Objective. To elucidate the role of microRNA (miRNA) in the pathogenesis of rheumatoid arthritis (RA), we analyzed synoviocytes from RA patients for their miRNA expression.Methods. Synoviocytes derived from surgical specimens obtained from RA patients were compared with those obtained from osteoarthritis (OA) patients for their expression of a panel of 156 miRNA with quantitative stem-loop reverse transcription-polymerase chain reaction. The miRNA whose expression decreased or increased in RA synoviocytes as compared with OA synoviocytes were identified, and their target genes were predicted by computer analysis. We used an in vitro system of enhancing the expression of specific miRNA by transfection of precursors into synoviocytes, and then we performed proliferation, cell cycle, and apoptosis assays, as well as enzyme-linked immunosorbent assays for cytokine production. The effects of transfection on predicted target protein and messenger RNA (mRNA) were then examined by Western blot analysis and luciferase reporter assay.Results. We found that miR-124a levels significantly decreased in RA synoviocytes as compared with OA synoviocytes. Transfection of precursor miR-124a into RA synoviocytes significantly suppressed their proliferation and arrested the cell cycle at the G 1 phase. We identified a putative consensus site for miR-124a binding in the 3 -untranslated region of cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein 1 (MCP-1) mRNA. Induction of miR-124a in RA synoviocytes significantly suppressed the production of the CDK-2 and MCP-1 proteins. Luciferase reporter assay demonstrated that miR-124a specifically suppressed the reporter activity driven by the 3 -untranslated regions of CDK-2 and MCP-1 mRNA.Conclusion. The results of this study suggest that miR-124a is a key miRNA in the posttranscriptional regulatory mechanisms of RA synoviocytes.MicroRNA (miRNA) are a well-established class of small (ϳ22 nucleotides) endogenous noncoding RNAs that influence the stability and translation of messenger RNA (mRNA) (1). Using various computational and experimental approaches, hundreds of miRNA have been identified in numerous animal species. The miRNA genes are transcribed by RNA polymerase II as primary miRNA (pri-miRNA) (2,3). The RNase III enzyme Drosha then processes the nuclear pri-miRNA, yielding a ϳ70-nucleotide molecule known as precursor miRNA (pre-miRNA) (4), which is exported from the nucleus. Maturation of the pre-miRNA into miRNA is then mediated by the cytoplasmic enzyme Dicer (5), after which the mature miRNA is loaded into the RNA-induced silencing complex (RISC)

Immature or reticulated platelets are known as a clinical marker of thrombopoiesis. Recently, an automatic method was established to detect reticulated platelets as immature platelet fraction (IPF) by means of hematology analyzer XE-2100. We assessed the effects of IPF detection after chemotherapy for various pediatric malignant disorders of 16 patients. Our results indicate that IPF should be considered a useful marker of imminent platelet recovery so that unnecessary platelet transfusion can be avoided.

Reactive oxygen species (ROS) and serum ferritin levels are both considered to be important biological factors in the pathogenesis of myelodysplastic syndrome (MDS). This study evaluated the levels of ROS in 40 patients with MDS (19 males and 21 females) using the Free Radical Analytical System, FRAS4, and derivatives of reactive oxygen metabolite kits. The patients' mean age was 67.3 years (range 58 - 86 years). The sera of 34 (85%) patients exhibited higher levels of oxidative stress than the reference range. There was a positive correlation between ROS levels and serum ferritin levels, and a negative correlation between ROS levels and haemoglobin levels. There was a negative relationship between serum haemoglobin and ferritin levels. The results indicated that iron accumulation or severe anaemia could contribute to oxidative stress in MDS patients. Iron chelation and antioxidant therapy may be suitable for the management of MDS.

Thus, our results suggest that hemin induced NETs in 15 minutes, a quicker reaction than NET induction by phorbol myristate acetate requiring 3 hours. Moreover, since RBC components, especially those with long-term storage, contained sufficient hemin concentration to induce NETs, special attention to hemolysis of stored RBC components is important.

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe3+ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress.NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs.Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.

Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10 minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50 μmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100 μmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis.

No abstract

Ratios of young platelets or reticulated platelets can be routinely obtained as an immature platelet fraction (IPF) with the XE-2100 automated hematology analyzer (Sysmex, Kobe, Japan). We combined IPF analysis of 31 patients with myelodysplastic syndrome (MDS) with a complete blood count, a bone marrow examination, and a chromosome analysis. The patients with >40 x 10(9)/L platelets were classified as group A, and those with > or =40 x 10(9)/L were placed in group B. The 2 groups were subclassified as A1 or B1 for patients with an IPF of <10% and as A2 or B2 for those with an IPF > or =10%. Categories A1, A2, B1, and B2 comprised 12 patients, 6 patients, 7 patients, and 6 patients, respectively. Patients with a relatively high IPF (>10%) (category A2 or B2) showed distinctive characteristics. Group B2 showed a higher frequency of chromosomal abnormalities than B1 (P = .029), and group A2 tended to show a higher incidence of clinical improvement than A1 (P = .08). IPF determination may be clinically useful for the assessment of prognosis for MDS patients.