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 construct an in vitro co-culture model of gastric cancer organoids and cancer-associated fibroblasts(CAFs),and investigate the role of cancer-associated fibroblasts in the proliferation and chemotherapy resistance of gastric cancer organoids.Methods Tumor tissues from 12 gastric cancer patients undergoing surgical treatment in Department of General Surgery of Second Affiliated Hospital of Army Medical University from February 2023 to March 2024 were collected to construct gastric cancer organoids using 3D culture.HE staining was used to observe the morphology,and immunohistochemical assay was employed to determine the expression of cytokeratin CK7,carcinoembryonic antigen(CEA),and proliferation marker Ki-67.After CAFs derived from the same patient were cultured,observed for their morphology under a light microscope,and detected for the phenotype by flow cytometry,the cells were co-cultured with gastric cancer organoids in a 1:1 ratio.Phase-contrast microscopy was applied to observe the growth of the organoids and analyze the number,average diameter,and total area.Then,organoids cultured alone served as the control group.After the control and co-culture groups were treated with chemotherapy drugs,5-fluorouracil and oxaliplatin,for 48 h,the viability and apoptosis of organoids were assessed with CellTiter-Glo??3D assay and CellEvent? Caspase 3/7 activity,respectively.Results Gastric cancer organoids and CAFs were successfully established from 10 gastric cancer patient-derived samples.The gastric cancer organoids exhibited morphological characteristics consistent with the corresponding primary tumors,and showed positive expression of CK7,CEA,and Ki-67.CAFs displayed typical spindle-shaped morphology and exhibited the phenotypic markers CD326-,CD45-,CD31-,α-SMA+,CD73+,CD90+,and CD105+.Compared to the organoids cultured alone,the organoids co-cultured with CAFs showed more formation of organoids,in larger average diameter,and taking larger total area(P<0.05).After the treatment of 5-fluorouracil and oxaliplatin,the half-maximal inhibitory concentration(IC50)was 10.66 and 3.26 μmol/L,respectively in the control group,while was 46.23 and 91.11 μmol/L in the co-culture group.Additionally,the number of CellEvent? Caspase 3/7 positive apoptotic cells was significantly less in the co-culture group than the control group.Conclusion Compared with individually cultured gastric cancer organoids,the co-culture model of gastric cancer organoids and CAFs better simulates the pro-tumor proliferation and drug resistance effects of in vivo tumor microenvironment.

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 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 distribution of B cells in both tumor and non-tumor tissues of gastric cancer patients,analyze their phenotypic characteristics and explore the impact on T cell proliferation.Methods Immunohistochemical staining was utilized to detect the expression of B cell surface marker CD 19 in tumor and non-tumor tissues from 33 gastric cancer patients.The expression levels of chemokine receptors and immunoglobulin molecules on B cells in both tumor and non-tumor tissues were measured using flow cytometry.Chemotaxis experiments were conducted to examine the role of the CXCL12-CXCR4 axis in B cell chemotaxis.B cells isolated and purified from both tissue types were co-cultured with autologous peripheral T cells to assess their effect on T cell proliferation.Results There were significantly more B cells infiltrated in tumor tissues than those infitrated in the non-tumor tissues of gastric cancer patients(P＜0.01),and CXCR4 was highly expressed on tumor-infiltrating B cells compared with B cells derived from non-tumor tissues(P＜0.05).The Cancer Genome Atlas(TCGA)analysis indicated that the expression level of CXCL12 in tumor tissues was positively correlated with the expression level of CD19 in gastric cancer patients(r=0.15,P＜0.01).And the expression level of CXCL12 in tumor tissues of the gastric cancer patients was also positively correlated with the number of B cells infiltrated in tumor tissues.Chemotaxis experiments confirmed that the CXCL12-CXCR4 axis was involved in promoting B cell chemotaxis(P＜0.05).Although B cells in tumor and non-tumor tissues had similar levels of IgM,IgG,and IgA expression,tumor-infiltrating B cells significantly inhibited the proliferation of T cells when compared with B cells derived from non-tumor tissues(P＜0.01).Conclusion There are more B cells infiltrated in gastric cancer tissues,which may be recruited to tumor tissues through the CXCL12-CXCR4 axis,and then inhibit T cell proliferation to promote the progression of gastric cancer.

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.