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B cells play a major role in regulating disease incidence through various factors, including spleen tyrosine kinase (Syk), which transmits signals to all hematopoietic lineage cells. Hypoxia-inducible factor (HIF)-1α accumulates under hypoxic conditions, which is also oxidative stress to induce nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responsible for gene expression of antioxidant enzymes. In the present study, we investigated whether B cells are regulated by crosstalk of HIF-1α and Nrf2 via reactive oxygen species (ROS)-mediated Syk activation. When B cells were incubated under hypoxic conditions, Syk phosphorylation, HIF-1α, and Nrf2 levels were increased. Hypoxia-inducible results were consistent with CoCl2 treatment, which mimics hypoxic conditions. Cell viability was reduced by the Syk inhibitor BAY 61-3606. Increased Nrf2 levels due to hypoxia or CoCl2 were inhibited by treatment with a HIF inhibitor. Hypoxia- or CoCl2-induced ROS increased HIF-1α and Nrf2 levels, which were attenuated by treatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. HIF-1α levels were reduced in doxycycline-treated shNrf2 cells. Clobetasol propionate, a Nrf2 inhibitor, also inhibited HIF-1α levels induced by hypoxia or CoCl2. ROS-mediated Syk phosphorylation at tyrosine 525/526 was confirmed by treatment with H2O2, hypoxia, and CoCl2, and attenuated with NAC treatment. Inhibition of Syk phosphorylation by BAY 61-3606 is consistent with a decrease in protein HIF-1α and Nrf2 levels. Taken together, HIF-1α levels might control Nrf2 levels and vice versa, and could be associated with Syk phosphorylation in B cells. The results indicate that B cells could be regulated by crosstalk of HIF-1α and Nrf2 through ROS-mediated Syk activation.

Cell survival is facilitated by the maintenance of mitochondrial membrane potential (MMP). B cell activating factor (BAFF) plays a role in survival, differentiation, and maturation of B cells. In the present study, we examined whether BAFF could attenuate oxidative stress-induced B cell death by the regulation of MMP collapse via spleen tyrosine kinase (Syk) activation using WiL2-NS human B lymphoblast cells. BAFF binds to receptors on WiL2-NS cells. When the cells were incubated in serum-deprived conditions with 1% fetal bovine serum (FBS), BAFF reduced the percentage of dead cells as determined through trypan blue staining and caspase 3 activity. BAFF also inhibited MMP collapse with 1% FBS, as indicated by a decrease in the number of cells with high-red fluorescence of MitoProbe™ JC-1 reagent or a decrease in the percentage of DiOC6-stained cells. Reactive oxygen species (ROS) production was reduced by incubation with BAFF in the presence of 10% or 1% FBS. BAFF inhibited MMP collapse, cell growth retardation, dead cell formation, and caspase 3 activation caused by treatment with H2O2. Syk phosphorylation on tyrosine (Y) 525/526 was increased in cells incubated with 1% FBS in the presence of BAFF than cells incubated with 1% FBS or BAFF alone. BAY61-3606, a Syk inhibitor reduced the effect of BAFF on MMP collapse, caspase 3 activation, cell growth retardation, and dead cell formation. Together, these data demonstrate that BAFF might attenuate oxidative stress-induced B cell death and growth retardation by the maintenance of MMP through Syk activation by Y525/526 phosphorylation. Therefore, BAFF and Syk might be therapeutic targets in the pathogenesis of B cell-associated diseases such as autoimmune disease.

We investigated the hemodynamic and mortality effects of continuous ketamine infusion in critically ill pediatric patients. We conducted a retrospective cohort study in a tertiary pediatric intensive care unit (PICU). Patients who used continuous sedative from 2015 to 2017 for 24 hours or more were included. We compared blood pressure, heart and respiratory rates, vasogenic medications, and sedation and pain scores for 12 hours before and after initiation of continuous ketamine. The mortality rates for continuous ketamine and Non-ketamine groups were compared by multivariate logistic regression. A total of 240 patients used continuous sedation, and 82 used continuous ketamine. The median infusion rate of ketamine was 8.1 mcg/kg/min, and the median duration was 6 days. Heart rates (138 vs. 135 beat/minute, P = .033) and respiratory rates (31 vs. 25 respiration/minute, P = .001) decreased, but blood pressure (99.9 vs. 101.1 mm Hg, P = .124) and vasogenic medications did not change after ketamine infusion. Continuous ketamine was not a significant risk factor for mortality (hazard ratio 1.352, confidence interval 0.458-3.996). Continous ketamine could be used in PICU without hemodynamic instability. Further studies in randomized controlled design about the effects of continuous ketamine infusion on hemodynamic changes, sedation, and mortality are required.

Nonylphenol (NP) as well-known "endocrine disrupter" influences sexual and reproductive development. Here, we investigated the effect of NP on M1-/M2-type macrophages and their role in lipopolysaccharide (LPS)-induced sepsis. Polarized macrophages of M1- and M2-types were obtained by the treatment with LPS and interleukin-4 (IL-4) to bone marrow-derived macrophages (BMDM), respectively. Coincubation of M1-macrophages with NP decreased COX-2, iNOS, IL-6, and TNF-α expression but no changes were detected in the production of nitric oxide (NO). Survival probability of LPS-induced sepsis mice was enhanced by the injection of NP-treated BMDM as compared to the injection of NP-untreated control BMDM. In the meanwhile, the expression of arginase 1(Arg1), a marker for M2-polarized macrophages was increased by the stimulation with LPS in BMDM. Arg1 expression was also enhanced by the treatment with IL-4 in BMDM, which was reduced by the coincubation with NP. Survival probability of LPS-induced sepsis mice was decreased by the injection of BMDM treated with IL-4 and NP as compared to the injection of IL-4-treated BMDM. It suggests that NP might inhibit macrophage function and the polarization to M2-macrophages. Taken together, data demonstrate that NP could differently affect immune responses of polarized macrophages resulted in the modulation of LPS-induced sepsis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2081-2089, 2016.

Autoimmune rheumatic lesions are often characterised by the immune cell recruitment including B lymphocytes and the presence of reactive oxygen species (ROS), which increase antioxidant gene transcription via nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Spleen tyrosine kinase (Syk) has a major role in the signal transmission of all haematopoietic lineage cells including B/T cells, mast cells, and macrophages. In this study, we investigated whether B cell survival is regulated by Nrf2 via ROS-mediated Syk activation in WiL2-NS human B lymphoblast cells. When WiL2-NS cells were incubated with 1% foetal bovine serum (FBS), the survival rate and mitochondrial membrane potential (MMP) were reduced. In addition, 1% FBS increased caspase 3 activity, cytochrome C release, nuclear localisation of Nrf2, and ROS production. N-acetylcysteine attenuated ROS production and nuclear translocation of Nrf2. It also inhibited cell death, caspase 3 activation, MMP collapse, and cytochrome C release. Results from the 1% FBS treatment were consistent with those of H

When defective or absent, Werner syndrome protein (WRN) causes a genetic premature aging disorder called Werner syndrome. Several studies have reported that defects in WRN function are responsible for not only progeria syndrome but also genomic instability via the deregulation of DNA repair, replication, recombination, and telomere stability. Given the importance of WRN in the repair process, we herein investigated the potential role of WRN in drug response by evaluating the DNA repair following exposure to cisplatin in human cancer cell lines. We found that the down-regulation of SIRT1 and inhibition of SIRT1 deacetylase activity blocked the translocation of WRN from the nucleolus to the nucleoplasm in response to genotoxic stresses. In addition, cells expressing low levels of WRN responded favorably to cisplatin, whereas cells expressing high levels responded poorly to cisplatin. The forced expression of WRN protein in chemosensitive cells resulted in an approximately two-fold increase in cell viability in response to cisplatin compared with vector controls and promoted DNA repair, while WRN-deficient cells accumulate unrepaired double-strand breaks following cisplatin exposure. These results suggest that WRN is regulated by SIRT1 and increased expression of WRN might be one of the determinants for the development of chemotherapeutic drug resistance.

Phthalates are widely used as plasticizers that influence sexual and reproductive development. Here, we investigated whether di-(2-ethylhexyl) phthalate (DEHP) affects macrophage polarization that are associated with tumor initiation and progression. No changes were observed in LPS- or ConA-stimulated in vitro spleen B or T cell proliferation for 48 h, respectively. In contrast, macrophage functions were inhibited in response to DEHP for 12 h as judged by LPS-induced H2O2 and NO production and zymosan A-mediated phagocytosis. When six weeks old male mice were pre-exposed to 4.0 mg/kg DEHP for 21 days before the injection of B16F10 melanoma cells and post-exposed to 4.0 mg/kg DEHP for 7 days, tumor nodule formation and the changes in tumor volume were higher than those in control group. Furthermore, when male mice were intraperitoneally pretreated with DEHP for 3 or 4 weeks and peritoneal exudate cells (PECs) or bone marrow-derived macrophages (BMDMs) were incubated with lipopolysaccharide (LPS), the expression of COX-2, TNF-α, and IL-6 was reduced in DEHP-pretreated cells as compared with that in LPS-stimulated control cells. While the production of nitric oxide (NO) for 18 h was reduced by LPS-stimulated PECs and M1-type BMDMs, IL-4 expression was enhanced in LPS-stimulated BMDMs. When BMDMs were incubated with IL-4 for 30 h, arginase 1 for M2-type macrophages was increased in transcriptional and translational level. Data implicate that macrophages were differentially polarized by DEHP treatment, which reduced M1-polarzation but enhanced M2-polarization. Taken together, these data demonstrate that DEHP could affect in vivo immune responses of macrophages, leading to the suppression of their tumor-preventing ability. This suggests that individuals at high risk for tumor incidence should avoid long-term exposure to various kind of phthalate including DEHP.

Trichostatin A (TSA) is an anticancer drug that inhibits histone deacetylases (HDACs). Hypoxia-inducible factor 1 (HIF-1) participates in tumor angiogenesis by upregulating target genes, such as vascular endothelial growth factor (VEGF). In the present study, we investigated whether TSA treatment increases HIF-1α stabilization via acetylation under normoxic conditions, which would lead to VEGF upregulation and resistance to anticancer drugs. TSA enhanced total HIF-1α and VEGF-HRE reporter activity under normoxic conditions. When cells were transfected with GFP-HIF-1α, treatment with TSA increased the number of green fluorescence protein (GFP)-positive cells. TSA also enhanced the nuclear translocation of HIF-1α protein, as assessed by immunoblotting and as evidenced by increased nuclear localization of GFP-HIF-1α. An increase in the interaction between HIF-1α and the VEGF promoter, which was assessed by a chromatin immunoprecipitation (ChIP) assay, led to activation of the VEGF promoter. TSA acetylated HIF-1α at lysine (K) 674, which led to an increase in TSA-induced VEGF-HRE reporter activity. In addition, TSA-mediated cell death was reduced by the overexpression of HIF-1α but it was rescued by transfection with a HIF-1α mutant (K674R). These data demonstrate that HIF-1α may be stabilized and translocated into the nucleus for the activation of VEGF promoter by TSA-mediated acetylation at K674 under normoxic conditions. These findings suggest that HIF-1α acetylation may lead to resistance to anticancer therapeutics, such as HDAC inhibitors, including TSA.

Patterns of adverse drug reactions (ADRs) in the medical intensive care unit (MICU) were analysed, and signals for detecting ADRs were developed from the analysis. A retrospective study was conducted in MICU wards at a tertiary care teaching hospital in Seoul, Korea. The areas included one general MICU and one cancer centre MICU. Two pharmacists evaluated ADRs in terms of length of stay, causality, severity, preventability, types, related organs, and incidence. Differences in ADR perception rates between physicians and pharmacists were also evaluated. ADR cases detected through the evaluation were reviewed to develop specific alerting signals for ICU ADRs. The study group included 346 patients admitted to the ICU over 4 months. The overall incidence of ADRs was 32%. ICU length of stay is closely related to ADRs (p = 0.014). Most ADR cases were mild, temporary, and harmful to the patient. Twenty percent of ADRs were preventable, and 74% were type A. Of the ADRs, 70% were noted by physicians; 80% required intervention. The most commonly implicated drug was amphotericin B, and the clinical presentation was a haematologic reaction. Data on the time required for pharmacists to identify ADRs indicated that they were not slower than physicians. Six signals for early detection of the ADRs were developed. The overall ADR incidence in the MICU was about one-third, and the length of stay of the ADR group was longer than that of those without this experience. Automated signal generation was developed. It seemed to be a valuable tool for faster and more efficient patient management, and possibly prevention of ADRs. A future study should scientifically evaluate the clinical relevance of this tool.

Background Curcumin, a major component of curry powder, which is a natural polyphenol product extracted from rhizoma curcumae longae, interacts with a specific binding site on microtubules. Vinblastine is an antitumor drug that induces microtubule depolymerization. Purpose We investigated whether curcumin influences the antitumor effect of vinblastine in HeLa human cervical cancer cells. Study design Changes in microtubule filaments were visualized by immuno-staining. Cell death was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) or water-soluble tetrazolium(WST) assay. Apoptotic cell formation was assessed by flow cytometry after staining cells with propidium iodide(PI) and/or Annexin V or with 6-diamidino-2-phenylindole(DAPI). Reactive oxygen species(ROS) were also measured by flow cytometry using dichloro-dihydro-fluorescein diacetate(DCF-DA). JC-1 was used to determine mitochondrial membrane potential (MMP). Results When cells were pretreated with curcumin, microtubule filaments were disordered. Vinblastine-induced microtubule depolymerization and cell death were reduced in HeLa human cervical cancer cells pretreated with curcumin compared to the control. The decrease in cell death was much greater in cells pretreated with curcumin compared to cotreatment or post-treatment. DNA condensation by vinblastine was also decreased in curcumin-pretreated cells. Curcumin reduced ROS production by vinblastine. However, no changes in vinblastine-mediated microtubule depolymerization were detected upon N-acetylcysteine(NAC) treatment. In contrast, vinblastine-induced MMP collapse was inhibited by pretreatment with curcumin or NAC. These findings suggest that vinblastine-induced tumor cell death might be inhibited by curcumin via ROS-independent microtubule dynamics and ROS-dependent MMP collapse. It also suggests that microtubule dynamics could be necessary for the optimal antitumor activity of vinblastine. Our results suggest that patients treated with vinblastine should not consume curcumin.

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.