Mapping of a hybrid insulin peptide in the inflamed islet ß-cells from NOD mice.

There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of ß-cell antigens, including hybrid insulin peptides (HIPs). The ligands for several CD4 T-cell clones derived from the NOD mouse are HIPs composed of a fragment of proinsulin joined to peptides from endogenous ß-cell granule proteins. The diabetogenic T-cell clone BDC-6.9 reacts to a fragment of C-peptide fused to a cleavage product of pro-islet amyloid polypeptide (6.9HIP). In this study, we used a monoclonal antibody (MAb) to the 6.9HIP to determine when and where HIP antigens are present in NOD islets during disease progression and with which immune cells they associate. Immunogold labeling of the 6.9HIP MAb and organelle-specific markers for electron microscopy were employed to map the subcellular compartment(s) in which the HIP is localized within ß-cells. While the insulin B9-23 peptide was present in nearly all islets, the 6.9HIP MAb stained infiltrated islets only in NOD mice at advanced stages of T1D development. Islets co-stained with the 6.9HIP MAb and antibodies to mark insulin, macrophages, and dendritic cells indicate that 6.9HIP co-localizes within insulin-positive ß-cells as well as intra-islet antigen-presenting cells (APCs). In electron micrographs, the 6.9HIP co-localized with granule structures containing insulin alone or both insulin and LAMP1 within ß-cells. Exposing NOD islets to the endoplasmic reticulum (ER) stress inducer tunicamycin significantly increased levels of 6.9HIP in subcellular fractions containing crinosomes and dense-core granules (DCGs). This work demonstrates that the 6.9HIP can be visualized in the infiltrated islets and suggests that intra-islet APCs may acquire and present HIP antigens within islets.

Novel diabetes autoantibodies and prediction of type 1 diabetes.

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.

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

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.

Identification of Autoantibodies to a Hybrid Insulin Peptide in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that attacks the insulin-producing b cells of the pancreatic islets. Autoantibodies to b cell proteins typically appear in the circulation years before disease onset, and serve as the most accurate biomarkers of T1D risk. Our laboratory has recently discovered novel b cell proteins comprising hybrid proinsulin:islet amyloid polypeptide peptides (IAPP). T cells from a diabetic mouse model and T1D patients are activated by these hybrid peptides. In this study, we asked whether these hybrid molecules could serve as antigens for autoantibodies in T1D and prediabetic patients. We analyzed sera from T1D patients, prediabetics and healthy age-matched donors. Using a highly sensitive electrochemiluminescence assay, sera were screened for binding to recombinant proinsulin:IAPP probes or truncated derivatives. Our results show that sera from T1D patients contain antibodies that bind larger hybrid proinsulin:IAPP probes, but not proinsulin or insulin, at significantly increased frequencies compared to normal donors. Examination of sera from prediabetic patients confirms titers of antibodies to these hybrid probes in more than 80% of individuals, often before seroconversion. These results suggest that hybrid insulin peptides are common autoantigens in T1D and prediabetic patients, and that antibodies to these peptides may serve as valuable early biomarkers of the disease.

Insulin B-chain hybrid peptides are agonists for T cells reactive to insulin B:9-23 in autoimmune diabetes.

Insulin is considered to be a key antigenic target of T cells in Type 1 Diabetes (T1D) and autoimmune diabetes in the NOD mouse with particular focus on the B-chain amino acid sequence B:9-23 as the primary epitope. Our lab previously discovered that hybrid insulin peptides (HIPs), comprised of insulin C-peptide fragments fused to other ß-cell granule peptides, are ligands for several pathogenic CD4 T cell clones derived from NOD mice and for autoreactive CD4 T cells from T1D patients. A subset of CD4 T cell clones from our panel react to insulin and B:9-23 but only at high concentrations of antigen. We hypothesized that HIPs might also be formed from insulin B-chain sequences covalently bound to other endogenously cleaved ß-cell proteins. We report here on the identification of a B-chain HIP, termed the 6.3HIP, containing a fragment of B:9-23 joined to an endogenously processed peptide of ProSAAS, as a strong neo-epitope for the insulin-reactive CD4 T cell clone BDC-6.3. Using an I-Ag7 tetramer loaded with the 6.3HIP, we demonstrate that T cells reactive to this B-chain HIP can be readily detected in NOD mouse islet infiltrates. This work suggests that some portion of autoreactive T cells stimulated by insulin B:9-23 may be responding to B-chain HIPs as peptide ligands.

Cathepsin D Drives the Formation of Hybrid Insulin Peptides Relevant to the Pathogenesis of Type 1 Diabetes.

Hybrid insulin peptides (HIPs) form in pancreatic ß-cells through the formation of peptide bonds between proinsulin fragments and other peptides. HIPs have been identified in pancreatic islets by mass spectrometry and are targeted by CD4 T cells in patients with type 1 diabetes (T1D) as well as by pathogenic CD4 T-cell clones in nonobese diabetic (NOD) mice. The mechanism of HIP formation is currently poorly understood; however, it is well established that proteases can drive the formation of new peptide bonds in a side reaction during peptide bond hydrolysis. Here, we used a proteomic strategy on enriched insulin granules and identified cathepsin D (CatD) as the primary protease driving the specific formation of HIPs targeted by disease-relevant CD4 T cells in T1D. We also established that NOD islets deficient in cathepsin L (CatL), another protease implicated in the formation of disease-relevant HIPs, contain elevated levels of HIPs, indicating a role for CatL in the proteolytic degradation of HIPs. In summary, our data suggest that CatD may be a therapeutic target in efforts to prevent or slow the autoimmune destruction of ß-cells mediated by HIP-reactive CD4 T cells in T1D.

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

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.

Mapping of conformational autoantibody epitopes in ZNT8.

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.

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

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.

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

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.

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

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.

Embryonic growth-associated protein is one subunit of a novel N-terminal acetyltransferase complex essential for embryonic vascular development.

N-terminal protein acetylation, catalyzed by N-terminal acetyltransferases (NATs) recognizing distinct N-terminal sequences, is gaining recognition as an essential regulator of normal cell function, but little is known of its role in vertebrate development. We previously cloned a novel gene, embryonic growth-associated protein (EGAP), the expression of which is associated with rapid vascular smooth muscle cell proliferation during development. We show herein EGAP is the mammalian/zebrafish homologue of yeast Mak10p, one subunit of the yeast NatC complex, and describe the cloning of its binding partners Mak3 and Mak31. The EGAP NAT forms a functional complex in mammalian cells, is evolutionarily conserved, and developmentally regulated. It is widely but not ubiquitously expressed during early zebrafish development but undetectable in later developmental stages. We demonstrate EGAP- and Mak3-deficient zebrafish fail to develop because of, in part, decreased cell proliferation, increased apoptosis, and poor blood vessel formation contributing to embryonic lethality. We examined the role of target of rapamycin (TOR), a highly conserved protein kinase controlling cell growth, as a physiological target of EGAP NAT acetylation. Compared with controls, TOR expression and signaling is significantly reduced in EGAP morphants. Pharmacological inhibition of TOR with rapamycin phenocopied the EGAP morpholino oligonucleotide-induced growth and vessel defects. Overexpression of constitutively active TOR rescued EGAP morphants, suggesting TOR is a direct or indirect endogenous substrate of the EGAP NAT complex. These data suggest the EGAP NAT complex is an essential regulatory enzyme controlling the function of a subset of proteins required for embryonic growth control and vessel development.

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.

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.

Perlecan-induced suppression of smooth muscle cell proliferation is mediated through increased activity of the tumor suppressor PTEN.

We were interested in the elucidation of the interaction between the heparan sulfate proteoglycan, perlecan, and PTEN in the regulation of vascular smooth muscle cell (SMC) growth. We verified serum-stimulated DNA synthesis, and Akt and FAK phosphorylation were significantly reduced in SMCs overexpressing wild-type PTEN. Our previous studies showed perlecan is a potent inhibitor of serum-stimulated SMC growth. We report in the present study, compared with SMCs plated on fibronectin, serum-stimulated SMCs plated on perlecan exhibited increased PTEN activity, decreased FAK and Akt activities, and high levels of p27, consistent with SMC growth arrest. Adenoviral-mediated overexpression of constitutively active Akt reversed perlecan-induced SMC growth arrest while morpholino antisense-mediated loss of endogenous PTEN resulted in increased growth and phosphorylation of FAK and Akt of SMCs on perlecan. Immunohistochemical and Western analyses of balloon-injured rat carotid artery tissues showed a transient increase in phosphoPTEN (inactive) after injury, correlating to high rates of neointimal cell replication; phosphoPTEN was largely limited to actively replicating SMCs. Similarly, in the developing rat aorta, we found increased PTEN activity associated with increased perlecan deposition and decreased SMC replication rates. However, significantly decreased PTEN activity was detected in aortas of perlecan-deficient mouse embryos, consistent with SMC hyperplasia observed in these animals, compared with E17.5 heterozygous controls that produce abundant amounts of perlecan at this developmental time point. Our data show PTEN is a potent endogenously produced inhibitor of SMC growth and increased PTEN activity mediates perlecan-induced suppression of SMC proliferation.

Unique, highly proliferative growth phenotype expressed by embryonic and neointimal smooth muscle cells is driven by constitutive Akt, mTOR, and p70S6K signaling and is actively repressed by PTEN.

BACKGROUND: At distinct times during embryonic development and after vascular injury, smooth muscle cells (SMCs) exhibit a highly proliferative, serum-independent growth phenotype. The aim of the present study was to evaluate the functional role of S6 ribosomal protein (S6RP) and upstream positive and negative regulators in the control of SMC serum-independent growth. METHODS AND RESULTS: We previously reported increased expression of S6RP mRNA was associated with this unique growth phenotype. Using immunohistochemistry and Western blot analysis, we report high levels of total and phospho-S6RP and increased levels of Akt and p70S6K phosphorylation, upstream positive regulators of S6RP, in rat embryonic aortas and adult balloon-injured carotid arteries compared with quiescent adult aortas and uninjured carotid arteries. Western blot analysis demonstrated that cultured embryonic and neointimal SMCs that exhibited serum-independent growth capabilities expressed high levels of S6RP and constitutively active Akt, mTOR, and p70S6K. Pharmacological and molecular inhibition of phosphatidylinositol 3-kinase (PI3K) signaling pathways, using PI3K inhibitors, rapamycin, or dominant-negative Akt adenovirus, suppressed embryonic and neointimal SMC serum-independent growth. Finally, decreased activity of PTEN, an endogenous negative regulator of PI3K signaling, was associated with high in vivo SMC growth rates, and morpholino-mediated loss of endogenous PTEN induced a serum-independent growth phenotype in cultured serum-dependent SMCs. CONCLUSIONS: The possibility exists that cells that exhibit a distinct embryonic-like growth phenotype different from traditional SMCs are major contributors to intimal thickening. Growth of SMCs that exhibit this phenotype is dependent on constitutive Akt and mTOR/p70S6K signaling and is actively inhibited through the timed acquisition of the endogenously produced growth suppressor PTEN.

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

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.

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

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.

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

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), ß-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 ß-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 ß-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 ß-cell mass and presence of IAA as inclusion criteria.

Zinc transporter 8 (ZnT8) and ß cell function.

Human pancreatic ß cells have exceptionally high zinc content. In ß 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 ß cells, its role in glucose metabolism based on knockout mouse studies, and current theories regarding the link between ZnT8 function and T2D.

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

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.