NLRP3 Inflammasome and Mineralocorticoid Receptors Are Associated with Vascular Dysfunction in Type 2 Diabetes Mellitus.

Aldosterone excess aggravates endothelial dysfunction in diabetes and hypertension by promoting the increased generation of reactive oxygen species, inflammation, and insulin resistance. Aldosterone activates the molecular platform inflammasome in immune system cells and contributes to vascular dysfunction induced by the mineralocorticoid hormone. It is unclear as to whether the NLRP3 inflammasome associated with the mineralocorticoid receptor contributes to vascular dysfunction in diabetic conditions. Here, we tested the hypothesis that an excess of aldosterone induces vascular dysfunction in type 2 diabetes, via the activation of mineralocorticoid receptors (MR) and assembly of the NLRP3 inflammasome. Mesenteric resistance arteries from control (db/m) and diabetic (db/db) mice treated with vehicle, spironolactone (MR antagonist) or an NLRP3 selective inhibitor (MCC950) were used to determine whether NLRP3 contributes to diabetes-associated vascular dysfunction. Db/db mice exhibited increased vascular expression/activation of caspase-1 and IL-1ß, increased plasma IL-1ß levels, active caspase-1 in peritoneal macrophages, and reduced acetylcholine (ACh) vasodilation, compared to db/m mice. Treatment of db/db mice with spironolactone and MCC950 decreased plasma IL-1ß and partly restored ACh vasodilation. Spironolactone also reduced active caspase-1-positive macrophages in db/db mice, events that contribute to diabetes-associated vascular changes. These data clearly indicate that MR and NLRP3 activation contribute to diabetes-associated vascular dysfunction and pro-inflammatory phenotype.

Post-transcriptional markers associated with clinical complications in Type 1 and Type 2 diabetes mellitus.

The delayed diagnosis and the inadequate treatment of diabetes increase the risk of chronic complications. The study of regulatory molecules such as miRNAs can provide expression profiles of diabetes and diabetes complications. We evaluated the mononuclear cell miRNA profiles of 63 Type 1 and Type 2 diabetes patients presenting or not microvascular complications, and 40 healthy controls, using massive parallel sequencing. Gene targets, enriched pathways, dendograms and miRNA-mRNA networks were performed for the differentially expressed miRNAs. Six more relevant miRNAs were validated by RT-qPCR and data mining analysis. MiRNAs associated with specific complications included: i) neuropathy (miR-873-5p, miR-125a-5p, miR-145-3p and miR-99b-5p); ii) nephropathy (miR-1249-3p, miR-193a-5p, miR-409-5p, miR-1271-5p, miR-501-3p, miR-148b-3p and miR-9-5p); and iii) retinopathy (miR-143-3p, miR-1271-5p, miR-409-5p and miR-199a-5p). These miRNAs mainly targeted gene families and specific genes associated with advanced glycation end products and their receptors. Sets of miRNAs were also defined as potential targets for diabetes/diabetes complication pathogenesis.

Review: MicroRNAS in ocular surface and dry eye diseases.

Since the first description of microRNAs (miRNAs) in the 1990s, more than 60 papers have described the role of miRNAs on the ocular surface and lacrimal gland (LG). MicroRNAs (miRNAs) have a role in several physiological events and in mediation of disease. They inhibit gene expression by blocking messenger RNA. Diseases such as Sjögren syndrome (SS), ocular surface neoplasias, and infections are known to increase or reduce the expression of specific miRNAs. These miRNAs play key roles in modulating inflammation, delaying or enhancing wound healing, cell differentiation metabolism, and survival. This review describes the current understanding of miRNAs as biomarkers, mediators of diseases, and potential therapeutic targets in ocular surface diseases.

Differential gene expression profiles may differentiate responder and nonresponder patients with rheumatoid arthritis for methotrexate (MTX) monotherapy and MTX plus tumor necrosis factor inhibitor combined therapy.

OBJECTIVE: We aimed to evaluate whether the differential gene expression profiles of patients with rheumatoid arthritis (RA) could distinguish responders from nonresponders to methotrexate (MTX) and, in the case of MTX nonresponders, responsiveness to MTX plus anti-tumor necrosis factor-α (anti-TNF) combined therapy. METHODS: We evaluated 25 patients with RA taking MTX 15-20 mg/week as a monotherapy (8 responders and 17 nonresponders). All MTX nonresponders received infliximab and were reassessed after 20 weeks to evaluate their anti-TNF responsiveness using the European League Against Rheumatism response criteria. A differential gene expression analysis from peripheral blood mononuclear cells was performed in terms of hierarchical gene clustering, and an evaluation of differentially expressed genes was performed using the significance analysis of microarrays program. RESULTS: Hierarchical gene expression clustering discriminated MTX responders from nonresponders, and MTX plus anti-TNF responders from nonresponders. The evaluation of only highly modulated genes (fold change > 1.3 or < 0.7) yielded 5 induced (4 antiapoptotic and CCL4) and 4 repressed (4 proapoptotic) genes in MTX nonresponders compared to responders. In MTX plus anti-TNF non-responders, the CCL4, CD83, and BCL2A1 genes were induced in relation to responders. CONCLUSION: Study of the gene expression profiles of RA peripheral blood cells permitted differentiation of responders from nonresponders to MTX and anti-TNF. Several candidate genes in MTX non-responders (CCL4, HTRA2, PRKCD, BCL2A1, CAV1, TNIP1, CASP8AP2, MXD1, and BTG2) and 3 genes in MTX plus anti-TNF nonresponders (CCL4, CD83, and BCL2A1) were identified for further study.

Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features.

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared-epitope, HLA-SE), autoimmune response [anti-cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS-28) and treatment (disease-modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3-labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3-monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA-SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety-one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti-CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty-eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.

Gene expression profiles stratified according to type 1 diabetes mellitus susceptibility regions.

The MHC region (6p21) aggregates the major genes that contribute to susceptibility to type 1 diabetes (T1D). Three additional relevant susceptibility regions mapped on chromosomes 1p13 (PTPN22), 2q33 (CTLA-4), and 11p15 (insulin) have also been described by linkage studies. To evaluate the contribution of these susceptibility regions and the chromosomes that house these regions, we performed a large-scale differential gene expression on lymphomononuclear cells of recently diagnosed T1D patients, pinpointing relevant modulated genes clustered in these regions and their respective chromosomes. A total of 4608 cDNAs from the IMAGE library were spotted onto glass slides using robotic technology. Statistical analysis was carried out using the SAM program, and data regarding gene location and biological function were obtained at the SOURCE, NCBI, and FATIGO programs. Three induced genes were observed spanning around the MHC region (6p21-6p23), and seven modulated genes (5 repressed and 2 repressed) were seen spanning around the 6q21-24 region. Additional modulated genes were observed in and around the 1p13, 2q33, and 11p15 regions. Overall, modulated genes in these regions were primarily associated with cellular metabolism, transcription factors and signaling transduction. The differential gene expression characterization may identify new genes potentially involved with diabetes pathogenesis.

Evaluation of cytokine production from peripheral blood mononuclear cells of type 1 diabetic patients.

This study aims to evaluate the production of cytokines, tumor necrosis factor (TNF), and interleukin 10 (IL-10) in peripheral blood mononuclear cells (PBMCs) from type 1 diabetic (T1D) patients by means of intracellular staining, flow cytometry, and ELISA and to correlate it with inadequate (IN) and adequate (A) metabolic controls. We studied 28 patients with T1D and 20 healthy individuals (C) paired by sex and age. T1D patients were divided in patients with IN and A metabolic control. PBMC cultures were stimulated with LPS to evaluate TNF or were stimulated with PMA/ionomycin or concanavalin A to evaluate IL-10. The TNF levels in supernatant of stimulated cultures, evaluated by ELISA, of diabetic patients were similar to those of healthy individuals, although the percentage of CD 33(+) cells that were positive for TNF was higher in the T1D IN group compared to the T1D A group (P= 0.01). Similarly, the IL-10 levels evaluated by ELISA in stimulated cultures of T1D patients were not different from those in the control group; moreover, the percentage of CD3(+) cells positive for intracellular IL-10 were higher in the T1D IN group compared to C groups (P= 0.007). The increased levels of cytokines in T1D IN diabetic patients, with reduction in the A group, suggests that hyperglycemia stimulates an inflammatory state that can result in a deficient immune cellular response. The data suggest that assessment by intracellular staining seems to be more accurate than the ELISA technique in evaluating diabetic patients.

Análise das seqüências de nucleotídios da regiäo 5' näo codificadora e dos genes das proteínas estruturais dos Flavivirus / Nucleotide sequence alignement of the 5' non coding and structural protein regions of the genome of Flaviviruses

Neste trabalho, analisaram-se comparativamente as seqüências de nucleotídios dos genes das proteínas estruturais C, prM e E de todos os Flavivirus, incluindo, também, a regiäo 5' näo codificadora, de 21 Flavivirus. Utilizou-se para a análise o programa de microcomputador DNAsis (Hitachi, Japäo) e construiu-se uma árvore filogenética, incluindo os vinte e um (21) vírus, após alinhamento de suas seqüências de nucleotídios. Na árvore filogenética obtida, observou-se uma ramificaçäo inicial, separando os vírus transmitidos por carrapatos daqueles transmitidos por mosquitos. Também, agruparam-se, em diferentes ramos, os vírus do dengue, os da febre amarela, e os da encefalite japonesa. Observou-se uma evidente relaçäo entre a árvore filogenética e os subgrupos e tipos virais, reconhecidos com base em relacionamento antigênico.