A High-throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19.

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.

Expression-based Genome-wide Association Study Links OPN and IL1-RA With Newly Diagnosed Type 1 Diabetes in Children.

CONTEXT: Islet autoantibodies (IAbs) are currently the most reliable indicators of islet autoimmunity. However, IAbs do not fully meet the need for the prediction and intervention of type 1 diabetes (T1D). Serological proteins should be great sources for biomarkers. OBJECTIVE: This work aimed to identify new proteomic biomarkers with the technology of an expression-based genome-wide association study (eGWAS) in children newly diagnosed with T1D. METHODS: In an attempt to identify additional biomarkers, we performed an eGWAS using microarray data from 169 arrays of the pancreatic islets of T1D rodents (78 T1D cases and 91 controls). We ranked all 16 099 protein-coding genes by the likelihood of differential expression in the pancreatic islets. Our top 20 secreted proteins were screened in 170 children including 100 newly diagnosed T1D, and 50 type 2 diabetes (T2D) and 20 age-matched healthy children. With 6 proteins showing significance, we further conducted a validation study using the second independent set of 400 samples from children including 200 newly diagnosed with T1D, 100 T2D, and 100 age-matched controls. RESULTS: We identified 2 serum proteins that were significantly changed in T1D vs both control and T2D, and 5 serum proteins were significantly changed both in T1D and T2D vs control. Serum osteopontin (OPN) levels were uniquely higher in T1D (T1D vs controls, P = 1.29E-13 ~ 9.38E-7, T1D vs T2D, P = 2.65E-8 ~ 1.58E-7) with no difference between T2D and healthy control individuals. Serum interleukin 1 receptor antagonist (IL-1RA) levels were lower in T1D compared both with T2D (P = 3.36E-9~0.0236) and healthy participants (P = 1.09E-79 ~ 2.00E-12). CONCLUSION: Our results suggest that OPN and IL1-RA could be candidates for useful biomarkers for T1D in children.

High-Throughput Multiplex Electrochemiluminescence Assay Applicable to General Population Screening for Type 1 Diabetes and Celiac Disease.

Objective: Large-scale screening of the general population for islet autoantibodies (IAbs) to detect type 1 diabetes (T1D) has started worldwide. The standard screening method of separate radio-binding assay (RBA) for each IAb is an inefficient bottleneck. Furthermore, most positive results by RBA in screening of general population individuals without a clinical diagnosis of T1D are low-affinity and not predictive of future diabetes. Research Design and Methods: We have developed and validated a novel 6-Plex assay based on electrochemiluminescence (ECL) technology that combines in a single well high-affinity IAbs (to insulin, GAD, IA-2, and ZnT8), transglutaminase autoantibodies for celiac disease, and severe acute respiratory syndrome coronavirus 2 antibodies. The Autoimmunity Screening for Kids (ASK) provided 880 serum samples, from 828 children aged 1-17 years without diabetes who were previously tested for IAbs using single ECL assays and RBA assays. Results: Levels of all six antibodies in the 6-Plex ECL assay correlated well with respective single ECL assay levels. Similar to single ECL assays, the 6-Plex ECL assay positivity was congruent with the RBA in 95% (35/37) of children who later developed T1D and in 88% (105/119) high-risk children with multiple IAbs. In contrast, only 56% (86/154, P < 0.0001) of children with persistent single IAb by RBA were found to be positive by 6-Plex ECL assay. Of 555 samples negative for all IAbs by RBA, few (0.2%-0.5%) were positive at low levels in the 6-Plex ECL assay. Conclusions: The study demonstrated that the 6-Plex ECL assay compares favorably to the standard RBAs in terms of disease specificity for general population screening in children. The 6-Plex ECL assay was therefore adopted as the primary screening tool in the general population screening ASK program with advantages of high efficiency, low cost, and low serum volume.

High-affinity ZnT8 Autoantibodies by Electrochemiluminescence Assay Improve Risk Prediction for Type 1 Diabetes.

CONTEXT: Single ZnT8 autoantibody (ZnT8A) positivity by standard radiobinding assay (RBA) is commonly seen in nondiabetes population-based screening and the risk of progression to type 1 diabetes (T1D) in subjects with single ZnT8A is unknown. OBJECTIVE: Identify the risk of progression to T1D in individuals positive only for ZnT8A. METHODS: We developed an electrochemiluminescence (ECL) assay to detect high-affinity ZnT8A and validated it in 3 populations: 302 patients newly diagnosed with T1D, 135 nondiabetic children positive for ZnT8A by RBA among 23 400 children screened by the Autoimmunity Screening for Kids (ASK) study, and 123 nondiabetic children multiple autoantibody positive or single ZnT8A positive by RBA participating in the Diabetes Autoimmunity Study in the Young (DAISY). RESULTS: In 302 patients with T1D at diagnosis, the positivity for ZnT8A was 62% both in RBA and ECL. Among ASK 135 participants positive for RBA-ZnT8A, 64 were detected ZnT8A as the only islet autoantibody. Of these 64, only 9 were confirmed by ECL-ZnT8A, found to be of high affinity with increased T1D risk. The overall positive predictive value of ECL-ZnT8A for T1D risk was 87.1%, significantly higher than that of RBA-ZnT8A (53.5%, P < .001). In DAISY, 11 of 2547 children who had no positivity previously detected for other islet autoantibodies were identified as single ZnT8A by RBA; of these, 3 were confirmed positive by ECL-ZnT8A and all 3 progressed to clinical T1D. CONCLUSION: A large proportion of ZnT8A by RBA are single ZnT8A with low T1D risk, whereas ZnT8A by ECL was of high affinity and high prediction for T1D development.

Improving clinical utility of GAD65 autoantibodies by electrochemiluminescence assay and clinical phenotype when identifying autoimmune adult-onset diabetes.

AIMS/HYPOTHESIS: It is important to differentiate the two major phenotypes of adult-onset diabetes, autoimmune type 1 diabetes and non-autoimmune type 2 diabetes, especially as type 1 diabetes presents in adulthood. Serum GAD65 autoantibodies (GADA) are the most sensitive biomarker for adult-onset autoimmune type 1 diabetes, but the clinical value of GADA by current standard radiobinding assays (RBA) remains questionable. The present study focused on the clinical utility of GADA differentiated by a new electrochemiluminescence (ECL) assay in patients with adult-onset diabetes. METHODS: Two cohorts were analysed including 771 diabetic participants, 30-70 years old, from the Action LADA study (n = 6156), and 2063 diabetic participants, 20-45 years old, from the Diabetes in Young Adults (DiYA) study. Clinical characteristics of participants, including requirement of early insulin treatment, BMI and development of multiple islet autoantibodies, were analysed according to the status of RBA-GADA and ECL-GADA, respectively, and compared between these two assays. RESULTS: GADA was the most prevalent and predominant autoantibody, >90% in both cohorts. GADA positivity by either RBA or ECL assay significantly discriminated clinical type 1 from type 2 diabetes. However, in both cohorts, participants with ECL-GADA positivity were more likely to require early insulin treatment, have multiple islet autoantibodies, and be less overweight (for all p < 0.0001). However, clinical phenotype, age at diagnosis and BMI independently improved positive predictive value (PPV) for the requirement of insulin treatment, even augmenting ECL-GADA. Participants with GADA detectable by RBA, but not confirmed by ECL, had a phenotype more similar to type 2 diabetes. These RBA-GADA positive individuals had lower affinity GADA compared with participants in which GADA was confirmed by ECL assay. CONCLUSIONS/INTERPRETATION: Detection of GADA by ECL assay, given technical advantages over RBA-GADA, identified adult-onset diabetes patients at higher risk of requiring early insulin treatment, as did clinical phenotype, together allowing for more accurate clinical diagnosis and management.

Large-Scale Screening in General Population Children for Celiac Disease with a Multiplex Electrochemiluminescence (ECL) Assay.

BACKGROUND: Autoimmunity Screening for Kids (ASK) study was launched to screen general population children for type 1 diabetes (T1D) and celiac disease (CD). METHODS: A total of 23,319 children from general population were screened. A high throughput multiplex electrochemiluminescence (ECL) assay to screen multiautoantibodies in a single well was applied, parallel with a standard radiobinding assay (RBA). All children with any positive autoantibodies in screening were revisited within one month for confirmation and followed every 6 months. RESULTS: Among 23,319 children, 2.6% (606/23,319) of children were tested positive for TGA. Multiplex ECL assay detected more TGA (584/23,319) in the initial screening than RBA (490/23,319, p = 0.004) and was able to detect TGA earlier than RBA in a subset of children by 0.8 to 34.8 months. Prevalence of TGA by either ECL or RBA in children with islet autoantibodies was found significantly higher than overall prevalence in general population screened. CONCLUSIONS: A multiplex ECL assay was more sensitive than standard RBA by identifying more TGA positivity and detecting TGA earlier in general population screening. It also provides a high efficient tool with its unique advantage of multiplexing measurements to screen for multiple autoimmune diseases simultaneously in general population.

High-throughput multiplexed autoantibody detection to screen type 1 diabetes and multiple autoimmune diseases simultaneously.

BACKGROUND: Islet autoantibodies (IAbs) are the most reliable biomarkers to assess risk of progression to clinical type 1 diabetes (T1D). There are four major biochemically defined IAbs currently used in clinical trials that are equally important for disease prediction. The current screening methods use a radio-binding assay (RBA) for single IAb measurement, which are laborious and inefficient for large-scale screening. More importantly, up to 40% of patients with T1D have other autoimmune conditions that can be identified through relevant autoantibody testing. Thus, there is a need to screen for T1D and other autoimmune diseases simultaneously. METHODS: Based on our well-established electrochemiluminescence (ECL) assay platform, we developed a multiplexed ECL assay that combines 7 individual autoantibody assays together in one single well to simultaneously screen T1D, and three other autoimmune diseases including celiac disease, autoimmune thyroid disease and autoimmune poly-glandular syndrome-1 (APS-1). The 7-Plex ECL assay was extensively validated against single antibody measurements including a standard RBA and single ECL assay. FINDINGS: The 7-Plex ECL assay was well correlated to each single ECL autoantibody assay and each RBA. INTERPRETATION: The multiplexed ECL assay provides high sensitivity and disease specificity, along with high throughput and a low cost for large-scale screenings of T1D and other relevant autoimmune diseases in the general population. FUND: JDRF grants 2-SRA-2015-51-Q-R, 2-SRA-2018-533-S-B, NIH grants DK32083 and DK32493. NSFC grants 81770777.

Screening children for type 1 diabetes-associated antibodies at community health fairs.

OBJECTIVE: The incidence of type 1 diabetes (T1D) is increasing, most notably in young children and in racial and ethnic minorities. Historically, screening for risk with T1D-associated antibodies has been limited to those with a family history, while up to 90% of newly diagnosed patients lack such a family history. To address the needs to screen diverse ethnic groups in the general population, we screened children for T1D-associated antibodies in the Denver, Colorado metro area at community health fairs. METHODS: Children attending health fairs from 2015 to 2018 were offered free T1D screening by measuring the four prototypical T1D-associated antibodies. A finger stick capillary puncture was performed to collect blood spots on filter paper. Dried blood spots (DBSs) were eluted and antibodies were measured using fluid-phase radio-binding assays. RESULTS: At 39 health fairs, children were educated on the signs and symptoms of diabetes, and screened for T1D-associated antibodies (n = 478), which represented 90% of those that attended. Median age was 9.0 years (range of 1-18) with diverse ethnic backgrounds: 37% Hispanic, 31% Caucasian, 20% African American, and 12% other. Nine children screened positive for antibodies, single n = 8 and multiple n = 1, and confirmation with serum samples showed excellent correlation to the measurements from DBSs for antibodies directed against GAD, IA-2, and ZnT8 (P < .01 for each). CONCLUSIONS: Screening for T1D risk at community health fairs using DBSs on filter paper is feasible and provides an avenue to screen children from ethnically diverse backgrounds.

Chimeric antigen receptor (CAR) T cells targeting a pathogenic MHC class II:peptide complex modulate the progression of autoimmune diabetes.

A primary initiating epitope in the NOD mouse model of Type 1 Diabetes (T1D) lies between residues 9 and 23 of the insulin B chain. The B:9-23 peptide can bind to the NOD MHC class II molecule (I-Ag7) in multiple registers, but only one, (register 3, R3), creates complexes able to stimulate the majority of pathogenic B:9-23-specific CD4+ T cells. Previously we generated a monoclonal antibody (mAb287) that targets this critical I-Ag7-B:9-23(R3) complex. When given weekly to pre-diabetic mice at either early or late stages of disease, mAb287 was able to delay or prevent T1D in the treated animals. Although the precise mechanism of action of mAb287 remains unclear, we hypothesized that it may involve deletion of antigen presenting cells (APCs) bearing the pathogenic IAg7-B:9-23(R3) complexes, and that this process might be rendered more efficient by re-directing cytotoxic T cells using a mAb287 chimeric antigen receptor (287-CAR). As anticipated, 287-CAR T cells secreted IFN-γ in response to stimulation by I-Ag7-B:9-23(R3) complexes expressed on artificial APCs, but not I-Ag7 loaded with other peptides, and killed the presenting cells in vitro. A single infusion of 287-CAR CD8+ T cells to young (5 week old) NOD mice significantly delayed the onset of overt hyperglycemia compared to untreated animals (p = 0.022). None of the 287-CAR CD8+ T cell treated mice developed diabetes before 18 weeks of age, while 29% of control-CAR T cell treated mice (p = 0.044) and 52% of the un-treated mice (p = 0.0001) had developed T1D by this time. However, the protection provided by 287-CAR CD8+ T cells declined with time, and no significant difference in overall incidence by 30 weeks between the 3 groups was observed. Mechanistic studies indicated that the adoptively transferred 287-CAR T cells selectively homed to pancreatic lymph nodes, and in some animals could persist for at least 1-2 weeks post-transfer, but were essentially undetectable 10-15 weeks later. Our study demonstrates that CAR T cells specific for a pathogenic MHC class II:peptide complex can be effective in vivo, but that a single infusion of the current iteration can only delay, but not prevent, the development of T1D. Future studies should therefore be directed towards optimizing strategies designed to improve the longevity of the transferred cells.

Determination of Autoantibodies to Transglutaminase by Electrochemiluminescence (ECL) Assay.

Appearance of autoantibodies to tissue transglutaminase (TGA) is the most reliable biomarker to identify celiac disease autoimmunity. A nonradioactive assay of determination of TGA was newly developed using electrochemiluminescence (ECL) technology. This ECL assay has been demonstrated to be more sensitive than current standard radio-binding assay (RBA) in detecting TGA and can detect TGA earlier among high-risk young children followed from birth.

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.

The Use of Electrochemiluminescence Assays to Predict Autoantibody and Glycemic Progression Toward Type 1 Diabetes in Individuals with Single Autoantibodies.

BACKGROUND: Electrochemiluminescence (ECL) assays have shown promise for enhancing the prediction of type 1 diabetes (T1D) with autoantibodies. We thus studied relatives of T1D patients to determine whether ECL assays can be used to refine risk assessments for T1D among individuals either positive for single GADA or single mIAA autoantibodies. SUBJECTS AND METHODS: TrialNet Pathway to Prevention (PTP) study participants with either GADA or mIAA single autoantibodies were tested for ECL positivity during their participation in the TrialNet PTP study. Those ECL positive (ECL+) were compared with those ECL negative (ECL-) for conversion to multiple autoantibodies, 6-month glycemic progression (PS6M), and the progression to T1D. RESULTS: The progression to multiple autoantibodies was significantly higher for those GADA/ECL+ (n = 107) than those GADA/ECL- (n = 78) (P = 0.001) and for those mIAA/ECL+ (n = 24) than those mIAA/ECL- (n = 63) (P < 0.001). The hazard ratios with 95% confidence intervals were 3.42 (1.58-7.39; P < 0.01) for GADA and 8.15 (3.02-22.00; P < 0.001) for mIAA. GADA/ECL+ and mIAA/ECL+ participants had significantly higher PS6M values than their ECL- counterparts (P = 0.001 for GADA and P = 0.009 for mIAA). Of those GADA/ECL+, 14% progressed to T1D; of those mIAA/ECL+, 17% progressed to T1D. Only 1 individual (positive for GADA) of the 141 who was ECL- progressed to T1D (median follow-up: 5 years). CONCLUSION: ECL measurements appear to have utility for natural history studies and prevention trials of individuals with single autoantibodies. Those ECL+ are at appreciable risk for developing multiple autoantibodies and for glycemic progression toward T1D, whereas those ECL- are at very low risk.

Islet Autoantibody Measurements from Dried Blood Spots on Filter Paper Strongly Correlate to Serum Levels.

Type 1 diabetes (T1D) is increasing in incidence and predictable with measurement of serum islet autoantibodies (iAb) years prior to clinical disease onset. Identifying iAb positive individuals reduces diabetic ketoacidosis and identifies individuals for T1D prevention trials. However, large scale screening for iAb remains challenging as assays have varying sensitivities and specificities, insulin autoantibodies remain difficult to measure and venipuncture is generally required to obtain serum. We developed an approach to reliably measure all four major iAb, including insulin autoantibodies, from dried blood spots (DBS) on filter-paper. By spiking iAb positive serum into iAb negative whole blood in a dose titration, we optimized the conditions for autoantibody elution from filter paper as measured by fluid phase radioimmunoassays. After assessing stability of measuring iAb from DBS over time, we then screened iAb from DBS and the corresponding serum in new-onset T1D (n = 52), and controls (n = 72) which included first-degree relatives of T1D patients. iAb measured from eluted DBS in new-onset T1D strongly correlated with serum measurements (R2 = 0.96 for mIAA, GADA = 0.94, IA-2A = 0.85, ZnT8A = 0.82, p<0.01 for each autoantibody). There were no false positives in control subjects, and 5/6 with previously unknown iAb positivity in sera were detected using DBS. With further validation, measuring iAb from DBS can be a reliable method to screen for T1D risk.

Do Electrochemiluminescence Assays Improve Prediction of Time to Type 1 Diabetes in Autoantibody-Positive TrialNet Subjects?

OBJECTIVE: To explore whether electrochemiluminescence (ECL) assays can help improve prediction of time to type 1 diabetes in the TrialNet autoantibody-positive population. RESEARCH DESIGN AND METHODS: TrialNet subjects who were positive for one or more autoantibodies (microinsulin autoantibody, GAD65 autoantibody [GADA], IA-2A, and ZnT8A) with available ECL-insulin autoantibody (IAA) and ECL-GADA data at their initial visit were analyzed; after a median follow-up of 24 months, 177 of these 1,287 subjects developed diabetes. RESULTS: Univariate analyses showed that autoantibodies by radioimmunoassays (RIAs), ECL-IAA, ECL-GADA, age, sex, number of positive autoantibodies, presence of HLA DR3/4-DQ8 genotype, HbA1c, and oral glucose tolerance test (OGTT) measurements were all significantly associated with progression to diabetes. Subjects who were ECL positive had a risk of progression to diabetes within 6 years of 58% compared with 5% for the ECL-negative subjects (P < 0.0001). Multivariate Cox proportional hazards models were compared, with the base model including age, sex, OGTT measurements, and number of positive autoantibodies by RIAs. The model with positivity for ECL-GADA and/or ECL-IAA was the best, and factors that remained significantly associated with time to diabetes were area under the curve (AUC) C-peptide, fasting C-peptide, AUC glucose, number of positive autoantibodies by RIAs, and ECL positivity. Adding ECL to the Diabetes Prevention Trial risk score (DPTRS) improved the receiver operating characteristic curves with AUC of 0.83 (P < 0.0001). CONCLUSIONS: ECL assays improved the ability to predict time to diabetes in these autoantibody-positive relatives at risk for developing diabetes. These findings might be helpful in the design and eligibility criteria for prevention trials in the future.

ECL-IAA and ECL-GADA Can Identify High-Risk Single Autoantibody-Positive Relatives in the TrialNet Pathway to Prevention Study.

BACKGROUND: Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies. MATERIALS AND METHODS: TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits. RESULTS: After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time. CONCLUSIONS: Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects.

Expression-Based Genome-Wide Association Study Links Vitamin D-Binding Protein With Autoantigenicity in Type 1 Diabetes.

Type 1 diabetes (T1D) is caused by autoreactive T cells that recognize pancreatic islet antigens and destroy insulin-producing ß-cells. This attack results from a breakdown in tolerance for self-antigens, which is controlled by ectopic antigen expression in the thymus and pancreatic lymph nodes (PLNs). The autoantigens known to be involved include a set of islet proteins, such as insulin, GAD65, IA-2, and ZnT8. In an attempt to identify additional antigenic proteins, we performed an expression-based genome-wide association study using microarray data from 118 arrays of the thymus and PLNs of T1D mice. We ranked all 16,089 protein-coding genes by the likelihood of finding repeated differential expression and the degree of tissue specificity for pancreatic islets. The top autoantigen candidate was vitamin D-binding protein (VDBP). T-cell proliferation assays showed stronger T-cell reactivity to VDBP compared with control stimulations. Higher levels and frequencies of serum anti-VDBP autoantibodies (VDBP-Abs) were identified in patients with T1D (n = 331) than in healthy control subjects (n = 77). Serum vitamin D levels were negatively correlated with VDBP-Ab levels in patients in whom T1D developed during the winter. Immunohistochemical localization revealed that VDBP was specifically expressed in α-cells of pancreatic islets. We propose that VDBP could be an autoantigen in T1D.

Apolipoprotein A-I Mimetic Peptide D-4F Reduces Cardiac Hypertrophy and Improves Apolipoprotein A-I-Mediated Reverse Cholesterol Transport From Cardiac Tissue in LDL Receptor-null Mice Fed a Western Diet.

Epidemiological studies have suggested that hypercholesterolemia is an independent determinant of increased left ventricular (LV) mass. Because high-density lipoprotein and its major protein apolipoprotein A-I (apoA-I) mediate reverse cholesterol transport (RCT) and have cardiac protective effects, we hypothesized that the apoA-I mimetic peptide D-4F could promote RCT in cardiac tissue and decrease cardiac hypertrophy induced by hypercholesterolemia. Low-density lipoprotein receptor-null mice were fed by a Western diet for 18 weeks and then randomized to receive water, or D-4F 0.3 mg/mL, or D-4F 0.5 mg/mL added to drinking water for 6 weeks. After D-4F administration, an increase in high-density lipoprotein cholesterol and a decrease in low-density lipoprotein cholesterol, total cholesterol, and triglyceride in a trend toward dose-responsivity were found in cardiac tissue. Ultrasound biomicroscopy revealed a reduction in LV posterior wall end-diastolic dimension, and an increase in mitral valve E/A ratio and LV ejection fraction. Hematoxylin-eosin staining showed reduced LV wall thickness and myocardial cell diameter. The protein levels of ABCA1 and LXRα were elevated in cardiac tissue of D-4F treated mice compared with the controls (P < 0.05). These results demonstrated that D-4F treatment reduced cardiac hypertrophy, and improved cardiac performance in low-density lipoprotein receptor-null mice fed a Western diet, presumably through the LXRα-ABCA1 pathway associated with enhanced myocardial RCT.

A multiplex assay combining insulin, GAD, IA-2 and transglutaminase autoantibodies to facilitate screening for pre-type 1 diabetes and celiac disease.

At the current time, multiple candidate interventions are being proposed to abrogate or slow progression to type 1 diabetes (T1D) among islet autoantibody (iAb) positive subjects, but mass screening for eligible subjects and the general population remains a laborious and inefficient process. We have recently developed and extensively validated nonradioactive iAb assays using electrochemiluminescense (ECL) detection with an excellent sensitivity and specificity compared to the gold-standard radioassays. Using ECL detection on a platform from MesoScale Discovery (MSD) allows the measurement of four antibodies in a single well using a small blood volume (6 µl). In the present study using a MSD QuickPlex 4-Spot plate, we successfully combined three iAb to insulin (IAA), GAD65 (GADA), and IA-2 (IA-2A) with tissue transglutaminase autoantibodies (TGA) in a single well of a 96 well plate. We tested 40 new onset T1D patients, all positive for at least one iAb and a half of them positive for TGA by radioassay, as well as 50 healthy controls. The multiplex assay retained 100% sensitivity and 100% specificity for all four autoantibodies in terms of positivity identified in patients versus normal controls compared to the corresponding standard radioassays and our single ECL assays. The multiplex ECL assay was able to identify more positivity than current radioassays for IAA and TGA. The development of this multiplex assay will facilitate high-throughput screening for T1D and celiac disease risk in the general population.

Higher Sensitivity and Earlier Identification of Celiac Disease Autoimmunity by a Nonradioactive Assay for Transglutaminase Autoantibodies.

Higher sensitive transglutaminase autoantibody (TGA) assay will detect the onset of celiac disease (CD) autoimmunity earlier. In developing a nonradioactive assay for TGA, we utilized electrochemiluminescence (ECL) technology and compared it to a high-performance radioimmunoassay (RIA) currently being used to screen patients with type 1 diabetes (T1D) and genetically at-risk individuals for CD. We selected 183 T1D patients with 60 patients having received biopsy and analyzed 396 sequential samples from 73 young children longitudinally followed up with TGA seroconversion, with 27 undergoing biopsy. In addition, 112 age-matched healthy control subjects were included in the study. With the 99th percentile of specificity, the ECL assay detected significantly more TGA positivity among patients with T1D (133/183) than RIA (114/183) and more of the sequential samples (34%) from 73 children than RIA (18%). The TGA assay performed by ECL was positive in all 59 subjects with villous atrophy. Among 73 longitudinally followed up children, ECL assay had earlier detection of TGA on 34 children by a mean of 2.5 years. In conclusion, the new TGA assay by ECL has a higher sensitivity than the current RIA assay and may better predict the onset of CD.

Electrochemiluminescence assays for insulin and glutamic acid decarboxylase autoantibodies improve prediction of type 1 diabetes risk.

We recently developed new electrochemiluminescence (ECL) insulin autoantibody (IAA) and glutamic acid decarboxylase 65 autoantibody (GADA) assays that discriminate high-affinity, high-risk diabetes-specific autoantibodies from low-affinity, low-risk islet autoantibodies (iAbs) detected by radioassay (RAD). Here, we report a further validation of the ECL-IAA and -GADA assays in 3,484 TrialNet study participants. The ECL assay and RAD were congruent in those with prediabetes and in subjects with multiple autoantibodies, but only 24% (P<0.0001) of single RAD-IAA-positive and 46% (P<0.0001) of single RAD-GADA-positive were confirmed by the ECL-IAA and -GADA assays, respectively. During a follow-up (mean, 2.4 years), 51% of RAD-IAA-positive and 63% of RAD-GADA-positive subjects not confirmed by ECL became iAb negative, compared with only 17% of RAD-IAA-positive (P<0.0001) and 15% of RAD-GADA-positive (P<0.0001) subjects confirmed by ECL assays. Among subjects with multiple iAbs, diabetes-free survival was significantly shorter if IAA or GADA was positive by ECL and negative by RAD than if IAA or GADA was negative by ECL and positive by RAD (P<0.019 and P<0.0001, respectively). Both positive and negative predictive values in terms of progression to type 1 diabetes mellitus were superior for ECL-IAA and ECL-GADA, compared with RADs. The prevalence of the high-risk human leukocyte antigen-DR3/4, DQB1*0302 genotype was significantly higher in subjects with RAD-IAA or RAD-GADA confirmed by ECL. In conclusion, both ECL-IAA and -GADA are more disease-specific and better able to predict the risk of progression to type 1 diabetes mellitus than the current standard RADs.