Objective To explore the effect and preliminary mechanism of the plant-derived immunostimulatory molecule,ophiopogonin,on enhancing the immune response of a subunit vaccine with the receptor-binding domain(RBD)of coronavirus spike protein as the antigen.Methods CCK-8 assay was used to determine the cytotoxicity of ophiopogonin D'(OPD')on bone marrow-derived dendritic cells(BMDCs).Female Balb/c mice were randomly divided into RBD,RBD/OPD',RBD/Alum,and control groups.The immunization dose was 5 μg of antigen per mouse and 100 μg of adjuvant per mouse,and immunization was carried out according to the intramuscular injection immunization procedure on days 0,21,and 42.The titers of specific IgG and its subtype antibodies were detected by ELISA.The cytokine levels in the supernatant of splenocytes were detected using ELISA.The number of splenocytes secreting IFN-γ was detected by ELISpot.Laser confocal microscopy was employed to observe the uptake of antigen by BMDCs.The phagocytic ability of BMDCs for antigen was quantitatively analyzed by flow cytometry.The mechanism of its enhanced immune effect was preliminarily explored using transcriptomics technology combined with bioinformatics research.Results When the concentration of OPD'was less than 5 μg/mL,the survival rate of BMDCs was 100%.After a single intramuscular injection in mice,except for a slight decrease in body weight,the other biochemical indicators were within corresponding normal ranges.After intramuscular injection immunization of the vaccine,the titers of serum-specific IgG,IgG1,and IgG2a in the RBD/OPD'group were significantly higher than those in the RBD group(P<0.05).Compared with the RBD group,the RBD/OPD'group induced a high-level Th1 cell immune response of IL-1β,TNF-α,and IFN-γ(P<0.01)and had more lymphocytes secreting IFN-γ(P<0.001).Laser confocal microscopy displayed that BMDCs took up more antigens after OPD'treatment,which was further confirmed with flow cytometry in quantitative analysis on antigen uptake rate(P<0.01).Transcriptomics results indicated that there was more significant enrichment of the PPAR signaling pathway in the RBD/OPD'group than the RBD group,suggesting that OPD'may activate the PPAR signaling pathway to exert its adjuvant effect.Conclusion OPD'effectively enhances the immune response of the RBD subunit vaccine,and its action mechanism may be related to the activation of the PPAR signaling pathway.

Objective To establish a mouse model of infection with the minimum lethal dose of Vibrio vulnificus(V.vulnificus)and to evaluate the protective efficacy of inactivated whole-cell(IWC)vaccine using this model.Methods A mouse model of lethal-dose infection was established by intraperitoneal injection of different doses of V.vulnificus.Bacterial colonization in the organs was detected with tissue homogenate plating,and pathological changes in the organs were observed after tissue section staining.Flow cytometry was used to detect immune cell responses after liver tissues were digested into single-cell suspension.IWC vaccine of V.vulnificus was prepared,and the mice were immunized through different routes to observe the protective efficacy of the vaccine.Results A mouse model of infection with the minimum lethal dose at 1×106 CFU of V.vulnificus was successfully established.After infection,the bacteria were mainly colonized in the liver of mice and caused severe pathological damages.Compared with the uninfected mice,the proportion of neutrophils in the liver was significantly increased in the infected mice,whereas the proportions of B cells and T cells were correspondingly decreased(P<0.05).A single intramuscular or intraperitoneal injection of the IWC vaccine could protect the mice effectively against lethal infection of V.vulnificus in 7 d later(P<0.01),although the level of serum IgG having no significant increase.Conclusion A mouse model of lethal-dose infection with V.vulnificus is successfully established,with histopathological characteristics.The IWC vaccine of V.vulnificus rapidly mediates immune protection in this model probably independent of IgG.

Objective To explore the mechanism which drives nimbolide(NIM)in treating acute pneumonia caused by Staphylococcus aureus(S.auteus).Methods A mouse model of acute pneumonia caused by S.auteus was constructed through endotracheal intubation.After NIM treatment,the survival rate was observed,the amount of bacteria in the lung was tested by plate culture,and the expression of inflammatory cytokines in the lung tissues was detected with ELISA.After primary cultured peritoneal macrophages(PM)were infected with S.auteus,the effect of NIM on the expression of inflammatory cytokines and activation of inflammatory pathway were studied with ELISA and Western blotting,respectively.The effect of RNF114 knockdown by lentiviral shRNA infection on inflammation responses in PM was explored with ELISA and Western blotting.Results Acute infection of S.auteus in the lung could cause acute death in the mice,while NIM treatment significantly improved the survival rate and down-regulated the levels of inflammatory cytokines in the lung.However,it had no effect on the lung colonization of S.auteus in the short term.The results of in vitro experiments indicated that NIM may regulate RNF114 function to down-regulate the phosphorylation level of ERK,inhibit the activation of MAPK pathway,and thus suppress the expression of inflammatory cytokines.Conclusion NIM may inhibit the activation of MAPK pathway by regulating the function of RNF114,and thus suppress the expression of inflammatory cytokines in the lung,and finally inhibit the death of mice with acute pulmonary hyperinflammation caused by S.auteus.

Tumor has become the major reasons cause of death,and its vaccine has become the effective tracts of treatment and prevention by enhancing the immune response of patients.However,most vaccines which are recombination subunit protein antigens are poorly immunogenic and difficult to induce a robust immune response in patients with compromised immune systems,resulting in poor marketing approval.The core component of the vaccine adjuvant can greatly enhance the strength,speed and duration of the immune response,thus becoming the key to the development of an ideal tumor vaccine.Most tumor vaccines are combined with tradition adjuvant such as aluminum,MF59 and AS adjuvant,but their products and patents are monopolized by large foreign companies.We found that natural adjuvants have many unique advantages,such as good biocompatibility and biodegradability,promoting the maturation of dendritic cell and the secretion of immune cytokines,significantly enhancing the tumor vaccine immune response,etc.In this paper,the application and future development of natural polysaccharides,saponins,flavonoid and plant virus-like particles in cancer vaccines were reviewed,which may lay a solid foundation for the development of the original and innovative adjuvants with domestic independent intellectual property rights.

Objective To investigate the efficacy and safety of 6-month and 12-month oral dual-antiplatelet therapy This work was supported by the National Key Technology Research and Development Program in the Twelfth Five-year Plan of China (2011BAIl1B07) and the Military Clinical Key Technology and Development Program (2010gxjs001)(DAPT) on patients implanted with biodegradable polymer-coated and drug-eluted long stents (BP-DES).Methods In the I-LOVE-IT 2 trial,574 patients implanted with biodegradable polymer-coated and sirolimus-eluted long stent (BP-SES) (total stent length ≥50mm) were randomized to accepting either 6-month (n=270) or 12-month (n=304) DAPT.The primary endpoint of present study was 12-month target lesion failure (TLF),including cardiac death,target vessel myocardial infarction and clinically indicated target lesion revascularization (CI-TLR).The major secondary endpoint was 12-month net adverse clinical events (NACE),including all-causes of death,myocardial infarction,stroke,all revascularization (CI-TLR plus clinically indicated nontarget lesion revascularization) and bleeding.Results For the patients implanted with BP-SES of total stent length≥ 50mm,the total stent length was 73.0 ± 22.5mm and 69.8 ± 19.4mm in the 6-month DAPT group and 12-month group,respectively (P=0.07).No significant difference existed in the incidence of 12-month TLF between 6-month DAPT group and 12-month DAPT group (11.1％ vs.9.2％,P=0.47).The incidence of NACE was similar between the 2 groups (21.9％ vs.19.7％,P=0.57).The incidence of revascularization was lower in 12-month DAPT group (5.6％) than in 6-month DAPT group (11.1％,P=0.01).Furthermore,6-month landmark analysis showed that 12-month DAPT was associated with significantly lower risk of TLF (2.6％ vs.6.3％,P=0.03) at a cost of slightly increased risk of all bleeding events (1.6％ vs.0.7％,Log-rank P=0.32) between 6 and 12-months compared to 6-month DAPT.Conclusions In patients treated with BP-SES of total stent length ≥ 50mm,12-month DAPT have similar impacts on 12-month clinical outcomes except for all revascularization.However,12 months DAPT decreased the incidence of TLF and total revascularization between 6 months to 12 months after PCI.

Objective To observe the effect of intensive insulin therapy (ⅡT) on prognosis of severe chest trauma (SCT) pateints.Methods 42 consecutive patients were randomly assigned to 2 groups:ⅡT group (n =21) and the conventional insulin therapy group (CIT group,n =21).Blood glucose was maintained at the level of 4.4 -6.1mmol/L in ⅡT group,and 10.0 -11.1mmol/L in CIT group.The 2 groups were observed in terms of fatality rate during hospital stay,infection rate,the duration of mechanical ventilation,ICU stay length,pleural drainage day and count of neutrophils.Results No death or hypoglycemic reaction happened during hospitalization.Compared to CIT group,infenction rate,mechanical ventilation time,ICU stay length,and the time required for white blood cell to resume normal in ⅡT group were decreased significantly ( P ＜ 0.05 ).The difference had no statistical significance between the 2 groups in duration of pleural drainage and incidence of hypoglycemia (P ＞ 0.05).Conclusion ⅡT improves the short-term prognosis of SCT patients.

AIM: To establish a method of simultaneously determining 5 components in Muxiang Fenqi Pill(Flos Caryophylli,Radix Aucklandiae,Cortex Magnoliae Officinalis). METHODS: Five components :eugenol,(costunolide),dehydrocostuslactone,magnolol and honokiol in Muxiang Fenqi Pill were determined simultaneously by HPLC,using a Kromasil C_(18) column(250 mm?4.6 mm,5.0 ?m),acetonitrile-menthanol-water(50∶8∶42) as a mobile phase.The detection wavelength was at 210 nm. RESULTS: The relationship between the concentrations and the peak areas of eugenol,costunolide,dehydrocostuslactone,magnolol and honokiol were linear respectively.The RSD of precision,repeatability and recovery were all less than 1.5%. CONCLUSION: The method is simultaneous determination for five components in Muxiang Fenqi Pill,and can be applied to the quality control of Muxiang Fenqi Pill.