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 prepare tubeimoside Ⅲ nanoemulsion(TBMⅢ-NE)and evaluate its adjuvant effect in vaccines.Methods TBMⅢ-NE was prepared using low-energy emulsification.Dynamic light scattering was used to characterize the particle size and polydispersity index of the obtained TBMⅢ-NE,and transmission electron microscopy(TEM)was employed to observe the morphology.CCK-8 assay was utilized to determine the cytotoxicity of TBMⅢ-NE on bone marrow-derived dendritic cells(BMDCs).The in vitro safety of TBMⅢ-NE was evaluated using a hemolysis assay.The ability of TBMⅢ-NE to promote the phagocytosis of antigens by DC2.4 cells was observed using confocal laser microscopy.After co-incubation of TBMⅢ-NE with BMDCs,the expression levels of CD40,CD86,MHC-Ⅰ,and CCR7 on the surface of BMDCs were detected using flow cytometry,and the levels of cytokines in the supernatant of BMDCs were measured using enzyme-linked immunosorbent assay(ELISA).After female BALB/c mice were immunized with the SARS-CoV-2 antigen RBD in combination with TBMⅢ-NE,ELISA was conducted to determine the serum levels of specific IgG,IgG2a,and IgG1 antibodies.The number of specific IFN-γ-secreting cells in mouse splenocytes was detected using enzyme-linked immunospot(ELISpot)assay.Results The prepared blank nanoemulsion(BNE)and TBMⅢ-NE were in a particle size of 25.46 and 25.89 nm,and a polydispersity index of 0.214 and 0.125,respectively.TEM displayed that TBMⅢ-NE was in uniform sphere and well dispersed.When the TBMⅢ-NE adjuvant was diluted by 400-fold,the survival rate of BMDCs was approximately 86%.Compared with free TBMⅢ,the hemolytic toxicity of TBMⅢ-NE was significantly reduced(P<0.01).TBMⅢ-NE promoted the phagocytosis of antigens by DC2.4 cells and significantly increased the expression of CCR7 on the surface of BMDCs(P<0.05),indicating its potential to promote more dendritic cells to effectively migrate to lymph nodes.TBMⅢ-NE also promoted the expression of IL-6 and IL-1β in the supernatant of BMDCs(P<0.05).When combined with RBD,TBMⅢ-NE significantly increased the levels of specific IgG,IgG2a,and IgG1 antibodies in mouse serum(P<0.01)and promoted the secretion of specific IFN-γ in splenocytes(P<0.01),indicating that TBM Ⅲ-NE could enhance specific cellular immune responses.Conclusion A stable and highly effective TBMⅢ-NE that can induce humoral and cellular immune responses is successfully prepared.

Objective To evaluate the stability,safety and immune enhancement and anti-tumor effects of Wilms'tumor gene 1(WT1)peptide combined with AddaVaxTM emulsion vaccine for acute myeloid leukemia.Methods The stability of WT1 peptide in the adjuvant vaccine was evaluated using MALDI-TOF-MS time-of-flight mass spectrometry.Female C57BL/6 mice were randomly divided into PBS group,WT1 peptide group,and WT1 peptide+AddaVaxTMemulsion adjuvant vaccine group.The immunization was performed at a dose of 50 μg/mouse for antigen and 50 μg/mouse for adjuvant,with intramuscular injection on days 0,14,and 28.HE staining was used to assess the toxicity of intramuscular vaccination on mouse organ tissues.Cytokine levels were detected by ELISA,and the number of IFN-γ-secreting splenocytes was measured by ELISpot.Flow cytometry was employed to detect the maturation of bone marrow-derived dendritic cells(BMDCs)promoted by the vaccine in vitro and the promotion for lymphocyte activation,and H-2Db WT1 tetramer was utilized to detect the proportion of specific CD8+T cells.After establishing a mouse leukemia tumor model using the C1498-mWT1 stable cell line,the anti-tumor effects of the vaccine for prevention and treatment were evaluated.Results The WT1 peptide stably existed in the vaccine without causing significant organ tissue changes in mice after intramuscular injection.Compared to the mice immunized with WT1 aqueous solution,the mice after intramuscular injection of the WT1 peptide emulsion adjuvant vaccine showed stronger immune responses of Th1 cells,including IFN-γ and TNF-α,as well as Th17 cells of IL-17A(P＜0.05),and the mice had not only promoted number of IFN-γ secreting splenocytes(P＜0.01)but also enhanced maturation of BMDCs,as indicated by an increase in the proportions of CD40+/CD11c+and CD86+CD80+/CD11c+ cells(P＜0.05).Additionally,there were increases in both the proportion of CD4+/CD3+T and CD69+/CD8+T cells(P＜0.05)and the proportion of specific CD8+T cells(P＜0.05).In the anti-tumor effect study using the C1498-mWT1 mouse model,the median survival time of the WT1+AddaVaxTM group was extended by 6 d compared to the WT1 aqueous solution group.At day 50,the survival rate of mice in the WT1+AddaVaxTM group was still 28.5％,while all mice in the other groups had died(P＜0.05).Conclusion The vaccine with the WT1 peptide and AddaVaxTM emulsion adjuvant exhibits good immunological and anti-tumor effects.

Objective To investigate the antibacterial effect and its preliminary mechanism of lavender essential oil on multi-drug resistant Acinetobacter baumannii.Methods Micro-dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)of lavender essential oil against multi-drug resistant Acinetobacter baumannii,and bactericidal kinetic study was employed to determine the onset and maintenance time of lavender essential oil.Meanwhile,the promoting and therapeutic effects of lavender essential oil on wound healing were observed in a mouse model of infection.Subsequently,crystal violet staining was used to determine the inhibition and clearance of multi-drug resistant Acinetobacter baumannii biofilm by lavender essential oil,and laser confocal microscopy was utilized to observe the survival of bacteria in biofilms.NanoDrop instrument was utilized to quantify the leakage of bacterial DNA nucleic acid and protein after intervention with 3 and 6 mg/mL lavender essential oil,and the leakage of bacterial potassium ion was measured by potassium ion test kit.Proteomics technology combined with bio-informatics were applied to explore the action mechanism of lavender essential oil against multi-drug resistant Acinetobacter baumannii.Results The MIC and MBC of lavender essential oil were both 6 mg/mL,which could kill almost all multi-drug resistant Acinetobacter baumannii at the time point of 120 min,and showed an obvious dose-and time-dependent manner.The overall animal model evaluation showed that both 3 and 6 mg/mL lavender essential oil could promote wound healing,and the curative effect was obvious.Further studies confirmed that 3 mg/mL lavender essential oil had a certain biofilm inhibitory effect on multi-drug resistant Acinetobacter baumannii,and 6 mg/mL also had a certain biofilm clearance effect under the same conditions.Meanwhile,when incubated at 37℃ for 1 h,the dose of 3 mg/mL could increase the leakage of DNA nucleic acid and protein,and significantly promote the efflux of potassium ions.Proteomic analysis suggested that the antibacterial effect of lavender essential oil may be related to affecting the oxidorereductase activity and cell metabolic process of multi-drug resistant Acinetobacter baumannii,and interfering with the biosynthesis of cell wall/membrane/envelope and other structures.Conclusion Lavender essential oil at 3 mg/mL can play an antibacterial effect against multi-drug resistant Acinetobacter baumannii,and its mechanism may be related to the destruction of bacterial biofilm and interference with bacterial metabolism.

Objective To design and develop an air purification system for intensive care ambulances,which can effectively purify the air in the compartment and sterilize the microbes in the air.Methods By integrating titanium dioxide photocatalysts with air filtration materials,an air purification system was designed that was capable of effective sterilization of microbes in the air via photocatalytic oxidation and decomposition,and of effective filtration of particles in the air through HEPA filters.The protection effect was evaluated by air filtration and sterilization tests in accordance with the related national standards.Results The air cleanliness could reach the 100 000 level with bacteria counts no more than 1 CFU per pan for 15 minutes in the ambulance compartment when the air purification system worked normally,which could meet the demands of environmental hygienic standards for intensive care units.Conclusion The air purification system can effectively purify and sterilize the air in the compartment of an intensive care ambulance.The effects of air purification meet the requirements of related hygienic standards,which can satisfy the demands of treatment and intensive care for the victims in the ambulance.

Objective:To study the effect of chemokines CCL20 and CCL22 combined with skin-induced Treg on survival time of grafted skin.Methods: Skin grafting mice were divided into four groups, three mice per group, namely Treg group, Treg+CCL20 group,Treg+CCL22 group and control group.C57BL/6 mice were used as donor and BALB/c as acceptor, and the Treg cells were isolated from the mice induced by skin allograft.After skin grafted,CCL20 and CCL22 were subcutaneous injection every day,which lasted for 10 day.Survival time of skin in each group were observed and recorded.The Treg colonzation experiments were performed as follows.We firstly isolated Treg with Magnetic cell sorting system( MACS) and then labled them with 99 Tcm.After that we intravenously injected them into the mice.3 hours later,the mice were sacrifced and the radioactivity of organs were detected by GC-2016γradioim-munoassay counter.Results:①After Treg treated the survival time of skin grafted in antigen-induced Treg group was signifiantly longer than control group,when treg were cooperated with CCL20 and CCL22,the skin grafted showed more longer survival time than Treg and control groups( P<0.001 ).②After injection of induced Treg, Treg in autologous and allogeneic skin grafts goups were mainly distributed in autologous and allogeneic skin,accounting for 60% and 98% respectively.When cooperated with CCL20 or CCL22,the Treg were mainly distributed in liver.Conclusion:Chemokines CCL20 and CCL22 synergistically improved the effects of skin antigen induced Treg on survival time of skin graft,which probably related with the Treg colonization into the liver.

Objective:To investigate the influence of acrylamide ( ACR) on adult neurogenesis and expression of GSK 3βin mouse.Methods:Method Adult male Kunming mice were used and divided into two groups:control group and experimental poisoning groups,that were exposured to acrylamide by intraperitoneal injection.Brdu labeling and immunohistochemistry were used to investigate the proliferation of adult neural stem cells in the subgranular zone ( SGZ).BrdU/NeuN/GFAP triple labeling to investigate the survival and differentiation of newly generated cells.Detecting GSK3βexpression and distribution in Neuro-2a cells,the expression of GSK3βwas examined by using Western blot.Results:Compared with control mice ,lower number of BrdU-positive cells and less differentiated into neurons in ACR mice.Less neural stem cells survived ,but more glia cells were generated in the subgranular zone of acrylamide mice.Moreover,higher phosphorylated GSK 3β( Ser9 ) were detected in Neuro-2a cells and mouse dentate gyrus in ACR mice respectively .Conclusion:These results suggested that acrylamide inhibits neural stem cells proliferation and influences the survival and differentiation of newly generated cells.Acrylamide inhibits neurogenesis maybe through GSK 3βsignaling pathway.

Objective To explore the CT appearance of thoracic lymphonodus in AIDS patients with immune reconstitution in-flammatory syndrome(IRIS)after highly active antiretroviral therapy (HAART).Methods The data of thoracic CT in 24 AIDS pa-tients after HAART with enlarged thoracic lymphonodus in IRIS were collected,and the chest CT appearance was analyzed.Results Of the 24 cases of AIDS patients with IRIS after HAART,1 9 cases were complicated with pulmonary tuberculosis,which includ-ed 5 cases with cervical tuberculous lymphadenitis,3 cases were co-infected with bacterium and fungi,1 case was infected by penicil-lium marneffei,1 case by pneumocystis carinii.The enlarged thoracic lymphonodus were primarily located in 4R region(20/24), secondly in 2R region(1 1/24)and 4L region(1 1/24),in which the density was uniform or non-uniform,edge clear or unclear,some parts of lymphonodus were fused together but not calcified.The minor axis of enlarged lymphonodus was 1 1.0-25.0 mm except X region,enhanced uniformly in 2 cases.Obstructive pneumonia and pulmonary consolidation were found in 2 cases with enlarged lym-phonodus,which were located in 10R region.Pleural effusion was found in 13 cases with greatest depth of about 22 mm,pericardial effusion was found in 5 cases with greatest depth of about 24 mm.Conclusion The enlarged thoracic lymphonodus in AIDS patients with IRIS affer HAART are mainly involved in the region of 4R,2R and 4L,with or without pleural effusion and pericardial effusion.

OBJECTIVE: To investigate the sleep monitoring feature of the MSMS in elderly patients with OSAHS. METHOD: One hundred and ninety patients diagnosed with OSAHS were divided into elderly group and non elderly group according to age, then the results of MSMS were analyzed. RESULT: Majority elderly patients were with mild to moderate OSAHS. The nocturnal mean blood oxygen and the lowest oxygen were higher than non elderly group, coupled with higher percentage of the total oxygen saturation < 90% monitoring time (TS90). There was no significant difference in sleep structure between two groups, but the total sleep time of elderly group is lower than the non elderly group, the difference is statistically significant. CONCLUSION The elderly patients with OSAHS were less severe in nature, but the nocturnal hypoxia last longer in the elderly group. There is no significant difference in the sleep structure between the two groups. But the total sleep time decrease in elderly group. With smaller interference, the MSMS is closer to the natural sleep stustus of the subjects.
Aged ; Humans ; Hypoxia ; diagnosis ; Oxygen ; physiology ; Polysomnography ; Sleep ; Sleep Apnea, Obstructive ; diagnosis