Objective:To investigate the mechanism of PE_PGRS60 protein in the pathogenesis of Mycobacterium tuberculosis infection. Methods:The cloned and purified PE_PGRS60 protein from Mycobacterium tuberculosis was used to stimulate RAW264.7 cells. The expression of cyclooxygenase 2(COX2) mRNA and protein was detected by qRT-PCR and Western blot, respectively. The signal pathways that may regulate the expression of COX2 were screened, and the expression of inflammatory cytokines induced by PE_PGRS60 was detected by ELISA. The level of cell death was measured by lactate dehydrogenase(LDH) release test and flow cytometry PI staining. Western blot was used to detect the expression of COX2 in Peripheral blood mononuclear cell(PBMC) from active tuberculosis patients. Results:PE_PGRS60 protein was found to promote the expression of COX2 in RAW264.7 cells and activate the three major members of the mitogen-activated protein kinase(MAPK) family: extracellular regulated protein kinase(ERK), p38 and c-Jun N-terminal kinase(JNK). Interestingly, only JNK-IN-7, the inhibitor of JNK was observed to suppress the up-regulation expression of COX2 induced by PE_PGRS60. This up-regulated expression of COX2 was also found in PBMCs from active tuberculosis patients. The COX2 inhibitor celecoxib can effectively block the expression of the inflammatory factors IL-1β, TNF-α and IL-6 induced by PE_PGRS60 and promote macrophage death.Conclusions:PE_PGRS60 can promote macrophages to release inflammatory factors by activating JNK/COX2 signal axis. Some macrophages still die under the protection of COX2.

Objective This retrospective study was based on 1 415 cases that had been done the flex-ible bronchoscopy examination.The data were analysed to investigate the value of flexible bronchoscope in the children's respiratory system diseases diagnosis,treatment and etiological study.Methods A total of 1 415 cases who admitted from June 2012 to December 2013 were included in the study and they were all met the inclusion criteria,had complete clinical data,done bronchoscope examinations,abnormal in the broncho-scope and diagnosed definitely.The endoscopic manifestation,clinical symptoms,X-ray film,laboratory data were analysed.Results In 1 415 cases,55.4% were boy,and 55.5% were younger than 5 years.Two cases (0.14%)were laryngeal cartilage soften,one case(0.07%)was epiglottic cyst,3 cases(0.21 %)were tra-cheomalacia,25 cases(1.8%)were bronchial foreign bodies,20 cases(1.4%)were tracheal bronchus de-formity,8 cases(5.7%)were tracheal stenosis,two cases(0.14%)were bronchial bridge,5 cases(0.35%) were bronchiolitis obliterans,6 cases (0.42%)were bronchiectasis,one case(0.07%)was immotile cilia syndrome,10 cases (0.71%)were bronchial tuberculosis,one case (0.07%)was aspergillosis,one case (0.07%)was pulmonary hemosiderosis,2 cases (0.14%)were pulmonary arteriovenous fistula,9 cases (0.63%)were plastic bronchitis,1 316 cases(93%)were founded tracheal intima inflammation,including the 350 cases(24.7%)of edema,mucosal folds form,279 cases(19.7%)of mucus plug obstruction,176 cases(12.4%)of suppurative obstruction,355 cases(25.1 %)of tracheal mucosal erosion necrosis,156 ca-ses(1 1.1 %)of wall fibrosis,stenosis,occlusion.Mycoplasma pneumoniae was the most common pathogen dectected in alveolar lavage.We also found that mycoplasma pneumonia easily combined the infection of bac-teria.A total of 1 19(22.7%)cases were no pathogens detected.In 1 415 cases,the main adverse reaction in the operations was hypoxemia caused by airway obstruction.Conclusion Flexible bronchoscopy examination is a very safe and reliable operation in diagnosis and treatment of respiratory diseases in pediat-rics,and plays an important role in the diagnosis of congenital developmental airway diseases,detection of pneumonia patho-gens and the treatment of lobe pneumonia.

Objective To investigate the advantages of pediatric electronic fiber bronchoscope ( FBO) in the infant bronchial foreign body,discuss the clinical features of infant bronchial foreign body,lung imaging characteristics and the kinds of microscopically position,the change of airway mucosa after stimulation by for-eign body under local anesthesia in 30 cases of infant bronchial foreign body. Methods Thirty cases,aged 0 to 3 years,were collected from September to December,2014. All of them were with foreign bodies examined by FBO in pediatric bronchoscopy room in Shengjing Hospital of China Medical University. Results In all infants, 6 cases (20. 0%) without history of inhaled foreign bodies and 24 cases (80. 0%) with a record history of in-haled. In the aspects of signs:normal breath sounds with a history of no choking cough in children were 2 cases (6. 7%) ,wheezing sounds were 3 patients (10. 0%) and weakened side breath sound was 1 case (3. 3%);with a history of choking cough in children,6 cases(20. 0%) with normal breath sounds,12 cases(40. 0%) with wheez-ing,6 cases(20. 0%) with lateral breath sounds less. Lung imaging characteristics was lack of specific perform-ance:only a case of all(n=30)show foreign body directly. Otherwise,other 29 cases had no specificity. Lung em-physema in 13 cases (43. 3%) is the main characteristic,while normal imaging findings in 2 cases (6. 7%). For-eign bodies in 19 cases were in the left lung (63. 4%) and 21 cases(70. 0%) of foreign body stimulated granula-tion inside airway,necrosis sputum bolt in distal obstruction of airway occured in 5 cases (16. 7%). Inhalled time of foreign body in airway was 4. 5 [2. 8,12. 5] day and inhalled time of foreign body in airway correlation coefficient with granulation hyperplasia(r=0. 688,P=0. 000),there was a significant correlation. Main adverse reaction was low oxygen in 6 cases (16. 7%). Conclusion The diagnostic accuracy of FBO under local anesthesia on children is high-er than other methods,and the FBO bronchial foreign bodies under local anesthesia is a safe and effective method.

Objective:To explore the mechanism of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the inflammatory response induced by Helicobacter pylori ( H. pylori) infection. Methods:From May 1, 2020 to January 31, 2021, 60 patients with gastritis (30 H. pylori positive and 30 H. pylori negative)and 30 healthy individuals, who initially visited the Department of Gastroenterology, Shiyan Taihe Hospital were collected, and their serum PCSK9 levels were detected. Normal gastric epithelial cell line GES-1 and macrophages induced from THP-1 cells, and GES-1 infected with H. pylori were selected to prepare different supernatant media. Phosphate buffer saline empty medium (negative control group), normal GES-1 cell supernatant medium ( H. pylori-uninfected GES-1 group), H. pylori infected GES-1 cell supernatant medium ( H. pylori infected GES-1 group), H. pylori infected GES-1 cell supernatant + PCSK9 neutralizing antibody medium (anti PCSK9 group), H. pylori infected GES-1 cell supernatant+ human immunoglobulin G medium (isotype control group) were established. The differences between H. pylori infected GES-1 group and H. pylori-uninfected GES-1 group, negative control group, anti PCSK9 group and isotype control group in number of migrated macrophages, relative expression level of CC chemokine receptor ( CCR2), the levels of released interleukin(IL)-6 and cell necrosis factor (TNF)- α, level of CD8 + T cell membrane phosphorylation, and the number of macrophage colonies were determined by Transwell assay, real time fluorescence quantitative polymerase chain reaction, plate colony assay, H. pylori and phagocytosis lysosome co-localization assay. The regulating mechanism of PCSK9 in H. pylori infection induced inflammation was analyzed. Independent sample t test was used for statistical analysis. Results:The serum level of PCSK9 of patients with H. pylori positive gastritis was higher than that of patients with H. pylori negative gastritis and healthy individuals ((384.00±57.57) g/L vs. (208.80±48.89) and (176.10±47.14) g/L), and the differences were statistically significant ( t=12.71 and 15.31; both P<0.001). Compared with negative control group, H. pylori standard strain and 4 isolated H. pylori strains could stimulate GES-1 to secrete PCSK9 ((1 267.00±287.50) g/L vs.(2 717.00±199.20), (4 858.00±302.40), (3 167.00±334.20), (6 075.00±597.30), (4 283.00±331.20) g/L), and the differences were statistically significant( t=10.15, 21.09, 10.56, 17.77, 16.85, all P<0.001). The number of migrated macrophages, CCR2 mRNA expression level in macrophage, expression levels of IL-6 and TNF-α, and the number of macrophage colonies of H. pylori-infected GES-1 group were all higher than those of H. pylori-uninfected GES-1 group and negative control group (132.20±5.67 vs.84.83±4.62, 39.83±4.12; 8.66±0.94 vs. 6.52±0.47 and 1.00±0.09, (281.00±8.56) ng/L vs. (115.00±7.72) and (64.00±5.44) ng/L, (619.80±18.47) ng/L vs.(373.30±12.85)and (225.70±6.44) ng/L, (357.00±16.31) colony forming unit (CFU) vs. (134.80±8.64) and (74.17±9.68) CFU), and the differences were statistically significant ( t=15.85, 32.27; 4.96, 19.79; 35.28, 52.43; 26.84, 49.37; 29.49, 36.53; all P<0.001). The percentage of co-localization of H. pylori and phagocytosis lysosome, and the expression of cell membrane CD3ζ Tyr142, granzyme B and perforin in CD8 + T cell of H. pylori-infected GES-1 group were lower than that of H. pylori-uninfected GES-1 group ((15.33±1.86)% vs. (34.50±3.72)% and (65.67±3.56)%, 464.20±120.80 vs. 1 924.00±262.10 and 2 390.00±484.10; (6.41±0.42)% vs.(17.37±0.73)% and (26.60±1.57)%; (6.84±1.37)% vs.(14.53±0.48)% and (26.22±1.21)%), and the differences were statistically significant( t=11.27 and 30.70, 12.39 and 9.45, 30.50 and 31.90, 25.96 and 13.00; all P<0.001). The number of migrated macrophages, the relative expression level of CCR2 mRNA, the expression levels of IL-6 and TNF-α, and the number of macrophage colonies of anti-PCSK9 group were all lower than those of isotype control group (72.50±4.97 vs. 128.30±6.74, 0.82±0.06 vs. 1.00±0.08, (85.50±4.37) ng/L vs. (277.70±8.98) ng/L, (291.80±13.69) ng/L vs. (615.30±12.65) ng/L, (111.50±10.21) CFU vs. (346.20±18.04) CFU), and the differences were statistically significant ( t=16.33, 4.40, 47.13, 42.50 and 27.73, all P<0.001). The percentage of co-localization of H. pylori and phagocytosis lysosome, the expression levels of CD3ζ Tyr142, granzyme B and perforin of anti-PCSK9 group were all higher than those of isotype control group ((51.05±3.03)% vs. (16.71±1.91)%, 2 948.00±384.00 vs. 1 156.00±178.60, (53.88±3.86)% vs. (5.88±0.93)%, (32.80±2.07)% vs. (6.83±0.54)%), and the differences were statistically significant ( t=23.49, 10.36, 29.60 and 29.76, all P<0.001). Conclusions:H. pylori can inhibit CD8 + T activation and cytotoxicity by inducing the release of PCSK9 from gastric epithelial cells, and can also recruit macrophages, activate nuclear factor-κB signal axis to up-regulate the level of released inflammatory factors from macrophages, inhibit the phagocytosis and killing effects of macrophages, so as to regulate the inflammatory response.

Objective:To evaluate the regulatory role and potential mechanism of Urease B(UreB) on macrophages.Methods:Bone marrow-derived macrophages (M0) were stimulated by recombinant UreB protein and then flowcytometry and ELISA were used to detect the apoptosis, polarization and antigen presentation-related biomarkers expression. CD4 + T cell co-culture assay, CFSE stain and flowcytometry were used to evaluate the impacts of UreB on antigen presentation capacity of macrophages. Truncated UreB protein, NanoBiT assay and co-immunoprecipitation were used to identify the binding sites of UreB to TLR2. Results:UreB promoted apoptosis and skewed macrophages from M1 to M2 in the presence of M1-inducer LPS. Moreover, UreB inhibited the expression of antigen presentation biomarkers, MHCⅡ and CD86 on macrophages, and further inhibited the proliferation and IFN-γ expression of CD4 + T cells. Molecular analyses revealed that the binding between seven carboxy-terminal amino acid residues of UreB and TLR2 were required for the UreB-mediated inhibitory effects. Conclusions:The findings in this study demonstrate that UreB mainly depends on the binding between seven carboxy-terminal amino acid residues and TLR2 to perform immune-suppressive activities, and which may provide valuable information for the design and optimization of UreB-based vaccines against Helicobacter pylori infection.