cells via adhesion molecules linking inflammation and thrombosis. Platelets of recent-onset insulin-dependent diabetic (IDDM) patients have been shown to be activated independent of metabolic control. This study evaluates the levels of circulating activated platelets exposing adhesion molecules in healthy subjects at increased risk of IDDM (surface markers were: P-selectin (CD62), thrombospondin, lysosomal GP53 (CD63). From the DENIS and the ENDIT screening programmes 19 identified islet cell antibody positive (titre > or = 20 Juvenile Diabetes Foundation units) first degree relatives of IDDM patients (male/female 9/10; age 22 +/- 15 years; body mass index (BMI): 20.0 +/- 4.3 kg/m2) with clearly normal metabolism (HbA1: 6.1 +/- 0.8%; fasting blood glucose: 4.95 +/- 0.67 mmol/l) were available for this investigation. Platelet CD62 as well as thrombospondin and CD63 expression were determined by flow cytometry. We matched 50 normal volunteers for age (29 +/- 6 years), anthropometric measures (male/female 26/24; BMI: 22.3 +/- 2.8 kg/m2) and metabolic parameters (HbA1: 5.8% +/- 0.3; fasting blood glucose: 4.41 +/- 0.53 mmol/1) served as control subjects. The mean number of CD62+ platelets was increased 3.2-times in prediabetic patients: 1.94 x 2.91 (+/- 1) vs 0.60 x 1.83 (+/- 1%), p < 0.0001. Thrombospondin+ and CD63+ platelet levels were concomitantly increased (1.45 x 2.38( +/- 1)/5.97 x 2.89 (+/- 1)% vs 0.52 x 2.01 (+/-1)/1.64 x 2.26 (+/-1)%, p < 0.0001 for both comparisons). Thus, intravasal platelet activation is already present in potentially prediabetic subjects representing an antecedent, potentially pathogenic feature of IDDM.

Systemic bias of cytokine production toward cell-mediated immune regulation in IDDM and toward humoral immunity in Graves' disease.

Disturbed immune regulation has been postulated to be crucial in the pathogenesis of IDDM and other autoimmune or allergic diseases. We therefore tested the hypothesis of a general bias in the peripheral immune system in patients with recent-onset IDDM or Graves' disease in comparison to healthy control subjects by studying whole blood cultures stimulated with phytohemagglutinin. Cells from IDDM patients (n = 53) produced significantly higher amounts of Th1 cytokines gamma-interferon (IFN-gamma) (P = 0.028) and tumor necrosis factor alpha (TNF-alpha) (P = 0.007) than normal control subjects (n = 56), while Th2 cytokine levels (interleukin [IL]-4, IL-10) were similar. Low levels of islet cell antibodies (ICAs) in IDDM patients were associated with high levels of Th1 and Th2 cytokines. Antibodies to GAD, ICA512, or insulin did not correlate with individual cytokine profiles. Also, HLA-DQ types did not significantly correlate with either Th1 or Th2 cytokine production. Conversely, whole blood cultures from patients with Graves' disease (n = 18) produced significantly less TNF-alpha and IL-4 than normal subjects (P = 0.001-0.006). However, when the balance between Th1 and Th2 cytokine production was analyzed in individuals, the ratio between IFN-gamma or TNF-alpha and IL-4 or IL-10 was clearly biased toward Th1 reactivity in patients with IDDM (P = 0.0001), while a dominance of Th2 cytokine production was seen in Graves' disease (P = 0.0001). The ratio of counterregulatory cytokines appeared to be the most reliable marker of the individual disease process. This study provides first evidence of a systemic bias in the immune regulation of humans, which might be either toward cell-mediated immunity (Th1) in IDDM or humoral immunity (Th2) in Graves' disease.

Combined screening for autoantibodies to IA-2 and antibodies to glutamic acid decarboxylase in first degree relatives of patients with IDDM. The DENIS Study Group. Deutsche Nikotinamid Interventions-Studie.

To determine the value of antibodies to the intracytoplasmic domain of the tyrosine phosphatase IA-2 (anti-IA-2ic) and glutamic acid decarboxylase (GADA) for identification of subjects at risk for insulin-dependent diabetes mellitus (IDDM) we investigated 1238 first degree relatives of patients with IDDM for the presence of anti-IA-2ic and GADA and compared the results with cytoplasmic islet cell antibodies (ICA). Anti-IA-2ic were observed in 54 (4.4%) first degree relatives, in 51 of 86 (59.3%) ICA positive relatives and in 3 of 4 individuals who developed overt IDDM within a follow-up period of 1 to 28 months. GADA were found in 78 of 1238 (6.3%) first degree relatives. They were detected in 22 of 35 (62.9%) sera with ICA alone and in 1 of 3 subjects with anti-IA-2ic in the absence of ICA. Of the 1238 subjects 37 (3.0%) sera were positive for all three antibodies. Both anti-IA-2ic and GADA were positively correlated with high levels of ICA. Anti-IA-2ic and GADA were detected in 39.1 and 47.8% of subjects with ICA of less than 20 Juvenile Diabetes Foundation units (JDF-U) but in 66.7 and 76.2% of individuals with ICA of 20 JDF-U or more, respectively (p < 0.05). The levels of ICA and GADA in first degree relatives with at least one additional marker were significantly higher than in subjects with ICA alone (p < 0.005) or GADA alone (p < 0.03). The combination of anti-IA-2ic and GADA identified 84.9% of all ICA positive subjects and 93.7% of individuals with high level ICA (> or = 20 IDF-U). All 4 individuals who progressed to IDDM had either IA-2ic or GADA. Our data indicate that primary screening for anti-IA-2ic and GADA provides a powerful approach with which to identify subjects at risk for IDDM in large-scale population studies which may represent the basis for the design of new intervention strategies.

Primary nonfunction of islet grafts in autoimmune diabetic nonobese diabetic mice is prevented by treatment with interleukin-4 and interleukin-10.

In isologous islet transplantation in spontaneously diabetic nonobese (NOD) mice, destruction of the islet graft is caused by recurrence of T helper (Th)1-driven insulitis[fnc,1. We established a model of transplantation in which female NOD recipients were rendered diabetic by a single injection of cyclophosphamide (250 mg/kg). Under these conditions, 500 freshly isolated islets from young NOD mice transplanted under the kidney capsule did not lead to normoglycemia within 3 day after transplantation, but underwent immediate impairment of function. This primary nonfunction was seen in > 80% of the recipients. Treatment of the recipients with the Th2-associated cytokine interleukin (IL)-4 alone did not prevent primary nonfunction, whereas treatment of the recipients with a combination of IL-4 and IL-10 restored immediate function of the grafts. Cytokine treatment did not prevent later rejection of grafts. Histological analysis of the grafts revealed less severely infiltrated islets, with well preserved islet architecture, in only normoglycemic animals treated with IL-4 or with IL-4 and IL-10. Staining for lymphocytes, macrophages, and tumor necrosis factor (TNF)-alpha did not show differences between the groups, but IFN-gamma was markedly less expressed in IL-4- and IL-10-treated grafts. Concomitantly, analysis of animals treated for 8 days after injection of cyclophosphamide, with IL-4 and IL-10, revealed a reduction of IL-12 mRNA in the pancreas. We conclude from these data that primary nonfunction of islet grafts is prevented by treatment of the recipients with a combination of IL-4 and IL-10, via downregulation of Th1 cytokines.

Insulitis and islet-cell antibody formation in rats with experimentally reduced beta-cell mass.

We studied the effect of severe reduction of beta-cell mass by 90% pancreatectomy on the immune tolerance to the endocrine pancreas. Four months after subtotal pancreatectomy all LEW.Han rats had developed mononuclear infiltration of islets and 9 of 14 rats were positive for islet-cell antibodies. Electron microscopy revealed lymphocytic invasion of endocrine tissue, lysis of beta cells and phagocytotic macrophages. None of these changes were seen 2 weeks after 90% pancreatectomy or 4 months after 10% pancreatectomy. Weekly substitution of islet antigens in the form of a homogenate of 100 islets into 90% pancreatectomized LEW.Han rats almost completely prevented the development of insulitis and autoantibodies. The dependence of insulitis on T cells was shown when 90% pancreatectomy in LEW.rnu rats (i.e., the congenic athymic nude strain), did not result in islet infiltration. The exocrine tissue remained normal in all experimental groups. During the observation period insulitis was not associated with overt diabetes but was accompanied by substantial enlargement of islets and of beta-cell mass, as shown by morphometry. Suppression of islet inflammation by injection of islet antigens abolished beta-cell regeneration, despite continuing metabolic stress in rats with 90% pancreatectomy. The findings indicate induction of islet autoimmunity in response to 90% but not to 10% pancreatectomy. We conclude that severe reduction of the islet-antigen mass allows the development of T-cell-dependent islet autoimmunity which indicates a loss of immune tolerance. In addition, the data suggest the existence of islet-antigen autoreactive immune cells in rats not genetically predisposed to autoimmune diabetes. Finally, we conclude that selective beta-cell regeneration occurs in association with insulitis.

Autoantibodies to the islet antigen ICA69 occur in IDDM and in rheumatoid arthritis.

Islet cell antigen (ICA) 69 is a newly-recognized islet cell antigen to which autoantibodies have been observed in prediabetic relatives of patients with insulin-dependent-diabetes mellitus (IDDM). Here we extend the earlier analysis of ICA69 antibodies to patients with recent-onset IDDM and to patients with other immune-mediated diseases. ICA69 antibodies were determined by Western blot using an affinity purified recombinant fusion protein of ICA69 and maltose binding protein. ICA69 antibody quantities were determined as titres using a titration curve of a standard serum as reference. Mean logarithmic ICA69 antibody titres were 3.4 (+/- 1.4) in 99 patients with acute IDDM compared to 2.8 (+/- 0.9) in 49 healthy blood donors (p < 0.001). A higher mean ICA69 antibody titre of 4.1 (+/- 0.8) was observed in 16 patients with rheumatoid arthritis in comparison to acute IDDM (p < 0.01) and healthy control subjects (p < 0.001). The percentage of sera with ICA69 antibody titres above the 2 SD level of normal subjects was 21% in IDDM, 31% in rheumatoid arthritis and 6% in healthy blood donors. None of the patients with autoimmune thyroid disease (n = 20), inflammatory bowel disease (n = 9) or multiple sclerosis (n = 7) had elevated ICA69 antibodies. In IDDM, presence of ICA69 antibodies persisted and the titre remained the same over 18 months of follow-up. The relationship of ICA69 antibodies to islet cell antibodies (ICA) or insulin autoantibodies (IAA) was tested. The production of ICA69 antibodies was not associated in diabetic patients with the presence of any of the two other autoantibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Regeneration of beta-cells in response to islet inflammation.

Subtotal pancreatectomy (90%) in Lewis rats induces chronic islet inflammation and tissue damage in the remaining pancreas 4 months after surgery. Concomitantly, significant enlargement of the islets of Langerhans was observed (90% pancreatectomy: islet/pancreas area: 25.6 +/- 9.6 x 10(-3), beta-cell/pancreas area: 12.4 +/- 4.4 x 10(-3); n = 4; controls without pancreatectomy: islet/pancreas area: 5.5 +/- 1.7 x 10(-3), beta-cell/pancreas area: 4.6 +/- 1.5 x 10(-3); n = 4; p < 0.05, respectively). Islet growth is mainly due to an increase in beta-cell mass. Beta-cell regeneration was not caused by the surgical manipulations or by metabolic stress. The former was ruled out by performing 10% pancreatectomy which did not cause islet enlargement after 4 months (islet/pancreas area: 13.6 +/- 11.3 x 10(-3), beta-cell/pancreas area: 7.1 +/- 2.0 x 10(-3); n = 3). An influence of metabolic stress was excluded by continuous substitution of syngenic islet antigens, which inhibits insulitis. In the absence of islet inflammation, despite persistent metabolic stress, beta-cell regeneration did not occur (islet/pancreas area: 7.0 +/- 5.5 x 10(-3), beta-cell/pancreas area: 5.5 +/- 4.1 x 10(-3); n = 4). Continuous treatment of animals after 90% pancreatectomy by insulin implants (1.5 U/day) avoided insulitis and beta-cell growth (islet/pancreas area: 9.2 +/- 1.1 x 10(-3), beta-cell/pancreas area: 6.8 +/- 1.0 x 10(-3), n = 3): All enlarged islets observed 4 months after 90% pancreatectomy without further treatment were infiltrated. Thus, beta-cell growth appears to be a response to insulitis. The stimulus for beta-cell growth could result from tissue damage caused by infiltrating cells or from cytokines secreted by the infiltrating cells, or both.

Inflammatory islet damage in patients bearing HLA-DR 3 and/or DR 4 haplotypes does not lead to islet autoimmunity.

The hypothesis was tested that islet autoimmunity is induced by ongoing islet cell destruction in subjects with susceptibility genes HLA-DR 3 and/or DR 4. Sixty-one patients with confirmed chronic pancreatitis were analysed, 30 of whom expressed HLA-DR 3 and/or DR 4. Electron microscopy studies in 10 patients showed that the inflammatory process also affected islets, as recognisable from islet cell lysis, intrainsular fibrosis and immune cell infiltrates. None of the sera tested contained any of three markers of islet autoimmunity, ICA, IAA or GAD antibodies. A correlation was seen between the loss of exocrine function, as determined by the ALTAB-test, and of beta-cell function, as determined by the C-peptide response to i.v. glucagon. However, there was no preferential loss of beta-cell function in patients with HLA-DR 3 and/or DR 4. We conclude that islet cell destruction occurs during chronic pancreatitis, but does not trigger islet autoimmunity, even in the presence of HLA-DR 3 and/or DR 4.

Linear loss of insulin secretory capacity during the last six months preceding IDDM. No effect of antiedematous therapy with ketotifen.

OBJECTIVE: To investigate the effect of an antiedematous therapy with the histamine antagonist ketotifen on beta-cell function in late prediabetes. RESEARCH DESIGN AND METHODS: In a randomized double-blind placebo-controlled study, ketotifen was administered for 3 months to 9 islet cell antibody positive (ICA+) prediabetic patients with a first-phase insulin response (FPIR) below the 2.5th percentile to preserve residual beta-cell function. Patients were followed by intravenous glucose tolerance tests (IVGTTs) every 4-6 weeks for determination of FPIR, HbA1, ICAs, and insulin autoantibodies. In 5 patients, the immune activation state was followed by determination of serum levels of tumor necrosis factor-alpha (TNF-alpha), beta 2-microglobulin, and C-reactive protein (CRP). RESULTS: Seven of nine patients developed diabetes within one year of follow-up. Irrespective of treatment with ketotifen, a slow and linear decline (P < 0.05) of 1 + 3-min insulin values was observed in sequential IVGTTs in those 7 patients who developed insulin-dependent diabetes mellitus (IDDM) during follow-up. The 2 other patients showed wide fluctuations of the insulin response with a threefold increase of initial insulin levels. HbA1 did not correlate with FPIR. Fasting blood glucose increased significantly during the study (P < 0.05). Individual levels of serum TNF-alpha, CRP, and beta 2-microglobulin did not change during the study. CONCLUSIONS: The study could not demonstrate preservation of beta-cell function by ketotifen in the late stage before manifestation of clinical diabetes. Manifestation is preceded in the last 6 months by a steady loss of the FPIR without rapid deterioration immediately before diagnosis and without signs of increased immune activity.

Transfer of insulin-dependent diabetes between HLA-identical siblings by bone marrow transplantation.

Insulin-dependent diabetes was observed in a woman, aged 29, 4 years after transplantation of bone marrow from her HLA-identical brother with insulin-dependent diabetes. Both had classic symptoms and insulin dependency from onset. At diagnosis of diabetes the recipient was positive for high-titre islet cell antibodies (ICA) whereas she had been ICA negative before transplantation. Chromosomal analyses verified that all circulating leucocytes were of male donor type. These findings suggest transfer of insulin-dependent diabetes by bone marrow cells and confirm the immune nature of the disease.

Intervention with nicotinamide in pre-type 1 diabetes: the Deutsche Nikotinamid Interventionsstudie-DENIS.

UNLABELLED: Nicotinamide is a prime candidate for clinical trials on preventing Type 1 diabetes. It is thought to protect Beta-cells mainly by increasing intracellular NADP levels via competitive inhibition of poly-(ADP-ribose)-polymerase. Thus, since nicotinamide protects target cells while under autoimmune attack it should be used at a time when sufficient numbers of beta-cells are still present, i.e. in the early stages of diabetes development. Increased diabetes risk can be identified by the presence of islet cell antibodies (ICA) and an increasing number of other more or less well defined antibodies in relatives of Type 1 diabetic patients. However, ICA is the best-evaluated marker and was therefore chosen for initial screening. The aim of the German Nicotinamide Intervention Study (DENIS) is to evaluate the potency of nicotinamide at protecting children with an increased risk of Type 1 diabetes from manifestation of the disease. The trial is placebo-controlled and double-blinded. A sustained-release preparation of nicotinamide is given at a dose of 1.2 g/m2 body surface. Entry criteria: ICA > or = 20 JDF units (at least two positive readings within 6 months, > or = 3 months apart, one measurement > or = 20 JDF units); siblings of Type 1 diabetic patients, 3-12 years of age; IVGTT performed. EXCLUSION CRITERIA: diabetes according to WHO criteria (including abnormal OGTT); no written consent; other chronic diseases. Definition of endpoint: Diabetes according to WHO criteria.(ABSTRACT TRUNCATED AT 250 WORDS)