OBJECTIVETo investigate the role of reactive oxygen species (ROS) and ROS-caused mitochondrial transmembrane potential loss in sodium selenite-induced apoptosis in NB4 cells.

METHODSROS production was measured by ROS-specific probe DCFH-DA. Sodium selenite mitochondrial transmembrane potential loss was evaluated by flow cytometry with Rh123 staining. Protein levels of cytochrome C, Bid, Bcl-xl, and Bax were measured by Western blot using protein-specific antibodies. NB4 cells were pre-incubated by MnTmPy or BSO before selenite treatment to further confirm the effects of ROS on NB4 cells.

RESULTS20 micromol/L sodium selenite induced ROS production and mitochondrial transmembrane potential loss in NB4 cells time-dependently. Cytochrome C accumulated in cytoplasm after selenite treatment. Sodium selenite also downregulated Bcl-xl and activated Bax and Bid at protein level. Pretreatment with antioxidant MnTmPy almost fully abrogated the proapoptotic effect of sodium selenite prevented the cleavage of Bid protein and in turn the mitochondrail transmembrane potential loss. On the contrary, pretreatment with BSO intensified the mitochondrail transmembrane potential loss induced by sodium selenite.

CONCLUSIONSSodium selenite may induce apoptosis by inducing ROS production in NB4 cells, which leads to the downregulation of Bcl-xl, upregulation of Bax, and cleavage and activation of Bid. Bax and tBid then agregate on mitochondrial membrane, which in turn causes a decrease of mitochondrial transmembrane potential and release of cytochrome C into cytoplasm.

Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; biosynthesis ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Reactive Oxygen Species ; metabolism ; Sodium Selenite ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis

OBJECTIVEIn the present study, we examined the expression changes of Bcl-2/Bax in C-reactive protein (CRP) treated human endothelium cells in vitro.

METHODSThe human umbilical vein endothelial cells (HUVEC) were cultured by digest method for 2 - 3 posterities and incubated with human CRP (0, 1, 5, 25 mg/L for 24 hours) and analyzed by flow cytometer for apoptosis ratio. The effects of CRP in various concentrations on Bcl-2/Bax mRNA and protein expression were examined by RT-PCR and Western Blotting.

RESULTSApoptosis ratio increased, downregulated Bcl-2 (gene promoting cell survival) and upregulated Bax (gene promoting apoptosis) at mRNA and protein levels in proportion to increased CRP concentrations.

CONCLUSIONThese results demonstrate that Bcl-2/Bax could be regulated by CRP in human HUVECs and might play a causal role in CRP-induced apoptosis.

Apoptosis ; drug effects ; C-Reactive Protein ; pharmacology ; Cell Line ; Endothelial Cells ; cytology ; metabolism ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Umbilical Veins ; cytology ; bcl-2-Associated X Protein ; biosynthesis

OBJECTIVETo study the clinical value of expression of peripheral blood neutrophil CD64 in adults with measles complicating pneumonia.

METHODS106 patients were divided into two groups by clinical manifestation and bacteria study: measles complicating bacterial pneumonia group and measles complicating viral pneumonia, using flow cytometry determination of CD64, C-reactive protein (CRP) and white blood cell (WBC) count.

RESULTSThe expression of CD64 in the bacterial pneumonia group with eruptive stage was (32.15 +/- 11.07) MFI, which was significantly higher than that in the group of with recovery stage (10.6 +/- 3.23) MFI (P < 0.01) and viral pneumonia (9.55 +/- 3.48) MFI (P < 0.01). These markers were considered positive if CD64 > or = 8.5 MFI, CRP > or = 10 mg/L and WBC > or = 9.05 x 10(9)/L. Their sensitivity was 78.12%, 80.48% and 59.37%. Their specificity was 76.19% ,67.67% and 64.28%. Their accuracy rate was 77.35%, 74.52%, 61.32%; CD64 has a positive relationship with CRP.

CONCLUSIONCompared to CRP, expression of peripheral blood neutrophil CD64 can be a better marker in the early diagnosis of patients with measles complicating bacterial pneumonia and as one of the indicators of disease conditions.

Adult ; Biomarkers ; blood ; metabolism ; C-Reactive Protein ; metabolism ; Female ; Humans ; Leukocyte Count ; methods ; Male ; Measles ; blood ; immunology ; microbiology ; Pneumonia, Bacterial ; blood ; immunology ; virology ; Receptors, IgG ; biosynthesis ; blood ; immunology

OBJECTIVETo investigate the effect of a tight control of blood glucose by intensive insulin therapy on human sepsis, and to explore the potential mechanism of the intensive insulin therapy.

METHODSEligible patients were randomized by a blinded pharmacist to receive tight control of blood glucose by intensive insulin therapy (maintenance of blood glucose at a level between 4.4 and 6.1 mmol/L) or to receive conventional treatment (maintenance of glucose at a level between 10.0 and 11.1 mmol/L). The expression of HLA-DR on peripheral monocytes was measured in 54 patients by flow cytometry on 24 h, 3 d, 5 d, 7 d, 10 d and 14 d of intensive care in parallel with serum c-reactive protein (CRP), severity of the disease (APACHE II score, SOFA score) and clinical data collection.

RESULTSPatients receiving intensive insulin therapy were less likely to require prolonged mechanical ventilation. Tight control of blood glucose significantly reduced the number of days during which leukopenia or leukocytosis and the days with hypo- or hyperthermia (P < 0.05). Hypoglycemia occurred in 3 patients (10.7%) in the tight control of blood glucose group. There were no instance of hemodynamic deterioration or convulsions. Compared with the conventional treatment, tight control of blood glucose also increased the HLA-DR expression of peripheral monocytes, and there were significantly difference on 3 d, 5 d and 7 d (P < 0.05). Whereas it suppressed the elevated serum CRP concentrations, there was significantly difference on 7 d (P < 0.05).

CONCLUSIONSTight control of blood glucose by intensive insulin therapy expedited healing of human sepsis, and increased the HLA-DR expression of peripheral and suppressed the elevated serum CRP. So, it is necessary to use insulin to strict control the glucose levels in human sepsis.

Blood Glucose ; metabolism ; C-Reactive Protein ; metabolism ; HLA-DR Antigens ; biosynthesis ; Humans ; Hyperglycemia ; drug therapy ; etiology ; metabolism ; Hypoglycemic Agents ; therapeutic use ; Insulin ; therapeutic use ; Sepsis ; complications

OBJECTIVE: To investigate the level of mRNA expression of C-reactive proteins (CRP) induced by LPS in human alveolar type epithelial cell line, A549. METHODS: Sputum and plasma specimens were obtained from patients with chronic obstructive pulmonary disease (COPD) diagnosed according to the national criteria. CRP was measured by enzyme-linked immunosorbent assays (ELISA). A549 cells were incubated with lipopolysaccharide (LPS) at different concentrations and for different time. CRP mRNA was extracted from cells and the expression was analyzed by RT-PCR. The CRP in the supernatant was measured by ELISA. RESULTS: The concentration of CRP in the sputum of patients with COPD was significantly higher than that in the plasma (P < 0.05). LPS at different concentrations and for different time induced the expression and secretion of CRP in A549 with a time-dependent increase and the expression of CRP were significantly higher than those of the control (P < 0.05 - 0.01). CONCLUSION Self-secretion of CRP in the respiratory tract may exist. A549 induced by LPS may express CRP mRNA.
C-Reactive Protein ; biosynthesis ; genetics ; metabolism ; Epithelial Cells ; cytology ; metabolism ; Female ; Humans ; Lipopolysaccharides ; Male ; Middle Aged ; Pulmonary Alveoli ; cytology ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Sputum ; metabolism

OBJECTIVETo observe the expression and localization of endogenous C-reactive protein (CRP) in cells from different tissues under different conditions.

METHODSMacrophages differentiated from THP-1 monocytes with phorbol ester (PMA) induction and human LO2 hepatocytes were stimulated with lipopolysaccharide (LPS). The culture supernatant of the LPS-stimulated THP-1 cells was collected and added into LO2 cell culture, and after incubation, the cells were lysed to extract the proteins for SDS-PAGE and Western blotting. The stimulated cells were also examined immunocytochemically for CRP expression.

RESULTSWestern blotting detected CRP in both of the unstimulated cell lysates, but in neither of the two cell supernatants. After LPS stimulation, CRP expression was significantly increased in the cell lysate of THP-1 cells with also a small amount present in the supernatant, but CRP expression and release in the LO2 cells showed no significant variation. Treatment of the LO2 cells with the culture supernatant of LPS-stimulated THP-1 cells resulted in positivity of CRP in the cell lysate and the culture supernatant. Immunocytochemistry identified CRP expression throughout the THP-1 cell body (most obvious in the nuclei), which increased after LPS stimulation. In LO2 hepatocytes, CRP expression was found only outside the nuclei and increased after stimulation with the culture supernatant of LPS-treated THP-1 cells, especially obvious around the membrane.

CONCLUSIONCRP can not be up-regulated directly by LPS treatment in LO2 cells, but can be induced by certain cytokines (IL-6) secreted from LPS-stimulated THP-1 cells. The localization of CRP represents the characteristics of secreted protein in LO2 cells, but in THP-1 cells, CRP is found mainly in the cell nuclei.

Blotting, Western ; C-Reactive Protein ; biosynthesis ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Immunohistochemistry ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; Monocytes ; cytology ; drug effects ; metabolism

We conducted a phase II multicenter trial to estimate the response and survival of patients with newly diagnosed multiple myeloma to high dose melphalan therapy followed by autologous peripheral blood stem cell transplantation. Eligible patients who had undergone induction with vincristine, adriamycin and dexamethasone (VAD) should have adequate cardiac, pulmonary and renal function (creatinine <2 mg/dL). Melphalan at 200 mg/m2 was used as a conditioning regimen. Eighty patients were enrolled from 13 centers. The median age of the patients was 53 yr (range; 20 to 68 yr). The initial stage was IA/IIA/IIB/IIIA/IIIB in 3/8/1/54/14 patients, respectively. Beta2-microglobulin, CRP and LDH were increased in 74, 42 and 34% of the patients examined. Cytogenetic data were available in 30 patients, and 6 patients showed numeric or structural abnormalities. Two therapy-related mortalities occurred from infection. Among the 78 evaluable patients, CR/PR/MR/NC/PD were achieved in 48/26/2/1/1patients, respectively. After a median follow-up of 30 months, the median overall and event-free survivals were 66 months (95% CI: 20-112) and 24 months (95% CI: 18-29), respectively. This study verifies the efficacy and feasibility of high dose melphalan therapy with autologous stem cell transplantation in newly diagnosed multiple myeloma.
Adult ; Aged ; Antigens, CD34/biosynthesis ; Antineoplastic Agents, Alkylating/*therapeutic use ; C-Reactive Protein/biosynthesis ; Cell Survival ; Combined Modality Therapy ; Cytogenetics ; Disease-Free Survival ; Female ; Human ; Korea ; L-Lactate Dehydrogenase/biosynthesis ; Male ; Melphalan/*therapeutic use ; Middle Aged ; Multiple Myeloma/*therapy ; Peripheral Blood Stem Cell Transplantation/*methods ; Time Factors ; Transplantation, Autologous/*methods ; beta 2-Microglobulin/blood

PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.
Anti-Bacterial Agents/pharmacology ; Apoptosis ; Blotting, Western ; C-Reactive Protein/biosynthesis/*genetics ; Cells, Cultured ; Endoplasmic Reticulum Stress/*drug effects/genetics ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Humans ; Polymerase Chain Reaction ; RNA, Messenger/*genetics ; Retinal Pigment Epithelium/*metabolism/pathology ; Serum Amyloid P-Component/biosynthesis/*genetics ; Tunicamycin/*pharmacology

The aim of this study was to investigate the effects of antidepressant treatment on serum cytokines and nutritional status in hemodialysis patients. Twenty-eight hemodialysis patients with a depressed mood were given 20 mg of fluoxetine for 8 weeks. The degree of depressive symptoms, the serum levels of interleukin-1beta, interleukin- 2, interleukin-6, tumor necrosis factor-alpha, c-reactive protein, and markers of nutritional status were assessed at baseline and after treatment. The outcome was assessed in terms of response to treatment (>50% reduction in the score of the Hamilton depression rating scale). Antidepressant treatment decreased the serum level of interleukin- 1 in both response and nonresponse groups, and increased the serum level of interleukin-6 only in the response group. At baseline, the level of interleukin-6 in the response group was lower than in the nonresponse group. Antidepressant treatment also increased fat distribution significantly in the response group which might have slightly improved the nutritional status. This study suggests that antidepressant treatment improve depressive symptoms and may affect immunological functions and nutritional status in chronic hemodialysis patients with depression.
Adult ; Antidepressive Agents, Second-Generation/*pharmacology ; C-Reactive Protein/biosynthesis ; Cytokines/*blood ; Depression/*drug therapy ; Electric Impedance ; Female ; Fluoxetine/*pharmacology ; Human ; Interleukin-1/blood ; Interleukin-2/blood ; Interleukin-6/blood ; Male ; Middle Aged ; Nutrition ; Renal Dialysis/*methods ; Support, Non-U.S. Gov't ; Treatment Outcome ; Tumor Necrosis Factor/biosynthesis

To study the molecular mechanisms of nitric oxide donor sodium nitroprusside (SNP) -induced apoptosis in K562 human leukemia cell line, the different concentrations of SNP and different time of culture were used to treat K562 cell. At the same time, potassium ferricyamide (PFC) was used as control, blank was designed in experiment. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantify in situ cell apoptosis. Reactive oxygen species (ROS) in cells and mitochondrial transmembrane potential (DeltaPsim) were labeled by dihydrorhodamin 123, 2', 7'-dichlorodihydrofluorescein diacetate and rhodamin 123/PI. bcl-2, bax, bad, p53 gene proteins and mitochondrial membrane protein were analysed by flow cytometry. The results showed that the K562 cell apoptosis was confirmed by typical cell morphology, DNA fragment, sub-G(1) phase, TUNEL and annexin-V/PI labeling. A majority of K562 cells were arrested in G(0)/G(1) phase. During the process of SNP-induced apoptosis in K562 cell, the mean fluorescence intensity of ROS in cells was significantly higher than those in blank and PFC control, while the DeltaPsim reduced. The expression of p53, bax, bad, Fas protein and mitochondrial membrane protein increased and bcl-2 protein decreased after SNP treatment. It is concluded that SNP induces K562 cell apoptosis through increasing ROS in cells, expressing the p53, bax, bad, Fas protein and mitochondrial membrane protein and decreasing bcl-2 protein, opening the mitochondrial permeability transition pore and reducing DeltaPsim. Furthermore, the Fas was activated during the apoptosis process.
Apoptosis ; drug effects ; Dose-Response Relationship, Drug ; Humans ; In Situ Nick-End Labeling ; K562 Cells ; Membrane Potential, Mitochondrial ; drug effects ; Nitric Oxide Donors ; pharmacology ; Nitroprusside ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Reactive Oxygen Species ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; biosynthesis