Radiobinding Research Articles

Population Screening for T1D and Celiac Disease—Autoimmunity Screening for Kids (ASK)

Early detection can prevent morbidity associated with type 1 diabetes (T1D) and celiac disease (CD). ASK is a 4-year program with the goal to screen Denver metro-area children for pre-symptomatic T1D and CD, increase public awareness of these diseases, and provide evidence for universal screening. We are reporting first-ever prevalence data for pre-symptomatic T1D and CD in the U.S. general population children aged 2-17 y. In 2017, ASK approached ∼15,000 children for consent, screened 5090 with results available for 4915. Study participant age, sex, and race/ethnicity closely reflected Denver’s general population; 4% had a T1D first-degree relative (FDR). Standard radiobinding assays (RBA) and more specific electrochemiluminescence (ECL) assays for autoantibodies to insulin, GAD, IA-2, ZnT8 and transglutaminase (TGA) were used for screening and confirmation. Children with confirmed persistent islet autoantibodies received follow-up with education to prevent DKA, psychological support, and referrals to prevention trials or clinical services. Multiple islet autoantibodies, predicting a 44% 5-y risk of T1D, were found in 32 (0.7%) children; of the 30 children retested to date, all remained persistently positive, six developed dysglycemia and one progressed to T1D. A single islet autoantibody, confirmed as high-affinity by ECL (29% 5-y risk of T1D), was found in 27 (0.5%); 18/19 children retested remained positive, 5 developed dysglycemia and one T1D. Over 86% (50/59) of the screening-detected children at risk for T1D did not have an FDR with T1D. The prevalence of TGA by both RBA and ECL was 2.2% and persistent in 74/76 (97%) children retested so far. This novel population-based screening program for the two most common autoimmune diseases of childhood reports high prevalence of pre-symptomatic T1D and CD in Denver children. Prospective follow-up of screening-detected cases for clinical outcomes and cost-effectiveness analysis will inform potential future universal screening. Disclosure C.R. Geno Rasmussen: None. M. Rewers: None. J. Baxter: None. K. Waugh: None. A. Steck: None. B.I. Frohnert: None. L. Yu: None. E. Liu: None.

Gut Microbiome Dysbiosis and Increased Intestinal Permeability in Australian Children with Islet Autoimmunity and Type 1 Diabetes

We aimed to characterise the gut microbiome and small intestinal permeability in children with islet autoimmunity and type 1 diabetes (T1D). 88 participants, median age 11.8 (IQR 8.1-13.3) years at baseline were followed 6 monthly for a median of 13 months (range 2-34). 18 had islet autoimmunity, 29 recent-onset T1D, and 41 were age and gender-matched unrelated or sibling controls. Stool samples were collected (Omnigene kit) for 16S rRNA gene sequencing. Small intestinal permeability was measured by blood lactulose:rhamnose ratio (L/R), 90 minutes after drinking a 5gm lactulose/1gm rhamnose solution. Antibodies to insulin, GAD, IA2 and ZnT8 were assayed using IASP standardized radiobinding assays. Statistical analysis used linear mixed models. Differential abundance analysis used limma with total-sum scaling. Children with islet autoimmunity or T1D had gut microbiome dysbiosis with reduced α diversity and different β diversity compared with controls. Progressors from islet autoimmunity to T1D had lower observed richness (OTUs) (p=0.01) and different β diversity (Adonis, P=0.004), compared with non-progressors. Genus Prevotella, with anti-inflammatory properties, was less abundant in children with T1D (p<0.005) or ≥ 2 islet antibodies (p<0.005) than in controls. Diet did not explain this difference. L/R was higher in T1D than sibling controls (mean difference +3.03±1.02, p=0.003). Progressors had higher L/R than non-progressors (+4.74±1.97, p=0.01) and sibling controls (+4.62±1.69, p=0.006). There was a trend towards a relationship between higher L/R and lower observed richness in the microbiome over time in children with ≥ 2 islet antibodies or T1D (coeff -0.0617, 95% CI -0.1254, 0.002 P=0.058). Australian children progressing from islet autoimmunity to T1D have gut dysbiosis and increased intestinal permeability. This observation supports a mechanism underlying progression to T1D. Disclosure J.E. Harbison: None. A.J. Roth-Schulze: None. S.C. Barry: None. C.D. Tran: None. K. Ngui: None. M.A. Penno: None. J. Wentworth: None. P.G. Colman: None. R. Thomson: None. M.E. Craig: None. A.T. Papenfuss: None. L. Giles: None. L.C. Harrison: None. J. Couper: None.

Electrochemiluminescence Assays for Human Islet Autoantibodies.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 °C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 °C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 °C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 °C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

First trimester enterovirus IgM and beta cell autoantibodies in mothers to children affected by type 1 diabetes autoimmunity before 7 years of age

BackgroundAutoimmune (type 1) diabetes (T1D) is a frequent chronic disease in children and adolescents globally. Gestational enterovirus (EV) infections have been associated with an increased risk for T1D in the offspring. We test the hypothesis that EV infections during the first trimester were associated with beta cell autoantibodies in mothers of children who developed islet autoantibodies before 7 years of age. Materials and methodsLocal registries were used to identify mothers to children born 2000–2007 who developed either beta cell autoantibodies or T1D during follow up. Serum samples from the first trimester were located in the Biobank. A total of 448 index mothers were identified and compared to 891 matched control mothers. EV-IgM was determined in a capture enzyme immunoassay. Beta cell autoantibodies were analyzed in standard radio binding assays. ResultsThe frequency of EV-IgM in index mothers was 20% (89/448), which did not differ from the control mothers 20% (175/891) (p = 0.922). Index mothers had multiple beta cell autoantibodies more often than control mothers (p = 0.037).Beta cell autoantibodies were increased during the November–April winter months in index compared to control mothers (p = 0.022). The observed difference was possibly explained by the months of February-April (p = 0.014). Concomitant EV-IgM and beta cell autoantibodies tended to be more common among index compared to control mothers (p = 0.039). ConclusionEV-IgM during the first trimester may be associated with beta cell autoantibodies in mothers to children who developed either beta cell autoantibodies or T1D before 7 years of age.

Osteoprotegerin autoantibodies do not predict low bone mineral density in middle-aged women

PurposeAutoantibodies against osteoprotegerin (OPG) have been associated with osteoporosis. The aim was to develop an immunoassay for OPG autoantibodies and test their diagnostic usefulness of identifying women general population with low bone mineral density. MethodsIncluded were 698 women at mean age 55.1years (range 50.4–60.6) randomly selected from the general population. Measurement of wrist bone mineral density (g/cm2) was performed of the non-dominant wrist by dual-energy X-ray absorptiometry (DXA). A T-score<−2.5 was defined as having a low bone mineral density. Measurements of OPG autoantibodies were carried by radiobinding assays. Cut-off levels for a positive value were determined from the deviation from normality in the distribution of 398 healthy blood donors representing the 99.7th percentile. ResultsForty-five of the 698 (6.6%) women were IgG-OPG positive compared with 2 of 398 (0.5%) controls (p<0.0001) and 35 of the 698 (5.0%) women had a T-score<−2.5. There was no difference in bone mineral density between IgG-OPG positive (median 0.439 (range 0.315–0.547) g/cm2) women and IgG-OPG negative (median 0.435 (range 0.176–0.652) g/cm2) women (p=0.3956). Furthermore, there was neither a correlation between IgG-OPG levels and bone mineral density (rs=0.1896; p=0.2068) nor T-score (rs=0.1889; p=0.2086). Diagnostic sensitivity and specificity of IgG-OPG for low bone mineral density were 5.7% and 92.9%, and positive and negative predictive values were 7.4% and 90.8%, respectively. ConclusionElevated OPG autoantibody levels do not predict low bone mineral density in middle-aged women selected from the general population.

Antibody Affinity Against 2009 A/H1N1 Influenza and Pandemrix Vaccine Nucleoproteins Differs Between Childhood Narcolepsy Patients and Controls.

Increased narcolepsy incidence was observed in Sweden following the 2009 influenza vaccination with Pandemrix®. A substitution of the 2009 nucleoprotein for the 1934 variant has been implicated in narcolepsy development. The aims were to determine (a) antibody levels toward wild-type A/H1N1-2009[A/California/04/2009(H1N1)] (NP-CA2009) and Pandemrix-[A/Puerto Rico/8/1934(H1N1)] (NP-PR1934) nucleoproteins in 43 patients and 64 age-matched controls; (b) antibody affinity in reciprocal competitive assays in 11 childhood narcolepsy patients compared with 21 age-matched controls; and (c) antibody levels toward wild-type A/H1N1-2009[A/California/04/2009(H1N1)] (H1N1 NS1), not a component of the Pandemrix vaccine. In vitro transcribed and translated 35S-methionine-labeled H1N1 influenza A virus proteins were used in radiobinding reciprocal competition assays to estimate antibody levels and affinity (Kd). Childhood patients had higher NP-CA2009 (p = 0.0339) and NP-PR1934 (p = 0.0246) antibody levels compared with age-matched controls. These childhood controls had lower NP-CA2009 (p = 0.0221) and NP-PR1934 (p = 0.00619) antibodies compared with controls 13 years or older. In contrast, in patients 13 years or older, the levels of NP-PR1934 (p = 0.279) and NP-CA2009 (p = 0.0644) antibodies did not differ from the older controls. Childhood antibody affinity (Kd) against NP-CA2009 was comparable between controls (68 ng/mL) and patients (74 ng/mL; p = 0.21) with NP-CA2009 and NP-PR1934 displacement (controls: 165 ng/mL; patients: 199 ng/mL; p = 0.48). In contrast, antibody affinity against NP-PR1934 was higher in controls with either NP-PR1934 (controls: 9 ng/mL; patients: 20 ng/mL; p = 0.0031) or NP-CA2009 (controls: 14 ng/mL; patients: 23 ng/mL; p = 0.0048). A/H1N1-NS1 antibodies were detected in 0/43 of the narcolepsy patients compared with 3/64 (4.7%) controls (p = 0.272). Similarly, none (0/11) of the childhood patients and 1/21 (4.8%) of the childhood controls had A/H1N1-NS1 antibodies. The higher antibody affinities against NP-PR1934 in controls suggest better protection against wild-type virus. In contrast, the reduced NP-PR1934 antibody affinities among childhood narcolepsy patients suggest poor protection from the wild-type A/H1N1 virus and possibly increased risk for viral damage.

T1D Autoantibodies: room for improvement?

Type 1 diabetes (T1D) is now predictable by measuring major islet autoantibodies (IAbs) against insulin and other pancreatic β cells proteins including GAD65 (GADA), islet antigen 2 (IA-2A), and zinc transporter 8 (ZnT8A). The assay technology for IAbs has made great progress; however, several important aspects still need to be addressed and improved. Currently a radio-binding assay has been well established as the 'gold' standard assay for all four IAbs. New generation of nonradioactive IAb assay with electrochemiluminescence technology has been shown to further improve sensitivity and disease specificity. Recently, multiplexed assays have opened the possibility of more efficient screening in large populations. Identification of potential new autoantibodies to neo-antigens or neo-epitopes posttranslational modification is a new important field to be explored. Individuals having a single positive autoantibody are at low risk for progression to T1D, whereas individuals expressing two or more positive autoantibodies, especially on multiple tests over time, have nearly 100% risk of developing clinical T1D when followed for over two decades. More efficient and cost effective IAb assays will hopefully lead to point-of-care screening in the general population.

Avoidance of Cow's Milk–Based Formula for At-Risk Infants Does Not Reduce Development of Celiac Disease: A Randomized Controlled Trial

Feeding during the first months of life might affect risk for celiac disease. Individuals with celiac disease or type 1 diabetes have been reported to have high titers of antibodies against cow's milk proteins. Avoidance of cow's milk-based formula for infants with genetic susceptibility for type 1 diabetes reduced the cumulative incidence of diabetes-associated autoantibodies. We performed a randomized controlled trial in the same population to study whether weaning to an extensively hydrolyzed formula reduced the risk of celiac disease autoimmunity or celiac disease. We performed a double-blind controlled trial of 230 infants with HLA-defined predisposition to type 1 diabetes and at least 1 family member with type 1 diabetes. The infants were randomly assigned to groups fed a casein hydrolysate formula (n= 113) or a conventional formula (control, n= 117) whenever breast milk was not available during the first 6-8 months of life. Serum samples were collected over a median time period of 10 years and analyzed for antibodies to tissue transglutaminase (anti-TG2A) using a radiobinding assay, to endomysium using an immunofluorescence assay, and antibodies to a deamidated gliadine peptide using an immunofluorometry assay. Duodenal biopsies were collected if levels of anti-TG2A exceeded 20 relative units. Cow's milk antibodies were measured during the first 2 years of life. Of the 189 participants analyzed for anti-TG2A, 25 (13.2%) tested positive. Of the 230 study participants observed, 10 (4.3%) were diagnosed with celiac disease. We did not find any significant differences at the cumulative incidence of anti-TG2A positivity (hazard ratio, 1.14; 95% confidence interval, 0.51-2.54) or celiac disease (hazard ratio, 4.13; 95% confidence interval, 0.81-21.02) between the casein hydrolysate and cow's milk groups. Children who developed celiac disease had increased titers of cow's milk antibodies before the appearance of anti-TG2A or celiac disease. In a randomized controlled trial of 230 infants with genetic risk factors for celiac disease, we did not find evidence that weaning to a diet of extensively hydrolyzed formula compared with cow's milk-based formula would decrease the risk for celiac disease later in life. Increased titers of cow's milk antibody before anti-TG2A and celiac disease indicates that subjects with celiac disease might have increased intestinal permeability in early life. ClinicalTrials.gov Number: NCT00570102.

An Increased Diagnostic Sensitivity of Truncated GAD65 Autoantibodies in Type 1 Diabetes May Be Related to HLA-DQ8.

N-terminally truncated (96-585) GAD65 (tGAD65) autoantibodies may better delineate type 1 diabetes than full-length GAD65 (fGAD65) autoantibodies. We aimed to compare the diagnostic sensitivity and specificity between fGAD65 and tGAD65 autoantibodies for type 1 diabetes in relation to HLA-DQ. Sera from children and adolescents with newly diagnosed type 1 diabetes (n = 654) and healthy control subjects (n = 605) were analyzed in radiobinding assays for fGAD65 (fGADA), tGAD65 (tGADA), and commercial 125I-GAD65 (RSRGADA) autoantibodies. The diagnostic sensitivity and specificity in the receiver operating characteristic curve did not differ between fGADA and tGADA. At the optimal cutoff, the diagnostic sensitivity for fGADA was lower than tGADA at similar diagnostic specificities. In 619 patients, 64% were positive for RSRGADA compared with 68% for fGADA and 74% for tGADA. Using non-DQ2/non-DQ8 patients as reference, the risk of being diagnosed with fGADA and tGADA was increased in patients with DQ2/2 and DQ2/8. Notably, logistic regression analysis suggested that DQ8/8 patients had an increased risk to be diagnosed with tGADA (P = 0.003) compared with fGADA (P = 0.09). tGADA had a higher diagnostic sensitivity for type 1 diabetes than both fGADA and RSRGADA. As DQ8/8 patients represent 10-11% of patients with newly diagnosed type 1 diabetes <18 years of age, tGADA analysis should prove useful for disease classification.

Role of proneurotensin as marker of paediatric coeliac disease.

Neurotensin (NT) is a gut hormone functioning proinflammatory through nuclear factor kappa B (NF-κB) and interleukin (IL)-8 secretion or anti-inflammatory through epidermal growth factor receptors. NT mRNA is down-regulated in duodenal biopsies of children with untreated coeliac disease. The aim of this study was to investigate if plasma pro-NT levels correlated with the degree of intestinal mucosal damage and tissue transglutaminase autoantibody (tTGA) levels in children with coeliac disease. Fasting plasma samples from 96 children with coeliac disease and 89 non-coeliac disease controls were analysed for NT precursor fragment pro-NT 1-117 by a chemiluminometric immunoassay. Pro-NT levels were compared with NT mRNA from duodenal biopsies, assessed previously with quantitative polymerase chain reaction (PCR). Illumina core exome arrays were used for human leucocyte antigen (HLA) typing and the Marsh criteria applied to score mucosal damage. Tissue TGA was measured by radio binding assay. A general linear model compared pro-NT levels with diagnosis of coeliac disease, Marsh score and HLA DQ haplotype. Spearman's rank test was used to compare pro-NT levels with tTGA, age and duodenal NT mRNA levels, respectively. Plasma pro-NT levels were elevated in children with coeliac disease (median 23 pmol/l higher, P = 0·003) and in those with severe intestinal mucosal damage (median 24 pmol/l higher for ≥ Marsh 3b versus not, P = 0·0004). Pro-NT levels correlated further with tTGA (r2 = 0·22, P = 0·002), but not with duodenal NTS mRNA levels (r2 = -0·12, P = 0·14). Pro-NT was not associated with any of the HLA risk-haplotypes. Elevated peripheral pro-NT levels reflect more severe forms of active coeliac disease, indicating a potential role of NT in intestinal inflammation.

3 Screen islet cell autoantibody ELISA: A sensitive and specific ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8

3 Screen, a new ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8, has been developed and evaluated.In the assay serum samples were incubated (overnight; 2–8°C) in ELISA plate wells coated with GAD65, IA-2 and ZnT8 followed by a wash step and incubation with biotinylated GAD65, IA-2 and ZnT8 (1h; 2–8°C,). The assay was completed by addition of streptavidin-peroxidase and tetramethylbenzidine. Samples tested in the 3 Screen were also analysed in ELISAs and radiobinding assays for the three individual autoantibodies.129/132 (98%) samples from newly diagnosed T1DM children and 1/100 non-diabetic children controls were positive in 3 Screen. There was good agreement between 3 Screen and the individual autoantibody assays. Dilution of positive samples showed good linearity characteristics. In the 2015 Islet Autoantibody Standardization Program 3 Screen achieved 94% sensitivity, 95.6% specificity and 0.948 area under curve by ROC analysis.3 Screen provides a simple and sensitive method for combined measurement of three major diabetes associated autoantibodies in a single sample. The assay should be a useful tool for large scale population screening for individuals at risk of developing T1DM.

3 Screen ELISA for High-Throughput Detection of Beta Cell Autoantibodies in Capillary Blood

Testing for beta cell autoantibodies is used for wide-scale identification of early stages of type 1 diabetes. This requires suitable screening assays. We aimed to establish screening that utilized a first step assay (3 Screen) able to detect autoantibodies to the target antigens glutamic acid decarboxylase-65 (GAD), insulinoma-associated antigen 2 (IA-2), and zinc transporter 8 (ZnT8) to identify children positive for multiple beta cell autoantibodies. An ELISA format was used where plates were coated with a mixture of recombinant GAD, IA-2, and ZnT8325W/R-dimer molecules. The performance was determined in venous blood from 686 first-degree relatives of patients with type 1 diabetes, and 200 patients at onset of type 1 diabetes, and applied as a screening assay in capillary blood from 33,639 general population children. The 3 Screen assay sensitivity for detecting autoantibody-positive patients at onset of type 1 diabetes was similar to that achieved by separate radiobinding assays (RBAs) for antibodies to GAD, IA-2, and ZnT8. Results in venous and capillary serum were correlated (R = 0.987). At a threshold corresponding to the 98th centile (29.1 U/mL) of all 33,639 capillary samples, the 3 Screen was positive in 123 samples with two or more RBA-positive antibodies to insulin, GAD, IA-2, or ZnT8, 146 with one antibody, and 479 that were RBA negative for beta cell autoantibodies. A 3 Screen ELISA was developed that was suitable for first step screening of multiple beta cell autoantibodies in capillary blood.

Islet autoantibodies present in association with Ljungan virus infection in bank voles (Myodes glareolus) in northern Sweden

Bank voles are known reservoirs for Puumala hantavirus and probably also for Ljungan virus (LV), a suggested candidate parechovirus in type 1 diabetes etiology and pathogenesis. The aim of this study was to determine whether wild bank voles had been exposed to LV and if exposure associated to autoantibodies against insulin (IAA), glutamic acid decarboxylase 65 (GADA), or islet autoantigen-2 (IA-2A). Serum samples from bank voles (Myodes glareolus) captured in early summer or early winter of 1997 and 1998, respectively, were analyzed in radio binding assays for antibodies against Ljungan virus (LVA) and Puumala virus (PUUVA) as well as for IAA, GADA, and IA-2A. LVA was found in 25% (189/752), IAA in 2.5% (18/723), GADA in 2.6% (15/615), and IA-2A in 2.5% (11/461) of available bank vole samples. LVA correlated with both IAA (P = 0.007) and GADA (P < 0.001), but not with IA-2A (P = 0.999). There were no correlations with PUUVA, detected in 17% of the bank voles. Compared to LVA negative bank voles, LVA positive animals had higher levels of both IAA (P = 0.002) and GADA (P < 0.001), but not of IA-2A (P = 0.205). Levels of LVA as well as IAA and GADA were higher in samples from bank voles captured in early summer. In conclusion, LVA was detected in bank voles and correlated with both IAA and GADA but not with IA-2A. These observations suggest that exposure to LV may be associated with islet autoimmunity. It remains to be determined if islet autoantibody positive bank voles may develop diabetes in the wild. J. Med. Virol. 89:24-31, 2017. © 2016 Wiley Periodicals, Inc.

Capillary blood islet autoantibody screening for identifying pre-type 1 diabetes in the general population: design and initial results of the Fr1da study

IntroductionType 1 diabetes can be diagnosed at an early presymptomatic stage by the detection of islet autoantibodies. The Fr1da study aims to assess whether early staging of type 1 diabetes...

Original Anticonvulsant Urea Derivative Alters the Properties of Benzodiazepine Receptors Central and Peripheral Types in the Cerebral Cortex of Heavy Drinkers Rats

Objective: studies of anticonvulsants have a stimulating effect on neuronal receptors; in particular GABAA/ benzodiazepine receptor complex (GABAA/BzDR) can be the basis for the development of new approaches to the treatment of alcohol withdrawal syndrome (AWS) and alcohol addiction. Benzodiazepine receptors (BzDRs) of the cerebral cortex of Wistar male rats with a different preference for alcohol and BzDRs in the brain “heavy drinkers” rats, treated with original anticonvulsant meta-chloro-benzhydryl urea (m-ch-BHU) were examined in these study. Materials and methods: Wistar male rats (n=250) were used in an experimental model of alcoholism. Properties of the BzDRs of the “central” (synaptic) and “peripheral” (mitochondrial) type were examined in membrane fractions obtained from the cerebral cortex of rats under various experimental groups using radio receptor binding assays (RRA) selective ligands: [3H]flunitrazepam and [3H] Ro5-4864 to these receptors respectively. Results: our study has shown that the binding affinity of [3H] flunitrazepam and [3H] Ro5-4864 with synaptosomal and mitochondrial membranes was decreased, but capacity of receptors was increased in the cerebral cortex of rats prefer alcohol. Administration of m-ch-BHU increased affinity of BzDRs in the cortex of “heavy drinkers” rats that can enhance the mediation of GABA in the brain of these animals. Conclusion: Our data showed that m-ch-BHU has a stimulating effect on GABAA/BzDRs in the brains of “heavy drinkers” rats and may provide a new pharmacotherapeutic approach to the treatment of alcohol addiction.

Antibodies to post-translationally modified insulin in type 1 diabetes.

Insulin is the most specific beta cell antigen and a potential primary autoantigen in type 1 diabetes. Insulin autoantibodies (IAAs) are the earliest marker of beta cell autoimmunity; however, only slightly more than 50% of children and even fewer adults newly diagnosed with type 1 diabetes are IAA positive. The aim of this investigation was to determine if oxidative post-translational modification (oxPTM) of insulin by reactive oxidants associated with islet inflammation generates neoepitopes that stimulate an immune response in individuals with type 1 diabetes. oxPTM of insulin was generated using ribose and various reactive oxygen species. Modifications were analysed by SDS-PAGE, three-dimensional fluorescence and MS. Autoreactivity to oxPTM insulin (oxPTM-INS) was observed by ELISA and western blotting, using sera from participants with type 1 or type 2 diabetes and healthy controls as probes. IAA was measured using the gold-standard radiobinding assay (RBA). MS of oxPTM-INS identified chlorination of Tyr16 and Tyr26; oxidation of His5, Cys7 and Phe24; and glycation of Lys29 and Phe1 in chain B. Significantly higher binding to oxPTM-INS vs native insulin was observed in participants with type 1 diabetes, with 84% sensitivity compared with 61% sensitivity for RBA. oxPTM-INS autoantibodies and IAA co-existed in 50% of those with type 1 diabetes. Importantly 34% of those with diabetes who were IAA negative were oxPTM-INS positive. Altogether, 95% of participants with type 1 diabetes presented with autoimmunity to insulin by RBA, oxPTM-INS or both. Binding to oxPTM-INS was directed towards oxPTM-INS fragments with slower mobility than native insulin. These data suggest that oxPTM-INS is a potential autoantigen in individuals with new-onset type 1 diabetes.

Islet autoantibody phenotypes and incidence in children at increased risk for type 1 diabetes.

Autoantibodies that precede type 1 diabetes frequently develop in early childhood and target distinct beta cell proteins. The aim of this study was to determine the heterogeneity of islet autoantibody development and fate. The ages of development of insulin autoantibodies (IAA) and GAD autoantibodies (GADA), followed by multiple islet autoantibodies and progression to diabetes were examined in 2,441 children participating in two German birth cohorts. In 218 children who developed islet autoantibodies, the first islet autoantibody-positive sample was characterised by single IAA in 80 (37%), multiple islet autoantibodies in 68 (31%) and single GADA in 63 (29%) children. Of the children who were single antibody positive at seroconversion, 35 (44%) IAA-positive and 15 (24%) GADA-positive children developed multiple islet autoantibodies. Single persistent antibodies had heterogeneous affinities; GADA were also heterogeneous in their binding to N-terminally truncated GAD65 and in an ELISA. Progression to diabetes occurred in >50% of children within 10 years in all groups that developed multiple islet autoantibodies and in 44% of children with persistent single high-affinity IAA or persistent single GADA that were positive in both a radiobinding assay and ELISA. The earliest autoantibody development was seen in children with single IAA that progressed to multiple islet autoantibodies or in those with persistent high-affinity single IAA, with a sharp peak in incidence observed at age 9 months. The peak incidence occurred at age 2 years for children who underwent seroconversion directly to multiple islet autoantibodies and at 5 years for children who first seroconverted to GADA and subsequently developed other autoantibodies. Seroconversion to low-affinity IAA or persistent single GADA occurred at a low incidence after the age of 9 months. Children of different ages have differing susceptibilities to autoimmunisation against specific beta cell autoantigens.

Reactivity to N-Terminally Truncated GAD65(96-585) Identifies GAD Autoantibodies That Are More Closely Associated With Diabetes Progression in Relatives of Patients With Type 1 Diabetes.

GAD autoantibodies (GADAs) identify individuals at increased risk of developing type 1 diabetes, but many people currently found to be GADA positive are unlikely to progress to clinical disease. More specific GADA assays are therefore needed. Recent international workshops have shown that the reactivity of sera from healthy donors varies according to assay type and indicated that the use of N-terminally truncated GAD65 radiolabels in GADA radiobinding assays is associated with higher specificity. To determine whether a radiobinding assay using radiolabeled GAD65(96-585) identified individuals who are at higher risk of developing diabetes, samples from recent-onset patients and GADA-positive first-degree relatives participating in the Bart's-Oxford type 1 diabetes family study were reassayed with full-length or N-terminally truncated GAD using the National Institute of Diabetes and Digestive and Kidney Diseases harmonized protocol. The sensitivity in patients was the same with both labels, but fewer relatives retested positive with truncated GAD. Among relatives who progressed to diabetes, similar proportions were found to be GADA positive when tested with either label, but because of their higher specificity the cumulative risk of diabetes was higher in those with autoantibodies to GAD65(96-585). Autoantibodies to GAD65(96-585) in relatives are more closely associated with diabetes risk than those to full-length GAD, suggesting that assays using N-terminally truncated GAD should be used to select participants for intervention trials.

Detection of Antibodies Directed to the N-Terminal Region of GAD Is Dependent on Assay Format and Contributes to Differences in the Specificity of GAD Autoantibody Assays for Type 1 Diabetes

GAD autoantibodies (GADAs) are sensitive markers of islet autoimmunity and type 1 diabetes. They form the basis of robust prediction models and are widely used for the recruitment of subjects at high risk of type 1 diabetes to prevention trials. However, GADAs are also found in many individuals at low risk of diabetes progression. To identify the sources of diabetes-irrelevant GADA reactivity, we analyzed data from the 2009 and 2010 Diabetes Autoantibody Standardization Program GADA workshop and found that binding of healthy control sera varied according to assay type. The characterization of control sera found positive by radiobinding assay (RBA), but negative by ELISA, showed that many of these sera reacted to epitopes in the N-terminal region of the molecule. This finding prompted development of an N-terminally truncated GAD65 radiolabel, 35S-GAD65(96–585), which improved the performance of most GADA RBAs participating in an Islet Autoantibody Standardization Program GADA substudy. These detailed workshop comparisons have identified a source of disease-irrelevant signals in GADA RBAs and suggest that N-terminally truncated GAD labels will enable more specific measurement of GADAs in type 1 diabetes.