Pseudomonas aeruginosa (PA) is an important pathogen that has been proven to colonize and cause infection in the respiratory tract of patients with structural lung diseases and to lead to bronchial fibrosis. The development of pulmonary fibrosis is a complication of PA colonization of the airway, resulting from repeated infection, damage and repair of the epithelium. Bronchial epithelial cell epithelial-mesenchymal transition (EMT) plays a vital role in bronchial fibrosis. To date, research on bronchial epithelial cell EMT caused by PA-secreted virulence factors has not been reported. Here, we found that PA3611 protein stimulation induced bronchial epithelial cell EMT with mesenchymal cell marker upregulation and epithelial cell marker downregulation. Moreover, integrin αvβ6 expression and TGF-β1 secretion were markedly increased, and p38 MAPK phosphorylation and NF-κB p65 subunit phosphorylation were markedly enhanced. Further research revealed that PA3611 promoted EMT via integrin αvβ6-mediated TGF-β1-induced p38/NF-κB pathway activation. The function of PA3611 was also verified in PA-infected rats, and the results showed that ΔPA3611 reduced lung inflammation and EMT. Overall, our results revealed that PA3611 promoted EMT via integrin αvβ6-mediated TGF-β1-induced p38/NF-κB pathway activation, suggesting that PA3611 acts as a crucial virulence factor in bronchial epithelial cell EMT and is a potential target for the clinical treatment of bronchial EMT and fibrosis caused by chronic PA infection.

BACKGROUND:The development of vaccines is a promising and cost-effective strategy to prevent emerging multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) infections. The purpose of this study was to prepare a multiepitope peptide nanovaccine and evaluate its immunogenicity and protective effect in BALB/c mice. METHODS:The B-cell and T-cell epitopes of Omp22 from A. baumannii were predicted using bioinformatics methods and identified by immunological experiments. The optimal epitopes were conjugated in series by 6-aminocaproic acid and chemically synthesized multiepitope polypeptide rOmp22. Then, rOmp22 was encapsulated by chitosan (CS) and poly (lactic-co-glycolic) acid (PLGA) to prepare CS-PLGA-rOmp22 nanoparticles (NPs). The immunogenicity and immunoprotective efficacy of the vaccine were evaluated in BALB/c mice. RESULTS:CS-PLGA-rOmp22 NPs were small (mean size of 272.83 nm) with apparently spherical structures, positively charged (4.39 mV) and nontoxic to A549 cells. A high encapsulation efficiency (54.94%) and a continuous slow release pattern were achieved. Compared with nonencapsulated rOmp22, CS-PLGA-rOmp22 immunized BALB/c mice induced higher levels of rOmp22-specific IgG in serum and IFN-γ in splenocyte supernatant. Additionally, lung injury and bacterial burdens in the lung and blood were suppressed, and potent protection (57.14%-83.3%) against acute lethal intratracheal A. baumannii challenge was observed in BALB/c mice vaccinated with CS-PLGA-rOmp22. CONCLUSION:CS-PLGA-rOmp22 NPs elicited specific IgG antibodies, Th1 cellular immunity and protection against acute lethal intratracheal A. baumannii challenge. Our results indicate that this nanovaccine is a desirable candidate for preventing A. baumannii infection.

Pseudomonas aeruginosa (PA) is one of the important pathogens, which has been proven to colonize and cause infection in the respiratory tract of patients with structural lung diseases, and further lead to bronchial fibrosis. Epithelial-Mesenchymal Transition (EMT) of bronchial epithelial cells plays a vital role in the process of bronchial fibrosis. Up to the present, the research on bronchial epithelial cells EMT caused by secreted virulence factors of PA has not been reported. In our present study, we found that PA3611 protein stimulation induced the bronchial epithelial cells EMT with up-regulation of mesenchymal cell markers and down-regulation of epithelial cell markers. Meantime, TGF-β1 secretion was markedly increased, IκBα expression was significantly decreased, and NF-κB p65 subunit phosphorylation was markedly enhanced, in addition, the levels of miR-3065-3p and miR-6802-3p expression and p38 MAPK phosphorylation were obviously increased in bronchial epithelial cells after PA3611 stimulation, further research revealed that PA3611 promoted EMT occur through TGF-β1 induced p38/miRNA/NF-κb pathway. The function of PA3611 was also verified in PA-infected rats and results showed that ΔPA3611 could reduce lung inflammation and EMT. Overall, our results revealed that PA3611 promotes EMT via simulating the production of TGF-β1 induced p38/miRNA/NF-κB pathway-dependent manner, suggesting that PA3611 acts as a crucial virulence factor in bronchial epithelial cells EMT process and has potential use as a target for clinical treatment of bronchial EMT and fibrosis caused by chronic PA infection. Author summary Structural lung disease can increase the chance of chronic infection, including infected by Pseudomonas aeruginosa, which can cause lung structure damages and affect lung functions in further, and forming a vicious circle of intertwining, ultimately, it leads to pulmonary fibrosis. EMT of bronchial epithelial cells plays a vital role in the process of bronchial fibrosis. However, the relationship and mechanism of PA infection leads to the destruction of lung structure and bronchial epithelial cells EMT are still not very clear. We found pseudomonas aeruginosa secreted protein PA3611 can stimulate bronchial epithelial cells EMT through up-regulation of mesenchymal cell markers α-SMA and Vimentin expression and down-regulation of epithelial cell markers E-cadherin and Zonula Occludens-1. Meantime, TGF-β1 secretion was markedly increased, IκBα expression was significantly decreased, and NF-κB p65 subunit phosphorylation was markedly enhanced, in addition, the levels of miR-3065-3p and miR-6802-3p expression and p38 MAPK phosphorylation were obviously increased in bronchial epithelial cells after PA3611 stimulation, further studies suggested that PA3611 was shown to promote EMT occur through TGF-β1 induced p38/miRNA/NF-Kb pathway. Our results revealed that PA3611 promotes EMT via simulating the production of TGF-β1 induced p38/miRNA/NF-κB pathway-dependent manner, suggesting that PA3611 acts as a crucial virulence factor in bronchial epithelial cells EMT process and as a potential target for the treatment of chronic structural lung diseases.

Background: Mtb exploits the 6 kDa early secreted antigenic target (ESAT-6) secretion system (ESX). Mtb exhibits membrane-perforating activity due to the secretion of ESAT-6. The ESAT-6-encoding gene is located in the RD1 region, which is conserved in virulent Mycobacterium bovis and Mtb strains, and is deleted from all BCG substrains. It has been shown that Mtb Rv2346c is a member of the ESAT-6-like family of proteins. The role of Rv2346c in Mtb pathogenesis and host immunity is still unclear. Objectives: Recombinant Rv2346c protein was produced to evaluate its immunomodulatory effect on BCG-infected macrophages and the related molecular mechanism. The function of Rv2346c was also verified in vivo. Methods: 1. Production of recombinant Rv2346c protein. 2. Transfection 3. Proliferation assay 4. Bacterial enumeration 5. ELISA assay6. Western blotting7. Microarray data analysis 8. Quantitative RT-PCR 9. Dual-luciferase reporter assay 10. Infection of mice 11. Bacterial load assessment in the lung 12. Histology examination and immunofluorescence detection. Results: 1. Rv2346c inhibits the proliferation of BCG-infected macrophages and enhances the survival of BCG in macrophages. 2. Rv2346c inhibition of cytokine production is accompanied by the downregulation of p65 and upregulation of p38 phosphorylation in BCG-infected macrophages. 3. Rv2346c upregulates miR-155 and miR-99b in BCG infected macrophages. 4. Rv2346c enhances the virulence of Mtb and inhibits the production of TNF-α and IL-6 in vivo. Conclusions: Rv2346c suppress the secretion of TNF-α and IL-6, and facilitates the survival of BCG through the p38/miR-155 and miR-99b/NF-κB pathways and has potential use as a target for anti-TB therapy.

目的:研究栀子苷对巨噬细胞系RAW264.7细胞活性和内吞功能的影响.方法:采用CCK-8法判定在不同浓度、不同时间下栀子苷对RAW264.7细胞增殖的影响;根据CCK-8检测结果,挑选适宜浓度栀子苷与RAW264.7细胞共孵育24h后,检测RAW264.7细胞对中性红溶液的胞饮活性;进一步以2 mg/mL栀子苷与RAW264.7细胞共孵育后,检测RAW264.7细胞对铜绿假单胞菌的吞噬活性及胞内细菌存活情况.结果:栀子苷的细胞毒性小,在0～8 mg/mL浓度内对RAW264.7细胞的增殖无明显影响;2 mg/mL的栀子苷与细胞共孵育24 h后可明显促进静息状态下RAW264.7细胞的胞饮活性;2 mg/mL的栀子苷抑制RAW264.7细胞对铜绿假单胞菌的吞噬及胞内杀菌作用.结论:栀子苷对RAW264.7细胞的胞饮和噬菌功能具有双向调节作用.

Background/aim8-Hydroxy-2’-deoxyguanosine (8-OHdG) is a biomarker of oxidative stress and has been implicated in many diseases. The aim of this study was to investigate the clinical value of plasma 8-OHdG level in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD).Materials and methodsA total of 154 subjects were enrolled in this study, including 20 healthy volunteers, 24 COPD patients in the stable phase, and 110 AECOPD patients. Peripheral blood samples, demographic information, and clinical characteristics were collected from all subjects at the time of being recruited into the study. Plasma 8-OHdG level was detected by enzyme-linked immunosorbent a ss ay.Results8-OHdG was increased in patients with AECOPD compared to healthy subjects and patients with stable COPD, especially in smokers. It also increased with the GOLD stage, mMRC grade, CAT score, and group level of combined COPD assessment. Additionally, further analysis revealed that 8-OHdG was negatively correlated with FEV1, FEV1% predicted, and FEV1/FVC and positively correlated with C-reactive protein, procalcitonin, and neutrophil CD64.Conclusion8-OHdG is associated with spirometric severity, symptomatic severity, exacerbation risk, and inflammatory biomarkers in AECOPD patients, suggesting it as a promising biomarker for reflecting disease severity and guiding the choice of optimal therapeutic decision.

Objective To investigate the Rv2346c gene function through constructing Rv2346c gene knockout strains of Mycobacterium tuberculosis (M . tuberculosis) mediated by bacteriophage and observing its virulence after infecting mice lung tissue in vivo .Methods The affinal exchange sites (AES) of the target gene was built ,and then integrated into the phage genomes of M .tuberculosis for harvesting the phagemids .The phagemids was imparted into Mycobacterium smegmatis to get recombinant phages with the same AES .A high titer of the recombinant phages was harvested through amplification in vitro . The M .tuberculosis was transfected and coated on solid medium with hygromycin resistance and cultured for 4 weeks at 37℃ .Single clone was picked out and gene knock-out was confirmed by PCR . Then C57BL/6J mice were infected with either wild type strain (WT ) or knockout strain (KO ) of M . tuberculosis .Mice mortality ,lung tissue inflammation and colony-forming units (CFU ) counts in vitro were observed 6 to 8weeks post infection with different strains . Paired-samples t test was used for comparison between groups ,chi-square test was used for comparison of rates .Results The products of PCR and inserted fragment sizes were consisted with the expectation and confirmed to be the target gene . The target fragment of Rv2346c was removed successfully and the mice were infected for 6-8 weeks .Themice infected with Rv2346c KO strain had reduced mortality (53％ vs 20％ ,χ2 =6 .1112 ,P<0 .05) ,lung tissue inflammation (1040 ± 89 vs 1960 ± 56 ,t=7 .1016 ,P<0 .05) and CFU count in vitro (15 .0 ± 0 .8 vs 90 .0 ± 1 .5 ,t=23 .0361 , P<0 .05) compared with WT strain 6-8 weeks post infection .Conclusion Rv2346c gene knockout strains of M . tuberculosis mediated by bacteriophageis are successfully constructed ,which establishes the foundation for the future gene function study of Rv2346c .

Pseudomonas aeruginosa (PA) has become one of the main gram-negative organisms responsible for hospital infections.PA secretes a variety of virulence factors,including pyocyanin,elastase,and virulence factors of the type Ⅲ secretion system,etc.The quorum sensing system and biofilm also play an important role in the pathogenesis of PA.In this paper,we briefly review research progress on the toxic protein in Pseudomonas aeruginosafor recent years.

Objective To investigate whether Pseudomonas aeruginosa recombinant protein PA3611 can induce the in vitro epithelial-mesenchymal transformation (EMT) of rat bronchial epithelial cells. Methods A series of processes including DNA synthesis, gene amplification, vector construction, induction of expression and protein purification was used to synthesize the recombinant protein PA3611. CCK-8 kit was used to detect the proliferation of bronchial epithelial cells after stimulation with the protein PA3611. Morphological changes in bronchial epithelial cells were observed. Western blot and qPCR were performed to measure the expression of E-cadherin (E-CAD) and α-smooth muscle actin (α-SMA). T test was used for statistical analysis. Results DNA sequencing verified the successful preparation of the recombinant protein PA3611. PA3611 inhibited the proliferation (P<0.05) and induced the EMT of bronchial epithelial cells. Moreover, it also inhibited the expression of E-CAD protein, but promoted the expression of α-SMA (P<0.05). Conclusion The recombinant protein PA3611 can induce the EMT of bronchial epithelial cells and the underlying mechanisms are worthy of further investigation. Key words: Recombinant protein PA3611; Pseudomonas aeruginosa; Epithelial-mesenchymal transformation

目的:构建和筛选鲍曼不动杆菌TonB蛋白抗原的B细胞、T细胞表位.方法:运用生物信息学软件预测TonB蛋白的小鼠B细胞及T细胞表位并人工合成相应肽段;构建重组质粒pET28a-tonB并原核表达、纯化获得TonB蛋白;分3次以TonB蛋白在皮下免疫BALB/c小鼠,5周后收集小鼠血清并分离小鼠脾脏细胞;间接ELISA法检测小鼠血清中B细胞表位抗体水平,双抗体夹心ELISA法检测经候选T细胞表位体外刺激后脾细胞分泌γ-干扰素(IFN-γ)量.结果:构建候选B细胞和T细胞表位各4条,分别为PB1、PB2、PB3、PB4和PT1、PT2、PT3、PT4;间接ELISA结果显示,PB3及PB4与TonB蛋白免疫组小鼠血清发生反应,其吸光度值与对照组相比显著升高(P＜0.05);双抗体夹心ELISA结果显示,PT2、PT3及PT4刺激免疫组小鼠脾脏细胞产生的IFN-γ量与对照组相比显著升高(P＜0.05).结论:成功构建并筛选出鲍曼不动杆菌TonB蛋白的2个B细胞表位PB3及PB4,3个T细胞表位PT2、PT3及PT4.

The intracellular survival of Mycobacterium tuberculosis (Mtb) has a central role in the pathogenesis of tuberculosis. Mtb Rv2346c is a member of 6-kDa early secreted antigenic target family of proteins, which are known to inhibit the host immune responses to promote bacillary persistence in macrophages. However, the mechanism through which Rv2346c participates in Mtb pathogenesis is unclear. In the present study, recombinant Rv2346c protein was synthesized and used to treat Bacillus Calmette–Guérin (BCG)-infected macrophages. The results showed that Rv2346c inhibited the proliferation of BCG-infected macrophages and enhanced the survival of BCG in macrophages. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were upregulated during BCG infection but downregulated by Rv2346c. Additional experiments showed that nuclear transcription factor-κB (NF-κB) in BCG-infected macrophages induced the production of TNF-α and IL-6. In addition, miR-155 and miR-99b had a suppressive effect on NF-κB, and the expression of these miRNAs was promoted by p38. Furthermore, Rv2346c was shown to decrease the activation of NF-κB, whereas it enhanced the phosphorylation of p38 and the expression of miR-155 and miR-99b. The function of Rv2346c was also verified in Mtb-infected mice. The results showed that Rv2346c increased the observed bacterial load and lung injury and downregulated TNF-α and IL-6 in vivo. Overall, our results reveal that Rv2346c enhances mycobacterial survival in macrophages via inhibiting the production of TNF-α and IL-6 in a p38/miRNA/NF-κB pathway-dependent manner, suggesting that Rv2346c acts as a crucial virulence factor in Mtb infection and has potential use as a target for anti-tuberculosis therapy.

Objective To screen B and T cell antigen epitopes on Acinetobacter baumannii outer membrane protein 33×103-36×103 (OMP33-36).Methods B and T cell epitopes on OMP33-36 of Acinetobacter baumannii were predicted by bioinformatics methods and synthesized.Recombinant expression plasmid pET-30a-OMP33-36 was cloned and used to express OMP33-36 in a prokaryotic expression system.The expressed OMP33-36 was used to immunize BALB/c mice after purification.Serum sample was collected from each mouse in immunization and negative control groups, and then analyzed by indirect ELISA with synthesized peptides to identify B cell epitopes.Splenocytes were separated from every mouse and then cultured with each of the synthesized peptides, respectively.Double sandwich ELISA was performed to detect IFN-γ secretion in the supernatant of cell cultures for screening of T cell epitopes.Results Candidates of B and T cell epitopes were constructed, which were PB1, PB2, PB3, PT1, PT2 and PT3.Results of the indirect ELISA showed that peptides PB1 and PB2 reacted with the serum samples collected from immunized mice and A450 values of the immunization group were significant higher than those of the negative control group.Compared with the negative control group, enhanced secretion of IFN-γ following peptide PT3 stimulation was observed in the immunization group as indicated by the double sandwich ELISA.Conclusion Two B cell epitopes PB1 and PB2, and one T cell epitope PT3 on the OMP33-36 of Acinetobacter baumannii were successfully constructed and screened out.

Objective To explore the expression level and clinical significance of 8-OHdG in patients with AECOPD.Methods The study selected 121 patients with AECOPD as the AECOPD group and 28 stable COPD patients as the COPD group from September 2015 to August 2016, and another 20 healthy people were taken as the control group.Their 8-OHdG, CD64 index, PCT and CRP in all groups were detected.The relationship of 8-OHdG with CAT score in the AECOPD group was analyzed.Results The levels of plasma 8-OHdG, CD64 index, PCT and CRP in the AECOPD group were significantly higher than those in the health control group (P<0.05).Compared with the stable group, the plasma levels of 8-OHdG and PCT were markedly elevated in the acute exacerbation group (P<0.05).In the acute exacerbation group, plasma 8-OHdG, CD64 index, PCT and CRP were significantly higher in patients with GOLD Ⅲ, Ⅳ grade than those with GOLD Ⅰ grade (P<0.05), and the levels of 8-OHdG, CD64 index and CRP were higher in GOLD Ⅳ grade than in GOLD Ⅱ grade (P<0.05).Plasma 8-OHdG level was positively correlated with CAT scores in the acute exacerbation group (r=0.419, P<0.01).Conclusion 8-OHdG can be used as the diagnostic marker for AECOPD patients and its level is associated with the severity of disease and quality of life.