Transcript Expression Profiles and MicroRNA Regulation Indicate an Upregulation of Processes Linked to Oxidative Stress, DNA Repair, Cell Death, and Inflammation in Type 1 Diabetes Mellitus Patients.

Type 1 diabetes (T1D) arises from autoimmune-mediated destruction of insulin-producing ß-cells leading to impaired insulin secretion and hyperglycemia. T1D is accompanied by DNA damage, oxidative stress, and inflammation, although there is still scarce information about the oxidative stress response and DNA repair in T1D pathogenesis. We used the microarray method to assess mRNA expression profiles in peripheral blood mononuclear cells (PBMCs) of 19 T1D patients compared to 11 controls and identify mRNA targets of microRNAs that were previously reported for T1D patients. We found 277 differentially expressed genes (220 upregulated and 57 downregulated) in T1D patients compared to controls. Analysis by gene sets (GSA and GSEA) showed an upregulation of processes linked to ROS generation, oxidative stress, inflammation, cell death, ER stress, and DNA repair in T1D patients. Besides, genes related to oxidative stress responses and DNA repair (PTGS2, ATF3, FOSB, DUSP1, and TNFAIP3) were found to be targets of four microRNAs (hsa-miR-101, hsa-miR148a, hsa-miR-27b, and hsa-miR-424). The expression levels of these mRNAs and microRNAs were confirmed by qRT-PCR. Therefore, the present study on differential expression profiles indicates relevant biological functions related to oxidative stress response, DNA repair, inflammation, and apoptosis in PBMCs of T1D patients relative to controls. We also report new insights regarding microRNA-mRNA interactions, which may play important roles in the T1D pathogenesis.

NLRP1 acts as a negative regulator of Th17 cell programming in mice and humans with autoimmune diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic ß cells. We show here that the protein NOD-like receptor family pyrin domain containing 1 (NLRP1) has a key role in the pathogenesis of mouse and human T1D. More specifically, downregulation of NLRP1 expression occurs during T helper 17 (Th17) differentiation, alongside greater expression of several molecules related to Th17 cell differentiation in a signal transducers and activators of transcription 3 (STAT3)-dependent pathway. These changes lead to a consequent increase in interleukin 17 (IL-17) production within the pancreas and higher incidence of diabetes in streptozotocin (STZ)-injected mice. Finally, in patients with T1D and a SNP (rs12150220) in NLRP1, there is a robust decrease in IL-17 levels in serum and in memory Th17 cells from peripheral blood mononuclear cells. Our results demonstrate that NLRP1 acts as a negative regulator of the Th17 cell polarization program, making it an interesting target for intervention during the early stages of T1D.

Disease activity index is associated with subclinical atherosclerosis in childhood-onset systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an independent risk factor for cardiovascular events. The present study determined the prevalence of subclinical atherosclerosis in childhood-onset SLE using the carotid intima-media thickness (CIMT) measurement and investigated associations between traditional and nontraditional risk factors for atherosclerosis, such as medications, SLE Disease Activity Index - SLEDAI-2 K and SLICC-ACR damage index and CIMT. METHODS: Cross-sectional prospective study between 2017 and 2018. CIMT was assessed by ultrasonography. Data were collected by chart review, nutritional evaluation and laboratory tests and analyzed by Fisher, Wilcoxon-Mann-Whitney tests, multiple linear and log binomial regression. RESULTS: Twenty-eight patients (mean age 13.9 years, SD 3) were enrolled. The prevalence of subclinical atherosclerosis was 32% (95% CI 14.8, 49.4). The mean CIMT was 0.43 ± 0.035 mm. The most common traditional risk factors observed were dyslipidemia (82.1%), uncontrolled hypertension (14.2%), obesity (14.3%), and poor diet (78.6%). Uncontrolled hypertension (p = 0.04), proteinuria (p = 0.02), estimated glomerular filtration rate < 75 ml /min/1.73 m2 (p = 0.02) and SLEDAI-2 K > 5 (P = 0.04) were associated with subclinical atherosclerosis. SLEDAI-2 K > 5 maintained association with CIMT after adjusting for control variables. CONCLUSION: Subclinical atherosclerosis is frequently observed in cSLE, mainly in patients with moderate to severe disease activity.

Increased O-GlcNAcylation of Endothelial Nitric Oxide Synthase Compromises the Anti-contractile Properties of Perivascular Adipose Tissue in Metabolic Syndrome.

Under physiological conditions, the perivascular adipose tissue (PVAT) negatively modulates vascular contractility. This property is lost in experimental and human obesity and in the metabolic syndrome, indicating that changes in PVAT function may contribute to vascular dysfunction associated with increased body weight and hyperglycemia. The O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of proteins (O-GlcNAcylation) is a unique posttranslational process that integrates glucose metabolism with intracellular protein activity. Increased flux of glucose through the hexosamine biosynthetic pathway and the consequent increase in tissue-specific O-GlcNAc modification of proteins have been linked to multiple facets of vascular dysfunction in diabetes and other pathological conditions. We hypothesized that chronic consumption of glucose, a condition that progresses to metabolic syndrome, leads to increased O-GlcNAc modification of proteins in the PVAT, decreasing its anti-contractile effects. Therefore, the current study was devised to determine whether a high-sugar diet increases O-GlcNAcylation in the PVAT and how increased O-GlcNAc interferes with PVAT vasorelaxant function. To assess molecular mechanisms by which O-GlcNAc contributes to PVAT dysfunction, thoracic aortas surrounded by PVAT were isolated from Wistar rats fed either a control or high sugar diet, for 10 and 12 weeks. Rats chronically fed a high sugar diet exhibited metabolic syndrome features, increased O-GlcNAcylated-proteins in the PVAT and loss of PVAT anti-contractile effect. PVAT from high sugar diet-fed rats for 12 weeks exhibited decreased NO formation, reduced expression of endothelial nitric oxide synthase (eNOS) and increased O-GlcNAcylation of eNOS. High sugar diet also decreased OGA activity and increased superoxide anion generation in the PVAT. Visceral adipose tissue samples from hyperglycemic patients showed increased levels of O-GlcNAc-modified proteins, increased ROS generation and decreased OGA activity. These data indicate that O-GlcNAcylation contributes to metabolic syndrome-induced PVAT dysfunction and that O-GlcNAcylation of eNOS may be targeted in the development of novel therapies for vascular dysfunction in conditions associated with hyperglycemia.

DNA damage increase in peripheral neutrophils from patients with rheumatoid arthritis is associated with the disease activity and the presence of shared epitope.

OBJECTIVES: Neutrophils play a major role in rheumatoid arthritis (RA) pathogenesis. We aimed to evaluate if neutrophil DNA damage in RA patients is associated with the disease activity, autoantibodies status, carriage of the RA shared epitope (SE) and treatment. METHODS: DNA damage was assessed by alkaline comet assay in peripheral blood (77 patients and 55 healthy controls) and in 10 RA synovial fluid neutrophils. Evaluation of the respiratory burst of 30 patients with RA and 30 healthy controls was done. RESULTS: Compared to controls, RA patients exhibited increased neutrophil DNA damage. RA synovial fluid cells DNA damage was increased when compared to OA synovial fluids cells. In addition, our study shows that anti-TNF-α therapy reduces the frequency of DNA damage. Patients with simple or double dose of shared epitope presented a higher frequency of DNA damage compared to patients without the allele. Positive correlation was found between neutrophil DNA damage and DAS-28 and ROS production. CONCLUSIONS: Our results suggest that an increase of respiratory burst of neutrophils reflects the higher levels of DNA damage in neutrophils and a positive correlation between DNA damage and disease activity shows the importance of oxidative stress in the pathogenesis of RA.

Integrative analysis of the transcriptome profiles observed in type 1, type 2 and gestational diabetes mellitus reveals the role of inflammation.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease, while type 2 (T2D) and gestational diabetes (GDM) are considered metabolic disturbances. In a previous study evaluating the transcript profiling of peripheral mononuclear blood cells obtained from T1D, T2D and GDM patients we showed that the gene profile of T1D patients was closer to GDM than to T2D. To understand the influence of demographical, clinical, laboratory, pathogenetic and treatment features on the diabetes transcript profiling, we performed an analysis integrating these features with the gene expression profiles of the annotated genes included in databases containing information regarding GWAS and immune cell expression signatures. METHODS: Samples from 56 (19 T1D, 20 T2D, and 17 GDM) patients were hybridized to whole genome one-color Agilent 4x44k microarrays. Non-informative genes were filtered by partitioning, and differentially expressed genes were obtained by rank product analysis. Functional analyses were carried out using the DAVID database, and module maps were constructed using the Genomica tool. RESULTS: The functional analyses were able to discriminate between T1D and GDM patients based on genes involved in inflammation. Module maps of differentially expressed genes revealed that modulated genes: i) exhibited transcription profiles typical of macrophage and dendritic cells; ii) had been previously associated with diabetic complications by association and by meta-analysis studies, and iii) were influenced by disease duration, obesity, number of gestations, glucose serum levels and the use of medications, such as metformin. CONCLUSION: This is the first module map study to show the influence of epidemiological, clinical, laboratory, immunopathogenic and treatment features on the transcription profiles of T1D, T2D and GDM patients.

MicroRNA expression profiling and functional annotation analysis of their targets in patients with type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing pancreatic ß-cells, leading to elimination of insulin production. The exact cause of this disorder is still unclear. Although the differential expression of microRNAs (miRNAs), small non-coding RNAs that control gene expression in a post-transcriptional manner, has been identified in many diseases, including T1DM, only scarce information exists concerning miRNA expression profile in T1DM. Thus, we employed the microarray technology to examine the miRNA expression profiles displayed by peripheral blood mononuclear cells (PBMCs) from T1DM patients compared with healthy subjects. Total RNA extracted from PBMCs from 11 T1DM patients and nine healthy subjects was hybridized onto Agilent human miRNA microarray slides (V3), 8x15K, and expression data were analyzed on R statistical environment. After applying the rank products statistical test, the receiver-operating characteristic (ROC) curves were generated and the areas under the ROC curves (AUC) were calculated. To examine the functions of the differentially expressed (p-value<0.01, percentage of false-positives <0.05) miRNAs that passed the AUC cutoff value ≥ 0.90, the database miRWalk was used to predict their potential targets, which were afterwards submitted to the functional annotation tool provided by the Database for Annotation, Visualization, and Integrated Discovery (DAVID), version 6.7, using annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. We found 57 probes, corresponding to 44 different miRNAs (35 up-regulated and 9 down-regulated), that were differentially expressed in T1DM and passed the AUC threshold of 0.90. The hierarchical clustering analysis indicated the discriminatory power of those miRNAs, since they were able to clearly distinguish T1DM patients from healthy individuals. Target prediction indicated that 47 candidate genes for T1DM are potentially regulated by the differentially expressed miRNAs. After performing functional annotation analysis of the predicted targets, we observed 22 and 12 annotated KEGG pathways for the induced and repressed miRNAs, respectively. Interestingly, many pathways were enriched for the targets of both up- and down-regulated miRNAs and the majority of those pathways have been previously associated with T1DM, including many cancer-related pathways. In conclusion, our study indicated miRNAs that may be potential biomarkers of T1DM as well as provided new insights into the molecular mechanisms involved in this disorder.

Patients with systemic sclerosis present increased DNA damage differentially associated with DNA repair gene polymorphisms.

OBJECTIVE: Patients with systemic sclerosis (SSc) exhibit increased toxicity when exposed to genotoxic agents. In our study, we evaluated DNA damage and polymorphic sites in 2 DNA repair genes (XRCC1 Arg399Gln and XRCC4 Ile401Thr) in patients with SSc. METHODS: A total of 177 patients were studied for DNA repair gene polymorphisms. Fifty-six of them were also evaluated for DNA damage in peripheral blood cells using the comet assay. RESULTS: Compared to controls, the patients as a whole or stratified into major clinical variants (limited or diffuse skin involvement), irrespective of the underlying treatment schedule, exhibited increased DNA damage. XRCC1 (rs: 25487) and XRCC4 (rs: 28360135) allele and genotype frequencies observed in patients with SSc were not significantly different from those observed in controls; however, the XRCC1 Arg399Gln allele was associated with increased DNA damage only in healthy controls and the XRCC4 Ile401Thr allele was associated with increased DNA damage in both patients and controls. Further, the XRCC1 Arg399Gln allele was associated with the presence of antinuclear antibody and anticentromere antibody. No association was observed between these DNA repair gene polymorphic sites and clinical features of patients with SSc. CONCLUSION: These results corroborate the presence of genomic instability in SSc peripheral blood cells, as evaluated by increased DNA damage, and show that polymorphic sites of the XRCC1 and XRCC4 DNA repair genes may differentially influence DNA damage and the development of autoantibodies.

Identifying common and specific microRNAs expressed in peripheral blood mononuclear cell of type 1, type 2, and gestational diabetes mellitus patients.

BACKGROUND: Regardless the regulatory function of microRNAs (miRNA), their differential expression pattern has been used to define miRNA signatures and to disclose disease biomarkers. To address the question of whether patients presenting the different types of diabetes mellitus could be distinguished on the basis of their miRNA and mRNA expression profiling, we obtained peripheral blood mononuclear cell (PBMC) RNAs from 7 type 1 (T1D), 7 type 2 (T2D), and 6 gestational diabetes (GDM) patients, which were hybridized to Agilent miRNA and mRNA microarrays. Data quantification and quality control were obtained using the Feature Extraction software, and data distribution was normalized using quantile function implemented in the Aroma light package. Differentially expressed miRNAs/mRNAs were identified using Rank products, comparing T1DxGDM, T2DxGDM and T1DxT2D. Hierarchical clustering was performed using the average linkage criterion with Pearson uncentered distance as metrics. RESULTS: The use of the same microarrays platform permitted the identification of sets of shared or specific miRNAs/mRNA interaction for each type of diabetes. Nine miRNAs (hsa-miR-126, hsa-miR-1307, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144, hsa-miR-199a-5p, hsa-miR-27a, hsa-miR-29b, and hsa-miR-342-3p) were shared among T1D, T2D and GDM, and additional specific miRNAs were identified for T1D (20 miRNAs), T2D (14) and GDM (19) patients. ROC curves allowed the identification of specific and relevant (greater AUC values) miRNAs for each type of diabetes, including: i) hsa-miR-1274a, hsa-miR-1274b and hsa-let-7f for T1D; ii) hsa-miR-222, hsa-miR-30e and hsa-miR-140-3p for T2D, and iii) hsa-miR-181a and hsa-miR-1268 for GDM. Many of these miRNAs targeted mRNAs associated with diabetes pathogenesis. CONCLUSIONS: These results indicate that PBMC can be used as reporter cells to characterize the miRNA expression profiling disclosed by the different diabetes mellitus manifestations. Shared miRNAs may characterize diabetes as a metabolic and inflammatory disorder, whereas specific miRNAs may represent biological markers for each type of diabetes, deserving further attention.

Shared and unique gene expression in systemic lupus erythematosus depending on disease activity.

Patients presenting with active systemic lupus erythematosus (SLE) manifestations may exhibit distinct pathogenetic features in relation to inactive SLE. Also, cDNA microarrays may potentially discriminate the gene expression profile of a disease or disease variant. Therefore, we evaluated the expression profile of 4500 genes in peripheral blood lymphocytes (PBL) of SLE patients. We studied 11 patients with SLE (seven with active SLE and four with inactive SLE) and eight healthy controls. Total RNA was isolated from PBL, reverse transcribed into cDNA, and postlabeled with Cy3 fluorochrome. These probes were then hybridized to a glass slide cDNA microarray containing 4500 human IMAGE cDNA target sequences. An equimolar amount of total RNA from human cell lines served as reference. The microarray images were quantified, normalized, and analyzed using the R environment (ANOVA, significant analysis of microarrays, and cluster-tree view algorithms). Disease activity was assessed by the SLE disease activity index. Compared to the healthy controls, 104 genes in active SLE patients (80 repressed and 24 induced) and 52 genes in nonactive SLE patients (31 induced and 21 repressed) were differentially expressed. The modulation of 12 genes, either induced or repressed, was found in both disease variants; however, each disease variant had differential expression of different genes. Taken together, these results indicate that the two lupus variants studied have common and unique differentially expressed genes. Although the biological significance of the differentially expressed genes discussed above has not been completely understood, they may serve as a platform to further explore the molecular basis of immune deregulation in SLE.

HLA-G expression in the skin of patients with systemic sclerosis.

OBJECTIVE: To determine HLA-G expression in skin biopsies from patients with systemic sclerosis (SSc), and its association with epidemiological, clinical, and laboratory variables and survival. METHODS: Paraffin-embedded skin biopsies obtained from 21 SSc patients (14 limited SSc, 7 diffuse SSc) and from 28 healthy controls were studied. HLA-G expression was evaluated by immunohistochemistry. RESULTS: HLA-G molecules were detected in 57% of skin biopsies from patients with SSc (9 from limited SSc, 3 from diffuse SSc), whereas no control sample expressed HLA-G (p=0.000004). In patients, HLA-G molecules were consistently observed within epidermal and some dermal cells. HLA-G expression was associated with a lower frequency of vascular cutaneous ulcers (p=0.0004), telangiectasias (p=0.008), and inflammatory polyarthralgia (p=0.02). After a 15-year followup, SSc patients who exhibited HLA-G survived longer than patients who did not. CONCLUSION: HLA-G is expressed in skin biopsies from patients with SSc, and this is associated with a better disease prognosis. This suggests a modulatory role of HLA-G in SSc, as observed in other skin disorders.

Profiling meta-analysis reveals primarily gene coexpression concordance between systemic lupus erythematosus and rheumatoid arthritis.

Consensus gene expression profiling by meta-analysis of 4,500 cDNA sequence microarray data obtained from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) was assembled and systematically analyzed. The normalized data were statistically analyzed by the significance analysis of microarray (SAM) program (false discovery rate

Metabolism genes are among the differentially expressed ones observed in lymphomononuclear cells of recently diagnosed type 1 diabetes mellitus patients.

The large-scale differential gene expression in lymphomononuclear cells of six patients with recently diagnosed type), and six normal individuals matched to patients for sex and age were studied. Glass slides containing 4608 cDNAs from the IMAGE library were spotted using robotic technology. Statistical analysis was carried out by the SAM program, and gene function assessed by the FATIGO program. Thirty differentially expressed genes (21 induced and 9 repressed) were disclosed when DM-1 patients were compared with controls. Although presenting with distinct biological function, most of the induced or repressed genes were related with protein, phosphate, DNA, RNA, carboxylic acid, and fatty acid metabolism. Although some of these genes have been previously associated with the pathogenesis of T1DM, many other genes were identified for further studies.

TNFa-e microsatellite, HLA-DRB1 and -DQB1 alleles and haplotypes in Brazilian patients presenting recently diagnosed type 1 diabetes mellitus.

TNF microsatellite and HLA class II polymorphisms were studied in 28 recently diagnosed Brazilian patients presenting type 1 diabetes mellitus (T1DM) and in 120 healthy controls. TNFa-e and HLA-DRB1/DQB1 alleles were identified using sets of sequence-specific primers. Compared to controls, the DRB1*03 and DQB1*02 allele groups, TNFa1 allele, and the TNFa4-b5-c1-d4-e3 and TNFa10-b5-c1-d4-e3 haplotypes were overrepresented in patients. TNF microsatellite together with HLA polymorphisms is associated with type 1 diabetes in Brazilian patients, corroborating the participation of the MHC genes in disease susceptibility.

Is HLA class II profile relevant for the study of large-scale differentially expressed genes in type 1 diabetes mellitus patients?

We have previously identified 30 differentially expressed genes when comparing recently diagnosed type 1 diabetes mellitus (DM-1) patients and controls paired for sex, age, and ethnic background. In this article we performed the hierarchical clustering of these genes taking into account the human-leukocyte-antigen (HLA)-DRB1/DQB1 profile. The dendrogram obtained using the Cluster program grouped patients and controls into three clusters, one including individuals with no susceptibility alleles, another including individuals with at least three susceptibility alleles, and a third intermingling susceptibility/protective alleles. In addition to other variables, the results of the present article suggest that the major histocompatibility complex (MHC) class II profile may be of relevance for the study of a large-scale differentially expressed genes.

Using cDNA microarrays to identify human CD19(+) B cell gene products (ESTs) originated from systemic lupus erythematosus susceptibility loci.

Systemic lupus erythematosus (SLE) is a prototype of autoimmune disease which arises from interactions between susceptibility genes and environmental factors. Despite the heterogeneous manifestations in this disease, all SLE patients present plasma autoantibodies recognizing nuclear components. Thus, auto reactive B cells represent key effectors to be investigated. Human linkage analysis is providing the localization of susceptibility loci distributed in chromosomes contributing to elucidate the manner in which interactions between these loci mediate SLE pathogenesis. We associate the cDNA microarray technology to investigate the differential gene expression of CD19(+) B cells with genetic linkage data. Bioinformatics programs served to evidentiate the differentially expressed sequences and the design of the microarray allowed hierarchical clustering of patients and controls. Sequencing allowed the identification of 8 new gene products differentially expressed (ESTs) that were co-localized in SLE or other autoimmune diseases susceptibility loci on chromosome 1p21, 2q21, 13q33, 16p12.1 and 16q12.1. These findings strongly suggest that chromosomal regions previously identified as SLE susceptibility loci are in fact transcribed in CD19(+) B cells of patients. In this review, we delineate a new possibility for the use of cDNA microarrays in studies focusing the control of gene expression of disease susceptibility loci identified by genetic linkage.