Sharad Purohit

A functional variant of SUMO4, a new I|[kappa]|B|[alpha]| modifier, is associated with type 1 diabetes

Previous studies have suggested more than 20 genetic intervals that are associated with susceptibility to type 1 diabetes (T1D), but identification of specific genes has been challenging and largely limited to known candidate genes. Here, we report evidence for an association between T1D and multiple single-nucleotide polymorphisms in 197 kb of genomic DNA in the IDDM5 interval. We cloned a new gene (SUMO4), encoding small ubiquitin-like modifier 4 protein, in the interval. A substitution (M55V) at an evolutionarily conserved residue of the crucial CUE domain of SUMO4 was strongly associated with T1D (P = 1.9 × 10−7). SUMO4 conjugates to IκBα and negatively regulates NFκB transcriptional activity. The M55V substitution resulted in 5.5 times greater NFκB transcriptional activity and ∼2 times greater expression of IL12B, an NFκB-dependent gene. These findings suggest a new pathway that may be implicated in the pathogenesis of T1D.

Extracellular High-Mobility Group Box 1 Acts as an Innate Immune Mediator to Enhance Autoimmune Progression and Diabetes Onset in NOD Mice

OBJECTIVE—The implication of innate immunity in type 1 diabetes development has long been proposed. High-mobility group box 1 (HMGB1), an evolutionarily conserved chromosomal protein, was recently recognized to be a potent innate inflammatory mediator when released extracellularly. We sought to test the hypothesis that HMGB1 acts as an innate immune mediator implicated in type 1 diabetes pathogenesis. RESEARCH DESIGN AND METHODS—Eight- and 12-week-old NOD mice were treated with an HMGB1 neutralizing antibody once a week until 25 weeks of age and monitored for insulitis progression and diabetes onset. The underlying mechanisms of HMGB1 regulation of autoimmune response were further explored. RESULTS—During autoimmunity, HMGB1 can be passively released from damaged pancreatic β-cells and actively secreted by islet infiltrated immune cells. Extracellular HMGB1 is potent in inducing NOD dendritic cell maturation and stimulating macrophage activation. Blockade of HMGB1 significantly inhibited insulitis progression and diabetes development in both 8- and 12-week-old NOD mice. HMGB1 antibody treatment decreased the number and maturation of pancreatic lymph node (PLN) CD11c++CD11b+ dendritic cells, a subset of dendritic cells probably associated with autoantigen presentation to naïve T-cells, but increased the number for PLN CD4+Foxp3+ regulatory T-cells. Blockade of HMGB1 also decreased splenic dendritic cell allo-stimulatory capability associated with increased tolergenic CD11c+CD8a+ dendritic cells. Interestingly, the number of CD8+interferon-γ+ (Tc1) T-cells was increased in the PLNs and spleen after blockade of HMGB1, which could be associated with retarded migration of activated autoreactive T-cells into the pancreatic islets. CONCLUSIONS—Extracellular HMGB1 functions as a potent innate immune mediator contributing to insulitis progression and diabetes onset.

Lack of correlation between the levels of soluble cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and the CT-60 genotypes.

BackgroundCytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulation of antigen-activated immune response and polymorphisms at the CTLA-4 gene have been shown to be associated with several autoimmune diseases including type-1 diabetes (T1D). The etiological mutation was mapped to the CT60-A/G single nucleotide polymorphism (SNP) that is believed to control the processing and production of soluble CTLA-4 (sCTLA-4).MethodsWe therefore determined sCTLA-4 protein levels in the sera from 82 T1D patients and 19 autoantibody positive (AbP) subjects and 117 autoantibody negative (AbN) controls using ELISA. The CT-60 SNP was genotyped for these samples by using PCR and restriction enzyme digestion of a 268 bp DNA segment containing the SNP. Genotyping of CT-60 SNP was confirmed by dye terminating sequencing reaction.ResultsHigher levels of sCTLA-4 were observed in T1D (2.24 ng/ml) and AbP (mean = 2.17 ng/ml) subjects compared to AbN controls (mean = 1.69 ng/ml) with the differences between these subjects becoming significant with age (p = 0.02). However, we found no correlation between sCTLA-4 levels and the CTLA-4 CT-60 SNP genotypes.ConclusionConsistent with the higher serum sCTLA-4 levels observed in other autoimmune diseases, our results suggest that sCTLA-4 may be a risk factor for T1D. However, our results do not support the conclusion that the CT-60 SNP controls the expression of sCTLA-4.

The Autoimmune Regulator Directly Controls the Expression of Genes Critical for Thymic Epithelial Function

The autoimmune regulator (Aire) gene plays an essential role in negative selection of T cells and deletion of autoreactive T cells in the thymus. The defect in thymic selection in Aire−/− mice was attributed to the repressed expression of tissue-specific Ags in the thymic epithelial cells and defective Ag presentation; however, the molecular mechanism underlying these functions has been elusive. Using the chromatin immunoprecipitation technique, we demonstrate here that Aire binds in vivo to specific DNA sequence motifs and directly regulates thymic expression of genes important for thymic functions including expression of autoantigens, cytokines, transcription factors, and posttranslational modifiers. These results unambiguously established Aire as a key transcriptional regulator of the immune system.

Proteomic technologies for the discovery of type 1 diabetes biomarkers

In this review, we discuss several important issues concerning the discovery of protein biomarkers for complex human diseases, with a focus on type 1 diabetes. Serum or plasma is the first choice of specimen due to its richness in biological information and relatively easy availability. It is a challenging task to comprehensively characterize the serum/plasma proteome because of the large dynamic range of protein concentration. Therefore, sample pretreatment is required in order to explore the low- to medium-abundance proteins contained in serum/ plasma. In this regard, enrichment of low-abundance proteins using random hexapeptide library beads has distinct advantages over the traditional immune-depletion methods, including higher efficiency, higher binding capacity, and lower cost. In-depth mining of serum/plasma proteome using different separation techniques have also been evaluated and are discussed in this review. Overall, the shotgun proteomics—multidimensional separation of digested peptides followed by mass spectrometry analysis—is highly Efficient and therefore has become a preferred method for protein biomarker discovery.

Molecular cloning and characterization of the mouse Acdp gene family

BackgroundWe have recently cloned and characterized a novel gene family named ancient conserved domain protein (ACDP) in humans. To facilitate the functional study of this novel gene family, we have cloned and characterized Acdp, the mouse homologue of the human ACDP gene family.ResultsThe four Acdp genes (Acdp1, Acdp2, Acdp3 and Acdp4) contain 3,631 bp, 3,244 bp, 2,684 bp and 2,743 bp of cDNA sequences, and encode deduced proteins of 951, 874, 713 and 771 amino acids, respectively. The mouse Acdp genes showed very strong homologies (>90%) in both nucleotide and amino acid sequences to their human counterparts. In addition, both nucleotide and amino acid sequences within the Ancient Conserved Domain (ACD) are highly conserved in many different taxonomic species. Particularly, Acdp proteins showed very strong AA homologies to the bacteria CorC protein (35% AA identity with 55% homology), which is involved in magnesium and cobalt efflux. The Acdp genes are widely expressed in all tissues tested except for Acdp1, which is only highly expressed in the brain with low levels of expression in kidney and testis. Immunostaining of Acdp1 in hippocampus neurons revealed a predominant localization on the plasma membrane.ConclusionThe Acdp genes are evolutionarily conserved in diverse species and ubiquitously expressed throughout development and adult tissues suggesting that Acdp may be an essential gene. Acdp showed strong homology to bacteria CorC protein and predominantly localized on the plasma membrane. These results suggest that Acdp is probably a family of proteins involved in ion transport in mammalian cells

Discovery and Validation of Serum Protein Changes in Type 1 Diabetes Patients Using High Throughput Two Dimensional Liquid Chromatography-Mass Spectrometry and Immunoassays

Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10−27), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10−20), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10−16); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10−5) and myeloperoxidase (OR = 0.51, p < 10−41). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10−37), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10−46), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10−16) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10−11) and myeloperoxidase (OR = 0.10, p < 10−43) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.

Mycophenolic Acid Inhibits Migration and Invasion of Gastric Cancer Cells via Multiple Molecular Pathways

Mycophenolic acid (MPA) is the metabolized product and active element of mycophenolate mofetil (MMF) that has been widely used for the prevention of acute graft rejection. MPA potently inhibits inosine monophosphate dehydrogenase (IMPDH) that is up-regulated in many tumors and MPA is known to inhibit cancer cell proliferation as well as fibroblast and endothelial cell migration. In this study, we demonstrated for the first time MPA’s antimigratory and anti-invasion abilities of MPA-sensitive AGS (gastric cancer) cells. Genome-wide expression analyses using Illumina whole genome microarrays identified 50 genes with ≥2 fold changes and 15 genes with > 4 fold alterations and multiple molecular pathways implicated in cell migration. Real-time RT-PCR analyses of selected genes also confirmed the expression differences. Furthermore, targeted proteomic analyses identified several proteins altered by MPA treatment. Our results indicate that MPA modulates gastric cancer cell migration through down-regulation of a large number of genes (PRKCA, DOCK1, INF2, HSPA5, LRP8 and PDGFRA) and proteins (PRKCA, AKT, SRC, CD147 and MMP1) with promigratory functions as well as up-regulation of a number of genes with antimigratory functions (ATF3, SMAD3, CITED2 and CEAMCAM1). However, a few genes that may promote migration (CYR61 and NOS3) were up-regulated. Therefore, MPA’s overall antimigratory role on cancer cells reflects a balance between promigratory and antimigratory signals influenced by MPA treatment.

Chemokine (C-C motif) ligand 2 (CCL2) in sera of patients with type 1 diabetes and diabetic complications.

Background Chemokine (C-C motif) ligand 2 (CCL2), commonly known as monocyte chemoattractant protein-1 (MCP-1), has been implicated in the pathogenesis of many diseases characterized by monocytic infiltration. However, limited data have been reported on MCP-1 in type 1 diabetes (T1D) and the findings are inconclusive and inconsistent. Methods In this study, MCP-1 was measured in the sera from 2,472 T1D patients and 2,654 healthy controls using a Luminex assay. The rs1024611 SNP in the promoter region of MCP-1 was genotyped for a subset of subjects (1764 T1D patients and 1323 controls) using the TaqMan-assay. Results Subject age, sex or genotypes of MCP-1 rs1024611SNP did not have a major impact on serum MCP-1 levels in either healthy controls or patients. While hemoglobin A1c levels did not have a major influence on serum MCP-1 levels, the mean serum MCP-1 levels are significantly higher in patients with multiple complications (mean = 242 ng/ml) compared to patients without any complications (mean = 201 ng/ml) (p = 3.5×10−6). Furthermore, mean serum MCP-1 is higher in controls (mean = 261 ng/ml) than T1D patients (mean = 208 ng/ml) (p<10−23). More importantly, the frequency of subjects with extremely high levels (>99th percentile of patients or 955 ng/ml) of serum MCP-1 is significantly lower in the T1D group compared to the control group (odds ratio = 0.11, p<10−33). Conclusion MCP-1 may have a dual role in T1D and its complications. While very high levels of serum MCP-1 may be protective against the development of T1D, complications are associated with higher serum MCP-1 levels within the T1D group.

Distribution of N‐ and O‐linked oligosaccharides on surface of spermatozoa from normal and infertile subjects

Sperm glycocalyx modifications are very important for gamete recognition and fertilisation in mammals. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. The purpose of this study was to determine the distribution of surface carbohydrates in human spermatozoa from normal and oligospermic subjects. Fifteen ejaculates each from normal fertile and oligospermic individuals were analysed. N‐linked and O‐linked surface carbohydrates were detected by fluorescence microscopy using fluorescein isothiocynate‐conjugated lectins. Triticum vulgaris agglutinin (WGA)‐binding sites were found to be decreased on acrosomal domain in spermatozoa from oligospermic individuals, while no changes were observed in the binding sites of Concanavalin ensiformis, Peanut agglutinin and Lens clunaris agglutinin. A reduction in binding sites for soyabean agglutinin and Ricinus communis agglutinin was observed on the acrosomal domains in spermatozoa from oligospermic individuals. Changes in sperm glycocalyx observed in this study provide new insights into molecular rearrangement of sperm membrane in infertility.