Differences in NF-κB activation and intracellular AA content between human leukemic and CB-CD34⁺ cells in the presence of high AA.

<p>A) Western blotting analysis of p-IκB in HL60 cells. Cells were treated with the vehicle or with high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There was a significant difference in the expression levels (*<i>P</i><0.001). Values represent the mean ± SD of triplicate samples. B) Immunocytochemical (left) and Western blotting (right) analyses of NF-κB in CB-CD34⁺ and HL60 cells. Cells were treated with vehicle or high AA for 1 h, then washed, cultured, and analyzed after 24 h. Note that translocation of NF-κB into the nucleus was markedly decreased in high AA-treated HL60 cells. Green and blue signals represent NF-κB and DAPI, respectively. Bars indicate 20 μm. There were significant differences in the expression levels (*<i>P</i><0.001, **<i>P</i><0.0001). The values represent the mean ± SD values of triplicate samples. C) Intracellular AA content of human leukemic cells and 2 different isolates of CB-CD34⁺ cells. Cells were treated with high AA for 1 h, washed in PBS, and analyzed immediately. There were significant differences in the content between leukemic and CB-CD34⁺ cells. *<i>P</i><0.001, as compared with CB-CD34⁺ cells (1) or (2). The values are mean ± SD values of triplicate samples.</p

In vitro effects of AA on human leukemic and CB-CD34⁺ cells, relative to catalase activity.

<p>A) Cell viability assay of various leukemic cell lines and 2 independent isolates of CB-CD34⁺ cells. Cells were treated with different concentrations of AA for 1 h, and then washed, cultured, and analyzed after 72 h. The viability of all cell lines reduced significantly in the presence of 280 and 2800 µM AA (*<i>P</i><0.0001, as compared with vehicle), but this finding was not obtained for CB-CD34⁺ cells (<i>P</i>>0.05). The values represent the mean ± SD values of quadruplicate samples. B) Flow cytometric measurement of apoptosis of HL60 cells. Cells were treated with vehicle or AA for 1 h, and then washed, cultured, and analyzed after 18 h. Representative profiles are shown. The annexin V⁺ propidium iodide (PI)⁺ cell fraction indicates apoptotic cells. Note that AA-induced apoptosis was almost completely abrogated by the addition of catalase. C) Intracellular catalase activity. Leukemic cells generally expressed lower catalase activities than did CB-CD34⁺ isolates (*<i>P</i><0.001, as compared with each cell line). The values represent the mean ± SD values of quadruplicate samples. D) Histochemical analysis demonstrated lower catalase activity in HL60 cells than in CB-CD34⁺ cells. The bars indicate 50 μm.</p

Expression of angiogenesis-related molecules in human leukemic and CB-CD34⁺ cells exposed to the vehicle or to high AA.

<p>A) Quantitative real-time PCR (qRT-PCR) analysis of <i>HIF-1α</i> mRNA in CB-CD34⁺ and HL60 cells. The cells were treated with vehicle or high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There were no significant differences in the expression levels for the 2 conditions (<i>P</i>>0.05) in CB-CD34⁺ cells. In contrast, there were significant differences in the expression levels between the 2 conditions (*<i>P</i><0.0001) in HL60 cells. The values represent the mean ± SD values of triplicate samples. B) Western blotting analysis of HIF-1α in CB-CD34⁺ and HL60 cells. The cells were treated with vehicle or high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels (*<i>P</i><0.01, **<i>P</i><0.0005). The values are mean ± SD values of triplicate samples. C) Sequential analysis of qRT-PCR results of <i>HIF-1α</i> and <i>VEGF</i> mRNA in HL60 cells. The cells were treated with high AA for 1 h, and then washed, cultured, and analyzed after 1, 3, 22, and 26 h. The expression of <i>VEGF</i> mRNA reduced along with that of <i>HIF-1α</i> over time. Compared with the expression levels at 0 h, there were significant differences in the expression levels (*<i>P</i><0.01, **<i>P</i><0.001, ***<i>P</i><0.0001). The values represent the mean ± SD values of triplicate samples.</p

Relationship between antileukemic effects of high AA and HIF-1α expression.

<p>A) Quantitative real-time PCR analysis of <i>HIF-1α</i> mRNA expression in K562 and K562-HIF1α cells. Cells were treated with the vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. After high AA exposure, <i>HIF-1α</i> mRNA expression significantly reduced in K562 (*<i>P</i><0.01), but not in K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. B) Western blotting analysis of HIF-1α in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. High AA exposure significantly reduced the HIF-1α protein level in both types of cells. However, the HIF-1α protein level in K562-HIF1α cells was significantly higher than that in K562 cells after vehicle or high AA exposure. *<i>P</i><0.01, **<i>P</i><0.0001, ***<i>P</i><0.00001. The values represent the mean ± SD values of triplicate samples. C) Flow cytometric measurement of apoptosis of K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 18 h. There was a significant difference in the number of apoptotic (annexin V⁺ propidium iodide (PI)⁺) cells between high AA-treated K562 and K562-HIF1α cells (*<i>P</i><0. 001). The values represent the mean ± SD values of triplicate samples. D) Flow cytometric measurement of cleaved caspase-3 expressed by K562 and K562-HIF1α cells. Cells were treated with vehicle (gray lines) or high AA (black lines) for 1 h, washed, cultured, and analyzed after 24 h. Activation of caspase-3 by high AA was lower in K562-HIF1α than in K562 cells. E) Western blotting analysis of Mcl-1, Bcl-x_L, and Bcl-2 in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.05) and between the vehicle-treated and high AA-treated K562 cells (**<i>P</i><0.0001). There was no significant difference between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. F) Western blotting analysis of Sp1, Sp3, Sp4, and VEGF. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels of these molecules between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.01, ** <i>P</i><0.0001). There were significant differences in the expression levels of Sp1, Sp3, and Sp4 between the vehicle-treated and high AA-treated K562 or K562-HIF1α cells (^†<i>P</i><0.01, ^††<i>P</i><0.001, ^†††<i>P</i><0.0001). There was a significant difference in the expression level of VEGF between the vehicle-treated and high AA-treated K562 (^†††<i>P</i><0.0001), but not between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05).</p

In vivo effects of high AA on progression of leukemia.

<p>A) High AA or the vehicle was injected intravenously for 6 days with a rest period of 2 days between 3 daily injections of mice transplanted with HL60 cells. Compared with vehicle (blue line), high AA (red line) significantly inhibited tumor growth (*<i>P</i><0.01). The values represent the mean ± SD values of 5 mice. B) Appearance of mice treated with vehicle (left) and high AA (right), 4 days after the final injection. C) Representative macroscopic appearance of tumors of mice treated with the vehicle (left) and high AA (right). Note that the tumors of high AA-treated mice were smaller and less erythematous than those of vehicle-treated mice. D) Immunohistochemical analysis of tumor neoangiogenesis in mice treated with the vehicle (left) and high AA (right). The green and blue signals represent CD31 and 4′,6-diamidino-2-phenylindole (DAPI), respectively. The bars indicate 100 μm.</p

Effects of high AA on tumor growth in the presence or absence of overexpression of <b><i>HIF-1α</i></b><b>.</b>

<p>A) Immunohistochemical analysis of tumor neoangiogenesis in vehicle-treated (left) and high AA-treated (right) mice transplanted with K562 (upper column) or K562-HIF1α cells (lower column). The green and blue signals represent CD31 and DAPI, respectively. The bars indicate 100 μm. Note that administration of high AA suppressed tumor neoangiogenesis in mice transplanted with K562 cells, but not in mice transplanted with K562-HIF1α cells. B) In the xenogeneic transplant model, high AA or vehicle was injected for 5 days. Administration of high AA significantly inhibited tumor growth of K562 cells (*<i>P</i><0.05) but not of K562-HIF1α cells (<i>P</i>>0.05). Tumor growth rate was estimated using the following equation: tumor volume on day 4 after high AA treatment/tumor volume just before high AA treatment. The values represent the mean ± SD values for 4 mice.</p

Table_1_A short decision time for transcatheter embolization can better associate mortality in patients with pelvic fracture: a retrospective study.docx

BackgroundEarly use of hemostasis strategies, transcatheter arterial embolization (TAE) is critical in cases of pelvic injury because of the risk of hemorrhagic shock and other fatal injuries. We investigated the influence of delays in TAE administration on mortality.MethodsPatients admitted to the Advanced Critical Care Center at Gifu University with pelvic injury between January 2008 and December 2019, and who underwent acute TAE, were retrospectively enrolled. The time from when the doctor decided to administer TAE to the start of TAE (needling time) was defined as “decision-TAE time.”ResultsWe included 158 patients, of whom 23 patients died. The median decision-TAE time was 59.5 min. Kaplan–Meier curves for overall survival were compared between patients with decision-TAE time above and below the median cutoff value; survival was significantly better for patients with values below the median cutoff value (p = 0.020). Multivariable Cox proportional hazards regression analysis revealed that the longer the decision-TAE time, the higher the risk of mortality (p = 0.031). TAE duration modified the association between decision-TAE time and overall survival (p = 0.109), as shorter TAE duration (procedure time) was associated with the best survival rate (p for interaction = 0.109).ConclusionDecision-TAE time may play a key role in establishing resuscitation procedures in patients with pelvic fracture, and efforts to shorten this time should be pursued.</p