In antibody-positive first-degree relatives of patients with type 1 diabetes, HLA-A*24 and HLA-B*18, but not HLA-B*39, are predictors of impending diabetes with distinct HLA-DQ interactions.

AIMS/HYPOTHESIS: Secondary type 1 diabetes prevention trials require selection of participants with impending diabetes. HLA-A and -B alleles have been reported to promote disease progression. We investigated whether typing for HLA-B*18 and -B*39 may complement screening for HLA-DQ8, -DQ2 and -A*24 and autoantibodies (Abs) against islet antigen-2 (IA-2) and zinc transporter 8 (ZnT8) for predicting rapid progression to hyperglycaemia. METHODS: A registry-based group of 288 persistently autoantibody-positive (Ab(+)) offspring/siblings (aged 0-39 years) of known patients (Ab(+) against insulin, GAD, IA-2 and/or ZnT8) were typed for HLA-DQ, -A and -B and monitored from the first Ab(+) sample for development of diabetes within 5 years. RESULTS: Unlike HLA-B*39, HLA-B*18 was associated with accelerated disease progression, but only in HLA-DQ2 carriers (p < 0.006). In contrast, HLA-A*24 promoted progression preferentially in the presence of HLA-DQ8 (p < 0.002). In HLA-DQ2- and/or HLA-DQ8-positive relatives (n = 246), HLA-B*18 predicted impending diabetes (p = 0.015) in addition to HLA-A*24, HLA-DQ2/DQ8 and positivity for IA-2A or ZnT8A (p ≤ 0.004). HLA-B*18 interacted significantly with HLA-DQ2/DQ8 and HLA-A*24 in the presence of IA-2 and/or ZnT8 autoantibodies (p ≤ 0.009). Additional testing for HLA-B*18 and -A*24 significantly improved screening sensitivity for rapid progressors, from 38% to 53%, among relatives at high Ab-inferred risk carrying at least one genetic risk factor. Screening for HLA-B*18 increased sensitivity for progressors, from 17% to 28%, among individuals carrying ≥ 3 risk markers conferring >85% 5 year risk. CONCLUSIONS/INTERPRETATION: These results reinforce the importance of HLA class I alleles in disease progression and quantify their added value for preparing prevention trials.

Screening for insulinoma antigen 2 and zinc transporter 8 autoantibodies: a cost-effective and age-independent strategy to identify rapid progressors to clinical onset among relatives of type 1 diabetic patients.

In first-degree relatives of type 1 diabetic patients, we investigated whether diabetes risk assessment solely based on insulinoma antigen 2 (IA-2) and zinc transporter 8 (ZnT8) antibody status (IA-2A, respectively, ZnT8A) is as effective as screening for three or four autoantibodies [antibodies against insulin (IAA), glutamate decarboxylase 65 kDa (GAD) glutamate decarboxylase autoantibodies (GADA) and IA-2A with or without ZnT8A] in identifying children, adolescents and adults who progress rapidly to diabetes (within 5 years). Antibodies were determined by radiobinding assays during follow-up of 6444 siblings and offspring aged 0-39 years at inclusion and recruited consecutively by the Belgian Diabetes Registry. We identified 394 persistently IAA(+) , GADA(+) , IA-2A(+) and/or ZnT8A(+) relatives (6·1%). After a median follow-up time of 52 months, 132 relatives developed type 1 diabetes. In each age category tested (0-9, 10-19 and 20-39 years) progression to diabetes was significantly quicker in the presence of IA-2A and/or ZnT8A than in their joint absence (P < 0·001). Progression rate was age-independent in IA-2A(+) and/or ZnT8A(+) relatives but decreased with age if only GADA and/or IAA were present (P = 0·008). In the age group mainly considered for immune interventions until now (10-39 years), screening for IA-2A and ZnT8A alone identified 78% of the rapid progressors (versus 75% if positive for ≥ 2 antibodies among IAA, GADA, IA-2A and ZnT8A or versus 62% without testing for ZnT8A). Screening for IA-2A and ZnT8A alone allows identification of the majority of rapidly progressing prediabetic siblings and offspring regardless of age and is more cost-effective to select participants for intervention trials than conventional screening.

Immune responses against islet allografts during tapering of immunosuppression--a pilot study in 5 subjects.

Transplantation of isolated islet of Langerhans cells has great potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of immunosuppression in successfully transplanted patients would lower patients' health risk. To identify immune biomarkers that may prove informative in monitoring tapering, we studied the effect of tapering on islet auto- and alloimmune reactivity in a pilot study in five transplant recipients in vitro. Cytokine responses to the graft were measured using Luminex technology. Avidity of alloreactive cytotoxic T Lymphocytes (CTL) was determined by CD8 blockade. The influence of immunosuppression was mimicked by in vitro replenishment of tacrolimus and MPA, the active metabolite of mycophenolate mofetil. Tapering of tacrolimus was generally followed by decreased C-peptide production. T-cell autoreactivity increased in four out of five patients during tapering. Overall alloreactive CTL precursor frequencies did not change, but their avidity to donor mismatches increased significantly after tapering (P = 0·035). In vitro addition of tacrolimus but not MPA strongly inhibited CTL alloreactivity during tapering and led to a significant shift to anti-inflammatory graft-specific cytokine production. Tapering of immunosuppression is characterized by diverse immune profiles that appear to relate inversely to plasma C-peptide levels. Highly avid allospecific CTLs that are known to associate with rejection increased during tapering, but could be countered by restoring immune suppression in vitro. Immune monitoring studies may help guiding tapering of immunosuppression after islet cell transplantation, even though we do not have formal prove yet that the observed changes reflect direct effects of immune suppression on immunity.

An important minority of prediabetic first-degree relatives of type 1 diabetic patients derives from seroconversion to persistent autoantibody positivity after 10 years of age.

AIMS/HYPOTHESIS: The appearance of autoantibodies (Abs) before diabetes onset has mainly been studied in young children. However, most patients develop type 1 diabetes after the age of 15 years. In first-degree relatives aged under 40 years, we investigated the frequency of seroconversion to (persistent) Ab positivity, progression to diabetes and baseline characteristics of seroconverters according to age. METHODS: Abs against insulin (IAA), glutamate decarboxylase (GADA), insulinoma-associated protein 2 (IA-2A) and zinc transporter 8 (ZnT8A) were measured during follow-up of 7,170 first-degree relatives. RESULTS: We identified 379 (5.3%) relatives with positivity for IAA, GADA, IA-2A and/or ZnT8A (Ab(+)) at first sampling and 224 (3.1%) at a later time point. Most seroconversions occurred after the age of 10 years (63%). During follow-up, Abs persisted more often in relatives initially Ab(+) (76%) than in seroconverters (53%; p < 0.001). In both groups diabetes developed at a similar pace and almost exclusively with Ab persistence (136 of 139 prediabetic individuals). For both groups, progression was more rapid if Abs appeared before the age of 10 years. Baseline characteristics at seroconversion did not vary significantly according to age. CONCLUSIONS/INTERPRETATION: Seroconversion to (persistent) Ab(+) occurs regardless of age. Although the progression rate to diabetes is higher under age 10 years, later seroconverters (up to age 40 years) have similar characteristics when compared with age-matched initially Ab(+) relatives and generate an important minority of prediabetic relatives, warranting their identification and, eventually, enrolment in prevention trials.

Hyperglycaemic clamp test for diabetes risk assessment in IA-2-antibody-positive relatives of type 1 diabetic patients.

AIMS/HYPOTHESIS: The aim of the study was to investigate the use of hyperglycaemic clamp tests to identify individuals who will develop diabetes among insulinoma-associated protein-2 antibody (IA-2A)-positive first-degree relatives (IA-2A(+) FDRs) of type 1 diabetic patients. METHODS: Hyperglycaemic clamps were performed in 17 non-diabetic IA-2A(+) FDRs aged 14 to 33 years and in 21 matched healthy volunteers (HVs). Insulin and C-peptide responses were measured during the first (5-10 min) and second (120-150 min) release phase, and after glucagon injection (150-160 min). Clamp-induced C-peptide release was compared with C-peptide release during OGTT. RESULTS: Seven (41%) FDRs developed diabetes 3-63 months after their initial clamp test. In all phases they had lower C-peptide responses than non-progressors (p < 0.05) and HVs (p < 0.002). All five FDRs with low first-phase release also had low second-phase release and developed diabetes 3-21 months later. Two of seven FDRs with normal first-phase but low second-phase release developed diabetes after 34 and 63 months, respectively. None of the five FDRs with normal C-peptide responses in all test phases has developed diabetes so far (follow-up 56 to 99 months). OGTT-induced C-peptide release also tended to be lower in progressors than in non-progressors or HVs, but there was less overlap in results between progressors and the other groups using the clamp. CONCLUSIONS/INTERPRETATION: Clamp-derived functional variables stratify risk of diabetes in IA-2A(+) FDRs and may more consistently identify progressors than OGTT-derived variables. A low first-phase C-peptide response specifically predicts impending diabetes while a low second-phase response may reflect an earlier disease stage. TRIAL REGISTRATION: ClinicalTrials.gov NCT00654121 FUNDING: The insulin trial was financially supported by Novo Nordisk Pharma nv.

Insulin treatment in IA-2A-positive relatives of type 1 diabetic patients.

AIMS: We examined whether parenteral regular insulin can prevent diabetes in IA-2 antibody-positive (IA-2A+) relatives of type 1 diabetic patients, using a trial protocol that differed substantially from that of the Diabetes Prevention Trial-1. METHODS: Twenty-five IA-2A+ relatives received regular human insulin twice a day for 36 months, during which time they were followed (median [interquartile range; IQR]: 47 [19-66] months) for glucose tolerance, HbA(1c) and islet autoantibodies, together with 25 IA-2A+ relatives (observation/control group) who fulfilled the same inclusion criteria, but were observed for 52 [27-67] months (P=0.58). RESULTS: Twelve (48%) insulin-treated relatives and 15 (60%) relatives in the control group developed diabetes. There was no difference in diabetes-free survival between the two groups (P=0.97). Five-year progression (95% confidence interval) was 44% (25-69) in the insulin-treated group and 49% (29-70) in the observation group. At inclusion, progressors tended to have a higher pro-insulin/C-peptide ratio than non-progressors when measured 2 hours after a standardized glucose load (median [IQR]: 2.7% [1.8-4.3] vs. 1.6% [1.1-2.1]; P=0.01). No major hypoglycaemic episodes or significant increases in body mass index or diabetes autoantibodies were observed. CONCLUSION: Prophylactic injections of regular human insulin were well tolerated, but failed to prevent type 1 diabetes onset in IA-2A+ relatives.

Relevance of cytotoxic alloreactivity under different immunosuppressive regimens in clinical islet cell transplantation.

Islet or beta cell transplantation provides a promising cure for type 1 diabetes patients, but insulin-independency decreases frequently over time. Immunosuppressive regimens are implemented attempting to cope with both auto- and alloimmunity after transplantation. We analysed the influence of different immunotherapies on autoreactive and alloreactive T cell patterns and transplant outcome. Patients receiving three different immunosuppressive regimens were analysed. All patients received anti-thymocyte globulin induction therapy. Twenty-one patients received tacrolimus-mycophenolate mofetil maintenance immunosuppression, whereas the other patients received tacrolimus-sirolimus (SIR, n = 5) or SIR only (n = 5). Cellular autoreactivity and alloreactivity (CTL precursor frequency) were measured ex vivo. Clinical outcome in the first 6 months after transplantation was correlated with immunological parameters. C-peptide levels were significantly different between the three groups studied (P = 0.01). We confirm that C-peptide production was correlated negatively with pretransplant cellular autoreactivity and low graft size (P = 0.001, P = 0.007 respectively). Combining all three therapies, cellular autoimmunity after transplantation was not associated with delayed insulin-independence or C-peptide production. In combined tacrolimus-SIR and SIR-treated patients, CTL alloreactivity was associated with less insulin independence and C-peptide production (P = 0.03). The percentage of donors to whom high CTLp frequencies were measured was lower in insulin-independent recipients (P = 0.03). In this cohort of islet cell graft recipients, clinical outcome in the first 6 months after transplantation correlates with the applied immunosuppressive regimen. An association exists between insulin-independence and lower incidence of CTL alloreactivity towards donor human leucocyte antigen. This observational study demonstrates the usefulness of monitoring T cell reactivity against islet allografts to correlate immune function with graft survival.

Allograft-specific cytokine profiles associate with clinical outcome after islet cell transplantation.

Islet cell transplantation can cure type 1 diabetes, but allograft rejection and recurrent autoimmunity may contribute to decreasing insulin independence over time. In this study we report the association of allograft-specific proliferative and cytokine profiles with clinical outcome. Peripheral blood mononuclear cells were obtained of 20 islet recipients. Cytokine values in mixed lymphocyte cultures (MLC) were determined using stimulator cells with graft-specific HLA class II. Qualitative and quantitative cytokine profiles were determined before and after islet transplantation, blinded from clinical outcome. Cytotoxic T Lymphocyte precursor (CTLp) assays were performed to determine HLA class I alloreactivity. Allograft-specific cytokine profiles were skewed toward a Th2 or regulatory (Treg) phenotype after transplantation in insulin-independent, but not in insulin-requiring recipients. IFNgamma/IL10 ratio and MLC proliferation decreased after transplantation in insulin-independent recipients (p = 0.006 and p = 0.01, respectively). Production of the Treg cytokine IL10 inversely correlated with proliferation in alloreactive MLC (p = 0.008) and CTLp (p = 0.005). Production of IL10 combined with low-MLC reactivity associated significantly with insulin independence. The significant correlation between allograft-specific cytokine profiles and clinical outcome may reflect the induction of immune regulation in successfully transplanted recipients. Islet donor-specific IL10 production correlates with low alloreactivity and superior islet function.

Adiponectin levels do not predict clinical onset of type 1 diabetes in antibody-positive relatives.

AIMS/HYPOTHESIS: Insulin resistance has been proposed as a risk factor for type 1 diabetes. We investigated whether adiponectin, an insulin sensitiser, can serve as an additional predictive marker for type 1 diabetes in first-degree relatives of known patients. METHODS: Adiponectin was followed in 211 persistently islet antibody-positive (Ab+) first-degree relatives of type 1 diabetic patients and in 211 age- and sex-matched persistently antibody-negative relatives, and correlated with antibody status, random proinsulin:C-peptide ratio and HLA-DQ genotype. During follow-up, 37 Ab+ relatives developed type 1 diabetes. RESULTS: In the group of 422 relatives, baseline adiponectin correlated inversely with age and BMI and was lower in male than in female participants, especially after 15 years of age (p < 0.001). There was no correlation with antibody status or later development of diabetes. In 24 Ab+ relatives sampled fasted, adiponectin levels correlated significantly with homeostasis model assessment of insulin sensitivity (p = 0.006). In Ab+ relatives (n = 211), adiponectin levels could not predict type 1 diabetes nor complement risk assessment based on islet antibodies, HLA-DQ genotype and pancreatic hormones in Cox regression analysis. CONCLUSIONS/INTERPRETATION: Adiponectin levels do not contribute to the prediction of type 1 diabetes in Ab+ relatives.

The age at diagnosis of type 1 diabetes continues to decrease in Belgian boys but not in girls: a 15-year survey.

BACKGROUND: The age at clinical onset of type 1 diabetes is decreasing. Preliminary Belgian data suggested that this anticipation occurred preferentially in boys. We investigated whether this gender-specific anticipation could be confirmed over a 15-year observation period. METHODS: In Antwerp, we studied incidence trends between 1989 and 2003 in 746 type 1 diabetic patients under age 40. For 2928 antibody-positive patients diagnosed nationwide during the same period, age at diagnosis was analysed according to gender and calendar year. RESULTS: In Antwerp, the incidence of type 1 diabetes under age 15 increased significantly with time from 10.9/100 000/year in 1989-1993 to 15.8/100 000/year in 1999-2003 (p = 0.008). The rising incidence in children was largely restricted to boys under age 10 where the incidence more than doubled during the 15-year period (6.8/100 000/year in 1989-1993 vs 17.2/100 000/year in 1999-2003; p < 0.001). Such an increase was not found in girls under age 10 (p = 0.54). This selective trend toward younger age at diagnosis in boys was confirmed in the larger group of Belgian patients where the median age at diagnosis decreased in boys-but not in girls-from 20 years in 1989-1993 to 15 years in 1999-2003 (p < 0.001). CONCLUSIONS: Over a 15-year observation period, a selective anticipation of clinical onset of type 1 diabetes was found in boys but not in girls. This suggests that an environmental factor may preferentially accelerate the sub-clinical disease process in young boys.

Identification of prediabetes in first-degree relatives at intermediate risk of type I diabetes.

Prevention trials of type I diabetes are limited by recruitment of individuals at high risk of the disease. We investigated whether demographic and biological characteristics can identify rapid progressors among first-degree relatives of known patients at intermediate (< 10%) 5-year risk. Diabetes-associated antibodies, random proinsulin : C-peptide (PI/C) ratio and HLA DQ genotype were determined (repeatedly) in 258 islet antibody-positive IA-2Antibody-negative (Abpos/IA-2Aneg) normoglycaemic first-degree relatives. During follow-up (median 81 months), 14 of 258 Abpos/IA-2Aneg relatives developed type I diabetes; 13 (93%) of them had persistent antibodies conferring a 12% [95% confidence interval (CI): 5-19%] 5-year risk of diabetes. In Abpos/IA-2Aneg relatives with persistent antibodies (n = 126), the presence of >/= 1 HLA DQ susceptibility haplotype in the absence of a protective haplotype (P = 0.033) and appearance on follow-up of a high PI/C ratio (P = 0.007) or IA-2A-positivity (P = 0.009) were identified as independent predictors of diabetes. In persistently antibody-positive relatives with HLA DQ risk a recurrently high PI/C ratio or development of IA-2A identified a subgroup (n = 32) comprising 10 of 13 (77%) prediabetic relatives and conferred a 35% (95% CI: 18-53%) 5-year risk. Under age 15 years, 5-year progression (95% CI) was 57% (30-84%) and sensitivity 62%. In the absence of IA-2A, the combination of antibody persistence, HLA DQ risk and elevated PI/C ratio or later development of IA-2A and young age defines a subgroup of relatives with a high risk of type I diabetes (>/= 35% in 5 years). Together with initially IA-2A-positive relatives these individuals qualify for standardized beta cell function tests in view of prevention trials.

Sex- and season-dependent differences in C-peptide levels at diagnosis of immune-mediated type 1 diabetes.

AIMS/HYPOTHESIS: The incidence of type 1 diabetes varies according to age, sex and season of diagnosis. We investigated whether these and other clinical, biological and anthropometric parameters were correlated with residual beta cell function in newly diagnosed patients, since it is possible that the nature of external and/or genetic disease accelerators may be (partly) reflected in the inaugural disease presentation. MATERIALS AND METHODS: The correlates of random C-peptide levels sampled shortly after diagnosis (median [interquartile range]: 3 [0-14] days) were studied by multivariate analysis in 1,883 islet-antibody-positive diabetic patients aged <40 years who were diagnosed between 1989 and 2000. RESULTS: Higher C-peptide levels (above percentile 50 of patients) were associated with older age at diagnosis, female sex, diagnosis in the high-incidence season (October to March), less-decreased BMI (expressed as a standard deviation score), lower insulin requirements after stabilisation, lower prevalence of ketonuria and a less-increased glycaemia at diagnosis (all p < 0.001). C-peptide levels were not correlated with calendar year at diagnosis, duration of symptoms prior to diagnosis, HLA-DQ2/DQ8 genotype or islet antibody status. CONCLUSIONS/INTERPRETATION: Sex- and season-dependent differences in residual functional beta cell mass and/or insulin resistance have been identified at diagnosis of type 1 diabetes. They may reflect differences in disease-precipitating external or lifestyle factors and should be further investigated longitudinally in prediabetes to further identify putative aetiological factors, which may provide targets for prevention.

Proinsulin levels and the proinsulin:c-peptide ratio complement autoantibody measurement for predicting type 1 diabetes.

AIMS/HYPOTHESIS: We investigated whether random proinsulin levels and proinsulin:C-peptide ratio (PI:C) complement immune and genetic markers for identifying relatives at high risk of type 1 diabetes. MATERIALS AND METHODS: During an initial sampling, random glycaemia, proinsulin, PI:C and HLA DQ genotype were determined in 561 non-diabetic first-degree relatives who had been positive for islet autoantibodies on one or more occasions and in 561 age- and sex-matched persistently antibody-negative relatives. RESULTS: During follow-up (median 62 months), 46 relatives with antibodies at entry developed type 1 diabetes. At baseline, antibody-positive relatives (n=338) had higher PI:C values (p<0.001) than antibody-negative subjects with (n=223) or subjects without (n=561) later seroconversion. Proinsulin and PI:C were graded according to risk of diabetes as expressed by positivity for (multiple) antibodies or IA-2 antibodies, especially in persons carrying the high-risk HLA DQ2/DQ8 genotype and in prediabetic relatives. In the presence of multiple or IA-2 antibodies, a PI:C ratio exceeding percentile 66 of all antibody-negative relatives at entry (n=784) conferred a 5-year diabetes risk of 50% and 68%, respectively (p<0.001 vs 13% for same antibody status with PI:C

Combined positivity for HLA DQ2/DQ8 and IA-2 antibodies defines population at high risk of developing type 1 diabetes.

AIMS/HYPOTHESIS: Prevention trials in first-degree relatives of type 1 diabetic patients are hampered by large interindividual differences in progression rate to diabetes. We investigated whether specific combinations of immune and genetic markers can identify subgroups with more homogeneous progression to clinical onset. METHODS: Antibodies against islet cell cytoplasm (ICA), insulin (IAA), glutamate decarboxylase (GADA) and IA-2 protein (IA-2A) were measured in 790 non-diabetic control subjects and 4,589 first-degree relatives under age 40. RESULTS: On first sampling, 11.1% of the siblings presented at least one antibody type (p<0.001 vs other relatives). During follow-up (median 52 months) 43 subjects developed type 1 diabetes (31 siblings, ten offspring of a diabetic father, two offspring of a diabetic mother). Using Kaplan-Meier survival analysis and Cox regression, IA-2A conferred the highest 5-year diabetes risk (>50%) irrespective of the number of antibodies present. In initially IA-2A-positive relatives (n=58) progression to hyperglycaemia depended more on HLA DQ status than on type of kinship (84% progression in the presence of DQ2/DQ8 vs 32% in its absence; p<0.003). In IA-2A-negative relatives (n=4,531) 5-year progression to diabetes increased with the number of other antibodies (ICA, GADA and/or IAA) (p<0.001) but overall did not exceed 10% even for two or more antibodies. Among relatives initially positive for one or more antibody type other than IA-2A (n=315), there was significantly more progression to diabetes (overall still <10%) in carriers of DQ2 (p<0.001 vs no DQ2), regardless of DQ8 status. CONCLUSIONS/INTERPRETATION: These observations suggest that the HLA-DQ-inferred risk of diabetes can proceed through two distinct pathways distinguished by IA-2A status. Combined positivity for DQ2/DQ8 and IA-2A defines a more homogeneous high-risk population for prevention trials than those used so far.

Seasonality in clinical onset of type 1 diabetes in belgian patients above the age of 10 is restricted to HLA-DQ2/DQ8-negative males, which explains the male to female excess in incidence.

AIMS/HYPOTHESIS: Type 1 diabetes arises from an interplay between environmental and genetic factors. The reported seasonality at diagnosis supports the hypothesis that currently unknown external triggers play a role in the onset of the disease. We investigated whether a seasonal pattern is observed at diagnosis in Belgian Type 1 diabetic patients, and if so whether seasonality varies according to age, sex and genetic risk, all known to affect the incidence of Type 1 diabetes. METHODS: The seasonal pattern at clinical diagnosis was assessed in 2176 islet antibody-positive diabetic patients aged 0 to 39 years diagnosed between 1989 and 2000. Additional stratification was performed for age, sex and HLA-DQ genotype. RESULTS: Overall, a significant seasonal pattern at clinical diagnosis of diabetes was observed (p<0.001). More subjects were diagnosed in the period of November to February (n=829) than during the period of June to September (n=619) characterised by higher averages of maximal daily temperature and daily hours of sunshine. However, the seasonal pattern was restricted to patients diagnosed above the age of 10 (0-9 years: p=0.398; 10-19 years: p<0.001; 20-29 years: p=0.003; 30-39 years: p=0.015). Since older age at diagnosis is associated with a male to female excess and a lower prevalence of the genetic accelerator HLA-DQ2/DQ8, we further stratified the patients aged 10 to 39 years (n=1675) according to HLA-DQ genotype and sex, and we found that the seasonal pattern was largely restricted to male subjects lacking DQ2/DQ8 (n=748; p<0.00 vs all others: n=927; p=0.031). CONCLUSIONS/INTERPRETATION: In a subgroup of male patients diagnosed over the age of 10, the later stages of the subclinical disease process may be more driven by sex- and season-dependent external factors than in younger, female and genetically more susceptible subjects. These factors may explain the male to female excess in diabetes diagnosed in early adulthood.

Epidemiology of type 1 and type 2 diabetes. The added value of diabetes registries for conducting clinical studies: the Belgian paradigm.

Diabetes registries have documented that the lifetime risk of diabetes amounts to at least 10% in the western world. Moreover the prevalence of type 2 diabetes is increasing worldwide especially in developing countries. Furthermore there is a secular trend toward earlier clinical manifestation of both type 1 and type 2 diabetes. In the absence of a permanent cure for primary diabetes the present estimated number of at least 150 million diabetic patients worldwide is expected to double within the next 20 years. Consequently a sharp increase in the global burden of chronic diabetes complications is to be feared in the coming decades. Therefore it is absolutely mandatory to intensify research efforts aiming at identifying the etiological factors involved and designing effective strategies for prediction and prevention of the disease and its devastating complications. Diabetes registries constitute instruments of choice to conduct such studies because they are able to collect standardised clinical, demographic and biological information from sufficiently large representative groups of patients and risk groups such as first degree relatives. Since 1989, the Belgian Diabetes Registry is studying all types of diabetes presenting before age 40 in Belgium and provides a paradigm of how diabetes registries may also contribute to the advancement of knowledge on disease heterogeneity, etiology, prediction and prevention.

IA-2 autoantibodies predict impending type I diabetes in siblings of patients.

AIMS/HYPOTHESIS: Multiple islet autoantibody positivity is currently believed to best predict progression to Type I (insulin-dependent) diabetes mellitus. We compared its predictive value with that of positivity for a particular type of islet autoantibody, directed against the IA-2 antigen. METHODS: Autoantibodies against islet cell cytoplasm (ICA), insulin (IAA), GAD (GADA) and IA-2 (IA-2A) were measured at initial sampling in 1724 non-diabetic siblings (median age [range]:16 [0-39] years) of Type I diabetic patients with a median follow-up of 50 months. RESULTS: On initial sampling 11% of siblings were positive for one antibody type or more and 2.1% for three of more types. During follow-up, 27 antibody-positive siblings developed diabetes. Using survival analysis, the risk for clinical onset within 5 years was 34% in subjects positive for three or more types compared with 13% in those with one type or more. Progression to diabetes amounted to 12% within 5 years among siblings positive for IAA, 20% for ICA, 19% for GADA but 59% for IA-2A (p<0.001 vs absence of the respective antibody). IA-2A were detected in 1.7% of all siblings and in 56% of the prediabetic subjects on first sampling. Initial positivity for two or three antibody markers was associated with a higher progression rate in IA-2A positive as compared to IA-2A negative siblings (p=0.001). In absence of IA-2A initial positivity for another antibody (IAA, ICA or GADA) conferred a low (<10% within 5 years) risk of diabetes compared to subjects lacking this antibody. CONCLUSIONS/INTERPRETATION: In siblings of Type I diabetic patients, IA-2A positivity is a more direct predictor of impending clinical onset than multiple antibody positivity per se. Assessment of IA-2A status allows us to select subjects with homogeneously high risk of diabetes for participation in prevention trials.

Prospects for predicting and stopping the development of type 1 of diabetes.

The prevention of diabetes and its devastating complications is the prime goal of diabetes care. In immune-mediated type 1 diabetes, beta cell destruction can be predicted with increasing confidence both before and after diagnosis, thus allowing the development of preventative strategies. Multicentre clinical trials with the natural products insulin and nicotinamide have been launched, but the results will only be available in a few years time. Meanwhile, observational studies in large representative risk groups can help to refine the selection of subjects with a more homogenous risk for beta cell destruction, thereby reducing the need for large sample sizes. The comparison between biological markers and disease progression will help to define surrogate disease end-points that can be monitored before the hard clinical end-points of hyperglycaemia or remission. These advances will facilitate the start of new pilot trials to identify relatively safe candidate interventions adapted to disease stage.

Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation.

Studies in Zucker diabetic fatty rats have led to the concept that chronically elevated free fatty acid (FFA) levels can cause apoptosis of triglyceride-laden pancreatic beta-cells as a result of the formation of ceramides, which induce nitric oxide (NO)-dependent cell death. This "lipotoxicity" hypothesis could explain development of type 2 diabetes in obesity. The present study examines whether prolonged exposure to FFA affects survival of isolated normal rat beta-cells and whether the outcome is related to the occurrence of triglyceride accumulation. A dose-dependent cytotoxicity was detected at 5-100 nmol/l of unbound oleate and palmitate, with necrosis occurring within 48 h and an additional apoptosis during the subsequent 6 days of culture. At equimolar concentrations, the cytotoxicity of palmitate was higher than that of oleate but lower than that of its nonmetabolized analog bromopalmitate. FFA cytotoxicity was not suppressed by etomoxir (an inhibitor of mitochondrial carnitine palmitoyltransferase I) or by antioxidants; it was not associated with inducible NO synthase expression or NO formation. An inverse correlation was observed between the percentage of dead beta-cells on day 8 and their cellular triglyceride content on day 2. For equimolar concentrations of the tested FFA, oleate caused the lowest beta-cell toxicity and the highest cytoplasmic triglyceride accumulation. On the other hand, oleate exerted the highest toxicity in islet non-beta-cells, where no FFA-induced triglyceride accumulation was detected. In conditions without triglyceride accumulation, the lower FFA concentrations caused primarily apoptosis, both in islet beta-cells and non-beta-cells. It is concluded that FFAs can cause death of normal rat islet cells through an NO-independent mechanism. The ability of normal beta-cells to form and accumulate cytoplasmic triglycerides might serve as a cytoprotective mechanism against FFA-induced apoptosis by preventing a cellular rise in toxic free fatty acyl moieties. It is conceivable that this potential is lost or insufficient in cells with a prolonged triglyceride accumulation as may occur in vivo.