Objective:To study the effect of TNF-? on the activation and fungicidal activity of mouse peritoneal exudate cells.Methods:The mouse peritoneal exudate cells and macrophage were stimulated with IL-12、IL-18、IFN-?、TNF-? or monoclonal antibody.Cytokines and nitric oxide(NO) levels in the medium supernatant were determined with ELISA and Griess method respectively.Detected the numbers of viable Cryptococcus neoformans in macrophage.Results:Combined with IL-12 and IL-18 synergistically induced the production of TNF-? by peritoneal exudate cells and this effect can be inhibited by neutralizing anti-IFN-? mAb.Combined with IFN-? and TNF-? synergistically induced the production of NO by macrophage and potentiate its activity against Cryptococcus neoformans.Conclusion:The result suggests that the TNF-? plays an important role in enhancement of the fungicidal activity of macrophages. [

AIM:To investigate the effect of dexamethasone-treated dendritic cells(DCs)on Th2 cytokine production from autologous T cells in asthmatic patients and explore the mechanisms by studying the effect of dexamethasone on differentiation,maturation and function of DCs from patients with asthma.METHODS:Human peripheral blood monocyte-derived DCs generated from asthmatic patients and healthy subjects were cultured in the absence or presence of dexamethasone.The phenotypic characterization of DCs was analyzed by flow cytometry.The mature DCs were harvested,washed,and then cocultured in vitro with autologous T cells purified by a nylon cotton column.The DC-T coculture supernatants were collected after 72 h incubation and analyzed for levels of IL-5 and IFN-? by ELISA.RESULTS:The concentrations of IL-5 in the culture supernatants of DC-T coculture were significantly up-regulated in patients with asthma compared with that in healthy controls [(145.13?89.76)ng/L vs(50.28?22.37)ng/L,P0.05)].There were significantly decreased levels of IL-5 by autologous T cells primed by dexamethasone-treated mature DCs from asthmatic patients [(45.39?19.61)ng/L vs(145.13?89.76)ng/L,P0.05).IFN-? production was decreased by autologous T cells primed by dexamethasone-treated mature DCs from both asthmatic patients and healthy controls [asthma group:(40.21?22.89)ng/L vs(197.58?76.32)ng/L,P

Objective:To explore the influence of disease severity and effectiveness of antituberculosis treatment on helper T-cells in peripheral blood from patients with pulmonary tuberculosis.Methods:Th1 cells and Th2 cells of peripheral blood of 62 pulmonary TB patients and 30 controls were marked by intracellular cytokine(INF-? and IL-4 representative of Th1 and Th2 subsets respectively) and counted by Flow Cytometry.The levels of Th1 and Th2 cell count from patients with different disease severities radiologically before antituberculosis therapy were compared and their correlation with effectiveness of different antituberculosis treatment radiologically was analyzed either.Results:The baseline levels of Th1 cell count and Th2 cell count in pulmonary tuberculosis group before antituberculosis therapy were significantly lower than those in the control group(P0.05,t test).The levels of Th1 cell count in the stable group were still significantly lower than those in the control group(P

Objective To assess antifungal activity of aspirin and fluconazole administered alone or in combination against Candida albicans biofilms in vitro,and to evaluate the combined effect of these two drugs.Methods The MIC50 of aspirin and fluconzole against biofilm-associated adherent cells were determined respectively,then the efficacy of combinations of aspirin and fluconazole were evaluated by calculating the fractional inhibitory concentration (FIC) index.The influence of aspirin on the mRNA expression of gene ALS3,HWP1 in biofilm cells were analyzed by fluorescent quantitative PCR assay.Results The MIC50 of aspirin for ATCC64550,clinical strains 14215,15346,15538,16335 were ＞ 1440 mg/L,＞ 1440 mg/L,1440 mg/L,720 mg/L and 1440 mg/L respectively,the MIC50 of fluconazole for biofilms cells of all the strains were ＞ 64 mg/L.Aspirin did not enhance the antifungal effect of fluconazole against biofilm formed by ATCC64550,but synergistic and additive effects were found for the combination of aspirin and fluconazole to block the biofilm formation by clinical isolates (FIC index =0.75,0.5,0.75,0.75).Quantitative real-time PCR analysis showed aspirin could reduce the transcript level of ALS3 and HWP1.Conclusion Aspirin could inhibit C.albicans biofilm formation; it may increase the sensitivity of biofilm cells of C.albicans to fluconazole.

Objective To provide a quantitative summary estimate on the efficacy and safety measures of the combination therapy.Methods We searched databases(Medline and Embase)from January 1997 to December 2009 using‘ Fluticasone and salmerterol' or‘ Seretide' or‘ Advair' or‘ budesonide/formoterol' or‘ Symbicort Turbuhaler' in combination with ‘ Randomised controlled trial'.The databases of GlaxoSmithKline Clinical Trial Register and Cochrane Controlled Trials Register,or relevant original studies and review articles were approached for additional studies or details of all relevant studies.Results Morning peak expiratory flow(PEF),evening PEF and clinic FEV1 were 17.59L/min,14.95L/min and 0.08L/min higher with combination therpy than with increasing ICSs by two fold or more at endpoint,respectively.The risk of asthmatic exacerbation was reduced in the combination therpy group compared with the increased doses of ICS groups,with OR being 0.61(95 ％ CI 0.53 ～ 0.70,P ＜ 0.01).Significantly increases in the percent of symptom-free days 6.30(95％ CI 3.52 ～9.10),asthma-control days 9.49(95％ CI 4.74 ～14.25)and relieve-free days 6.59(95％ CI 6.10 ～ 7.08)were observed in patients treated with ICS/LABA compared with increased doses of ICSs at endpoint.Reduction in reliever medication use(inhalations/day)0.22(95 ％ CI 0.11 ～0.33)with significant difference was also observed at endpoint.Conclusion Combination products of ICS/LABA were more effective than a high dose of ICSs in improving lung function,reducing asthmatic exacerbation and use of reliever medication and improving control of asthma.

Objective To investgate role of TLR2 in the activation, the innate immune and inflammation of human platelets. Methods Human washed platelets were separated from healthy people(n=5) and were stimulated with different concentrations(1μg/ml, 5μg/ml, 10μg/ml) of TLR2 agonistPam3CSK4(a synthetic bacterial lipoproteins). Then the platelet aggregation rate, the expression of CD62p and TLR2 on the platelet surface were measured. Results The platelet aggregation rate were (28.32±5.67)%, (52.56±8.54)% and (76.24±11.23)%, respectively, at concentration of 1μg/ml, 5μg/mland 10μg/ml of Pam3CSK4, more than (12.83±2.43)% at 0μg/ml of it. In addition, the expression of CD62p were (18.45±2.66)%, (22.45±2.04)%, (29.53±4.08)%, respectively at above concentration of Pam3 CSK4, more than (11.20±1.67)% of CD62p at control group(P<0.01). The expression of TLR2 was not significantly increased at a lower concentration of Pam3CSK4(1μg/ml) with (16.85±6.10)% compared with(10.81±3.99)% at the control group. However, it were (21.15±9.90)% and (22.52±9.26)%, respectively, at a higher concentration(5μg/ml, 10μg/ml)more than(10.81±3.99)% at the control group(P<0.05). Conclusion Pam3CSK4 induce aggregation, activation and the up-regulation of TLR2 of platelet by stimulating TLR2 receptor of it. Thereafter, TLR2 play an important role in the innate immune of platelet.

[Objective] To reduce misdiagnosis and underdiagnosis rate of pulmonary embolism,the prediction of the revised Geneva score and Wells score for pulmonary embolism were compared and analyzed by receiver operating characteristic curves.[Methods] Sixty-five cases with suspected pulmonary embolism (PE) were collected in the Third Affiliated Hospital of SUN Yat-sen University from January 1998 to October 2008.Of which 44 cases with PE were clinically confirmed.Relevant clinical data were recorded,summarized and the analysis variables were input to SPSS11.0 for statistical analysis.ROC curves was used to evaluate the probability of PE predicted by the Wells and the revised Geneva scores.[Results] Twenty-four patients had a low clinical probability of PE (Wells score ＜ 2 points ),of which 8 (33.3%) had proven PE.The prevalence of PE was 87.2% in the 39 patients with intermediate probability (2-6 points) and 100% in the 2 patients with high probability (＞ 6 points) (P ＝ 0.000).The confirmed PE was 22.2% in the 18 patients with a low probability (Geneva score 0-3 points),82.1% (32/39) in intermediate probability (4-10 points),100% (8/8) in high probability (score ≥11 points) (P ＝ 0.000).The area under curve (AUC) of the ROC curve in the Wells and Geneva scores was 0.785 ± 0.060 and 0.900 ± 0.038,respectively (P ＝ 0.000).The optimal cutoff value was 2 points in the Wells score and 6.5 points in the Geneva score.The Wells score more than 2 points predicted PE with a sensitivity of 81.8% and specificity of 76.2%.The Geneva score more than 6 points predicted PE with a sensitivity of 72.7% and specificity of 100%.The comparison of the area under curve between the Wells and the Geneva score had a significant difference statistically (P ＜ 0.05).[Conclusion] The Wells score and the revised Geneva score are beneficial to predict pulmonary embolism.The revised Geneva score is roughly superior to the Wells score both in sensitivity and specificity.

Objective To investigate the lipoteichoic acid(LTA) induced apoptosis and the expression of inflammatory cytokines in human alveolar macrophage (AM) and the anti-apoptotic and anti-inflamatory effect of moxifloxacin (MXF).Methods Obtained human AM from bronchoalveolar lavage and used MTT assay to observe the effects of LTA and MXF on cell activity,optical microscope to investigate the change of the cell morphology,flow cytometry to assess cell apoptosis,RT-PCR to detect the mRNA levels of TLR2,IL-1 β,IL-8 and TNF-α,ELISA for the production of IL-8 to exam RT-PCR.Results LTA showed cytotoxicity on AM in a dose-dependent manner ( P＜0.05 ) ; MXF inhibited the effect of LTA without cytotoxicicy ( P＜0.05 ).LTA promoted apoptosis ( P＜0.05 ) and the mRNA expressions of TRL2,IL-1 β,IL-8 and TNF-α significantly in AM (P＜0.05),the peaks and peak time ofthe above factors were (3.56±0.03) at 12 h,(46.63±7.06) at 6 h,(28.07±1.24) at 12 h and (2.34 ±0.50) at 3 h respectively and increased the release of IL-8 protein level at 24 h (P＜0.05).MXF inhibited the cell apoptosis and the above mRNA expression at 12h ( P＜0.05 ),and inhibited the IL-8 protein level at 24 h( P＜0.05 ).Conclusion LTA showed cytotoxicity on AM,induced AM apoptosis and increased the expression of TLR2,IL-I β,IL-8 and TNF-α of AM ; MXF could protect AM through inhibiting of the above effects and may play a key role beside bactericidal effect in gram-positive bacteria pneumonia.

AIM:Panton-Valentine leukocidin(PVL)is a pore-forming toxin secreted by Staphylococcus aureus epidemiologieally associated with the often-lethal necrotizing pneumonia.Until now,the mechanisms of pathogene-sis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult.In the present study，we obtain recombinant staphylococcal F and S components of the Panton-Valentine leukocidin by gene engineering and evaluate its biological activity in vitro,which provides an experimental basis for the further studies of its biological func-tion and its toxicity in pneumonia.METHODS:The full-length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by hiSh-fidelity PCR was cloned into prokaryotic expression vector pET22b(+),and the vector was transformed into BL21(DE3)plysS to construct a prokaryotie expression system.The integrity of the opening-reading frame of each construct was verified by DNA sequencing.The recombinant PVL(rPVL)was induced by1.0 mmol/L IPTG.The expressed products were identified by SDS-PAGE and the fusion proteins(6His-LukS-PV and 6His-LukF-PV)were purified from lysates of transfeeted E.coli cells by affinity chromatography on nitrilotriacetic acid columns.The eytolytie activity was tested by incubation of rPVL with human polymorphonuclear neutrophils(PMNs)in vitro.RESULTS:The nueleotide sequence of the cloned PVL gene was the same as that of reported in GenBank.E coli BL21(DE3)plysS containing recombinant vectors grow at 37℃causes some proteins to accumulate as inclusion bodies.while incubation at 30℃led to a significant amount of soluble active proteins which accounted for about 31.7％ of the total bacterial protein.The relative molecular weight showed on SDS-PAGE profile was consistent with the expected value which the LukS-PV protein was about 34 kD.and the LukF-PV protein was about 35 kD.The purified rPVL was obtained and its cytolytic activity to PMNs was demonstrated.CONCLUSION:The genes of 1ukS-PV and lukF-PV are successfully cloned into plasmid pET22b(+)and expressed in E coli respectively.which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.

Objective To study influencing factor of mechanical ventilation (MV) time in patients withchronic obstructive pulmonary disease (COPD). Method Totally 128 patients with COPD and respiratory failurerequiting ventilation support were retrespectively investigated. The clinical characteristics of patients before and af-ter intuhation during the respiratory intensive care unit (RICU) stay were recorded and analyzed, t- test, X2 testand stepwise logistic regression analysis were used for statistical analysis. Results In the 128 patients, 61%,20%, and 9% of the patients required MV longer than 7, 14 and 21 days, respectively. There were no significantdifferences in the history of COPD, smoking, lung function, and complications between patients with MV>7 d, 14d, 21 d and patients with MV<7 d, 14 d, 21 d. Multiple regression analysis showed APACHE Ⅱ score (OR:2.3; 95% Ca: 1.2-5.7, P = 0.02) was an independent factor for MV > 7 days, while shock (OR: 0.7;95% CI: 1.0-1.9, P = 0.04) and decreased serum albumin (OR: 0.4, 95% CI: 0.2-0.8, P = 0.003)were an independent factors for MV > 21 days. The ventilator-associated pneumonia (VAP) was the most impor-rant risk factor for the time of MV. Conclusions APACHE Ⅱ score, serum albumin levels, hock and VAP arethe main factors affecting MV time in patients with COPD.