Effects of ferric citrate on intracellular oxidative stress markers after hydrogen peroxide treatment of human U937 monocytes

Phosphate binders, such as iron （III） citrate hydrate （FCH）, are essential medications for hemodialysis patients. Some in vivo studies have demonstrated that FCH prevented induction of oxidative stress in the presence of transferrin. However, how FCH affects iron-related oxidative stress in the absence of transferrin remains unclear. In the current study, we investigated the effects of ferric citrate （FC） on oxidative stress in the absence of transferrin in vitro to address this question. Human U937 monocytes were pretreated with FC, iron （II） chloride tetrahydrate （FeCl2・4H2O）, iron （III） chloride hexahydrate （FeCl3・6H2O）, or saccharated ferric oxide for 24 h and then treated with 10-mM hydrogen peroxide （H2O2） for 30 min. The final Fe concentrations were adjusted to approximately 200µg/dl. Iron concentration, intracellular reactive oxygen species （ROS） levels, and intracellular lipid peroxidation of the cell membrane were measured. After treatment with FC, iron concentration and ROS levels increased. Change in lipid peroxidation after treatment with FC was not observed. However, after treatment with H2O2, no change was observed in the intracellular ROS levels in FC-pretreated cells, whereas lipid peroxidation of the cell membrane was decreased. Despite the high iron concentration in FC-pretreated cells, neither intracellular ROS nor cell membrane lipid peroxidation levels were increased with H2O2 treatment. Their results might represent antioxidative effects of FC. The results of this study may contribute to a better understanding of the effects of oxidative stress in hemodialysis patients treated with FCH

Linagliptin inhibits lipopolysaccharide-induced inflammation in human U937 monocytes

Abstract Background Atherosclerosis and inflammation are more common in patients with diabetes than in patients without diabetes, and atherosclerosis progression contributes to inflammation. Therefore, anti-inflammatory therapy is important for the prognosis of patients with diabetes. Linagliptin is the only bile-excreted, anti-diabetic oral dipeptidyl peptidase-4 (DPP-4) inhibitor. Although the anti-inflammatory effects of DPP-4 inhibitors in vivo and in vitro have been reported, few in vitro studies have examined the effects of linagliptin using monocytes, which play a central role in arteriosclerosis-related inflammation. Herein, we assessed the anti-inflammatory effects of linagliptin in human U937 monocytes. Methods U937 cells at densities of 1 × 106 cells/mL were cultured in Roswell Park Memorial Institute medium supplied with 10% fetal bovine serum and treated with 100 nM phorbol myristate acetate for 48 h for differentiation into macrophages. The media were replaced, and the cells were pretreated with 1, 5, 10, 50, and 100 nM linagliptin for 1 h or were left untreated. The media were then replaced again, and the cells were treated with 1 μg/mL lipopolysaccharide (LPS) or 10 nM interleukin (IL)-1β only, in combination with 1, 5, 10, 50, and 100 nM linagliptin or were left untreated. The extracted media were used to measure IL-6 and tumor necrosis factor (TNF)-α levels using enzyme-linked immunosorbent assay kits. Results LPS alone significantly increased IL-6 and TNF-α production compared with the control treatment. The treatment of cells with linagliptin at all concentrations significantly inhibited the LPS-stimulated IL-6 and TNF-α production. Meanwhile, IL-1β alone significantly increased IL-6 production compared with the control treatment. No significant difference in IL-6 production was noted between the cells treated with IL-1β and simultaneous treatment with IL-1β and linagliptin. Conclusions Linagliptin inhibited LPS-induced inflammation in human monocytic U937 cells

Size-Based Isolation of Circulating Tumor Cells in Lung Cancer Patients Using a Microcavity Array System

<div><p>Background</p><p>Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method.</p><p>Methods</p><p>Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer’s protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems.</p><p>Results</p><p>CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0–291 cells/7.5 mL) than by the CellSearch system (median 0, range 0–37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system.</p><p>Conclusions</p><p>The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.</p></div

CTC recovery efficiency and average cell diameter.

<p>Cells were spiked into 1 mL of normal blood and recovered using the MCA system.</p

Gallery of cells captured on the MCA from blood of advanced lung cancer patients.

<p>Cells were stained with Hoechst 33342, FITC-labeled anti-cytokeratin antibody, and PE-labeled anti-CD45 antibody.</p

CTC count using the MCA system.

<p>CTC count/7.5 mL blood is shown for 6 healthy donors, 22 NSCLC patients and 20 SCLC patients.</p

Patient characteristics.

<p>Patient characteristics.</p

MCA system for size-based isolation of CTCs.

<p>(a) Schematic diagram of the structure of the MCA system. (b) Scanning electron microscope image of a cultured tumor cell line trapped on the MCA system. (c–f) Cells isolated from SCLC patient blood stained with Hoechst 33342 (c) and fluorescent-labeled antibodies that target cytokeratin (d) and CD45 (e). Merging of the images (f) allowed for identification of CTCs and hematologic cells. Scale bar = 60 µm.</p

Gallery of CTCs captured on a transparent MCA from SCLC patient blood.

<p>May-Grünwald–Giemsa-stained cells showed a high nucleus–cytoplasm ratio and nuclear molding (×40). Black arrow indicates 9-µm microcavity. Scale bar = 60 µm.</p