ObjectiveTo construct an Enterococcus faecium （Ef）-based recombinant Ef-PA0057 vaccine of Pseudomonas aeruginosa（Pa）and to study its protective immune mechanism in mice. MethodsThe PA0057 gene was cloned from the genomic DNA of PA01 strain ATCC9027 by PCR and inserted into pGEX-1λT to construct pGEX-PA0057. The recombinant plasmid was electroporated into TX0016 strain to construct rEf-PA0057 vaccine. The plasmid was extracted from rEf for PCR. The rEf vaccine was expressed through IPTG induction， and the expression of protein was analyzed by SDS-PAGE and Western blot. BALB/c mice were immunized intragastrically with 5×10⁸ CFU rEf-PA0057 vaccine 3 times per week for 3 weeks. 4 weeks after the first immunization， mice were challenged intranasally with 5×10⁷ CFU of PA01 strain. 2 weeks after challenge， mice were sacrificed， and their lungs were separated. Bacteria in lungs were incubated and colonies were counted. Sera were collected at 0， 4， and 6 weeks after the first immunization. The IgG and its subclasses， and IgE were detected by ELISA. ResultsThe 900 bp PA0057 gene was successfully cloned by PCR. PCR showed that PA0057 gene was amplified when the extracted plasmid from rEf as template； the relative molecular mass （Mr） of the expressed PA0057-GST fusion protein was approximately 58 ku， detected by SDS-PAGE. The amount of the expressed protein was 18% of the total bacterial proteins. Western blot showed that the target protein could be recognized by Pa sera. The colony numbers of lung tissue in rEf-PA0057 vaccine group， blank vector group and Ef control group were （0.297±0.011）×10⁸ CFU， （7.576±0.206）×10⁸ CFU and （7.551±0.185）×10⁸ CFU， respectively， the difference was statistically significant （P0.01）. The levels of IgG， IgG1， IgG2b， IgG3 and IgE increased. At the same time point， there was a significant difference compared with the two control groups （P0.01）. ConclusionThe rEf-PA0057 vaccine is successfully constructed. It may induce mice to produce humoral response against challenge with PA01.

Objective:To construct a mouse asthma model induced by mugwort pollen and to explore endotyping,providing methods for subsequent precision treatment.Methods:BALB/c mice were intraperitoneally injected with mugwort pollen extract(MPE)to sensitize,following MPE intranasal challenge to construct MPE allergic asthma murine model.Mice were randomly divided into PBS sensitization and PBS challenge(P-P),MPE sensitization and PBS challenge(M-P),MPE sensitization and MPE challenge model(M-M)groups.24 h after final challenge,mice were performed to examine airway responsiveness;bronchoalveolar lavage fluid(BALF)was harvested for cell counting and statistical classification of inflammatory cells through flow cytometry analysis.Pulmonary slides were collected for pathological examination,including HE,PAS,Masson and α-SMA immunohistochemical staining.ELISA was used to detect levels of IFN-γ,IL-4,IL-5,IL-13,IL-17A in lung tissue and serum,as well as serum total IgE and MPE-specific IgE,IgG1,IgG2a levels.Results:Pathological examination showed higher airway reactivity,more inflammatory cells infiltration around airway,obvious goblet metaplasia,thickening of airway smooth muscles and dramatical fibrin deposition around airway in model group.Total cell numbers of BALF were increased from<1×105 cells/ml in P-P group to>5×105 cells/ml in model group,in which eosinophils were predominant cellular type,levels of IL-4,IL-13,IL-17A in lung and IL-5,IL-13 levels in serum were significantly increased,as well as significant increasing levels of total IgE and MPE-specific IgE,IgG1,IgG2a.Conclusion:MPE-sensitized and challenged mice induces typical eosinophilic asthma featured with elevated eosinophils,as well as secretion of inflammatory factors of type 2 and type 17,IgE,IgG1 and IgG2a subtypes soars to high levels.

Objective:To construct Lactococcus lactis (LL) based EmⅡ/3 vaccine of Echinococcus multilocularis and observe its expression efficiency. Methods:EmⅡ/3 gene was obtained through PCR amplification using the pCD-EmⅡ/3 as a template, the gene was cloned into pMG36e to construct pMG36e-EmⅡ/3 and transformed into Escherichia coli BL21 (DE3) competent cell, the recombinant plasmid was identified by double restriction endonuclease digestion, then was electroporated into LL MG1363 to construct recombinant (r) LL-EmⅡ/3 vaccine. After screening for roxithromycin resistance, the plasmid was extracted for PCR identification, and the expression was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Results:The pMG36e-EmⅡ/3 recombinant plasmid was identified by double restriction endonuclease digestion, and there was an approximately 3 600 bp carrier band and a 1 759 bp EmⅡ/3 gene band. The plasmid extracted from roxithromycin resistant rLL strain was used as a template, and 1 759 bp EmⅡ/3 gene was amplified by PCR; SDS-PAGE demonstrated that the rLL-EmⅡ/3 vaccine could express EmⅡ/3 protein with a relative molecular weight of 66 × 10 3, and the expressed protein accounted for about 20% of the total bacteria protein after 3 days of culture; Western blot showed that the expressed protein could be recognized by mouse serum infected with alveolar hydatid cyst. Conclusion:We have successfully constructed the rLL-EmⅡ/3 vaccine of Echinococcus multilocularis, which expresses Em Ⅱ/3 protein with specific antigenicity.

Objective:To establish an irritant contact dermatitis(ICD)model induced by sodium dodecyl sulfate(SDS)in mice,and explore its endotype to provide an experimental and theoretical basis for subsequent precise treatment.Methods:Mice were randomly divided into two groups(model group and control group),4%SDS was topically applied to induce ICD in mice,saline was used on control group,the dose and frequency were consistent with model group,and the skin lesions of mice were observed.Epidermal thickness and inflammatory cell infiltration were analyzed by HE staining,toluidine blue staining and immunofluorescence staining.Real-time quantitative PCR was performed to investigate mRNA expression levels of cytokines.Results:Compared with control group,mice in ICD model group showed epidermal thickness on the back of neck,and the numbers of inflammatory cells were increased in dermis.The number of neutrophils,macrophages and T cells were increased.Expressions of Il17a and Il17f mRNA levels were increased.Conclusion:SDS-induced ICD model is successfully established,with the elevated infiltration of neutrophils,macrophages and T cells,and secretion of type 17 cytokines.

Objective:To construct a recombinant Efs-EmⅡ/3-Em14-3-3 vaccine against Echinococcus multilocularis (Em) using Enterococcus faecalis (Efs) as a vector, and investigate its antigenicity. Methods:The recombinant plasmid pGEX-EmⅡ/3-Em14-3-3 was transformed into Efs ATCC47077 strain using electroporation method, and the recombinant Efs-EmⅡ/3-Em14-3-3 vaccine was constructed. The plasmid was extracted for PCR amplification and identification. The recombinant Efs-EmⅡ/3-Em14-3-3 vaccine was expressed through isopropyl-β-D-thiogalactoside (IPTG) induction, and the recombinant protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and the proportion of expressed proteins in the total proteins of the bacteria was analyzed by thin layer scanning technology.Results:Using the plasmid extracted from recombinant Efs bacteria as a template, the EmⅡ/3-Em14-3-3 fusion gene with a size of about 2 554 bp could be amplified by PCR. The relative molecular mass ( Mr) of the expressed EmⅡ/3-Em14-3-3 fusion protein was approximately 119 × 10 3 by SDS-PAGE; after 5 h induction by IPTG, the expression level of target protein was high, accounting for about 9% of the total bacterial protein. Western blotting showed that the expressed protein could be recognized by mouse serum infected with alveolar hydatid cyst. Conclusion:The recombinant Efs-EmⅡ/3-Em14-3-3 vaccine is successfully constructed, and the expressed fusion protein shows specific antigenicity.

Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.

Objective To construct Lactococcus lactis(LL)-based recombinant LL-Eg95(rLL-Eg95)vaccine for Echinococcus granulosus(Eg)and to examine its expression efficiency.Methods Eg95 gene was obtained by PCR from the template of pCD-Eg95.Then,pMG36e was inserted in the Eg95 gene after double cleaving with restriction endonucleases Xba Ⅰ and Hind Ⅲ to construct recombinant plasmid pMG36e-Eg95,which was transformed into E.coli BL2(DE3)competent cells.The recombinant plasmid was extracted and identified by double restriction endonuclease digestion and was then electroporated into LL MG1363 to construct rLL-Eg95 vaccine.Then,the plamid was extracted and identified by PCR.Results Examination of the recombinant plasmid by double restriction endonuclease digestion showed that the segment was of the expected length.PCR showed that 471 base pairs of Eg95 gene were amplified when the plasmid extracted from roxithromycin-resistant recombinant LL was used as the template.Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the relative molecular mass of the Eg95 protein expressed was approximately 16.5×103 and that the amount of the expressed protein was 17％of the total bacterial proteins.Western blot findings suggested that the expressed protein could be recognized by mice serum infected with hydatid cyst.Conclusion The rLL-Eg95 vaccine was successfully constructed,expressing Eg95 protein that has specific antigenicity.

Objective:To investigate the in vitro inhibitory activity of a novel class Ⅰ and Ⅱb selective histone deacetylase (HDAC) inhibitor, purinostat mesylate (PM) , in diffuse large B-cell lymphoma and its mechanism.Methods:The 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide method was used to detect the effect of PM on cell proliferation. The effects of PM on cell cycle and apoptosis were detected by flow cytometry. The acetylation levels of HDAC substrate, cell cycle protein, apoptosis-related protein, and oncogene protein expression were detected by Western blot.Results:PM significantly inhibited the proliferation of lymphoma SUDHL-4 and SUDHL-6 cells and increased the acetylation levels of HDAC substrates H3, H4, and α-tubulin. In cell cycle experiments, PM induced G 0/G 1 phase arrest in SUDHL-4 and SUDHL-6 cells. Western blot experiment showed that PM could significantly downregulate the expression of cyclin-dependent kinases Cdk2, Cdk4, Cdk6, cyclin D1, and cyclin E and upregulate the expression of CDK inhibitor protein p21. In the apoptosis experiment, PM could induce the apoptosis of SUDHL-4 and SUDHL-6 cells. Western blot experiment demonstrated that PM promoted endogenous apoptosis by activating caspase-3 kinase and affecting antiapoptotic protein Bcl-2. In addition, PM could downregulate the expression of oncogene marker proteins MYC, IKZF1, and IKZF3. Conclusion:PM has an efficient biological activity in vitro for diffuse large B-cell lymphoma, including double-hit lymphoma, and provides valuable experimental evidence for PM in clinical treatment.

Purpose: Accumulating evidence has suggested that toll-like receptor 4 (TLR4) is critically involved in the pathogenesis of asthma. The aim of this study was to investigate the role of TLR4 in toluene diisocyanate (TDI)-induced allergic airway inflammation. Methods: TLR4−/− and wild-type (WT) C57BL/10J mice were sensitized and challenged with TDI to generate a TDI-induced asthma model. B-cell lymphoma 2 (Bcl-2) inhibitors, ABT-199 (4 mg/kg) and ABT-737 (4 mg/kg), were intranasally given to TDI-exposed TLR4−/− mice after each challenge. Results: TDI exposure led to increased airway hyperresponsiveness (AHR), granulocyte flux, bronchial epithelial shedding and extensive submucosal collagen deposition, which were unexpectedly aggravated by TLR4 deficiency. Following TDI challenge, TLR4−/− mice exhibited down-regulated interleukin-17A and increased colony-stimulating factor 3 in bronchoalveolar lavage fluid (BALF), while WT mice did not. In addition, TLR4 deficiency robustly suppressed the expression of NOD-like receptor family pyrin domain containing 3 and NLR family CARD domain containing 4, decreased caspase-1 activity in TDI-exposed mice, but had no effect on the level of high mobility group box 1 in BALF. Flow cytometry revealed that TDI hampered both neutrophil and eosinophil apoptosis, of which neutrophil apoptosis was further inhibited in TDI-exposed TLR4−/− mice, with marked up-regulation of Bcl-2. Moreover, inhibition of Bcl-2 with either ABT-199 or ABT-737 significantly alleviated neutrophil recruitment by promoting apoptosis. Conclusions These data indicated that TLR4 deficiency promoted neutrophil infiltration by impairing its apoptosis via up-regulation of Bcl-2, thereby resulting in deteriorated AHR and airway inflammation, which suggests that TLR4 could be a negative regulator of TDI-induced neutrophilic inflammation.

Purpose: Accumulating evidence has suggested that toll-like receptor 4 (TLR4) is critically involved in the pathogenesis of asthma. The aim of this study was to investigate the role of TLR4 in toluene diisocyanate (TDI)-induced allergic airway inflammation. Methods: TLR4−/− and wild-type (WT) C57BL/10J mice were sensitized and challenged with TDI to generate a TDI-induced asthma model. B-cell lymphoma 2 (Bcl-2) inhibitors, ABT-199 (4 mg/kg) and ABT-737 (4 mg/kg), were intranasally given to TDI-exposed TLR4−/− mice after each challenge. Results: TDI exposure led to increased airway hyperresponsiveness (AHR), granulocyte flux, bronchial epithelial shedding and extensive submucosal collagen deposition, which were unexpectedly aggravated by TLR4 deficiency. Following TDI challenge, TLR4−/− mice exhibited down-regulated interleukin-17A and increased colony-stimulating factor 3 in bronchoalveolar lavage fluid (BALF), while WT mice did not. In addition, TLR4 deficiency robustly suppressed the expression of NOD-like receptor family pyrin domain containing 3 and NLR family CARD domain containing 4, decreased caspase-1 activity in TDI-exposed mice, but had no effect on the level of high mobility group box 1 in BALF. Flow cytometry revealed that TDI hampered both neutrophil and eosinophil apoptosis, of which neutrophil apoptosis was further inhibited in TDI-exposed TLR4−/− mice, with marked up-regulation of Bcl-2. Moreover, inhibition of Bcl-2 with either ABT-199 or ABT-737 significantly alleviated neutrophil recruitment by promoting apoptosis. Conclusions These data indicated that TLR4 deficiency promoted neutrophil infiltration by impairing its apoptosis via up-regulation of Bcl-2, thereby resulting in deteriorated AHR and airway inflammation, which suggests that TLR4 could be a negative regulator of TDI-induced neutrophilic inflammation.