Antibodies to influenza virus A/H1N1 hemagglutinin in Type 1 diabetes children diagnosed before, during and after the SWEDISH A(H1N1)pdm09 vaccination campaign 2009-2010.

We determined A/H1N1-hemagglutinin (HA) antibodies in relation to HLA-DQ genotypes and islet autoantibodies at clinical diagnosis in 1141 incident 0.7-to 18-year-old type 1 diabetes patients diagnosed April 2009-December 2010. Antibodies to (35) S-methionine-labelled A/H1N1 hemagglutinin were determined in a radiobinding assay in patients diagnosed before (n = 325), during (n = 355) and after (n = 461) the October 2009-March 2010 Swedish A(H1N1)pdm09 vaccination campaign, along with HLA-DQ genotypes and autoantibodies against GAD, insulin, IA-2 and ZnT8 transporter. Before vaccination, 0.6% patients had A/H1N1-HA antibodies compared with 40% during and 27% after vaccination (P < 0.0001). In children <3 years of age, A/H1N1-HA antibodies were found only during vaccination. The frequency of A/H1N1-HA antibodies during vaccination decreased after vaccination among the 3 < 6 (P = 0.006) and 13 < 18 (P = 0.001), but not among the 6 < 13-year-olds. HLA-DQ2/8 positive children <3 years decreased from 54% (15/28) before and 68% (19/28) during, to 30% (9/30) after vaccination (P = 0.014). Regardless of age, DQ2/2; 2/X (n = 177) patients had lower frequency (P = 0.020) and levels (P = 0.042) of A/H1N1-HA antibodies compared with non-DQ2/2; 2/X (n = 964) patients. GADA frequency was 50% before, 60% during and 51% after vaccination (P = 0.009). ZnT8QA frequency increased from 30% before to 34% during and 41% after vaccination (P = 0.002). Our findings suggest that young (<3 years) along with DQ2/2; 2/X patients were low responders to Pandemrix(®) . As the proportion of DQ2/8 patients <3 years of age decreased after vaccination and the frequencies of GADA and ZnT8QA were enhanced, it cannot be excluded that the vaccine affected clinical onset of type 1 diabetes.

Thyroid autoimmunity in relation to islet autoantibodies and HLA-DQ genotype in newly diagnosed type 1 diabetes in children and adolescents.

AIMS/HYPOTHESIS: The aim of this work was to investigate, in children newly diagnosed with type 1 diabetes: (1) the prevalence of autoantibodies against thyroid peroxidase (TPOAb) and thyroglobulin (TGAb); and (2) the association between TPOAb, TGAb or both, with either islet autoantibodies or HLA-DQ genes. METHODS: Blood samples from 2,433 children newly diagnosed with type 1 diabetes were analysed for TPOAb and TGAb in addition to autoantibodies against arginine zinc transporter 8 (ZnT8RA), tryptophan zinc transporter 8 (ZnT8WA), glutamine zinc transporter 8 (ZnT8QA), glutamic acid decarboxylase (GADA), insulin (IAA), insulinoma-associated protein-2 (IA-2A), HLA-DQA-B1 genotypes, thyroid-stimulating hormone (TSH) and free thyroxine (T4). RESULTS: At type 1 diabetes diagnosis, 12% of the children had thyroid autoantibodies (60% were girls; p < 0.0001). GADA was positively associated with TPOAb (p < 0.001) and with TGAb (p < 0.001). In addition, ZnT8A was associated with both TPOAb (p = 0.039) and TGAb (p = 0.015). DQB1*05:01 in any genotype was negatively associated with TPOAb (OR 0.55, 95% CI 0.37, 0.83, p value corrected for multiple comparisons (p c) = 0.012) and possibly with TGAb (OR 0.55, 95% CI 0.35, 0.87, p c = 0.07). Thyroid autoimmunity in children newly diagnosed with type 1 diabetes was rarely (0.45%) associated with onset of clinical thyroid disease based on TSH and free T4. CONCLUSIONS/INTERPRETATION: GADA and ZnT8A increased the risk for thyroid autoimmunity at the time of clinical diagnosis of type 1 diabetes, while HLA-DQB1*05:01 reduced the risk. However, the associations between thyroid autoimmunity and HLA-DQ genotype were weak and did not fully explain the co-occurrence of islet and thyroid autoimmunity.

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.

Changes in GAD65Ab-specific antiidiotypic antibody levels correlate with changes in C-peptide levels and progression to islet cell autoimmunity.

CONTEXT: The previously reported absence of 65-kDa glutamate decarboxylase antibody (GAD65Ab)-specific antiidiotypic antibodies (anti-Id) in type 1 diabetes (T1D) patients at clinical onset could be due to an inability to mount an antibody response to GAD65Ab or a longitudinal decline in anti-Id levels. OBJECTIVE AND DESIGN: We investigated anti-Id levels in longitudinal samples obtained from T1D patients (n = 41) (clinical diagnosis - 12 months), and latent autoimmune diabetes in adults (LADA) patients (n = 32) who received alum-formulated human recombinant GAD65 (baseline - 12 months). We also determined anti-Id levels in a small cohort of Type 2 diabetes patients during their development of autoimmune T cell responses. RESULTS: At clinical onset T1D patients presented no or low anti-Id levels. However, 22/41 T1D patients showed ≥50% increase in GAD65Ab-specific anti-Id levels during follow-up; peaking at 3 (n = 1), 6 (n = 10), 9 (n = 10), or 12 (n = 1) months. Increasing anti-Id levels marked patients who experienced a temporary increase in C-peptide levels. Anti-Id levels correlated significantly with glycated hemoglobin and C-peptide levels at 6 and 9 months (P values ranged from <0.001 to <0.05). In LADA patients receiving placebo, anti-Id levels declined in seven of nine patients, whereas four of five patients receiving 20 µg alum-formulated human recombinant GAD65 showed increasing anti-Id levels. Changes in anti-Id and C-peptide levels closely correlated (P < 0.0001). The significant decline in anti-Id levels (P = 0.03) in T2D patients developing T cell autoimmune responses supports our hypothesis that declining anti-Id levels are associated with developing islet autoimmunity. CONCLUSIONS: The close association between GAD65Ab-specific anti-Id levels and ß-cell function may provide a novel marker for the progression of autoimmune diabetes.

Temporal trends of HLA genotype frequencies of type 1 diabetes patients in Sweden from 1986 to 2005 suggest altered risk.

The aim of this study was to compare the frequency of human leukocyte antigen (HLA) genotypes in 1-18-year-old patients with type 1 diabetes newly diagnosed in 1986-1987 (n = 430), 1996-2000 (n = 342) and in 2003-2005 (n = 171). We tested the hypothesis that the HLA DQ genotype distribution changes over time. Swedish type 1 diabetes patients and controls were typed for HLA using polymerase chain reaction amplification and allele specific probes for DQ A1* and B1* alleles. The most common type 1 diabetes HLA DQA1*-B1*genotype 0501-0201/0301-0302 was 36% (153/430) in 1986-1987 and 37% (127/342) in 1996-2000, but decreased to 19% (33/171) in 2003-2005 (P \ 0.0001). The 0501-0201/0501-0201 genotype increased from 1% in 1986-1987 to 7% in 1996-2000 (P = 0.0047) and to 5% in 2003-2005 (P > 0.05). This study in 1-18-year-old Swedish type 1 diabetes patients supports the notion that there is a temporal change in HLA risk.

Autoantibody epitopes to the smaller isoform of glutamate decarboxylase do not differ in Swedish and Japanese type 1 diabetes patients and may be associated with high-risk human leucocyte antigen class II alleles.

Type 1 diabetes (T1D) is an autoimmune disease with a strong human leucocyte antigen (HLA) class II association. Depending on geographic locations, the disease-associated HLA class II alleles vary. We evaluated the beta cell-specific autoimmunity reflected in autoantibodies directed to the smaller isoform of glutamate decarboxylase (GAD65) in Japanese and Swedish T1D patients. GAD65Ab epitope specificities were assessed using GAD65-specific recombinant Fab. GAD65Ab epitope specificities did not differ between Swedish and Japanese patients. Only recognition of the MICA-4-defined middle epitope was significantly stronger in the Japanese T1D patient group compared to the Swedish T1D patients (P = 0.001). Binding to the b96.11-defined middle epitope was substantial in both groups and showed significant associations with high-risk HLA class II haplotypes. In the Japanese T1D group the association was with haplotype DRB1*0802-DQB1*0302 (P = 0.0008), while in the Swedish T1D patients binding to the b96.11-defined epitope as associated with the presence of high-risk HLA genotypes DR3-DQB1*0201 and/or DR4-DQB1*0302 (P = 0.02). A significant association between reduction in binding in the presence of recombinant Fab (rFab) DPD and high-risk allele DQB1*0201 was found (P = 0.008) in the Swedish T1D patients only. We hypothesize that epitope-specific autoantibodies effect the peptide presentation on HLA class II molecules by modulating antigen uptake and processing. Molecular modelling of the high-risk HLA class II molecules will be necessary to test whether these different molecules present similar peptide-binding specificities.

Assessment of an empowerment education programme. A randomized study in teenagers with diabetes.

AIMS: To determine the effects of an empowerment programme on glycaemic control and empowerment, and to study the role of parental involvement. METHODS: The wait-list design is a randomized controlled trial lasting for 6 months, after which the control group participate in the same education programme as the intervention group. After 6 months, data from the two groups are analysed together (pre/post). Thirty-two teenagers with Type 1 diabetes (12-17 years) completed an empowerment group education programme, meeting weekly for 6 weeks. They were also offered an extra meeting together with their parents, which resulted in three groups: together with parents, only parents and no parent involvement at all. HbA(1c) was measured before intervention and after 6, 12, 18 and 24 months, and empowerment before, and 6 and 12 months after. RESULTS: HbA(1c) and empowerment were similar in the intervention group and the control group 6 months after intervention. In pre/post analysis, HbA(1c) was significantly higher 6 and 12 months after intervention in teenagers > 14 years (from 8.4% to 9.3%; P < 0.05 to 9.6%; P < 0.01), but returned to baseline 18 months after the programme. In teenagers < or = 14 years of age, HbA(1c) did not change during the study. The teenagers felt more ready for changes after the programme than before (3.9 sd = 0.5 to 4.1 sd = 0.5; P < 0.05). In the teenagers in the group that involved their parents, there was a significant decrease in HbA(1c) 12 and 24 months after intervention, from 8.9% (sd = 1.1) to 7.6% (sd = 1.3; P < 0.05, confidence interval 0.37, 2.26). CONCLUSION: This empowerment programme for teenagers with diabetes showed no positive glycaemic or empowerment effects. Empowerment programmes for diabetic teenagers in early and middle adolescence should include parental involvement.

Longitudinal epitope analysis of insulin-binding antibodies in type 1 diabetes.

Autoantibodies to insulin (IAA) are one of the first markers of the autoimmune process leading to type 1 diabetes (T1D). While other autoantibodies in T1D have been studied extensively, relatively little is known about IAA and their binding specificities, especially after insulin treatment is initiated. We hypothesize that insulin antibodies (IA) that develop upon initiation of insulin treatment differ in their epitope specificities from IAA. We analysed insulin antibody binding specificities in longitudinal samples of T1D patients (n = 49). Samples were taken at clinical diagnosis of disease and after insulin treatment was initiated. The epitope specificities were analysed using recombinant Fab (rFab) derived from insulin-specific monoclonal antibodies AE9D6 and CG7C7. Binding of radiolabelled insulin by samples taken at onset of the disease was significantly reduced in the presence of rFab CG7C7 and AE9D6. rFab AE9D6 competed sera binding to insulin significantly better than rFab CG7C7 (P = 0.02). Binding to the AE9D6-defined epitope in the initial sample was correlated inversely with age at onset (P = 0.005). The binding to the AE9D6-defined epitope increased significantly (P < 0.0001) after 3 months of insulin treatment. Binding to the CG7C7-defined epitope did not change during the analysed period of 12 months. We conclude that epitopes recognized by insulin binding antibodies can be identified using monoclonal insulin-specific rFab as competitors. Using this approach we observed that insulin treatment is accompanied by a change in epitope specificities in the emerging IA.

Epitope analysis of insulin autoantibodies using recombinant Fab.

Autoantibodies to insulin are often the first autoantibodies detected in young children with type 1 diabetes and can be present before the onset of clinical diabetes. These autoantibodies and their epitopes are, however, not well characterized. We explored the use of monoclonal antibodies and their recombinant Fab as reagents for epitope analysis. In this study we cloned and characterized the recombinant Fab of the insulin-specific monoclonal antibody CG7C7. We found the epitope of this antibody to be located predominantly at the A-chain loop of the insulin molecule. The recombinant Fab was then used to compete for insulin binding against insulin autoantibodies present in sera from patients with type 1 or type 1.5 diabetes. In competition experiments with sera positive for autoantibodies to insulin the recombinant Fab significantly reduced the binding to [125I]-insulin by sera of type 1 (n = 35) and type 1.5 diabetes [latent autoimmune diabetes in adults (LADA)] (n = 14) patients (P < 0.0001). We conclude that competition between insulin-specific monoclonal antibodies or their recombinant Fab and insulin autoantibodies should prove useful in the epitope analysis of autoantibodies to insulin.

Multiplicity of the antibody response to GAD65 in Type I diabetes.

Type I diabetes (TID) is an autoimmune disease characterized in part by the presence of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65), among other pancreatic islet antigens. We investigated the independent epitope specificities of these GAD65 antibodies (GAD65Ab) and their combinations in the sera of new onset TID patients and first-degree relatives positive for GAD65Ab. For our analysis, we used four GAD65-specific recombinant Fabs (rFabs) that recognize different conformational determinants of GAD65 located throughout the molecule, including the N-terminal, the middle and the C-terminal regions. We used these epitope-specific rFabs in competition assays to determine the binding specificity of the autoantibodies found in patient sera. Among the 61 sera from newly diagnosed GAD65Ab-positive TID patients GAD65 binding was competed for 23 sera by all four rFabs, 29 by at least two rFabs, and in nine sera were displaced by one or no rFab. In contrast, none of the 24 sera from GAD65Ab-positive first-degree relatives of TID patients were displaced by all four rFabs. When using all four rFabs simultaneously to compete with GAD65Ab binding, binding of sera from TID patients was reduced by an average of 70%. A significantly weaker competition was observed when evaluating sera of GAD65Ab-positive first-degree relatives (P < 0.0001).

Recognition of glutamic acid decarboxylase (GAD) by autoantibodies from different GAD antibody-positive phenotypes.

Autoantibodies against the smaller isoform of glutamic acid decarboxylase (GAD) are markers for Type 1 diabetes. GAD65 autoantibody (GAD65Ab)-positive individuals in the general population are, however, mostly at low risk of developing Type 1 diabetes, suggesting that GAD65Ab phenotypes may be associated with different underlying pathogenic processes. The aim of this study was to test the hypothesis that Type 1 diabetes patients (n = 243; group I), GAD65Ab-positive healthy individuals (n = 28; group II), and healthy first-degree relatives of Type 1 diabetes patients (n = 41; group III) have antibody phenotypes that recognize different GAD65 epitopes. Sera from groups I-III were tested for their binding to GAD65 and GAD67, as well as six different GAD65/67 fusion proteins. Regardless of group, sera reactive to both GAD65 and GAD67 showed broader epitope reactivity than GAD65-specific sera. Furthermore, Type 1 diabetes patients showed a more restricted epitope binding than healthy individuals and first-degree relatives, demonstrating significantly less binding to the N-terminal part of GAD65 and to GAD67. Our analysis demonstrates that the N-terminal part is essential for full antibody binding to GAD65, in particular, to the middle epitope. It is suggested that Type 1 diabetes is associated with restricted GAD65Ab epitope specificity.

Glutamate decarboxylase (GAD) autoantibody epitope shift during the first year of type 1 diabetes.

Autoantibodies in Type 1 diabetes patients may differentiate between glutamate decarboxylase (GAD65) cloned from human, mouse and rat with a significant better binding to the human antigen. A subgroup of 15% (27/183) patients showed significantly better binding to rodent than to human GAD65. The aim of this study was to determine whether the autoantibody specificity would remain anti-rodent during longitudinal follow-up for one year. We observed 1) that the average slope of the difference between human and mouse GAD65 autoantibodies binding increased between onset and after one year, which demonstrates reduced binding to rodent GAD65 and 2) that, in a group followed every third month, 9/11 (80%) children with rodent specific GAD65 autoantibodies at onset converted within one year to preference against human GAD65. This shift in preference was confirmed by significantly lower EC50 values in the initially anti-rodent GAD65 autoantibodies compared to samples taken one year after clinical diagnosis as determined in displacement studies with unlabeled human GAD65. We speculate that the evolution of GAD65 autoantibodies in Type 1 diabetes includes reactivity to a non-human GAD65 N-terminal end conformation. Progression towards Type 1 diabetes is, however, associated with a maturation of the immune response towards human GAD65 autoreactivity.

Leptin concentrations in cord blood in normal newborn infants and offspring of diabetic mothers.

Leptin has been implicated in the regulation of body weight and energy balance; Leptin is produced by adipocytes and placental tissue. Chronic fetal hyperinsulinemia and accelerated fetal growth with increased amounts of body fat are frequent findings in the offspring of diabetic mothers. In this study, we examined whether leptin levels in cord blood of infants of type 1 diabetic mothers (n = 29), gestational diabetic mothers (n = 6 and controls (n = 96) correlated with level of maternal glucose control, maternal leptin level at delivery, gender, fetal and placental size, and C-peptide in cord blood at birth. Leptin was significantly elevated in infants of type 1 diabetic (24.7 ng/ml) and gestational diabetic mothers (29.3 ng/ml) as compared to controls (7.9 ng/ml). C-peptide was also significantly higher in infants of type 1 diabetic (0.91 nmol/l) and gestational diabetic mothers (0.99 nmol/l) vs controls (0.34 nmol/l). Infants of type 1 diabetic mothers with a leptin level in cord blood above the upper normal range, i.e. > 30 ng/ml (n = 13), had an average maternal HbA1c level of 5.4% (normal < 5.5%) that was not different from 5.2% in infants with a leptin level < 30 ng/ml (n = 15). In both neonatal groups of diabetic mothers, leptin in cord blood did not correlate with maternal leptin concentrations, placental weight, birthweight, gender and cord blood C-peptide. In controls, leptin in cord blood was higher in girls than in boys (p = 0.044) and correlated significantly with birthweight (p = 0.41, p < 0.001) and cord blood C-peptide (p = 0.44, p < 0.001) but not with maternal leptin level or placental weight. The 3-4 times higher leptin levels in the offspring of diabetic mothers than normal could reflect increased adipose tissue mass and/or increased contribution from other sources such as placental tissue.

Species-specific autoantibodies in type 1 diabetes.

GAD65 autoantibodies (GAD65Ab) are important markers for type 1 (insulin-dependent) diabetes mellitus. Although most patients have GAD65Ab at the time of clinical diagnosis, there are also GAD65Ab-positive individuals in the population at low risk of developing type 1 diabetes. The aim of this study was to test the hypothesis that the GAD65Ab reactivity to GAD65 cloned from human, mouse, and rat in newly diagnosed type 1 diabetic patients differ from antibody-positive healthy individuals. Sera from 254 new-onset 0- to 34-yr-old type 1 diabetic patients and 270 controls were assayed for their reactivity to human, mouse, and rat GAD65. Among the type 1 diabetic patients there was a significant better binding of human GAD65 compared to either mouse (P = 0.03) or rat GAD65 (P = 0.0005). The preference for human GAD65 increased with increasing age at onset (P = 0.0002). This differentiation was not observed in 88 GAD65Ab-positive control subjects. Our data indicate that recognition of epitopes by GAD65Ab in type 1 diabetes is different from that in nontype 1 diabetes, GAD65Ab-positive individuals.

Age governs gender-dependent islet cell autoreactivity and predicts the clinical course in childhood IDDM.

Most IDDM patients temporarily restore some of their beta-cell function following the initiation of insulin therapy. The aim of this study was to analyse the influence of age, gender, metabolic state at diagnosis and presence of autoantibodies (GAD65 antibodies and ICA) on the duration of the clinical partial remission. In total, 149 consecutively diagnosed IDDM children, 0-16 y old (70F, 79M, mean age 9.5 y) were studied. Partial remission was arbitrarily defined as the period when the insulin dose was below 0.5 U/BW 24 h-1 and HbA1c below 7.5%, and occurred in 119/149 patients with a duration between 1 and 38 months. Cox's regression analysis showed that the factors significantly associated with the duration of remission were age, gender, interaction between age and gender, ICA and a high initial HbA1c, whereas GAD65Ab had no influence. Young boys had the shortest remission period, while adolescent boys had the longest, as compared to young and adolescent girls. The ICA-negative patients (n = 42) had a longer remission period (median 9.7 months) than the ICA-positive children (n = 107; 5.0 months; p = 0.0001), regardless of GAD65Ab status. We speculate that the relative insulin resistance, which is more pronounced in pubertal girls than in boys, may be associated with a more rapid increase of exogenous insulin requirement. These findings are important when evaluating the effect of islet cell autoreactivity on the clinical course of IDDM in children.

Transient hypertriglyceridemia of infancy.

A premature boy who had suffered from IRDS, bronchopulmonary dysplasia and retinopathy of prematurity developed massive hypertriglyceridemia (48.1 mmol/L) together with moderate hypercholesterolemia (12.6 mmol/L) at 5 months of age. Lipoprotein electrophoresis revealed a marked elevation of the level of the very low density lipoprotein fraction. There was a moderate decrease in the activity of a lipolytic enzyme, lipoprotein lipase (LPL). The child had neither liver or renal disorder nor any inflammatory disease. The hyperlipidemia disappeared spontaneously at the age of 3 years. The cause of the decreased LPL activity could not be established. A partial genetic deficiency in lipoprotein lipase appears the most likely explanation, since no signs of secondary lowering of LPL activity could be found.

Radioimmunoassays for glutamic acid decarboxylase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human ligands.

Autoantibodies are an important marker of human autoimmune diseases and the development of simple, precise and reproducible immunoassays to detect autoantibodies is important to our understanding of human autoimmunity. GAD65 autoantibodies occur frequently in insulin-dependent diabetic patients and is a useful marker for IDDM. A RIA to detect immunoreactive GAD65 has not been described. In the present study we describe a semi-automated fluid-phase immunoassay for the rapid detection of GAD65 autoantibodies in human serum. We also developed a sensitive RIA to determine immunoreactive human GAD65 in biological fluids and in vitro cell systems. Using in vitro translated recombinant human GAD65 in a multiwell-adapted procedure, our GAD65Ab RIA combines high specificity and sensitivity with a high capacity to analyze a large number of samples. In this report the three critical steps in the GAD65Ab RIA, DNA preparation, in vitro translation and immunoprecipitation, have been optimized. In our RIA, GAD65Ab were detected in 116/155 (75%) new onset Swedish IDDM children and in 1/85 (1.2%) healthy controls. In an immunoassay to detect autoantibodies against the proinsulin converting enzyme 2 (PC-2) no such antibodies were detected in IDDM patients. In the GAD65 RIA the lower detection limit was 2 ng/ml (31 fmol/ml). Our data demonstrate that autoantigen radioligands produced by in vitro translation are useful in RIA for autoantibodies and autoantigens in studies of human autoimmunity.

Lipoprotein alterations in children with bacterial meningitis.

Abnormalities in serum lipids, including hypertriglyceridemia, are common during infectious disorders. However, the lipoprotein pattern during infections, particularly in children, has been investigated to only a limited extent. We have monitored alterations in serum lipoproteins in eight children with a severe bacterial infection (meningitis) by a quantitating method measuring cholesterol and triglycerides in each major class. The levels of triglycerides in serum and in low-density lipoproteins were markedly elevated during the infection, whereas the amount of cholesterol in high-density lipoproteins was decreased. The cholesterol to triglyceride ratio was decreased in low-, as well as in high-density lipoproteins. These lipoprotein abnormalities may, at least in part, be explained by a depressed lipolytic activity of lipoprotein lipase, the key enzyme for removal of triglycerides in man. Serum triglycerides and the levels of cholesterol in high-density lipoproteins, as well as the ratio between these parameters, may be used as indicators of inflammatory activity.