Publications by authors named "Janet Wenzlau"

37 Publications

Novel autoantibodies to the β-cell surface epitopes of ZnT8 in patients progressing to type-1 diabetes.

J Autoimmun 2021 Aug 12;122:102677. Epub 2021 Jun 12.

Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD, USA. Electronic address:

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by autoimmune destruction of insulin-producing β-cells in pancreatic islets. Seroconversions to islet autoantibodies (IAbs) precede the disease onset by many years, but the role of humoral autoimmunity in the disease initiation and progression are unclear. In the present study, we identified a new IAb directed to the extracellular epitopes of ZnT8 (ZnT8ec) in newly diagnosed patients with T1D, and demonstrated immunofluorescence staining of the surface of human β-cells by autoantibodies to ZnT8ec (ZnT8ecA). With the assay specificity set on 99th percentile of 336 healthy controls, the ZnT8ecA positivity rate was 23.6% (74/313) in patients with T1D. Moreover, 30 children in a longitudinal follow up of clinical T1D development were selected for sequential expression of four major IAbs (IAA, GADA, IA-2A and ZnT8icA). Among them, 10 children were ZnT8ecA positive. Remarkably, ZnT8ecA was the earliest IAb to appear in all 10 children. The identification of ZnT8ec as a cell surface target of humoral autoimmunity in the earliest phase of IAb responses opens a new avenue of investigation into the role of IAbs in the development of β-cell autoimmunity.
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http://dx.doi.org/10.1016/j.jaut.2021.102677DOI Listing
August 2021

Cluster of Differentiation 4 T Cells and Neoantigens in Autoimmune Diabetes.

Crit Rev Immunol 2020 ;40(5):441-446

Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO.

Our investigations of antigens for pathogenic T cells in autoimmune diabetes led to the discovery of hybrid insulin peptides as T cell epitopes. T cells reactive to hybrid insulin peptides can be found at high frequency in the nonobese diabetic mouse model of type 1 diabetes and are also present in human patients. Hybrid insulin peptides can also be administered to mice in a tolerogenic form, thereby suppressing the autoimmune response.
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http://dx.doi.org/10.1615/CritRevImmunol.2020034833DOI Listing
January 2020

Autoantibodies Toward ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase Are Reliable Serological Pre-endoscopic Markers of Corpus Atrophic Gastritis.

Clin Transl Gastroenterol 2020 10;11(10):e00240

Medical-Surgical Department of Clinical Sciences and Translational Medicine, Sant'Andrea Hospital, University Sapienza, Rome, Italy.

Introduction: Noninvasive assessment of corpus atrophic gastritis (CAG), a condition at increased risk of gastric cancer, is based on the measurement of pepsinogens, gastrin, and Helicobacter pylori antibodies. Parietal cell autoantibodies (PCAs) against the gastric proton pump (ATP4) are potential serological biomarkers of CAG. The purpose of this study was to compare the diagnostic performance of PCA and pepsinogen I tests in patients with clinical suspicion of CAG with the histopathological evaluation of gastric biopsies as reference standard.

Methods: A prospective case-finding study was performed on 218 naive adult patients (131 women, median age 65 years) who underwent gastric biopsies to confirm/exclude CAG. Patients with histopathological CAG were defined as cases, conversely as controls. Autoantibodies against the individual alpha (ATP4A) and beta (ATP4B) subunits of ATP4 were measured by luciferase immunoprecipitation, and global PCA and pepsinogen I by enzyme-linked immunosorbent assay.

Results: Histopathology classified 107 subjects (49%) as cases (CAG+, autoimmune 81.2%, and multifocal extensive 18.8%) and 111 subjects (51%) as controls (CAG-). In cases, ATP4A, ATP4B, and PCA titers were increased compared with controls, whereas pepsinogen I was reduced (P < 0.0001 for all). ATP4B, ATP4A, and pepsinogen I tests showed sensitivities of 77%, 75%, and 73% and specificities of 88%, 88%, and 80%, respectively. The receiver operating characteristic (ROC) area under the ROC curve (AUC) of these serological biomarkers confirmed their ability to discriminate cases from controls (ATP4B = 0.838, ATP4A = 0.826, pepsinogen I = 0.775, and PCA = 0.805), whereas the partial ROC-pAUC90 analysis showed that the ATP4B test had the best diagnostic performance (P = 0.008 vs ATP4; P = 0.0002 vs pepsinogen I). The presence of autoimmune or extensive gastritis was not significantly different between ATP4B positive or negative cases (P = 0.217).

Discussion: PCAs are promising serological biomarkers for the identification of CAG in high-risk individuals, particularly in an autoimmune pattern but also in an extensive-multifocal atrophy pattern.
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http://dx.doi.org/10.14309/ctg.0000000000000240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544173PMC
October 2020

Measurement of Autoantibodies to Gastric H+,K+-ATPase (ATP4A/B) Using a Luciferase Immunoprecipitation System (LIPS).

Methods Mol Biol 2019 ;1901:113-131

Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy.

The Luciferase Immuno Precipitation System (LIPS) enables the detection of specific serum antibodies by immunoprecipitation of recombinant antigens tagged with a luciferase reporter. Here we describe LIPS assays for the quantification of autoantibodies to the H+, K+-ATPase A (ATP4A) and B (ATP4B) subunits, two serological markers of autoimmune atrophic gastritis and pernicious anemia. In particular, we will describe the expression of luciferase-tagged recombinant ATP4A and ATP4B, their immunoprecipitation with test sera, the recovery and washing of immune-complexes with a protein-A coated resin, and the quantification of autoantibodies by addition of a luciferase substrate and the measurement of the light output from captured luciferase-tagged antigens.
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http://dx.doi.org/10.1007/978-1-4939-8949-2_10DOI Listing
June 2019

ATP4A autoimmunity in pediatric patients with type 1 diabetes and its relationship to blood count, iron metabolism, and vitamin B12.

Pediatr Diabetes 2018 02 12;19(1):80-84. Epub 2017 Apr 12.

University of Colorado Denver, Barbara Davis Center for Diabetes, Aurora, Colorado.

Objective: We aimed to assess the prevalence of autoantibodies against the 4A subunit of the gastric proton pump (ATP4A) in pediatric type 1 diabetes (T1D) patients and explore the relationship between ATP4A positivity and blood cell count, iron turnover, and vitamin B12 concentration.

Subjects: The study included 94 (59% female) T1D children (aged 12.5 ± 4.1 years, T1D duration 4.2 ± 3.6 years, HbA1c 7.3 ± 1.5% (57 ± 12.6 mmol/mol) with no other autoimmune diseases.

Methods: ATP4A antibodies were measured in T1D patients using a radioimmunoprecipitation assay. Blood cell count, iron concentration, total iron binding capacity, ferritin, transferrin, hepcidin, and vitamin B12 concentration were measured in all the study participants.

Results: A total of 16 (17%) children were ATP4A positive. Serum concentrations of ferritin were significantly lower in ATP4A positive than in antibody negative subjects (P = .034). Overall the levels of ATP4A antibodies (ATP4A Index) correlated positively with the age at T1D diagnosis (r = 0.228, P = .026) and negatively with ferritin levels (r = -0.215, P = .037). In ATP4A positive patients, the ATP4A Index correlated positively with age at diagnosis (r = 0.544, P = .032) and negatively with vitamin B12 levels (r = -0.685, P = .004).

Conclusions: ATP4A antibodies were present in a significant proportion of children with T1D. Higher ATP4A levels in T1D children are associated with lower, yet still fitting within the normal range, levels of vitamin B12, and ferritin. Routine screening of T1D children for gastric autoimmunity (ATP4A) should be considered with follow-up of those positive for vitamin B12 and iron deficiency.
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http://dx.doi.org/10.1111/pedi.12528DOI Listing
February 2018

Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase in Atrophic Body Gastritis Patients.

Clin Transl Gastroenterol 2017 Jan 19;8(1):e215. Epub 2017 Jan 19.

Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Objectives: Circulating autoantibodies targeting the H+/K+-ATPase proton pump of gastric parietal cells are considered markers of autoimmune gastritis, whose diagnostic accuracy in atrophic body gastritis, the pathological lesion of autoimmune gastritis, remains unknown. This study aimed to assess autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in atrophic body gastritis patients and controls.

Methods: One-hundred and four cases with atrophic body gastritis and 205 controls were assessed for serological autoantibodies specific for ATP4A or ATP4B subunits using luminescent immunoprecipitation system (LIPS). Recombinant luciferase-reporter-fused-antigens were expressed by in vitro transcription-translation (ATP4A) or after transfection in Expi293F cells (ATP4B), incubated with test sera, and immune complexes recovered using protein-A-sepharose. LIPS assays were compared with a commercial enzyme immunoassay (EIA) for parietal cell autoantibodies.

Results: ATP4A and ATP4B autoantibody titers were higher in cases compared to controls (P<0.0001). The area under the receiver-operating characteristic curve was 0.98 (95% CI 0.965-0.996) for ATP4A, and 0.99 (95% CI 0.979-1.000) for ATP4B, both higher as compared with that of EIA: 0.86 (95% CI 0.809-0.896), P<0.0001. Sensitivity-specificity were 100-89% for ATP4A and 100-90% for ATP4B assay. Compared with LIPS, EIA for parietal cell autoantibodies showed a lower sensitivity (72%, P<0.0001) at a similar specificity (92%, P=0.558).

Conclusions: Positivity to both, ATP4A and ATP4B autoantibodies is closely associated with atrophic body gastritis. Both assays had the highest sensitivity, at the cost of diagnostic accuracy (89 and 90% specificity), outperforming traditional EIA. Once validated, these LIPS assays should be valuable screening tools for detecting biomarkers of damaged atrophic oxyntic mucosa.
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http://dx.doi.org/10.1038/ctg.2016.71DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288605PMC
January 2017

Anti-parietal cell antibodies - diagnostic significance.

Adv Med Sci 2016 Sep 13;61(2):175-179. Epub 2016 Jan 13.

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver Aurora, United States.

Anti-parietal cell antibodies (APCA) are an advantageous tool for screening for autoimmune atrophic gastritis (AAG) and pernicious anemia (PA). The target for APCA is the H+/K+ ATP-ase. It has been demonstrated, that APCA target both, the alpha, and beta subunits of the proton pump, although the major antigen is the alpha subunit. Circulating serum APCA can be detected by means of immunofluorescence, enzyme-linked immunosorbent assay - currently the most commonly used method, and radioimmunoprecipitation assay (RIA) - the 4A subunit has been optimized as a molecular-specific antigen probe. RIA is the most accurate method of antibody assessment, characterized by highest sensitivity. APCA can be found in 85-90% of patients with PA. Their presence is not sufficient for diagnosis, because they are not specific for PA as they are also found in the circulation of individuals with other diseases. APCA are more prevalent in the serum of patients with T1D, autoimmune thyroid diseases, vitiligo, celiac disease. People with autoimmune diseases should be closely screened for AAG/PA. The anemia develops longitudinally over many years in APCA-positive patients, symptomless, slowly promotes atrophy of the gastric mucosa and parietal cells. APCA are present in 7.8-19.5% of the general healthy adult population. A fraction of these sero-positive people, will never develop AAG or PA. An interesting and not fully explained question is whether APCA presence is related to Helicobacter pylori infection. APCA are found in up to 20.7% of these patients. H. pylori is implicated as one of the candidates causing AAG.
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http://dx.doi.org/10.1016/j.advms.2015.12.004DOI Listing
September 2016

ATPase4A Autoreactivity and Its Association With Autoimmune Phenotypes in the Type 1 Diabetes Genetics Consortium Study.

Diabetes Care 2015 Oct;38 Suppl 2:S29-36

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, CO.

Autoantibodies targeting the H+/K+-ATPase proton pump of the gastric parietal cell (parietal cell antibodies [PCA]) are diagnostic of atrophic body gastritis (ABG) leading to pernicious anemia (PA). PCA, ABG, and PA occur in increased frequency in patients with type 1 diabetes and their relatives and are considered "minor" components of forms of autoimmune polyglandular syndrome (APS). A customized radioimmunoprecipitation assay was applied to 6,749 samples from the Type 1 Diabetes Genetics Consortium to measure ATP4A autoreactivity. Autoantibody prevalence was correlated with variants in HLA class II, PTPN22, and CTLA4 genes. With an ATP4A radioimmunoprecipitation assay, PCA were detected in sera from 20.9% of affected individuals. PCA prevalence increased with age and was greater in females (25.3%) than males (16.5%) and among Hispanics (36.3%) and blacks (26.2%) compared with non-Hispanic whites (20.8%) and Asians (16.7%). PCA and other organ-specific autoantibodies GAD65, IA-2, thyroid peroxidase (TPO), 21-hydroxylase (21-OH), and transglutaminase (TG) clustered within families with heritability estimates from 71 to 95%. PCA clustered with TPO, 21-OH, and persistent GAD65 autoantibodies but not with celiac (TG) or IA-2 autoantibodies. PCA-positive subjects showed an increased frequency of DRB1*0404, DPB1*0201, and PTPN22 R620W (rs2476601-T) and a decreased frequency of DRB1*0101, DPB1*0301, and CTLA4 CT60 (rs3087243-T). Genetic variants accounted for 4-5% of the heritable risk for PCA. The same alleles were associated with other autoantibody phenotypes in a consistent pattern. Whereas most of the heritable risk for PCA and other antibodies reflects genetic effects that are tissue specific, parietal cell autoimmunity is a major pathogenetic contributor in APS2.
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http://dx.doi.org/10.2337/dcs15-2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582907PMC
October 2015

Changes in Zinc Transporter 8 Autoantibodies Following Type 1 Diabetes Onset: The Type 1 Diabetes Genetics Consortium Autoantibody Workshop.

Diabetes Care 2015 Oct;38 Suppl 2:S14-20

Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO

Zinc transporter 8 autoantibodies (ZnT8A) were analyzed in sera from 1,504 subjects as part of the Type 1 Diabetes Genetics Consortium (T1DGC) Autoantibody Workshop. For these participants with type 1 diabetes (T1D), samples were collected within 3 years of T1D diagnosis. ZnT8A were detected in 862 subjects (57.3%), with the highest frequencies and median titers being associated with the shortest duration of disease. ZnT8A were present at similar frequencies in non-Hispanic whites, non-Hispanic blacks, and Hispanics, but significantly less prevalent in those of Asian ancestry. Sera containing ZnT8A selectively recognizing at least one of the SLC30A8 single nucleotide polymorphisms (encoding ZnT8A) were detected in all populations; however, Trp-specific sera were much less frequent in non-Hispanic blacks, consistent with the anticipated lower frequency of the SLC30A8 rs13266634 T allele in African American populations. ZnT8A positivity was associated with HLA-DQ8, but this was primarily due to the DRB1*0404-DQ8 haplotype. This was in contrast to autoantibodies to IA-2 that were strongly associated with DRB1*0401-DQ8. These effects appeared essentially independent of racial or ethnic background. The DRB1*0401-DQ8 and DRB1*0404-DQ8 haplotypes were associated with T1D subjects positive for GAD65, IA-2, and ZnT8A. In contrast to DRB1*0401-DQ8, there was no significant association of DRB1*0404-DQ8 with single or dual autoantibody positivity. The DRB1*0404-DQ8 haplotype was also associated with T1D subjects whose sera recognized both polymorphic variants of zinc transporter 8, an effect not seen for DRB1*0401-DQ8.
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http://dx.doi.org/10.2337/dcs15-2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582909PMC
October 2015

Novel Association Between Immune-Mediated Susceptibility Loci and Persistent Autoantibody Positivity in Type 1 Diabetes.

Diabetes 2015 Aug 31;64(8):3017-27. Epub 2015 Mar 31.

Department of Pediatrics, Herlev University Hospital, Herlev, Denmark

Islet autoantibodies detected at disease onset in patients with type 1 diabetes are signs of an autoimmune destruction of the insulin-producing β-cells. To further investigate the genetic determinants of autoantibody positivity, we performed dense immune-focused genotyping on the Immunochip array and tested for association with seven disease-specific autoantibodies in a large cross-sectional cohort of 6,160 type 1 diabetes-affected siblings. The genetic association with positivity for GAD autoantibodies (GADAs), IA2 antigen (IA-2A), zinc transporter 8, thyroid peroxidase, gastric parietal cells (PCAs), tissue transglutaminase, and 21-hydroxylase was tested using a linear mixed-model regression approach to simultaneously control for population structure and family relatedness. Four loci were associated with autoantibody positivity at genome-wide significance. Positivity for GADA was associated with 3q28/LPP, for IA-2A with 1q23/FCRL3 and 11q13/RELA, and for PCAs with 2q24/IFIH1. The 3q28 locus showed association after only 3 years duration and might therefore be a marker of persistent GADA positivity. The 1q23, 11q13, and 2q24 loci were associated with autoantibodies close to diabetes onset and constitute candidates for early screening. Major susceptibility loci for islet autoantibodies are separate from type 1 diabetes risk, which may have consequences for intervention strategies to reduce autoimmunity.
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http://dx.doi.org/10.2337/db14-1730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4512221PMC
August 2015

Preexisting insulin autoantibodies predict efficacy of otelixizumab in preserving residual β-cell function in recent-onset type 1 diabetes.

Diabetes Care 2015 Apr 12;38(4):644-51. Epub 2015 Jan 12.

Diabetes Research Center and University Hospital Brussels (UZ Brussel), Vrije Universiteit Brussel, Brussels, Belgium.

Objective: Immune intervention trials in recent-onset type 1 diabetes would benefit from biomarkers associated with good therapeutic response. In the previously reported randomized placebo-controlled anti-CD3 study (otelixizumab; GlaxoSmithKline), we tested the hypothesis that specific diabetes autoantibodies might serve this purpose.

Research Design And Methods: In the included patients (n = 40 otelixizumab, n = 40 placebo), β-cell function was assessed as area under the curve (AUC) C-peptide release during a hyperglycemic glucose clamp at baseline (median duration of insulin treatment: 6 days) and every 6 months until 18 months after randomization. (Auto)antibodies against insulin (I[A]A), GAD (GADA), IA-2 (IA-2A), and ZnT8 (ZnT8A) were determined on stored sera by liquid-phase radiobinding assay.

Results: At baseline, only better preserved AUC C-peptide release and higher levels of IAA were associated with better preservation of β-cell function and lower insulin needs under anti-CD3 treatment. In multivariate analysis, IAA (P = 0.022) or the interaction of IAA and C-peptide (P = 0.013) independently predicted outcome together with treatment. During follow-up, good responders to anti-CD3 treatment (i.e., IAA(+) participants with relatively preserved β-cell function [≥ 25% of healthy control subjects]) experienced a less pronounced insulin-induced rise in I(A)A and lower insulin needs. GADA, IA-2A, and ZnT8A levels were not influenced by anti-CD3 treatment, and their changes showed no relation to functional outcome.

Conclusions: There is important specificity of IAA among other diabetes autoantibodies to predict good therapeutic response of recent-onset type 1 diabetic patients to anti-CD3 treatment. If confirmed, future immune intervention trials in type 1 diabetes should consider both relatively preserved functional β-cell mass and presence of IAA as inclusion criteria.
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http://dx.doi.org/10.2337/dc14-1575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370324PMC
April 2015

Zinc transporter 8 (ZnT8) and β cell function.

Trends Endocrinol Metab 2014 Aug 18;25(8):415-24. Epub 2014 Apr 18.

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Human pancreatic β cells have exceptionally high zinc content. In β cells the highest zinc concentration is in insulin secretory granules, from which it is cosecreted with the hormone. Uptake of zinc into secretory granules is mainly mediated by zinc transporter 8 (ZnT8), the product of the SLC30A8 [solute carrier family 30 (zinc transporter), member 8] gene. The minor alleles of several single-nucleotide polymorphisms (SNPs) in SLC30A8 are associated with decreased risk of type 2 diabetes (T2D), but the precise mechanisms underlying the protective effects remain uncertain. In this article we review current knowledge of the role of ZnT8 in maintaining zinc homeostasis in β cells, its role in glucose metabolism based on knockout mouse studies, and current theories regarding the link between ZnT8 function and T2D.
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http://dx.doi.org/10.1016/j.tem.2014.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112161PMC
August 2014

Clinical and biological characteristics of diabetic patients under age 40 in Cameroon: relation to autoantibody status and comparison with Belgian patients.

Diabetes Res Clin Pract 2014 Jan 20;103(1):97-105. Epub 2013 Nov 20.

Diabetes Research Center, Brussels Free University - VUB, Laarbeeklaan 103, B-1090 Brussels, Belgium. Electronic address:

Aims: We investigated the prevalence of diabetes autoantibodies (Abs) in Cameroonian patients and controls, assessed their contribution in disease classification and compared results with data from Belgium.

Methods: Abs against GAD (GADA), IA-2 (IA-2A) and zinc transporter 8 (ZnT8A) were assessed in 302 recently diagnosed Cameroonian patients with diabetes and 184 control subjects without diabetes aged below 40 years.

Results: Only 27 (9%) Cameroonian patients were younger than 15 years. Overall, 29% of patients presented at least one diabetes-associated antibody vs 9% in healthy controls (24% vs 7% for GADA (p<0.001), 10% vs 3% for IA-2A (p<0.006), 4% vs 2% for ZnT8A). Ab(+) patients had lower C-peptide levels (p<0.001), were more often insulin-treated (p<0.002) and were as frequently diagnosed with type 1 diabetes as Ab(-) patients. Only 43% of Ab(+) patients aged 15-39 years were clinically classified as having type 1 diabetes in Cameroon vs 96% in Belgium (p<0.001). Not one Ab(+) Cameroonian patient carried HLA-DQ2/DQ8 genotype vs 23% of Belgian Ab(+) patients (p<0.001). Younger age at diagnosis and antibody positivity were independent predictors of insulin therapy. Ab(+) Cameroonian patients were older (p<0.001), had higher BMI (p<0.001) and lower Ab titers than Belgian Ab(+) patients. In ketonuric patients, prevalence of autoantibodies was similar as in non-ketonuric patients.

Conclusions: In Cameroonian patients with diabetes aged under 40 years, antibody-positivity is not clearly related to disease phenotype, but may help predict the need for insulin treatment.
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http://dx.doi.org/10.1016/j.diabres.2013.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4120294PMC
January 2014

Novel diabetes autoantibodies and prediction of type 1 diabetes.

Curr Diab Rep 2013 Oct;13(5):608-15

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 1775 Aurora Ct, Aurora, CO, 80045, USA,

Autoantibodies are currently the most robust biomarkers of type 1 diabetes and are frequently used to establish entry criteria for the participation of genetically at-risk individuals in secondary prevention/intervention clinical trials. Since their original description almost 40 years ago, considerable efforts have been devoted toward identifying the precise molecular targets that are recognized. Such information can have significant benefit for developing improved metrics for identifying/stratifying of at-risk subjects, developing potential therapeutic targets, and advancing understanding of the pathophysiology of the disease. Currently, four major molecular targets ([pro]insulin, GAD65, IA-2, and ZnT8) have been confirmed, with approximately 94% of all subjects with a clinical diagnosis of type 1 diabetes expressing autoantibodies to at least one of these molecules at clinical onset. In this review, we summarize some of the salient properties of these targets that might contribute to their autoantigenicity and methods that have been used in attempts to identify new components of the humoral autoresponse.
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http://dx.doi.org/10.1007/s11892-013-0405-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887556PMC
October 2013

Proinsulin/Insulin autoantibodies measured with electrochemiluminescent assay are the earliest indicator of prediabetic islet autoimmunity.

Diabetes Care 2013 Aug 19;36(8):2266-70. Epub 2013 Feb 19.

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, Colorado, USA.

Objective: We evaluated a novel electrochemiluminescent assay for insulin/proinsulin autoantibodies (ECL-IAA) as a new marker of the onset of islet autoimmunity and as a predictor of type 1 diabetes.

Research Design And Methods: The Diabetes Autoimmunity Study in the Young (DAISY) prospectively follows children at increased genetic risk for development of islet autoimmunity (defined as presence of autoantibodies to insulin, GAD65, IA-2, or zinc transporter 8 [ZnT8]) and type 1 diabetes (general population of children and first-degree relatives). Serial serum samples from subjects who progressed to type 1 diabetes and who had their first islet autoantibodies measured by age 18 months (N = 47) were tested using ECL-IAA.

Results: Almost all prediabetic children tested positive for ECL-IAA (46 of 47, 98%) during follow-up. ECL-IAA was almost always the first autoantibody to appear (94% total; 21% very first [by itself]; 23% with only mIAA; 19% with another islet autoantibody [GAD or ZnT8]; and 30% with ≥ 2 other antibodies [mIAA, GAD, IA-2, or ZnT8]). Among the 46 subjects who were ECL-IAA positive, ECL-IAA antedated the onset of other islet autoantibodies by a mean of 2.3 years (range, 0.3-7.2 years). Both the age of appearance of autoantibody and IAA levels (but not GAD65, IA2, or ZnT8 levels) are major determinants of the age of diabetes onset.

Conclusions: This new ECL-IAA assay defines more precisely the onset of prediabetic autoimmunity and may help identify events triggering islet autoimmunity, as well as allow earlier intervention for type 1 diabetes. Nearly all young children progressing to diabetes are insulin autoantibody positive.
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http://dx.doi.org/10.2337/dc12-2245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3714529PMC
August 2013

HLA-A*24 is an independent predictor of 5-year progression to diabetes in autoantibody-positive first-degree relatives of type 1 diabetic patients.

Diabetes 2013 Apr 16;62(4):1345-50. Epub 2012 Nov 16.

Diabetes Research Center, Brussels Free University-VUB, Brussels, Belgium.

We investigated whether HLA-A*24 typing complements screening for HLA-DQ and for antibodies (Abs) against insulin, GAD, IA-2 (IA-2A), and zinc transporter-8 (ZnT8A) for prediction of rapid progression to type 1 diabetes (T1D). Persistently Ab(+) siblings/offspring (n = 288; aged 0-39 years) of T1D patients were genotyped for HLA-DQA1-DQB1 and HLA-A*24 and monitored for development of diabetes within 5 years of first Ab(+). HLA-A*24 (P = 0.009), HLA-DQ2/DQ8 (P = 0.001), and positivity for IA-2A ± ZnT8A (P < 0.001) were associated with development of T1D in multivariate analysis. The 5-year risk increased with the number of the above three markers present (n = 0: 6%; n = 1: 18%; n = 2: 46%; n = 3: 100%). Positivity for one or more markers identified a subgroup of 171 (59%) containing 88% of rapid progressors. The combined presence of HLA-A*24 and IA-2A(+) ± ZnT8A(+) defined a subgroup of 18 (6%) with an 82% diabetes risk. Among IA-2A(+) ± ZnT8A(+) relatives, identification of HLA-A*24 carriers in addition to HLA-DQ2/DQ8 carriers increased screening sensitivity for relatives at high Ab- and HLA-inferred risk (64% progression; P = 0.002). In conclusion, HLA-A*24 independently predicts rapid progression to T1D in Ab(+) relatives and complements IA-2A, ZnT8A, and HLA-DQ2/DQ8 for identifying participants in immunointervention trials.
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http://dx.doi.org/10.2337/db12-0747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609594PMC
April 2013

Extrapancreatic autoantibody profiles in type I diabetes.

PLoS One 2012 21;7(9):e45216. Epub 2012 Sep 21.

Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, and National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.

Type I diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-producing β-cells in the pancreas. Although several islet cell autoantigens are known, the breadth and spectrum of autoantibody targets has not been fully explored. Here the luciferase immunoprecipitation systems (LIPS) antibody profiling technology was used to study islet and other organ-specific autoantibody responses in parallel. Examination of an initial cohort of 93 controls and 50 T1D subjects revealed that 16% of the diabetic subjects showed anti-gastric ATPase autoantibodies which did not correlate with autoantibodies against GAD65, IA2, or IA2-β. A more detailed study of a second cohort with 18 potential autoantibody targets revealed marked heterogeneity in autoantibody responses against islet cell autoantigens including two polymorphic variants of ZnT8. A subset of T1D subjects exhibited autoantibodies against several organ-specific targets including gastric ATPase (11%), thyroid peroxidase (14%), and anti-IgA autoantibodies against tissue transglutaminase (12%). Although a few T1D subjects showed autoantibodies against a lung-associated protein KCNRG (6%) and S100-β (8%), no statistically significant autoantibodies were detected against several cytokines. Analysis of the overall autoantibody profiles using a heatmap revealed two major subgroups of approximately similar numbers, consisting of T1D subjects with and without organ-specific autoantibodies. Within the organ-specific subgroup, there was minimal overlap among anti-gastric ATPase, anti-thyroid peroxidase, and anti-transglutaminase seropositivity, and these autoantibodies did not correlate with islet cell autoantibodies. Examination of a third cohort, comprising prospectively collected longitudinal samples from high-risk individuals, revealed that anti-gastric ATPase autoantibodies were present in several individuals prior to detection of islet autoantibodies and before clinical onset of T1D. Taken together, these results suggest that autoantibody portraits derived from islet and organ-specific targets will likely be useful for enhancing the clinical management of T1D.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448600PMC
February 2013

Genetic determinants of 21-hydroxylase autoantibodies amongst patients of the Type 1 Diabetes Genetics Consortium.

J Clin Endocrinol Metab 2012 Aug 20;97(8):E1573-8. Epub 2012 Jun 20.

Barbara Davis Center for Childhood Diabetes, Aurora, Colorado 80045, USA.

Background: Autoantibodies to 21-hydroxylase (21OH-AA) precede the onset of autoimmune Addison's disease (AD) and are found in 1.5% of individuals with type 1 diabetes mellitus (T1DM). The greatest genetic risk for both disorders is found in the major histocompatibility complex (MHC), suggesting a common pathophysiology between AD and T1DM. Screening for 21OH-AA in newly diagnosed T1DM patients is a valuable prognostic tool, made stronger when MHC genotype is considered.

Methods: The Type 1 Diabetes Genetics Consortium has collected genotype data in T1DM subjects with tissue-specific autoantibody typing. Genotype and phenotype data in individuals positive and negative for 21OH-AA are compared.

Results: Major genetic risk for 21OH-AA is in the MHC haplotypes DRB1*04-DQB1*0302 (primarily DRB1*0404) and DRB1*0301-DQB1*0201. Protective effects in class II MHC haplotypes DRB1*0101-DQB1*0501 and DRB1*0701-DQB1*0202 also were detected. There is no difference in the presence of HLA-B15 and little difference in the presence of HLA-B8 (after class II effects are accounted for) in T1DM patients with 21OH-AA compared with known associations (HLA-B8 positive and HLA-B15 negative) in AD.

Conclusions: In 21OH-AA(+) subjects, genetic risk is found mainly in MHC class II haplotypes DR3 and DR4 but not class I alleles (HLA-B8 or HLA-B15). This suggests a difference between autoantibody formation (class II dependent) and progression to overt disease (class I dependent) in AD.
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http://dx.doi.org/10.1210/jc.2011-2824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410257PMC
August 2012

Zinc transporter-8 autoantibodies improve prediction of type 1 diabetes in relatives positive for the standard biochemical autoantibodies.

Diabetes Care 2012 Jun 23;35(6):1213-8. Epub 2012 Mar 23.

Barbara Davis Center for Childhood Diabetes, University of Colorado at Denver, Aurora, Colorado, USA.

Objective: We assessed diabetes risk associated with zinc transporter-8 antibodies (ZnT8A), islet cell antibodies (ICA), and HLA type and age in relatives of people with type 1 diabetes with the standard biochemical autoantibodies (BAA) to insulin (IAA), GAD65 (GAD65A), and/or insulinoma-associated protein 2 antigen (IA-2A).

Research Design And Methods: For this analysis, 2,256 relatives positive for at least one BAA, of whom 142 developed diabetes, were tested for ZnT8A, ICA, and HLA genotype followed by biannual oral glucose tolerance tests. ZnT8A were also tested in 911 randomly chosen antibody-negative relatives.

Results: ZnT8A were associated with the other BAA (548 of 2,256 [24.3%] BAA(+) vs. 8 of 911 [0.8%] BAA(-), P < 0.001) and BAA number (177 of 1,683 [10.5%] single-, 221 of 384 [57.6%] double-, and 150 of 189 [79.4%] triple-BAA positivity, P < 0.001). The 4-year diabetes risk was higher in single BAA(+) relatives with ZnT8A than ZnT8A(-) relatives (31 vs. 7%, P < 0.001). In multivariable analysis, age ≤ 20 years (hazard ratio 2.13, P = 0.03), IA-2A (2.15, P = 0.005), IAA (1.73, P = 0.01), ICA (2.37, P = 0.002), and ZnT8A (1.87, P = 0.03) independently predicted diabetes, whereas HLA type (high and moderate vs. low risk) and GAD65A did not (P = 0.81 and 0.86, respectively).

Conclusions: In relatives with one standard BAA, ZnT8A identified a subset at higher diabetes risk. ZnT8A predicted diabetes independently of ICA, the standard BAA, age, and HLA type. ZnT8A should be included in type 1 diabetes prediction and prevention studies.
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http://dx.doi.org/10.2337/dc11-2081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357246PMC
June 2012

Mapping of conformational autoantibody epitopes in ZNT8.

Diabetes Metab Res Rev 2011 Nov;27(8):883-6

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, CO 80045, USA.

Background: Identification of the major humoral epitopes in zinc transporter 8 (ZnT8) will expand the range of biomarkers for human type 1 diabetes and may provide clues to the mechanisms governing disease progression. Our initial studies suggested that most ZnT8-reactive sera recognize conformational epitopes in the final 100aa region of the molecule. Subsequently we identified residue 325 as a major determinant in two epitopes linked to a genetic polymorphism with high minor allele frequency (rs13266634). The goal of the current study was to extend this analysis to identify non-polymorphic epitopes in ZnT8.

Methods: Although the carboxy-terminal domains of human and mouse ZnT8 are ∼80% identical, the mouse probe is not precipitated by the majority of human type 1 diabetes sera. Thus to identify key residues we systematically 'humanized' the mouse probe at each position that differs and evaluated the probes in radio-immunoassays.

Results: As previously reported, only the alteration of Q>R325 by itself showed any restoration of binding to human sera. However, when clusters of structurally adjacent variant residues were also changed an additional region of antigenicity was revealed that depended on residues R332, E333, K336, and K340. Using a panel of 112 sera from recent onset subjects tested with the hC325Q and m-R325R332E333K336K340 probes, 39.3% of the subjects were ZnT8(Q)A+ , of which 38.6% (17/44) also recognized the mouse probe.

Conclusions: We conclude that the mR-REKK probe identifies a third major epitope in ZnT8 that may add to the diagnostic utility of measuring autoantibodies to this molecule.
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http://dx.doi.org/10.1002/dmrr.1266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664522PMC
November 2011

Diabetes antibody standardization program: first proficiency evaluation of assays for autoantibodies to zinc transporter 8.

Clin Chem 2011 Dec 6;57(12):1693-702. Epub 2011 Oct 6.

Genomic Unit for the Diagnosis of Human Pathologies, Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy.

Background: Zinc transporter 8 (ZnT8) is a recently identified major autoantigen in type 1 diabetes, and autoantibodies to ZnT8 (ZnT8A) are new markers for disease prediction and diagnosis. Here we report the results of the first international proficiency evaluation of ZnT8A assays by the Diabetes Antibody Standardization Program (DASP).

Methods: After a pilot workshop in 2007, an expanded ZnT8A workshop was held in 2009, with 26 participating laboratories from 13 countries submitting results of 63 different assays. ZnT8A levels were measured in coded sera from 50 patients with newly diagnosed type 1 diabetes and 100 blood donor controls. Results were analyzed comparing area under the ROC curve (ROC-AUC), sensitivity adjusted to 95% specificity (AS95), concordance of sample ZnT8A positive or negative designation, and autoantibody levels.

Results: ZnT8A radio binding assays (RBAs) based on combined immunoprecipitation of the 2 most frequent ZnT8 COOH-terminal domain polymorphic variants showed a median ROC-AUC of 0.848 [interquartile range (IQR) 0.796-0.878] and a median AS95 of 70% (IQR 60%-72%). These RBAs were more sensitive than assays using as antigen either 1 ZnT8 variant only or chimeric constructs joining NH(2)- and COOH-terminal domains, assays based on immunoprecipitation and bioluminescent detection, or assays based on immunofluorescent staining of cells transfected with full-length antigen.

Conclusions: The DASP workshop identified immunoprecipitation-based ZnT8A assays and antigen constructs that achieved both a high degree of sensitivity and specificity and were suitable for more widespread clinical application.
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http://dx.doi.org/10.1373/clinchem.2011.170662DOI Listing
December 2011

Contribution of antibodies against IA-2β and zinc transporter 8 to classification of diabetes diagnosed under 40 years of age.

Diabetes Care 2011 Aug 29;34(8):1760-5. Epub 2011 Jun 29.

Brussels Free University, Brussels, Belgium.

Objective: We investigated whether measuring autoantibodies against zinc transporter 8 (ZnT8A) and IA-2β (IA-2βA) may improve classification of new-onset type 1 diabetic patients based on detection of autoantibodies against insulin (IAA), GAD (GADA), and IA-2 (IA-2A). In addition, we studied the correlation of IA-2βA and ZnT8A with other biological and demographic variables.

Research Design And Methods: Circulating autoantibodies were determined by liquid-phase radiobinding assays from 761 healthy control subjects and 655 new-onset (<1 week insulin) diabetic patients (aged 0-39 years) with clinical type 1 diabetes phenotype consecutively recruited by the Belgian Diabetes Registry.

Results: At diagnosis, IA-2βA and ZnT8A prevalences were 41 and 58%, respectively. In IAA-negative, GADA-negative, and IA-2A-negative patients, one IA-2βA-positive and eleven ZnT8A-positive individuals were identified at the expense of eight and seven additional positive control subjects (1%), respectively, for each test. ZnT8A or IA-2βA screening increased (P < 0.001; McNemar) the number of patients with ≥2 antibodies both under (from 78 to 87% for ZnT8A and 82% for IA-2βA) and above age 15 (from 51 to 63% for ZnT8A and 56% for IA-2βA) versus 0% in control subjects. IA-2βA and ZnT8A were preferentially associated with IA-2A, and with younger age at diagnosis. Unlike ZnT8A, IA-2βA levels were positively correlated with HLA-DQ8 and negatively with HLA-DQ2. ZnT8A could replace IAA for classification of patients above age 10 without loss of sensitivity or specificity.

Conclusions: ZnT8A, and to a lesser degree IA-2βA, may usefully complement GADA, IA-2A, and IAA for classifying insulin-treated diabetes under age 40 years.
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http://dx.doi.org/10.2337/dc10-2268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3142046PMC
August 2011

Human type 1 diabetes is associated with T cell autoimmunity to zinc transporter 8.

J Immunol 2011 May 6;186(10):6056-63. Epub 2011 Apr 6.

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, CO 80045, USA.

Recently we demonstrated that zinc transporter 8 (ZnT8) is a major target of autoantibodies in human type 1 diabetes (T1D). Because the molecules recognized by T1D autoantibodies are typically also targets of autoreactive T cells, we reasoned that this would likely be the case for ZnT8. To test this hypothesis, IFN-γ-producing T cells specific for ZnT8 in the peripheral blood of 35 patients with T1D (<6 mo after onset at blood draw) and 41 age-matched controls were assayed by ELISPOT using a library of 23 overlapping dipeptide pools covering the entire 369 aa primary sequence. Consistent with our hypothesis, patients showed significantly higher T cell reactivity than the matched controls, manifest in terms of the breadth of the overall response and the magnitude of responses to individual pools. Therefore, the median number of pools giving positive responses (stimulation index ≥ 3) in the control group was 1.0 (range, 0-7) compared with 6.0 (range, 1-20; p < 0.0001) for the patients. Similarly, the median stimulation index of positive responses in controls was 3.1 versus 5.0 in the patients (p < 0.0001). Individually, 7 of 23 pools showed significant disease association (p < 0.001), with several of the component peptides binding the disease associated HLA-DR3 (0301) and -DR4 (0401) molecules in vitro. We conclude that ZnT8 is also a major target of disease-associated autoreactive T cells in human T1D, and we suggest that reagents that target ZnT8-specific T cells could have therapeutic potential in preventing or arresting the progression of this disease.
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http://dx.doi.org/10.4049/jimmunol.1003815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088514PMC
May 2011

[Value of zinc transporter 8 autoantibody in the diagnostic classification of acute-onset diabetics].

Zhonghua Yi Xue Za Zhi 2010 Sep;90(36):2536-40

Diabetes Center, Institute of Metabolism & Endocrinology, Department of Endocrinology, Second Xiangya Hospital, Central South University, Changsha 410011, China.

Objective: to explore the application significance of zinc transporter 8 autoantibody (ZnT8A) in the diagnostic classification of acute-onset diabetics.

Methods: according to the status of glutamic acid decarboxylase antibody (GADA) and tyrosine phosphatase antibody (IA2-A), 453 acute-onset diabetics were divided into A+ subgroup (any antibody positive) and A- subgroup (both antibodies negative). A total of 555 type 2 diabetics and 405 healthy controls were recruited. The distribution and correlated factors of ZnT8A were analyzed in the acute-onset diabetic group and two subgroups (A+ and A-). The clinical characteristics were compared between the patients with ZnT8A positive alone and patients without any antibody. All these islet antibodies were measured by radioligand assay.

Results: the prevalence of ZnT8A in acute-onset diabetics was 24.3% and it was significantly higher than that in type 2 diabetics (1.8%) and healthy controls (1.0%) (both P < 0.01). The frequency of ZnT8A in A+ subgroup was much higher than A- subgroup (29.7% vs 15.8%, P < 0.01). The positive rates of ZnT8A were much higher in all the subgroups with age at onset of < 30 yr than those with ≥ 30 yr (0 - 9, 34.9%; 10 - 19, 26.7%; 20 - 29, 26.3% vs ≥ 30 yr, 18.3%; all P < 0.05); furthermore, the rates were also higher in BMI < 21.0 kg/m(2) and 21.0 - 25.0 kg/m(2) subgroups than in BMI > 25.0 kg/m(2) subgroup (25.5% and 25.9% vs 8.7%, both P < 0.05). The ZnT8A level was only positively correlated with IA2-A titer (r = 0.165, P = 0.01), but not related to such factors as GADA titer, age at onset, duration, body mass index, HbA1c and CP levels (all P > 0.05). As compared with Ab- patients, the patients with ZnT8A positive alone had much higher insulin injection dosage [(35.5 ± 9.3) U/d vs (29.8 ± 14.7) U/d, P < 0.05], and much lower systolic blood pressures [(107 ± 15) mm Hg vs (113 ± 16) mm Hg, P < 0.05] and diastolic blood pressures [(69 ± 12) mm Hg vs (73 ± 12) mm Hg, P < 0.05].

Conclusion: ZnT8A testing may be applied in the diagnostic classification of acute-onset diabetics, especially in those without an evidence of GADA and IA2-A since it helps to identify a clinical phenotype which is more similar to the classic type 1 diabetes.
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September 2010

The diagnostic value of zinc transporter 8 autoantibody (ZnT8A) for type 1 diabetes in Chinese.

Diabetes Metab Res Rev 2010 Oct;26(7):579-84

Diabetes Center, Institute of Metabolism and Endocrinology, Second Xiangya Hospital, Central South University, Changsha, China.

Background: Zinc transporter-8 (ZnT8) was recently identified as a novel autoantigen in human type 1 diabetes (T1D). Autoantibody to ZnT8 (ZnT8A) was detected in up to 80% of patients with new-onset T1D and 26% of patients with T1D otherwise classified as negative on the basis of existing markers. As no data of ZnT8A in Chinese have been reported, we aim to evaluate the utility of ZnT8A for diagnosis of autoimmune T1D in Chinese relative to other autoantibody markers.

Methods: Radioligand binding assays were performed on 539 T1D sera using human ZnT8 carboxyterminal 325Arg construct or a dimer incorporating 325Arg and 325Trp alongside antibodies to glutamic acid decarboxylase (GADA) or insulinoma-associated protein 2 (IA-2A). The antigenic specificity was analysed in the context of clinical characteristics of the patients.

Results: ZnT8A were present in 24.1% (130 of 539) of patients with T1D versus 1.8% (10 of 555; P < 0.001) in type 2 diabetes. At diagnosis, ZnT8A and IA-2A were less prevalent in Chinese subjects with T1D than in Caucasian populations (both P < 0.001) but similar to Japanese. The diagnostic sensitivity of combined GADA, IA-2A and ZnT8A measurements reached 65.5% with ZnT8A detected in 13.5% (29 of 215) of GADA and/or IA-2A-negative subjects. ZnT8A prevalence was lower in older and fatter patients. ZnT8A+ alone patients were distinguished from Ab- ones (P < 0.05-0.001) on the basis of higher insulin requirement and lower systolic blood pressure level.

Conclusion: ZnT8A is an independent marker for T1D in Chinese and combined with GADA and IA-2A enhances diagnostic sensitivity. ZnT8A may be associated with different clinical phenotypes than GADA or IA-2A.
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http://dx.doi.org/10.1002/dmrr.1128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2962924PMC
October 2010

Triple chimeric islet autoantigen IA2-ZnT8WR to facilitate islet autoantibody determination.

J Immunol Methods 2010 Feb 24;353(1-2):20-3. Epub 2009 Dec 24.

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, CO, United States.

Type 1A diabetes is strongly associated with the presence of islet autoantibodies. Large scale population screening of islet autoantibodies is essential for many different national and international studies related to defining subtypes of diabetes, the natural history of the disease, and for trials of prevention. Testing for relevant autoantibodies has become more difficult as the number of important autoantibodies/epitopes increases. In the present study, we created a chimeric protein, IA2-ZnT8WR, with two major islet autoantigens, IA-2 and the recent Zinc transporter 8 (ZnT8). The chimeric molecule included both common polymorphisms of the ZnT8 molecule, arginine or tryptophan at position 325. Serum samples from 284 patients with newly diagnosed diabetes, 10 prediabetics, and 110 age-matched normal controls were analyzed for islet autoantibodies reacting with the IA2-ZnT8WR molecule. Autoantibodies to the chimeric molecule were compared to reactivity with individual assays detecting autoantibodies reacting with the separate molecules (IA-2, ZnT8-R and ZnT8-W). With this chimeric protein antigen, IA2-ZnT8WR, one radioassay is able to detect autoantibodies to IA-2 and to both major forms of ZnT8 (100% sensitivity, 100% unchanged specificity, relative to individual molecules). The chimeric assay provides an efficient and economical technique to screen for islet autoantibodies reacting with IA-2 and ZnT8.
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http://dx.doi.org/10.1016/j.jim.2009.12.004DOI Listing
February 2010

Novel antigens in type 1 diabetes: the importance of ZnT8.

Curr Diab Rep 2009 Apr;9(2):105-12

Barbara Davis Center for Childhood Diabetes, University of Colorado at Denver and Health Sciences Center, PO Box 6511, 1775 North Ursula Court, Aurora, CO 80045, USA.

The presence of circulating islet cell autoantibodies distinguishes type 1A diabetes (T1D) from other diabetic syndromes and determination of autoantigen genes and proteins is instrumental in understanding T1D as a clinical entity and in investigating the pathogenesis of the disease. ZnT8 was recently defined as a candidate autoantigen based on a -bioinformatics analysis focused on discovery of beta-cell-specific proteins associated with the regulatory pathway of secretion. The native molecule does not lend itself easily to solution-phase autoantibody assays, but ligands based on the predicted domain structure and molecular modeling have led to robust diagnostic procedures showing high specificities and sensitivities that complement current T1D autoantibody assays and add to the predictive value of their measurement. The incorporation of genetic and structural epitope analysis into ZnT8A determinations adds a further dimension to its diagnostic value and understanding of its role in the autoimmune disease process.
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http://dx.doi.org/10.1007/s11892-009-0019-4DOI Listing
April 2009

SlC30A8 is a major target of humoral autoimmunity in type 1 diabetes and a predictive marker in prediabetes.

Ann N Y Acad Sci 2008 Dec;1150:256-9

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, Colorado 80045, USA.

Type 1A diabetes (T1D) results from autoimmunity targeted at a limited number of molecules that are expressed in the pancreatic beta cell. Putative novel autoantigen candidates were identified from microarray expression profiling of human and rodent islet cells. The highest ranking candidate was Slc30A8 (zinc transporter 8; ZnT8), which was screened by radioimmunoprecipitation assays against new-onset T1D and prediabetic sera. Such assays detected 63% of subjects with new-onset diabetes, but fewer than 2% of controls, 3% of those with type 2 diabetes, and 10% of patients with other autoimmune disorders. ZnT8 autoantibodies were found, however, in 26% of T1D subjects previously classified as autoantibody-negative on the basis of existing markers (GADA, IA2 A, IAA, and ICA). We conclude that SLC30A8 provides an important additional and independent predictive marker for T1D.
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http://dx.doi.org/10.1196/annals.1447.029DOI Listing
December 2008

Identification of a major humoral epitope in Slc30A8 (ZnT8).

Ann N Y Acad Sci 2008 Dec;1150:252-5

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, Colorado 80045, USA.

The human zinc transporter Slc30A8 (ZnT8) is a major target of humoral autoimmunity in human type 1A diabetes. However, despite extensive conservation, the majority of human autoimmune sera fail to recognize the murine ortholog. Moreover, Slc30A8 appears not to be a significant target of humoral autoimmunity in the NOD mouse. We therefore "humanized" the murine protein by site-directed mutagenesis. Only conversion of Q324 to arginine (equivalent to R325 in the human protein) partially restored reactivity to a pool of sera selected for high titers to the human probe. Additionally, the reciprocal mutation (human R325 to Q) abolished reactivity for 38/103 (36.9%) of ZnT8(+) sera. We conclude that the C-terminal domain of human ZnT8 contains at least two discrete epitopes, one of which is critically dependent upon the arginine residue at position 325.
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http://dx.doi.org/10.1196/annals.1447.028DOI Listing
December 2008
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