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

Improving the hyperpolarization of (31)p nuclei by synthetic design

Traditional (31)P NMR or MRI measurements suffer from low sensitivity relative to (1)H detection and consequently require longer scan times. We show here that hyperpolarization of (31)P nuclei through reversible interactions with parahydrogen can deliver substantial signal enhancements in a range of regioisomeric phosphonate esters containing a heteroaromatic motif which were synthesized in order to identify the optimum molecular scaffold for polarization transfer. A 3588-fold (31)P signal enhancement (2.34% polarization) was returned for a partially deuterated pyridyl substituted phosphonate ester. This hyperpolarization level is sufficient to allow single scan (31)P MR images of a phantom to be recorded at a 9.4 T observation field in seconds that have signal-to-noise ratios of up to 94.4 when the analyte concentration is 10 mM. In contrast, a 12 h 2048 scan measurement under standard conditions yields a signal-to-noise ratio of just 11.4. (31)P-hyperpolarized images are also reported from a 7 T preclinical scanner

Insulin receptor based lymphocyte trafficking in the progression of type 1 diabetes

The insulin receptor (IR) is a transmembrane receptor which recognizes and binds the hormone insulin. We describe two models that were devised to explore the role of IR over-expression on T-lymphocytes and their chemotactic motility in the progression of type 1 diabetes. FVB/NJ-CD3-3×FLAG-mIR/MFM mice were generated to selectively over-express 3×FLAG tagged murine IR in T-lymphocytes via an engineered CD3 enhancer and promoter construct. Insertion of the 3×FLAG-mIR transgene into FVB/NJ mice, a known non-autoimmune prone strain, lead to a minor population of detectable 3×FLAG-mIR tagged T-lymphocytes in peripheral blood and the presence of a few lymphocytes in the pancreas of the Tg+/- compared to age matched Tg-/- control mice. In order to induce stronger murine IR over-expression then what was observed with the CD3 enhancer promoter construct, a second system utilizing the strong CAG viral promoter was generated. This system induces cell specific IR over-expression upon Cre-Lox recombination to afford functional 3×FLAG tagged murine IR with an internal eGFP reporter. The pPNTlox2-3×FLAG-mIR plasmid was constructed and validated in HEK-Cre-RFP cells to ensure selective Cre recombinase based 3×FLAG-mIR expression, receptor ligand affinity towards insulin, and functional initiation of signal transduction upon insulin stimulation

Humoral Autoimmunity against the Extracellular Domain of the Neuroendocrine Autoantigen IA-2 Heightens the Risk of Type 1 Diabetes

The objective of this study was to determine whether antigenic determinants localized within the extracellular domain of the neuroendocrine autoantigen tyrosine phosphatase-like protein IA-2 are targets of humoral responses in type 1 diabetes (T1DM). Previous studies indicated that the immunodominant region of IA-2 is localized within its intracellular domain (IA-2ic; amino acids 601–979). We analyzed 333 subjects from the Children's Hospital of Pittsburgh study, 102 of whom progressed to insulin-requiring diabetes (prediabetics). Autoantibodies from these individuals were initially assayed for ICA512bdc (Barbara Davis Center amino acids 257–556; 630–979), IA-2ic (amino acids 601–979), and IA-2 full-length (amino acids 1–979) in addition to islet cell antibody (ICA), glutamic acid decarboxylase, 65-kDa isoform, and insulin autoantibodies. We identified an autoantibody response reactive with the extracellular domain of IA-2 that is associated with very high risk of T1DM progression. Relatives with no detectable autoantibodies against ICA512bdc (or IA-2ic) exhibited antibody responses against the IA-2 full-length peptide (log rank, P = 0.008). This effect was also observed in first-degree relatives who were positive for glutamic acid decarboxylase, 65–kDa isoform (log rank, P = 0.026) or at least two islet autoantibodies but were negative for ICA512bdc (log rank, P = 0.022). Competitive binding experiments and immunoprecipitation of the IA-2 extracellular domain (amino acid residues 26–577) further lend support for the presence of autoantibodies reactive with new antigenic determinants within the extracellular domain of IA-2. In summary, the addition of measurements of autoantibodies reactive with the IA-2 extracellular domain to assays geared to assess the progression of autoimmunity to clinical T1DM may more accurately characterize this risk. This has considerable implications not only for stratifying high diabetes risk but also facilitating the search for pathogenic epitopes to enable the design of peptide-based immunotherapies that may prevent the progression to overt T1DM at its preclinical stages

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