Objective:To study the interaction of the inflammatory cytokines expression between CD4+ T cells and primary human umbilical vein endothelial cells (HUVECs) infected by dengue virus (DENV-2).Methods:PBMC was extracted from white blood cells by density gradient centrifugation,CD4+ T cells were sorted by immunomagnetic beads.The expression of CD3 and CD4 molecules on the surface of cells was detected by flow cytometry to identify the purity of CD4+ T cells.First,HUVECs were pretreated by specific-S1 P1 receptor agonist CYM-5442 for 24 h,second,infected by DENV-2 on the titer of 103 TCID50,then co-culturing with CD4+ T cells,The relative expression of NS1 partial sequence and IL-6,L-8 mRNA of HUVECs,and IL-4,IL-17,TNF-α,IFN-γ of CD4+ T cells detected by Real-time RT-PCR.IL-6 and IL-8 secreted in cultured supematant analyzed by ELISA.Results:The purity of CD4+ T cells was (98.02±0.32) ％.The expression of NS1 gradually increased to 24 h (3.03±0.26,P＜0.001),decreased after reaching the peak.The relative expression of NS1 in the group of co-cultured with CD4+ T cells was lower than other groups.After infection,the expression of IL-6 and IL-8 were up-regulated,and the expression of IL-6 and IL-8 at each time point was significantly increased after co-culturing with CD4+ T (P＜0.01).IL-6 of CYM-5442 pretreatment group,in 24 h (28.91±2.34,P＜0.05),36 h(19.36±0.1,P＜ 0.05) and 72 h(13.84±0.82,P＜0.05) was significantly decreased,the expression of IL-8 also decreased significantly.The mRNA expression of IL-4,IL-17,TNF-α and IFN-γ in CD4+ T cells was significantly increased after co-culturing with HUVECs.After the treatment with CYM-5442 group,the expression was decreased.Conclusion:DENV-2 could infect the primary HUVECs,and the expression of NS1 was inhibited after co-culturing with CD4+ T cells.CD4+ T cells can not only enhance the activation of HUVECs infected by DENV-2,but also can be activated by the infected HUVECs infected with DENV-2.

Objective To investigate the effects of interaction between human umbilical vein endothelial cells (HUVECs) which were infected with dengue virus type 2 (DENV-2) and CD4+T cells on the expression of ICAM-1 (intercellular adhesion molecule 1),VCAM-1 (vascular cell adhesion molecule 1),IL-10 and TGF-β1 at mRNA level for further understanding the immunological mechanism of DENV infection.Methods HUVECs were treated with CYM-5442,a selective agonist for sphingosine-1-phosphate receptor 1 (S1P1),for 24 hours and then infected with 103 TCID50 (50% tissue culture infective dose) of DENV-2 before co-culturing with CD4+T cells.Changes in the expression of NS1 (DENV-2 nonstructural protein),SPHK1 (sphingosine kinase 1,phosphorylating sphingosine to S1P),ICAM-1,VCAM-1,IL-10 and TGF-β1 at mRNA level were detected by real-time PCR after 4,8,12,24,48 and 72 hours of co-culturing.Results There was a certain timeliness in the expression of NS1 at mRNA level after infecting HUVECs with DENV-2 and the expression reached a peak at 24 h.Treating HUVECs with or without CYM-5442 had no significant influence on the expression of DENV-2 NS1 at mRNA level.The expression of SPHK1 at mRNA level was significantly increased after treating HUVECs with CYM-5442 and DENV-2 (P<0.05).Compared with DENV-2-infected or untreated HUVECs,Co-culturing DENV-2-infected HUVECs with CD4+T cells increased the expression of ICAM-1 and VCAM-1 in HUVECs at mRNA level (P<0.01) as well as the expression of IL-10 in CD4+T cells at mRNA level (P<0.05),but had no significant influence on the expression of TGF-β1 in CD4+T cells at mRNA level.Conclusion This study shows that DENV-2 can replicate and proliferate in HUVECs,but CD4+T cells inhibit the replication and proliferation.CD4+T cells play an important role in promoting the expression of VCAM-1 and ICAM-1 in DENV-2-infected HUVECs at mRNA level,activating HUVECs and increasing inflammation,which may be associated with increased vascular permeability induced by DENV-2 infection.Co-culturing CD4+T cells with DENV-2-infected HUVECs promotes the expression of IL-10 in CD4+T cells at mRNA level,but has no significant effect on TGF-β1.

To investigate the inhibitory effect of Pluronic on P-glycoprotein (P-gp) drug efflux pump, Caco-2 cells and animal models were established to study the influence of Pluronic on celiprolol transport across Caco-2 cell monolayer and intestinal mucous membrane with verapamil set as a positive control. Drug concentration was measured by HPLC and the apparent permeability coefficient (P(app)), absorption rate constant (k(a)) and the effective permeability coefficient (P(eff)) were calculated. P(app) of basolateral to apical side and apical to basolateral side was (2.10 +/- 0.13) x 10(-6) and (0.333 +/- 0.018) x 10(-6) cm x s(-1), respectively. Transports of celiprolol across Caco-2 cell monolayer were influenced by both verapamil and Pluronic. The absorption constants (k(a)) of celiprolol at duodenum, jejunum, ileum, and colon were (0.09 +/- 0.03), (0.14 +/- 0.04), (0.11 +/- 0.03) and (0.05 +/- 0.02) h(-1), k(a) of celiprolol in verapamil group were (0.14 +/- 0.03), (0.24 +/- 0.02), (0.25 +/- 0.03) and (0.23 +/- 0.02) h(-1), and k(a) of celiprolol in Pluronic group were (0.13 +/- 0.02), (0.22 +/- 0.02), (0.22 +/- 0.03) and (0.20 +/- 0.03) h(-1), respectively. Pluronic showed significant effect on inhibiting P-gp of Caco-2 cell and intestinal mucosa in rats.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Animals ; Biological Transport ; drug effects ; Caco-2 Cells ; Celiprolol ; pharmacokinetics ; Excipients ; Humans ; Intestinal Absorption ; drug effects ; Intestinal Mucosa ; metabolism ; Jejunum ; metabolism ; Male ; Permeability ; Poloxamer ; administration & dosage ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the outcome of mometasone furoate nasal spray (MFNS) used for 3 months on non-allergic rhinitis (NAR).

METHODSIn this multicenter study, NAR patients were enrolled from eight hospitals and received MFNS 200 microgram once daily for 3 months. The patients were followed-up for three times (at baseline, month 1 and month 3) to record the symptom scores and nasal endoscopic appearances. At the same time, the adverse events frequency was recorded and analyzed.

RESULTSA total of 188 NAR cases were enrolled in the study. The total nasal symptom score assessment descended significantly at month 1 (1.70 ± 0.75) and month 3 (0.95 ± 0.79) visits versus at baseline (2.67 ± 0.68, Z value were from -11.603 to -10.491, all P < 0.01). The individual symptoms, including nasal stuffiness, nasal discharge, nasal stuffiness-related dizziness or headache, hyposmia, sleep quality, daily life activity, work or study efficiency, mental status, and whole body fatigue, also showed less scores at month 1 and month 3 visits versus at baseline (Z value were from -11.313 to -6.802, all P < 0.01). At the same time, nasal mucosal appearances assessed by endoscopy had lower scores at month 1 (1.40 ± 0.62) and month 3 (0.75 ± 0.71) visits versus at baseline (2.27 ± 0.73, Z value were from -11.484 to -10.002, all P < 0.01). Additionally, adverse events were only observed in 5.3% cases with light rhinorrhagia and nasal dryness. No other side effect was found.

CONCLUSIONSA 3-months administration of intranasal mometasone can effectively and safely improve NAR patients' clinical symptom and nasal mucosal appearances.

Adolescent ; Adult ; Anti-Allergic Agents ; administration & dosage ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Mometasone Furoate ; Nasal Sprays ; Pregnadienediols ; administration & dosage ; therapeutic use ; Rhinitis ; classification ; drug therapy ; Treatment Outcome ; Young Adult

OBJECTIVETo explore the influence of occupational stress and negative life events on low birth weight (LBW).

METHODS1:1 matched case-control study was employed, in which 438 singleton LBW infants with birth weight less than 2500 g (their pregnancy term being 28 to 42 weeks) served as case group while 438 with singleton term normal birth weight served as control group matched by sex, delivery time and hospital. All of their mothers were inquired by well trained investigators about their socio-demographic characteristics, occupational stress, and negative life events occurring in different pregnancy term. After controlling for mother's age, occupation, education level and family income, conditional logistic regression was employed to asses the influence of occupational stress and negative life events on LBW.

RESULTSCompared with those with low technical skill utilization and low job decision, mothers with high technical skill utilization (OR=0.62; 95% CI=0.43 approximately 0.91) and high job decision (OR=0.67; 95% CI=0.46 approximately 0.97) significantly decreased the risk of laboring LBW. Compared with those not exposed to negative life events, mothers with negative life event score being=3 in the middle three months of pregnancy (OR=18.85; 95% CI=1.58 approximately 225.02), with negative life event score being 1 in the later three months of pregnancy (OR=2.67; 95% CI=1.14 approximately 6.28), with negative life event score being 2 (OR=2.80; 95% CI=1.04 approximately 7.52) and=3 in the whole time of pregnancy (OR=2.94; 95% CI=1.22 approximately 7.09) were the risk factors of LBW.

CONCLUSIONNegative life events might affect LBW and negative life events occurring in the different term of pregnancy impact LBW differently.

Adult ; Burnout, Professional ; Case-Control Studies ; Female ; Humans ; Infant, Low Birth Weight ; Infant, Newborn ; Life Change Events ; Logistic Models ; Male ; Pregnancy ; Young Adult

OBJECTIVETo investigate the change of early serum TNF-alpha and IL-6 levels in acute cerebral infarction and its significances.

METHODSSerum TNF-alpha and IL-6 levels in 30 health subjects and 35 patients with acute cerebral infarction (ACI) within 6 hours of onset were measured by enzyme linked immunosorbent assay (ELISA); neurological deficits scores (NDS) in all cases were determined, and Spearman test was used for correlation.

RESULTSThe serum levels of TNF-alpha and IL-6 in ACI group were markedly higher than those in health subjects and there was a positive correlation of TNF-alpha and IL-6 levels with 6 h NDS (rs=0.89 and 0.93, P<0.001) and with NDS progression (rs=0.90 and 0.91, P<0.001). Early serum TNF-alpha and IL-6 levels in progressive cerebral infarction (PCI) group were evidently higher than those in stable cerebral infarction (SCI)[(49.56+/-12.12) pg/L compared with (24.30+/-7.4) ng/L and (39.76+/-7.88) ng/L compared with (20.78+/-6.28) ng/L, respectively, P<0.01)].

CONCLUSIONThe early serum levels of TNF-alpha and IL-6 in ACI markedly increase and are closely correlated with disease severity; which may be of value in PCI risk evaluation.

Acute Disease ; Adult ; Aged ; Biomarkers ; blood ; Cerebral Infarction ; blood ; Early Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interleukin-6 ; blood ; Male ; Middle Aged ; Time Factors ; Tumor Necrosis Factor-alpha ; blood

Objective To explore the influence of occupational stress and negative life events that occur during pregnancy on different types of low birth weight (LBW). Methods 438 singleton LBW infants (birth weight of less than 2500 g and their pregnancy term from 28 to 42 weeks) were selected as case group, and they were further divided into symmetric LBW infants (337 cases) and asymmetric LBW infants (101 cases). According to situation of each LBW infant, a singleton with full term and normal birth weight was selected as control group matched by sex, pregnancy term, time during delivery and types of hospital.All of their mothers were inquired by well trained investigators on their socio-demographic characteristics, occupational stress, and negative life events that occurred in different pregnancy term. After controlling for mother's age, occupation,education level and family income, multinomial logistic regression was employed to asses the influence of occupational stress and negative life events on symmetric LBW and asymmetric LBW. Results Compared with those using low technical skills, mothers with high technical skill utilization significantly decreased the risk of laboring both symmetric LBW ( OR = 0.69, 95 ％ CI:0.49-0.98) and asymmetric LBW (OR = 0.53,95％CI: 0.31-0.89). Compared with those without exposure to negative life events, mothers with negative life event score ≥ 3 in the whole duration of pregnancy had significantly increased the risk of delivering symmetric LBW ( OR = 2.30, 95％ CI : 1.08-4.88), mothers with negative life event score ≥3 in the middle three months of pregnancy, ≥3 in the last three months of pregnancy, = 2 and ≥3 in the whole duration of pregnancy had significantly increased the risk of delivering asymmetric LBW, and their OR (95 ％ CI ) was 8.85 (1.97-39.68), 3.80 ( 1.40-10.29 ),3.58(1.33-9.66) and 3.48 (1.32-9.13), respectively. Conclusion Occupational stress and negative life events might produce different influence on symmetric LBW and negative life events that occurr in the different terms of pregnancy had different impact on symmetric LBW and asymmetric LBW.

OBJECTIVE: To investigate the effect of overexpression of glycosylphosphatidyl-inositol-specific phospholipase D (GPI-PLD) on the biological character of hepatocellular carcinoma cell line HepG2. METHODS: The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/ GPI-PLD was transiently transfected into HepG2 cell by lipid-media transfection. The untransfected HepG2 and HepG2 transfected with pcDNA3.1(+) were used as controls. After screening with G418, the single clone was obtained. The expression level of GPI-PLD mRNA in HepG2 was identified by reverse transcription polymerase chain reaction (RT-PCR). GPI-PLD activities were analyzed quantitatively by triton-X-114 partition with GPI anchored placental alkaline phosphatase (PLAP) as a substrate. Cell count was used to detect the proliferation of the 3 groups, and complement dependent cytotoxicity (CDC) effects were observed by the staining of trypan blue. Apoptosis cells were analyzed by flow cytometry. Carcinoembryonic antigen (CEA)was detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with HepG2 and pcDNA3.1(+)/HepG2 cell, the levels of GPI-PLD activities and its mRNA from pcDNA3.1(+)/GPI-PLD/HepG2 were increased with almost 2 to 5 times,respectively. The GPI anchored PLAP and CEA released into the medium by GPI-PLD, and the rate of CDC killing on the cells were significantly increased. However, the proliferative capacity was obviously decreased, and the typical apoptosis cells were presented in positive clones and its apoptosis rates were increased significantly. CONCLUSION The stable cell line with overexpression of GPI-PLD has been constructed. The overexpression of GPI-PLD in these cells increases the sensitivity of these cells to CDC killing and impairs the proliferative capacity of cells, and promotes the apoptosis.
Apoptosis ; genetics ; Carcinoma, Hepatocellular ; genetics ; pathology ; Complement Activation ; genetics ; Cytotoxicity, Immunologic ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; pathology ; Phospholipase D ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

Objective: To explore the expression and prognostic significance of miR-223 in patients with mantle cell lymphoma (MCL) and to investigate the possible mechanism. Methods: Twenty-one newly diagnosed MCL patients with bone marrow involvement were enrolled in the present study, 20 healthy donors as normal control. The expression level of miR-223 and SOX11 mRNA was determined by RQ-PCR. CCK-8 and flow cytometer assays were used to analyze cell proliferation, cell cycle and apoptosis of the constructed miR-223 overexpressing MCL cell line, Granta519 cells. SOX11 protein expression level was determined by Western blot. The target gene of miR-223 was confirmed by dual luciferase reporter assay. Results: ①Of the 21 newly diagnosed MCL patients, 15 were male and 6 female, the median age was 58 (37-72) years. The expression level of miR-223 was significantly down regulated in MCL patients compared with that of healthy donors (14.7±10.5 vs 1 244.1±1 935.2, P<0.001). The lower expression of miR-223 was inversely correlated with high-risk mantle international prognostic index (P=0.001), elevated LDH (P=0.001), ECOG score ≥2 (P=0.035). ②Using the median relative expression level of miR-223 as the cutoff value, 21 MCL patients were divided into high-expression group (n=10) and low-expression group (n=11) and found that the high-expression group had a significantly superior OS (median OS: 36 vs 12 months, P=0.021). ③In vitro results showed that compared with the control group, the proliferation of miR-223 overexpressed Granta519 cells was inhibited (the most significant reduction on 96h, P<0.001), manifested by lower proportion of cells in G2/M phase (P<0.001) and increased apoptosis (P<0.001), and the expression level of SOX11 protein in Granta519 cells was significantly lower than that of the control group. ④miR-223 could inhibited the 3' untranslated region of SOX11, and the expression level of miR-223 was significantly negatively correlated with mRNA level of SOX11 in MCL patients (r=-0.81, P<0.001). Conclusions: The expression of miR-223 was repressed in MCL and was associated with poor clinical outcomes, which may be probably attributed to its direct targeting SOX11.
Adult ; Aged ; Female ; Humans ; Lymphoma, Mantle-Cell ; Male ; MicroRNAs ; Middle Aged ; Prognosis ; RNA, Messenger ; SOXC Transcription Factors

OBJECTIVETo screen out effective lung cancer associated antigens for early diagnosis in order to improve the level of early diagnosis.

METHODSA T7 phage display cDNA library of human early lung cancer was developed. And then differential phage clones were picked out to be sequenced and bioinformatically analyzed. With the 8 screened differential phage clones a lung cancer associated antigen microarray was established to evaluate the single or combined roles of all the selected antigens in the diagnosis of lung cancer by the reaction of the antigens plus serum from normal subjects and patients with lung cancer, respectively.

RESULTSThe titer of the constructed cDNA library was 3.71×10 (6); pfu/ml and the number of phage was 1.11×10 (6); pfu, with a recombination rate of cDNA library over 90%. Nine differential phage clones were initially screened out, but the genes of two antigens (A42 and A83) were found the same. Bioinformatics analyses showed that the genes of the 8 antigens were known before and they were all proven to be related with tumor except A64. The positive reaction rates of the 8 antigens with serum from lung cancer patients were significantly higher than that with serum from normal subjects (Ps<0.05). When keeping specificity no less than 60%, the sensitivity of each antigen in predicting lung cancer alone was under 70% and the areas under curve (AUC) of the antigens were all under 0.8. However, when all the antigens were combined to detect lung cancer, the sensitivity and specificity was 90.8% and 94.1%, respectively, and AUC reached up to 0.969.

CONCLUSIONA T7 phage display cDNA library with a good quality of capacity, recombination rate and representativeness of human early lung cancer was successfully developed, and 8 lung cancer associated antigens were screened out. A combination of the 8 antigens can greatly improve their value to diagnose lung cancer with a higher sensitivity and specificity (both above 90%)．

Antigens, Neoplasm ; genetics ; Early Detection of Cancer ; methods ; Gene Library ; Humans ; Lung Neoplasms ; diagnosis ; genetics ; Sensitivity and Specificity