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 analyze the changes in the expression of Toll-like receptors 4 (TLR4) and 7 on the surface of murine bone marrow-derived dendritic cells following dengue virus type 2 (DEN2) infection.Methods DEN2 NGC strain was infected into BALB/c suckling mice through intracranial injection and injected into C6/36 cells to induce the in vivo and in vitro proliferation of DEN2, respectively.RT-PCR was performed to identify DEN2 RNA.Reed-Muench method was used to determine the 50% tissue culture infective dose (TCID50) of DEN2.Dendritic cells (DCs) were prepared by stimulating bone marrow cells isolated form C57BL/6 mice with IL-4 and GM-CSF and then identified by flow cytometry.The prepared murine bone marrow-derived DCs were infected with DEN2 and observed with direct immunofluorescence assay.Dynamic changes in the expression of CD11c, CD86 and I-A/I-E molecules on DCs after DEN2 infection were detected by flow cytometry.Levels of DEN2 RNA and the expression of TLR4 and TLR7 at mRNA level were dynamically detected by real-time quantitative PCR.Results The TCID50 of DEN2 to C6/36 cells was 10-5.8.Murine bone marrow-derived DCs were acquired with a purity of 70% and could be infected with DEN2 in vitro.The percentages of CD86 and I-A/I-E molecules on the surface of DCs infected with 1×105 TCID50 of DEN2 were statistically different from those of the negative control group.Neither of the two groups showed a significant difference in the percentages of membrane molecules over time.However, the percentages of membrane molecules on DCs increased with increasing viral load.Compared with the negative control group, the levels of DEN2 RNA in infection groups were increased with increasing virus load, while the expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level was decreased with increasing viral load.Conclusion DEN2 infection promotes the maturation of DCs.Expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level decreases with increasing viral load, which suggests that TLR4 and TLR7 are closely related to viral infection and play a certain role in the pathogenesis of DEN infection.

Vitamine E in peanut oil was determined by means of f Juorometric method (Ex = 285nm, Em=324nm) and TLC scanning method (? = 292nm). Both methods include saponification of the oil, isolation o'f the unsaponifiable matters and the respective final measurements. The result of the former method indicates the total vitamine E in terms of ?-vitamine E and the latter method may indicate their ?,?,? and 6 analogues separately. These two methods are simple, time-saving and reliable. The recovery of VE and the standard deviation of replication of fluorometric method and TLC scanning method are 99.3%, ? 0.587mg/100g sample and 103.04%, ?0.959mg/100g sample respectively. Both methods are also applicable to other foods such as fortified cakes, confectionaries, infant foods etc., but the oil in these samples must be extracted before saponification.

To study the value of high-fluorescent cell for the screening of malignant hydrothorax or ascites. The clinical diagnosis results and hydrothorax or ascites specimens of 400 outpatients registered from January to December in 2014 were collected, and high-fluorescent cells were counted with Sysmex XE-5000 blood cell analyzer, and then stained for microscopy. Receiver operating characteristic (ROC) curve was used to explore the sensitivity, speci-ficity, positive likelihood ratio and negative likelihood ratio of high-fluorescent cell for screening malignant hydrothorax or ascites. The numbers of high-fluorescent cells of the cancer patients were significantly higher than those of the non-cancer patients, with Z equal to -7.372 and P less than 0.05. The values of the area under curve (AUC), sensitivity, specificity, positive likelihood ratio and negative likelihood ratio were 0.801, 70.67%, 84.31%, 4.50 and 0.35 respective-ly. Detection of high-fluorescent may be used to screen malignant hydrothorax or ascites, and further exami-nation and follow-up have to be performed in case the number of high-fluorescent cells in the hydrothorax or ascites is not less than 68.

Clinical data of 53 patients with lower caliceal stone during August 2005 and March2007 were analyzed retrospectively.The stones were 11-35 mm in diameter.Under the guidance of X-ray.single renal tract parallel to the lower caliceal for percutaneous nephrolithotomy was established.The procedures were successful in all the patients.Fifty patients were stone free after first minimally invasive pereutaneous nephrolithotomy(MPCNL),2 were stone free following second MPCNL,1 saw residual small stones clear off spontaneously during the follow-up period.Operative time was 65-162 minutes.and blood loss was 10-200 ml.No severe complications or death occurred.MPCNL may be related with minimal invasion and fewer complications,thus provides an effective and safe way of lower caliceal stone treatment.

Objective To investigate the role of norepinephrine in the down-regulated visceral sensitivity of rats deprived of rapid eye movement(REM)sleep.Methods Twenty-four male Sprague- Dawley rats were randomly divided into 3 groups:cage-yoked rats as control(YC),rats with REM sleep deprivation(SD)and rats with yohimbine administered intraperitoneally after REM sleep deprivation(YSD).Flower pot technique was employed to make sleep deprivation model.YSD group was given vohimbine intraperitoneally at the 48th hour after REM sleep deprivation.After both SD and YSD groups had completed these processes,rats of all the three groups were given colorectal distension(CRD)and electromyogram (EMG)was recorded at the same time.The number of discharges of EMG and the threshold of Dain perception of the rats were observed to evaluate the visceral sensitivity.The thalamus,rectum and distal colon were taken after CRD;MAO-mRNA and TH-mRNA in three tissues were detected with RT-PCR.Resuits On 48th hour,the number of discharges of EMG in 10 seconds responding to CRD in group SD was significantly less than that in group YC and the threshold of pain perception in group SD was higher than that in group YC(P＜0.05).The number of discharge of EMG in group YSD was significantly more than that in group SD(P＜0.05).The expression of MAO-mRNA in group SD was lower than that in group YC(P＜0.05)and the expression of TH-mRNA in group SD was higher than that in group YC(P＜ 0.05).Conclusions The visceral sensitivity in rats is down-regulated by REM sleep deprivation,which can increase synthesis of norepinephrine.Norepinephrine can modulate visceral sensitivity.

Twenty-three patients with pain from osteoporotic vertebral fractures,aged 65-90 yr,weighing 51-78 kg,received an image intensifier-assisted nerve-root block with a 6-8 ml mixture of 0.5 ％ lidocaine,mecobalamine 0.5 mg and betamethasone sodium phosphate injection 5.26 mg in a prone or lateral position.The VAS scores before operation,at 0,1 week,1 and 3 months after operation were 8.6 ± 0.9,1.5 ± 0.7,2.8 ± 0.9,1.6 ± 0.5 and 2.5 ± 0.7,respectively.VAS scores were significantly lower at each time pint after operation than before operation (P ＜ 0.05).According to modified MacNab standard,the effectiveness of treatment was rated as excellent/good in 87％ of the patients.No complication such as bleeding,hematoma,infection,pneumothorax,hemopneumothorax,headache was found during or after operation.Selective nerve-root block is effective in the treatment of pain resulting from osteoporotic vertebral fractures in patients.

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.

Objective Membranous nephropathy ( MN) is rarely complicated by crescents.This study was to observe the clinicopathologic characteristics of MN with crescents. Methods This retrospective study included 53 cases of biopsy-proven idiopathic MN with crescents in the absence of immunologic and clinical etiologic factors and another 100 MN patients without histological crescents as controls.The clinicopathologic features, treatment response, and out-comes were analyzed and compared between the two groups of pa-tients. Results Significantly higher percentages of hypertension and decreased eGFR were observed in the MN +crescents group than in the control (47.2%vs 19.0%, P<0.05;28.3%vs 40.%, P<0.05).Circulating autoantibodies against the M-type phospholipase A2 receptor (PLA2R) were found in 66.7%(30/45) of the patients.The glomeruli exhibited a median of 4.6%(1.8%-35.3%) involvement of crescents.Compared with the controls, the MN +crescents group showed remarkably higher rates of segmental glomer-ulosclerosis lesions (16.0%vs 49.1%, P<0.05), capillary loops necrosis (0.0%vs 11.3%, P<0.05), interstitial fibrosis/tubu-lar atrophy (IFTA) (54.0%vs 86.8%, P<0.05) and afferent arterial lesions (65.0%vs 92.5%, P<0.05).No significant differ-ences were found in the outcomes of the two groups of patients. Conclusion MN with crescents is rare, and secondary MN and cres-centic glomerulonephritis should be considered.Crescentic MN usually presents with hypertension and renal dysfunction clinically and high rates of severe segmental and global glomerulosclerosis, capillary loops necrosis, and IFTA histologically.The condition has a fa-vorable short-term prognosis.

Objective:To observe the effect of different concentration HMGB1 on the secretion of TNF-αand NO from Ana-1 infected with DEN2 and virus copy.Methods:DEN2 were proliferated and identified by conventional methods.The adherence of DEN2 to Ana-1 was observed by direct immunofluorescence and RT-PCR.The level of virus mRNA were detected by qRT-PCR.The concentration of TNF-αwas detected by ELISA.The concentration of NO was detected with Griess reagent.Results:Ana-1 was able to adhered for DEN2.Compared with DEN group,the inhibition ratio(%) of the level of virus mRNA in D-HMGB1-1 group,D-HMGB1-10 group,D-HMGB1-100 group,D-HMGB1-1000 group was 41.53 ±2.12,55.30 ±1.59,74.75 ±1.12,86.35 ±1.42.Compared with DEN group,the level of TNF-αand NO decreased in D-HMGB1 groups(P<0.05).Conclusion:HMGB1 can be effectively regulated of Ana-1 secreted inflammation factor of infected with DEN2,and inhibited DEN2 replication.