OGTT Metrics Surpass Continuous Glucose Monitoring Data for T1D Prediction in Multiple-Autoantibody-Positive Individuals.

CONTEXT: The value of continuous glucose monitoring (CGM) for monitoring autoantibody (AAB)-positive individuals in clinical trials for progression of type 1 diabetes (T1D) is unknown. OBJECTIVE: Compare CGM with oral glucose tolerance test (OGTT)-based metrics in prediction of T1D. METHODS: At academic centers, OGTT and CGM data from multiple-AAB relatives were evaluated for associations with T1D diagnosis. Participants were multiple-AAB-positive individuals in a TrialNet Pathway to Prevention (TN01) CGM ancillary study (n = 93). The intervention was CGM for 1 week at baseline, 6 months, and 12 months. Receiver operating characteristic (ROC) curves of CGM and OGTT metrics for prediction of T1D were analyzed. RESULTS: Five of 7 OGTT metrics and 29/48 CGM metrics but not HbA1c differed between those who subsequently did or did not develop T1D. ROC area under the curve (AUC) of individual CGM values ranged from 50% to 69% and increased when adjusted for age and AABs. However, the highest-ranking metrics were derived from OGTT: 4/7 with AUC ∼80%. Compared with adjusted multivariable models using CGM data, OGTT-derived variables, Index60 and DPTRS (Diabetes Prevention Trial-Type 1 Risk Score), had higher discriminative ability (higher ROC AUC and positive predictive value with similar negative predictive value). CONCLUSION: Every 6-month CGM measures in multiple-AAB-positive individuals are predictive of subsequent T1D, but less so than OGTT-derived variables. CGM may have feasibility advantages and be useful in some settings. However, our data suggest there is insufficient evidence to replace OGTT measures with CGM in the context of clinical trials.

CGM Metrics Identify Dysglycemic States in Participants From the TrialNet Pathway to Prevention Study.

OBJECTIVE: Continuous glucose monitoring (CGM) parameters may identify individuals at risk for progression to overt type 1 diabetes. We aimed to determine whether CGM metrics provide additional insights into progression to clinical stage 3 type 1 diabetes. RESEARCH DESIGN AND METHODS: One hundred five relatives of individuals in type 1 diabetes probands (median age 16.8 years; 89% non-Hispanic White; 43.8% female) from the TrialNet Pathway to Prevention study underwent 7-day CGM assessments and oral glucose tolerance tests (OGTTs) at 6-month intervals. The baseline data are reported here. Three groups were evaluated: individuals with 1) stage 2 type 1 diabetes (n = 42) with two or more diabetes-related autoantibodies and abnormal OGTT; 2) stage 1 type 1 diabetes (n = 53) with two or more diabetes-related autoantibodies and normal OGTT; and 3) negative test for all diabetes-related autoantibodies and normal OGTT (n = 10). RESULTS: Multiple CGM metrics were associated with progression to stage 3 type 1 diabetes. Specifically, spending ≥5% time with glucose levels ≥140 mg/dL (P = 0.01), ≥8% time with glucose levels ≥140 mg/dL (P = 0.02), ≥5% time with glucose levels ≥160 mg/dL (P = 0.0001), and ≥8% time with glucose levels ≥160 mg/dL (P = 0.02) were all associated with progression to stage 3 disease. Stage 2 participants and those who progressed to stage 3 also exhibited higher mean daytime glucose values; spent more time with glucose values over 120, 140, and 160 mg/dL; and had greater variability. CONCLUSIONS: CGM could aid in the identification of individuals, including those with a normal OGTT, who are likely to rapidly progress to stage 3 type 1 diabetes.

Autoantibodies Directed Toward a Novel IA-2 Variant Protein Enhance Prediction of Type 1 Diabetes.

We identified autoantibodies (AAb) reacting with a variant IA-2 molecule (IA-2var) that has three amino acid substitutions (Cys27, Gly608, and Pro671) within the full-length molecule. We examined IA-2var AAb in first-degree relatives of type 1 diabetes (T1D) probands from the TrialNet Pathway to Prevention Study. The presence of IA-2var-specific AAb in relatives was associated with accelerated progression to T1D in those positive for AAb to GAD65 and/or insulin but negative in the standard test for IA-2 AAb. Furthermore, relatives with single islet AAb (by traditional assays) and carrying both IA-2var AAb and the high-risk HLA-DRB1*04-DQB1*03:02 haplotype progress rapidly to onset of T1D. Molecular modeling of IA-2var predicts that the genomic variation that alters the three amino acids induces changes in the three-dimensional structure of the molecule, which may lead to epitope unmasking in the IA-2 extracellular domain. Our observations suggest that the presence of AAb to IA-2var would identify high-risk subjects who would benefit from participation in prevention trials who have one islet antibody by traditional testing and otherwise would be misclassified as "low risk" relatives.

CD19+IgM+ cells demonstrate enhanced therapeutic efficacy in type 1 diabetes mellitus.

We describe a protective effect on autoimmune diabetes and reduced destructive insulitis in NOD.scid recipients following splenocyte injections from diabetic NOD donors and sorted CD19+ cells compared with NOD.scid recipients receiving splenocytes alone. This protective effect was age specific (only CD19+ cells from young NOD donors exerted this effect; P < 0.001). We found that the CD19+IgM+ cell is the primary subpopulation of B cells that delayed transfer of diabetes mediated by diabetogenic T cells from NOD mice (P = 0.002). Removal of IgM+ cells from the CD19+ pool did not result in protection. Notably, protection conferred by CD19+IgM+ cotransfers were not dependent on the presence of Tregs, as their depletion did not affect their ability to delay onset of diabetes. Blockade of IL-10 with neutralizing antibodies at the time of CD19+ cell cotransfers also abrogated the therapeutic effect, suggesting that IL-10 secretion was an important component of protection. These results were strengthened by ex vivo incubation of CD19+ cells with IL-5, resulting in enhanced proliferation and IL-10 production and equivalently delayed diabetes progression (P = 0.0005). The potential to expand CD19+IgM+ cells, especially in response to IL-5 stimulation or by pharmacologic agents, may be a new therapeutic option for type 1 diabetes.

Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development.

OBJECTIVE: The characterization of diverse subtypes of diabetes is a dynamic field of clinical research and an active area of discussion. The objective of this study was to identify new antigenic determinants in the neuroendocrine autoantigen IA-2 (ICA512) and assess whether circulating autoantibodies directed to new IA-2 epitopes identify autoimmune diabetes in young and adult populations with diabetes. RESEARCH DESIGN AND METHODS: Clinically diagnosed patients with type 2 diabetes (n = 258; diabetes duration: 0.01-31 years) were evaluated using a new biomarker detecting autoantibodies directed to the extracellular domain of the neuroendocrine autoantigen IA-2 (IA-2ec). The proportion of IA-2ec autoantibodies was also evaluated in newly diagnosed patients with type 1 diabetes (n = 150; diabetes duration: 0.04-0.49 years). In addition, IA-2 (intracellular domain), GAD65, and zinc transporter 8 autoantibodies were assayed. RESULTS: IA-2ec autoantibodies were detected in patients with type 1 diabetes and, surprisingly, in 5% of patients with type 2 diabetes without serologic responses to other IA-2 antigenic epitopes or other islet autoantigens. We also assessed the ability of IA-2ec-derived peptides to elicit CD4+ T-cell responses by stimulating peripheral blood mononuclear cells from patients with type 1 diabetes (n = 18) and HLA-matched healthy subjects (n = 13) with peptides and staining with the peptide/DQ8-specific tetramers, observing disease-associated responses to previously unreported epitopes within IA-2ec. CONCLUSIONS: We developed a new antibody biomarker identifying novel antigenic determinants within the N terminus of IA-2. IA-2ec autoantibodies can be detected in patients with type 1 diabetes and in a subgroup of adult autoimmune patients with type 2 diabetes phenotype negative for conventional islet autoantibody testing. These observations suggest that islet autoimmunity may be more common in clinically diagnosed type 2 diabetes than previously observed.

Islet autoimmunity identifies a unique pattern of impaired pancreatic beta-cell function, markedly reduced pancreatic beta cell mass and insulin resistance in clinically diagnosed type 2 diabetes.

There is a paucity of literature describing metabolic and histological data in adult-onset autoimmune diabetes. This subgroup of diabetes mellitus affects at least 5% of clinically diagnosed type 2 diabetic patients (T2DM) and it is termed Latent Autoimmune Diabetes in Adults (LADA). We evaluated indexes of insulin secretion, metabolic assessment, and pancreatic pathology in clinically diagnosed T2DM patients with and without the presence of humoral islet autoimmunity (Ab). A total of 18 patients with at least 5-year duration of clinically diagnosed T2DM were evaluated in this study. In those subjects we assessed acute insulin responses to arginine, a glucose clamp study, whole-body fat mass and fat-free mass. We have also analyzed the pancreatic pathology of 15 T2DM and 43 control cadaveric donors, using pancreatic tissue obtained from all the T2DM organ donors available from the nPOD network through December 31, 2013. The presence of islet Ab correlated with severely impaired ß-cell function as demonstrated by remarkably low acute insulin response to arginine (AIR) when compared to that of the Ab negative group. Glucose clamp studies indicated that both Ab positive and Ab negative patients exhibited peripheral insulin resistance in a similar fashion. Pathology data from T2DM donors with Ab or the autoimmune diabetes associated DR3/DR4 allelic class II combination showed reduction in beta cell mass as well as presence of autoimmune-associated pattern A pathology in subjects with either islet autoantibodies or the DR3/DR4 genotype. In conclusion, we provide compelling evidence indicating that islet Ab positive long-term T2DM patients exhibit profound impairment of insulin secretion as well as reduced beta cell mass seemingly determined by an immune-mediated injury of pancreatic ß-cells. Deciphering the mechanisms underlying beta cell destruction in this subset of diabetic patients may lead to the development of novel immunologic therapies aimed at halting the disease progression in its early stage.

Sequence variation in promoter of Ica1 gene, which encodes protein implicated in type 1 diabetes, causes transcription factor autoimmune regulator (AIRE) to increase its binding and down-regulate expression.

ICA69 (islet cell autoantigen 69 kDa) is a protein implicated in type 1 diabetes mellitus in both the non-obese diabetic (NOD) mouse model and humans. ICA69 is encoded by the Ica1 gene on mouse chromosome 6 A1-A2. We previously reported reduced ICA69 expression in the thymus of NOD mice compared with thymus of several non-diabetic mouse strains. We propose that reduced thymic ICA69 expression could result from variations in transcriptional regulation of the gene and that polymorphisms within the Ica1 core promoter may partially determine this transcriptional variability. We characterized the functional promoter of Ica1 in NOD mice and compared it with the corresponding portions of Ica1 in non-diabetic C57BL/6 mice. Luciferase reporter constructs demonstrated that the NOD Ica1 promoter region exhibited markedly reduced luciferase expression in transiently transfected medullary thymus epithelial (mTEC(+)) and B-cell (M12)-derived cell lines. However, in a non-diabetic strain, C57BL/6, the Ica1 promoter region was transcriptionally active when transiently transfected into the same cell lines. We concomitantly identified five single nucleotide polymorphisms within the NOD Ica1 promoter. One of these single nucleotide polymorphisms increases the binding affinity for the transcription factor AIRE (autoimmune regulator), which is highly expressed in thymic epithelial cells, where it is known to play a key role regulating self-antigen expression. We conclude that polymorphisms within the NOD Ica1 core promoter may determine AIRE-mediated down-regulation of ICA69 expression in medullary thymic epithelial cells, thus providing a novel mechanistic explanation for the loss of immunologic tolerance to this self-antigen in autoimmunity.

Cytoplasmic islet cell antibodies remain valuable in defining risk of progression to type 1 diabetes in subjects with other islet autoantibodies.

The discovery of islet cell antibodies (ICAs) was the prelude to the understanding that type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease. The issue regarding whether or not the measurement of ICAs should be completely replaced by biochemical markers detecting islet autoantibodies (AAs) for the prediction of T1DM has been the subject of endless international debates. In light of this controversy, we assessed the current role of ICAs as a predictive marker for T1DM progression. We examined a cohort of 1484 first-degree relatives (FDRs) of T1DM probands from the Children's Hospital of Pittsburgh Registry. These relatives were consecutively enrolled between 1979 through 1984 and followed up to 22 yr. Serum obtained at the time of enrollment was assayed for ICAs, glutamic acid decarboxylase (GAD)65, insulin A (IA)-2 AA, and insulin AAs (IAAs). In FDRs who had ICAs in addition to GAD65 and IA-2 AAs, the cumulative risk of developing insulin-requiring diabetes was 80% at 6.7 yr of follow-up, whereas this risk in those with GAD65 and IA-2 AAs without ICAs was only 14% at 10 yr of follow-up (log rank: P < 0.00001). Cox regression analysis showed that diabetes risk was significantly associated with the presence of ICAs in both subjects with low titer and high titer GAD65 and IA-2 AAs. The addition of IAAs in GAD65 and IA-2 AA-positive relatives did not increase the cumulative risk for conversion to insulin-treated diabetes. We provide evidence that a subgroup of ICAs predicts a more rapid progression to insulin-requiring diabetes in GAD65 and IA-2 AA-positive relatives and should remain part of the assessment of T1DM risk for intervention trials. In addition, these findings provide impetus for efforts to identify a novel islet autoantigen(s) reactive with this ICA subset.

Anti-insulin receptor autoantibodies are not required for type 2 diabetes pathogenesis in NZL/Lt mice, a New Zealand obese (NZO)-derived mouse strain.

The New Zealand obese (NZO) mouse strain shares with the related New Zealand black (NZB) strain a number of immunophenotypic traits. Among these is a high proportion of B-1 B lymphocytes, a subset associated with autoantibody production. Approximately 50% of NZO/HlLt males develop a chronic insulin-resistant type 2 diabetes syndrome associated with 2 unusual features: the presence of B lymphocyte-enriched peri-insular infiltrates and the development of anti-insulin receptor autoantibodies (AIRAs). To establish the potential pathogenic contributions of B lymphocytes and AIRAs in this model, a disrupted immunoglobulin heavy chain gene (Igh-6) congenic on the NZB/BlJ background was backcrossed 4 generations into the NZO/HlLt background and was then intercrossed to produce mice that initially segregated for wild-type versus the mutant Igh-6 allele and thus permitted comparison of syndrome development. A new flow cytometric assay (AIRA binding to transfected Chinese hamster ovary cells stably expressing mouse insulin receptor) showed IgM and IgG subclass AIRAs in serum from Igh-6 intact males, but not in Igh-6null male serum. However, the absence of B lymphocytes and antibodies distinguishing mutant from wild-type males failed to significantly affect diabetes-free survival. The Igh-6null males gained weight less rapidly than wild-type males, probably accounting for a retardation, but not prevention, of hyperglycemia. Thus, AIRA and the B-lymphocyte component of the peri-insulitis in chronic diabetics were not essential either to development of insulin resistance or to eventual pancreatic beta cell failure and loss. A new substrain, designated NZL, was generated by inbreeding Igh-6 wild-type segregants. Currently at the F10 generation, NZL mice exhibit the same juvenile-onset obesity as NZO/HlLt males, but develop type 2 diabetes at a higher frequency (> 80%). Also, unlike NZO/HlLt mice that are difficult to breed, the NZL/Lt strain breeds well and thus offers clear advantages to obesity/diabetes researchers.

Alternative core promoters regulate tissue-specific transcription from the autoimmune diabetes-related ICA1 (ICA69) gene locus.

Islet cell autoantigen 69-kDa (ICA69), protein product of the human ICA1 gene, is one target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional promoters within the 5'-regulatory region of ICA1. 5'-RNA ligase-mediated rapid amplification of cDNA ends evaluation of ICA1 transcripts expressed in human islets, testis, heart, and cultured neuroblastoma cells reveals that three 5'-untranslated region exons are variably expressed from the ICA1 gene in a tissue-specific manner. Surrounding the transcription initiation sites are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters, including consensus Sp1/GC boxes, an initiator element, cAMP-responsive element-binding protein (CREB) sites, and clusters of other putative transcription factor sites within a genomic CpG island. Luciferase reporter constructs demonstrate that the first two ICA1 exon promoters reciprocally stimulate luciferase expression within islet- (RIN 1046-38 cells) and brain-derived (NMB7) cells in culture; the exon A promoter exhibits greater activity in islet cells, whereas the exon B promoter more efficiently activates transcription in neuronal cells. Mutation of a CREB site within the ICA1 exon B promoter significantly enhances transcriptional activity in both cell lines. Our basic understanding of expression from the functional core promoter elements of ICA1 is an important advance that will not only add to our knowledge of the ICA69 autoantigen but will also facilitate a rational approach to discover the function of ICA69 and to identify relevant ICA1 promoter polymorphisms and their potential associations with disease.