rs401681 and rs402710 confer lung cancer susceptibility by regulating TERT expression instead of CLPTM1L in East Asian populations.

Lung cancer is a common cancer in human and has presented significant genetic predisposition. Previous genome-wide association study observed that rs401681 within CLPTM1L (CLPTM1 like) was significantly associated with lung cancer. By analyzing 1000 genomes data for East Asian, we identified only one SNP in nearby region, rs402710, in high linkage disequilibrium with rs401681, which was also associated with lung cancer. However, the real causal SNP and mechanism for the association were still not clear. The following plasmid construction, mutagenesis, transient transfection and luciferase reading indicated that both SNPs could regulate gene expression in lung/bronchial epithelium Beas-2B cell line. By chromosome conformation capture, it was identified that the segment containing these two SNPs could interact with TERT (telomerase reverse transcriptase) promoter, thus indicating that these SNPs confer lung cancer risk by regulating TERT expression instead of CLPTM1L. Through chromatin immunoprecipitation, the transcript factors HNF4A (hepatocyte nuclear factor 4 alpha) and MAF1 (MAF1 homolog, negative regulator of RNA polymerase III) were recognized for the regions spanning rs401681 and rs402710, respectively. Our results uncovered a complete link between these two SNPs and lung cancer.

Extracellular heat shock protein 90α mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling.

BACKGROUND: The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. METHODS: To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90ß respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and ß-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. RESULTS: The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and ß-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90ß that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. CONCLUSIONS: The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.

Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis protects against lung fibrosis by inhibiting the MAPK/NF-κB pathway.

Accumulating evidence has demonstrated that up-regulation of the angiotensin (Ang)-converting enzyme (ACE)/AngII/AngII type 1 receptor (AT1R) axis aggravates pulmonary fibrosis. The recently discovered ACE2/Ang-(1-7)/Mas axis, which counteracts the activity of the ACE/AngII/AT1R axis, has been shown to protect against pulmonary fibrosis. However, the mechanisms by which ACE2 and Ang-(1-7) attenuate pulmonary fibrosis remain unclear. We hypothesized that up-regulation of the ACE2/Ang-(1-7)/Mas axis protects against bleomycin (BLM)-induced pulmonary fibrosis by inhibiting the mitogen-activated protein kinase (MAPK)/NF-κB pathway. In vivo, Ang-(1-7) was continuously infused into Wistar rats that had received BLM or AngII. In vitro, human fetal lung-1 cells were pretreated with compounds that block the activities of AT1R, Mas (A-779), and MAPKs before exposure to AngII or Ang-(1-7). The human fetal lung-1 cells were infected with lentivirus-mediated ACE2 before exposure to AngII. In vivo, Ang-(1-7) prevented BLM-induced lung fibrosis and AngII-induced lung inflammation by inhibiting the MAPK phosphorylation and NF-κB signaling cascades. However, exogenous Ang-(1-7) alone clearly promoted lung inflammation. In vitro, Ang-(1-7) and lentivirus-mediated ACE2 inhibited the AngII-induced MAPK/NF-κB pathway, thereby attenuating inflammation and α-collagen I production, which could be reversed by the Mas inhibitor, A-779. Ang-(1-7) inhibited AngII-induced lung fibroblast apoptotic resistance via inhibition of the MAPK/NF-κB pathway and activation of the BCL-2-associated X protein/caspase-dependent mitochondrial apoptotic pathway. Ang-(1-7) alone markedly stimulated extracellular signal-regulated protein kinase 1/2 phosphorylation and the NF-κB cascade. Up-regulation of the ACE2/Ang-(1-7)/Mas axis protected against pulmonary fibrosis by inhibiting the MAPK/NF-κB pathway. However, close attention should be paid to the proinflammatory effects of Ang-(1-7).

Interrelationship of circulating matrix metalloproteinase-9, TNF-α, and OPG/RANK/RANKL systems in COPD patients with osteoporosis.

Previous studies have shown that matrix metalloproteinase-9 (MMP-9) and its cognate inhibitor TIMP-1, inflammatory cytokine TNF-α, and the OPG/RANK/RANKL system may each play individual roles in the pathogenesis of osteoporosis in patients with COPD. In the present study, we investigated the interrelationships of these factors in male COPD patients with and without osteoporosis. The serum levels of MMP-9, MMP-9/TIMP-1 ratio, TNF-α, RANKL, OPG, and the RANKL/OPG ratio were higher in COPD patients with osteoporosis than in individuals with normal or low bone mineral density (BMD) (N = 30, all P < 0.05 or < 0.01). The lung function FEV1%Pre and the BMD of the lumbar spine and femoral neck were found to be negatively correlated with MMP-9 serum level (r = -0.36, P < 0.05, r = -0.58, P < 0.001, and r = -0.62, P < 0.01, respectively), RANKL serum level (r = -0.21, P < 0.05, and r = -0.25, P < 0.05, and r = -0.26, P < 0.05, respectively), and RANKL/OPG ratio (r = -0.23, P < 0.05, r = -0.33, P < 0.05, and r = -0.38, P < 0.05, respectively). However, they had no correlation with TIMP-1, TNF-α, OPG, or RANK. The MMP-9 serum level was found to be positively correlated with TNF-α level (r = 0.35, P < 0.05) and RANKL/OPG ratio (r = 0.27, P < 0.05) but not associated with RANKL. These results suggest that MMP-9, TNF-α, and the OPG/RANK/RANKL system may be closely interrelated and may play interactive roles in pathogenesis of osteoporosis in COPD.

[Anti-fibrotic effects of angiotensin1-7 on bleomycin-induced pulmonary fibrosis in rats].

OBJECTIVE: To explore the anti-fibrotic effects of angiotensin (Ang) 1-7 on bleomycin (BLM) -induced pulmonary fibrosis in rats. METHODS: Eighteen Wistar male rats were randomly divided into 3 groups, including control group (intratracheal instillation with physiological saline and subcutaneous micro-pump with bi-distilled water at the rate of 0.29 µl/h), BLM group (intratracheal instillation with bleomycin and subcutaneous micro-pump with bi-distilled water at the same rate) and BLM+Ang1-7 group (intratracheal instillation with bleomycin and subcutaneous micro-pump with Ang1-7 at a dose of 25 µg·kg(-1) · h(-1) at the same rate). At Day 28, lung tissues were collected. Histological changes of lungs were evaluated by hematoxylin and eosin and Masson's trichrome stains. Collagen content of lung tissues was assessed by hydroxyprolin concentration. Then the products of protein and RNA were collected. And Western blot and realtime polymerase chain reaction (RT-PCR) were used to detect the protein or mRNA of TGF-ß1 and α-collagenI. Human embryonic lung fibroblast (HFL-1) was divided into 5 groups: (1) control group: no stimulation; (2) AngII group: stimulation of AngII (10(-7)mol/L) ; (3) Ang1-7 group: stimulation of Ang1-7 (10(-7)mol/L); (4) Ang1-7 plus AngII group: stimulation by AngII (10(-7)mol/L) with Ang1-7 (10(-7)mol/L) pre-treatment; (5) Ang1-7+AngII+A-779 group: stimulation by AngII and Ang1-7 (10(-7) mol/L) with Mas receptor inhibitor A-779 (10(-6)mol/L) pre-treatment. Then the products of protein and RNA were collected. And QuantiGene and RT-PCR were used to detect the activation of TGF-ß1, and α-collagenI mRNA. RESULTS: Compared with control group, fibrosis score and hydroxyproline concentrations increased significantly in BLM group, but declined in BLM+Ang1-7 group. The difference was statistically significant (P < 0.05). TGF-ß1 mRNA, α-collagenI mRNA and α-collagenI protein level were up-regulated by BLM (4.45 ± 0.45 vs 1.00 ± 0.20, 5.14 ± 0.55 vs 1.00 ± 0.08, 1.48 ± 0.34 vs 0.23 ± 0.11) (all P < 0.05); while compared with BLM group, those of BLM+Ang1-7 group were down-regulated (2.80 ± 0.35, 3.10 ± 0.52, 0.49 ± 0.11) (all P < 0.05). In vitro: TGF-ß1 mRNA and α-collagen I mRNA level were up-regulated by AngII (1.67 ± 0.26 vs 1.00 ± 0.10, 4.86 ± 1.36 vs 1.46 ± 0.54) (all P < 0.05); while those of AngII+Ang1-7 group were down-regulated (0.91 ± 0.30, 1.57 ± 0.27) compared with AngII group (all P < 0.05); no significant difference existed between the AngII+Ang1-7+A-779 group (1.25 ± 0.14, 1.29 ± 0.49) and AngII+Ang1-7 group (P > 0.05). CONCLUSION: Ang1-7 has anti-fibrous effect upon bleomycin-induced pulmonary fibrosis in rats and such an effect of Ang1-7 may be associated with AngII-induced expression of TGF-ß1.

[Risk factors for in-hospital mortality in patients with acute exacerbation of chronic obstructive pulmonary disease].

OBJECTIVE: To explore the risk factors for hospitalization case fatality of patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). METHODS: A retrospective review of medical records was performed for 182 hospitalized AECOPD patients at Nanfang Hospital from January 2010 to August 2012. Their general information, condition in stable stage, the results of spirometry, blood routine test, blood gas analysis and C-reactive protein (CRP) were collected and analyzed. The risk factors for mortality were analyzed by multivariable Logistic regression. RESULTS: Among them, 42 died during hospitalization. Univariate analysis revealed that 8 factors had significant differences between two groups (all P < 0.05): high exacerbation risk (death vs improvement group, 90.4% vs 70.0%) , low peripheral absolute lymphocyte count (73.8% vs 47.1%), high CRP (50.0% vs 17.1%), concurrent anemia (50.0% vs 27.1%), hypoproteinemia (71.4% vs 46.4%), hypercapnia (64.3% vs 30.7%), chronic pulmonary heart disease (76.1% vs 40.7%) and ischemic heart disease (19.0% vs 7.0%). By multiple Logistic regression analysis, high CRP (OR = 3.226, P = 0.009), hypercapnia (OR = 2.928, P = 0.013), chronic pulmonary heart disease (OR = 2.510, P = 0.045), low peripheral absolute lymphocyte count (OR = 2.488, P = 0.045) were the independent risk factors for hospitalization case fatality of AECOPD patients. CONCLUSION: Low peripheral absolute lymphocyte count, high CRP, hypercapnia and chronic pulmonary heart disease were the independent risk factors for mortality in hospitalized AECOPD patients.

[Expression and significance of Toll-like receptor-4 in rats lung established by passive smoking or associated with intratracheal instillation of lipopolysaccharide].

OBJECTIVE: To explore the expression and effect of Toll-like receptor 4 (TLR-4) in the lung tissue of rats established by passive smoking or intratracheal instillation of lipopolysaccharide (LPS). METHODS: Eight-week-old male Sprague-Dawley rats (n = 15) were randomly divided into 3 groups, including: (1) group A: conventional breeding; (2) group B: the rats were placed into a 120-L organic glass box with twice-daily exposure to cigarette smoking plus an intratracheal instillation of water at Day 1 and 14; (3) Group C: exposure to cigarette smoking the same as group B plus intratracheal instillation of lipopolysaccharide (1 mg/kg) at Day 1 and 14. Four weeks later, general status, arterial blood gas, pulmonary function and histopathology were analyzed. The expressions of TLR-4 and nuclear factor kappa-B (NF-κB) were determined by immunohistochemistry. Western blot was used to measure the protein contents of TLR-4, NF-κB, p-Iκ-Kα/ß, Iκ-Kα/ß, IκB-α. And real-time polymerase chain reaction (PCR) was employed to detect the mRNA levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6). RESULTS: Rats in Groups B and C were marantic with intermittent cough and dyspnea. Peak expiratory flow (PEF) and 50% expiratory flow-volume (EP50) were much lower in Group C ((10.6 ± 1.4), (0.77 ± 0.14) ml/s) than that in Groups A ((13.5 ± 2.0), (1.01 ± 0.08) and B (12.3 ± 0.9), (0.91 ± 0.10) ml/s) (all P < 0.05). Accumulated volume (AV) and carbon dioxide pressure (PCO2) were much higher in Groups B ((4358 ± 1501) ml, (52.77 ± 1.97) mm Hg) (1 mm Hg = 0.133 kPa) and C ((10 077 ± 1866) ml, (51.03 ± 4.96) mm Hg) than that in Group A ((1735 ± 798) ml, (39.57 ± 1.43) mm Hg) (all P < 0.05). Hematoxylin and eosin stain showed chronic bronchitis and emphysema in Groups B and C. Besides, quantitative analysis demonstrated that in unit area, mean lining interval (MLI) and destruction index (DI) in Group B ((84 ± 13) µm, 0.228 ± 0.047) and Group C ((86 ± 10) µm, 0.294 ± 0.060) significantly increased versus Group A ((65 ± 6) µm, 0.036 ± 0.012) (all P < 0.05). Immunohistochemical staining indicated that the expression of TLR-4 in cytoplasm and cytomembrane and NF-κB in nucleus markedly increased in Groups B and C versus Group A. Relative expressions of TLR-4 and NF-κB assayed by Western blot increased in Group B (0.68 ± 0.03, 0.21 ± 0.08) and Group C (1.12 ± 0.11, 0.59 ± 0.06) than that in Group A (1.36 ± 0.07, 1.04 ± 0.08). Compared with Group A, the expression levels of TLR-4, NF-κB and IκB-α and the phosphorylation levels of Iκ-Kα/ß in Group B and C significantly increased (all P < 0.05). The mRNA levels of TNF-α and IL-6 increased in Group B (3.95 ± 0.29, 5.04 ± 0.28) and C (5.33 ± 0.26, 7.23 ± 0.39) versus that in Group C (1.00 ± 0.37, 1.00 ± 0.25) (all P < 0.05). CONCLUSIONS: Both passive smoking and intratracheal instillation of LPS may cause lung injury analogous to chronic obstructive pulmonary disease via NF-κB signaling pathway. And TLR4 plays an important role in this process.

[Post-therapeutic change of cathelicidin LL-37 in asthmatics of different inflammatory phenotypes].

OBJECTIVE: To explore the post-therapeutic change of cathelicidin LL-37 in asthmatics of different inflammatory phenotypes. METHODS: Thirty-four patients with initially diagnosed asthma (asthma group) and 14 normal subjects (control group) were recruited at Nanfang Hospital from August 2009 to August 2010 for this prospective study. Sputum and venous blood samples were collected and analyzed for cell differential. Eosinophilic asthma was defined as the count of sputum eosinophils ≥ 3%. The LL-37 concentrations in plasma and sputum supernatant were measured by enzyme-linked immunosorbent assay (ELISA) kit. The subjects were treated with budesonide/formoterol (160/4.5 µg) one inhalation twice daily and re-examined after 1 month. RESULTS: Prior to treatment, there were no differences between the asthma and control groups in the levels of LL-37 in plasma and sputum supernatant (P = 0.427,0.427). The plasma concentrations of LL-37 in asthma group were negatively correlated with baseline forced expiratory volume in one second (FEV(1), r = -0.470, P = 0.005), percent predicted of FEV(1) (FEV(1)%pred, r = -0.421, P = 0.013) and forced vital capacity (FVC, r = -0.367, P = 0.033). After treatment, the plasma and sputum supernatant concentrations of LL-37 (M (Q(R))) in the asthma group (5.6 (16.2), 65.6 (184.0) µg/L) were significantly higher than those baseline concentrations (5.03 (9.21), 28.40(109.76) µg/L, P = 0.005, 0.015). In the eosinophilic asthma subgroup, the plasma and sputum supernatant concentrations of LL-37 (M (Q(R))) after treatment (5.3 (19.3), 65.6 (185.2) µg/L) were significantly higher than those baseline concentrations (6.7 (8.9) L, 35.3 (102.0) µg/L, P = 0.021,0.014). And in the non-eosinophilic asthma subgroup, the changes of plasma and sputum supernatant concentrations of LL-37 showed no significant differences (P = 0.139, 0.386). In the asthma group, the correlations between plasma concentrations of LL-37 and FEV(1), FEV(1)%pred, FVC were not statistically significant (P = 0.283, 0.706,0.272) after treatment. CONCLUSIONS: LL-37 may participate in the aggravation of asthma. The elevated concentrations of LL-37 in eosinophilic asthma is probably due to the resolved suppression of LL-37 expression by eosinophilic inflammation. But its mechanism needs further researches.

Successful treatment of disseminated nocardiosis diagnosed by metagenomic next-generation sequencing: A case report and review of literature.

BACKGROUND: Nocardia paucivorans is an infrequently found bacterium with the potential to cause severe infection, with a predilection for the central nervous system, both in immunocompromised and immunocompetent individuals. Rapid etiological diagnosis of nocardiosis can facilitate timely and rational antimicrobial treatment. Metagenomic next-generation sequencing (mNGS) can improve the rate and reduce the turnaround time for the detection of Nocardia. CASE SUMMARY: A 49-year-old man was admitted to hospital with cough and hemoptysis. Imaging revealed pulmonary consolidation as well as multiple brain lesions. Nocardia asiatica and Nocardia beijingensis were rapidly detected by mNGS of bronchoalveolar lavage fluid (BALF) while bacterial culture of BALF and pathological biopsy of lung tissue were negative. In early stages, he was treated with trimethoprim-sulfamethoxazole (TMP-SMZ) and linezolid by individual dose adjustment based on serum concentrations and the adverse effects of thrombocytopenia and leukopenia. The treatment was then replaced by TMP-SMZ and ceftriaxone or minocycline. He was treated with 8 mo of parenteral and/or oral antibiotics, and obvious clinical improvement was achieved with resolution of pulmonary and brain lesions on repeat imaging. CONCLUSION: mNGS provided fast and precise pathogen detection of Nocardia. In disseminated nocardiosis, linezolid is an important alternative that can give a better outcome with the monitoring of linezolid serum concentrations and platelet count.

Up-regulation of METCAM/MUC18 promotes motility, invasion, and tumorigenesis of human breast cancer cells.

BACKGROUND: Conflicting research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as both a tumor promoter and a tumor suppressor in the development of breast cancer. To resolve this, we have re-investigated the role of this CAM in the progression of human breast cancer cells. METHODS: Three breast cancer cell lines were used for the tests: one luminal-like breast cancer cell line, MCF7, which did not express any METCAM/MUC18, and two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which expressed moderate levels of the protein.MCF7 cells were transfected with the human METCAM/MUC18 cDNA to obtain G418-resistant clones which expressed the protein and were used for testing effects of human METCAM/MUC18 expression on in vitro motility and invasiveness, and in vitro and in vivo tumorigenesis. Both MDA-MB-231 and MDA-MB-468 cells already expressed METCAM/MUC18. They were directly used for in vitro tests in the presence and absence of an anti-METCAM/MUC18 antibody. RESULTS: In MCF7 cells, enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, anchorage-independent colony formation (in vitro tumorigenesis), and in vivo tumorigenesis. In both MDA-MB-231 and MDA-MB-468 cells, the anti-METCAM/MUC18 antibody inhibited both motility and invasiveness. Though both MDA-MB-231 and MDA-MB-468 cells established a disorganized growth in 3D basement membrane culture assay, the introduction of the anti-METCAM/MUC18 antibody completely destroyed their growth in the 3D culture. CONCLUSION: These findings support the notion that human METCAM/MUC18 expression promotes the progression of human breast cancer cells by increasing their motility, invasiveness and tumorigenesis.

[Production of connective tissue growth factor by angiotensin II in human embryonic lung fibroblast via RhoA-ROCK pathway].

OBJECTIVE: To explore the production of connective tissue growth factor (CTGF) by Angiotensin II (AngII) in human embryonic lung fibroblast via the RhoA-ROCK pathway. METHODS: Human embryonic lung fibroblast (HFL-1) was divided into 4 groups: (1) control group: no stimulation; (2) AngII group: stimulation of AngII (10(-7) mol/L) ; (3) Irbesartan plus AngII group: stimulation by AngII (10(-7) mol/L) with AT-1 receptor antagonist irbesartan (10(-6) mol/L) pre-treatment; (4) Irbesartan plus AngII group: stimulation by AngII (10(-7) mol/L) with ROCK inhibitor Y27632 (10(-6) mol/L) pre-treatment. Then the products of protein and RNA were collected. Western blot and QuantiGene were used to detect the activation of RhoA-Rock pathway and CTGF. RESULTS: Exploring the affect of irbesartan on AngII through the Western blot analysis of CTGF and RhoA protein expression: the CTGF level was up-regulated by AngII (0.89 ± 0.05 vs control 0.48 ± 0.10, P < 0.01). Such an effect was markedly blocked by a pretreatment of irbesartan (0.72 ± 0.05, P < 0.05). After the use of AngII, the expression of RhoA protein was significantly enhanced (3.40 ± 0.46 vs control 1.77 ± 0.37, P < 0.01) and blunted by a pretreatment of irbesartan (2.27 ± 0.45, P < 0.05). The Western blot analysis of CTGF protein expression showed that AngII caused a robust increase in CTGF (0.62 ± 0.15 vs control 0.16 ± 0.05, P < 0.01). Such an effect was markedly blocked by a pretreatment of Y27632 (0.17 ± 0.04, P < 0.01). The result was similar at the gene level. AngII significantly increased the expression of CTGF mRNA (1.16 ± 0.06 vs control 1.00 ± 0.01, P < 0.01). And it was markedly blocked by a pretreatment of irbesartan (0.99 ± 0.07, P < 0.01) or Y27632 (1.04 ± 0.08, P < 0.05). AngII significantly increased the expression of RhoA mRNA (1.21 ± 0.07 vs control 1.00 ± 0.06, P < 0.01). And it was markedly blocked by a pretreatment of irbesartan (1.00 ± 0.12, P < 0.05) but not Y27632 (1.10 ± 0.05, P > 0.05). CONCLUSION: Ang II activates HFL-1 to produce CTGF through the AT-1 receptor. And the RhoA-Rock pathway is involved.

Differential proteomic analysis of children infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) infection is the main cause of lower respiratory tract infection in children. However, there is no effective treatment for RSV infection. Here, we aimed to identify potential biomarkers to aid in the treatment of RSV infection. Children in the acute and convalescence phases of RSV infection were recruited and proteomic analysis was performed to identify differentially expressed proteins (DEPs). Subsequently, promising candidate proteins were determined by functional enrichment and protein-protein interaction network analysis, and underwent further validation by western blot both in clinical and mouse model samples. Among the 79 DEPs identified in RSV patient samples, 4 proteins (BPGM, TPI1, PRDX2, and CFL1) were confirmed to be significantly upregulated during RSV infection. Functional analysis showed that BPGM and TPI1 were mainly involved in glycolysis, indicating an association between RSV infection and the glycolysis metabolic pathway. Our findings provide insights into the proteomic profile during RSV infection and indicated that BPGM, TPI1, PRDX2, and CFL1 may be potential therapeutic biomarkers or targets for the treatment of RSV infection.

[Lung cancer in pregnancy: report of two cases and review of literature].

OBJECTIVE: to raise awareness about lung cancer in pregnancy. METHODS: the clinical presentations, diagnosis and treatment of 2 cases of lung cancer in pregnancy were reported, and related literatures were reviewed. RESULTS: The first case was a 31-year-old pregnant woman at 34(th)-week gestation, who presented with right sided pleural effusion on a Chest X-ray film. A boy was delivered by Cesarean section at 35 weeks of gestation. Biopsy of the right-supraclavicular lymph node was performed simultaneously, and histopathological examination showed metastatic large cell lung cancer. Her respiratory condition worsened after the Caesarian section, and so mechanical ventilation, antibiotics and gefitinib were administered, but the treatment failed. She died on the 28(th) day after Caesarian section. The second case was a 28-year-old pregnant woman at the 27 week of gestation. PET showed right lung cancer with metastases to the pericardium, right pleura, liver and pelvic cavity. Bronchoscopic biopsy showed small-cell lung cancer. After pregnancy termination, the patient received 2 cycles of chemotherapy consisting of cisplatin and etoposide. The size of lesions decreased and the patient returned to the local hospital. CONCLUSIONS: lung cancer in pregnancy is a rare condition with poor prognosis. To improve the prognosis and prevent the metastasis to the fetus, systemic therapy should be considered, and meanwhile maternal advantage must be always weighed against possible embryo-fetal risks.

[Effect of ribavirin upon secretion of thymic stromal lymphopoietin in respiratory syncytial virus-induced asthma exacerbation of mice].

OBJECTIVE: To investigate the role of ribavirin (RIB) in treating respiratory syncytial virus (RSV)-induced asthma exacerbation of mice. METHODS: (1) Cell experiment: 32 flasks of human airway epithelial cell 16HBEs were randomly divided into four groups: RSV group, RSV/RIB group, RIB group and control group. 16HBEs were infected with RSV at a multiplicity of infection (MOI) of 2. RIB 50 microg/ml was added in culture medium and Western blot used to detect the production of thymic stromal lymphopoietin (TSLP) protein; (2) Animal experiment: 32 female BALB/c mice were randomly divided into four groups: Ovalbumin (OVA) group, OVA/RSV group, OVA/RSV/RIB group and control group. Mice were sensitized by OVA and stimulated with nebulized OVA. RSV was inoculated into murine nasal cavity and RIB 10 mg/kg intramuscularly administered. BUXCO noninvasive murine lung function detection instrument was used to examine the airway response to metacholine; ELISA was used to detect IL-4, IL-5, IL-13 and IFN-gamma in murine serum and TSLP in supernatants of bronchoalveolar lavage fluid (BALF); murine lung specimens were stained with HE to observe inflammation and immunohistochemical technique was employed to observe the production of TSLP in murine airway epithelial cells. RESULTS: The cell experiment demonstrated the productions of TSLP protein in RSV group, RSV/RIB group, RIB group and control group were 1.97 +/- 0.22, 1.16 +/- 0.19, 0.99 +/- 0.17 and 0.89 +/- 0.08 respectively, and the production of TSLP in RSV/RIB group was lower than that in RSV group (P < 0.01). The animal experiment demonstrated that the murine airway responsiveness in RSV/OVA/RIB group was lower than that in OVA/RSV group (P < 0.01). The levels of IL-4 [(109.7 +/- 41.9) pg/ml], IL-5 [(220.8 +/- 30.9) pg/ml], IFN-gamma [(13.0 +/- 3.4) pg/ml] in murine serum and TSLP [(1945 +/- 82) pg/ml] in BALF of RSV/OVA/RIB group were significantly lower than those in OVA/RSV group [(274.2 +/- 103.7), (293.3 +/- 46.1), (30.1 +/- 5.7) and (2127 +/- 46) pg/ml respectively, all P < 0.01]; less infiltration of airway inflammatory cells in OVA/RSV/RIB group was observed than that in OVA/RSV group. Immunohistochemical staining of TSLP also showed a lower production of TSLP in airway epithelial cells of OVA/RSV/RIB group than OVA/RSV group. CONCLUSION: Ribavirin can inhibit the elevated production of TSLP after RSV infection and relieve RSV-induced asthma exacerbation in mice.

[Effect of mechanical ventilation with different tidal volume on small intestine in dogs suffering from acute lung injury].

OBJECTIVE: To compare the effect of different tidal volume (VT) on intestinal tissue in oleic acid-induced acute lung injury (ALI) dogs undergoing mechanical ventilation (MV). METHODS: ALI was induced with oleic acid in dogs. While all of them were undergoing MV, they were randomized into two groups: low VT group (n=6), with VT 6 ml/kg, positive end-expiratory pressure (PEEP) 10 cm H2O (1 cm H2O= 0.098 kPa), and large VT group (n=6), with VT 20 ml/kg, PEEP 10 cm H2O. MV with different VT was maintained for 6 hours. After 6 hours, dogs were sacrificed by exsanguination. Pathological changes in small bowel tissues were observed with hematoxylin and eosin (HE) staining, and cellular apoptosis detected with terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) in situ apoptosis determination. RESULTS: After 6-hour MV, the degree of flatulence was severe in the large VT group, but no flatulence was observed in the low VT group. Bowel injury score was lower in the low VT group than in large VT group [(3.17+/-0.75) scores vs. (2.00+/-0.89) scores]. There was significant difference between two groups (P<0.01). Apoptosis-positive cells were rare in the small bowel tissues in both groups. CONCLUSION: MV with large VT can induce bowel dysfunction, in contrast low VT MV does not produce small bowel dysfunction.

[Disturbed shear stress affects disposition of caveolin-1 in endothelial cells.].

OBJECTIVE: To observe the distribution and expression changes of caveolin-1 under shear stress in a turbulence flow channel model mimicking susceptible sites of atherosclerosis in vitro. METHODS: Human umbilical vein endothelial cells (HUVECs) were inoculated to flow channel and then exposed to disturbed shear stress for 6, 12, 24 and 48 hours, respectively. HUVECs inoculated to flow channel without shear stress served as controls. Immunofluorescence staining was performed with SABC-FITC to detect the caveolin-1 distribution and RT-PCR was used to determine the mRNA expression of caveolin-1 gene in these cells. RESULTS: Confocal microcopy revealed ellipse-like shaped HUVECs under disturbed shear stress and caveolin-1 didn't move toward to the upstream edge of HUVECs but moved toward to the edge of HUVECs, and expression of caveolin-1 gene in HUVECs was significantly downregulated after exposure to shear stress for 48 hours compared to HUVECs without shear stress stimulation. CONCLUSION: Disturbed shear stress affected disposition and expression of caveolin-1 which might be responsible for shear stress induced endothelial cell pathological atherosclerotic changes.

Characteristics of PLGA-gelatin complex as potential artificial nerve scaffold.

The segmentation lesion of peripheral nerve will seriously impair the motion and sensation of the patients, and the satisfactory recovery of segmented peripheral nerve by autograft or allograft is still a great challenge posing to the neurosurgery. Apart from autograft for nerve repair, different allograft has been studying. In this study, a scaffold fabricated with polylactic acid-co-glycolic acid (PLGA) copolymer and gelatin was evaluated to be a potential artificial nerve scaffold in vitro. The effect of different mass ratio between PLGA and gelatin upon the characteristics of PLGA-gelatin scaffolds such as microstructure, mechanical property, degradation behavior in PBS, cell adhesion property were investigated. The results showed the homogeneity and mechanical property of the scaffolds became poor with the increase of gelatin, and the rate of max water-uptake and the mass loss of scaffolds increases with the increase of gelatin, and the cells could adhere to the scaffolds. Those indicated the scaffolds fabricated by the PLGA-gelatin complex had excellent biocompatibility, suitable mechanical property and sustained-release characteristics, which would meet the requirements for artificial nerve scaffold.

[Role of epidermal growth factor receptor in house dust mite-induced airway epithelial barrier dysfunction].

OBJECTIVE: To investigate the role of epidermal growth factor receptor (EGFR) signaling pathway in bronchial epithelial actin stress fiber (F-actin) rearrangement induced by house dust mite (HDM). METHODS: Normal human bronchial epithelial cells (16HBE) were stimulated with HDM with or without pretreatment with AG-1478, an EGFR inhibitor. The levels of phospho(p)-EGFR, F-actin, E-cadherin and ß-catenin in the cell cultures were detected with Western blotting. The localizations of F-actin, E-cadherin and ß-catenin in the bronchial epithelial cells were determined with immunofluorescence assay, and the transmembrane electrical resistance (TER) and FITC-dextran flux (FITC-DX) in the cells were measured to assess the barrier function of the bronchial epithelia. RESULTS: HDM stimulation of the cells for 10 min resulted in significantly increased p-EGFR expression (P<0.05) without causing obvious changes in the expression of E-cadherin (P>0.05) or ß-catenin (P>0.05). Immunofluorescence assay revealed delocalization of E-cadherin and ß-catenin in HDM-treated 16HBE cells, shown by their diffusion from the cell membrane to the cytoplasm. In HDM-treated cells, the TER was significantly decreased to (70.00∓4.33)% and the FITC-DX was significantly increased to (115.98∓4.34)%; Inhibition of EGFR reversed the delocalization of E-cadherin and ß-catenin, improved the TER to (90.00∓3.75)% and lowered the FITC-DX to (101.10∓2.10)%. HDM induced increased expression and rearrangement of F-actin, which was obviously inhibited by pretreatment of the cells with AG-1478 (P<0.05). CONCLUSION: EGFR signaling pathway mediates HDM-induced F-actin rearrangement in human bronchial epithelial cells to contribute to epithelial barrier dysfunction.

[Receptor for advanced glycation end products upregulates MUC5AC expression and promotes mucus overproduction in mice with toluene diisocyanate-induced asthma].

OBJECTIVE: To explore the role of the receptor for advanced glycation end products (RAGE) in regulating the expression of MUC5AC and mucus production in a mouse model of toluene diisocyanate (TDI)?induced asthma. METHODS: BALB/c mice were randomly divided into control group, vehicle (AOO) group, TDI?induced asthma group and RAGE inhibitor (FPS?ZM1) group. PAS staining, Western blotting, and immunohistochemistry were used to analyze the changes in mucus production and MUC5AC expression in the airway of the mice, and the expression of p?ERK was detected with Western blotting. In vitro cultured human bronchial epithelial cell line 16HBE was transfected with lentiviral vector carrying short hairpin RNA targeting RAGE (shRNA?RAGE) and subsequently challenged with a TDI?human serum albumin (TDI-HSA) conjugate, and the changes in cellular MUC5AC mRNA expression as detected using RT-PCR; the protein expressions of ERK and p?ERK in the cells were examined with Western blotting. The effect of ERK inhibitor U0126 pretreatment on MUC5AC mRNA expression was also analyzed in the cells. RESULTS: Compared with the control mice, TDI-induced asthmatic mice showed significantly higher rates of PAS positivity and increased MUC5AC and p?ERK expressions in the airway (P<0.05). Treatment with FPS?ZM1 significantly decreased PAS positivity and lowered MUC5AC and p?ERK expressions in the airway of the asthmatic mice (P<0.05). Exposure of 16HBE cells to TDI?HSA caused a significant increase in MUC5AC mRNA expression and p?ERK protein expression (P<0.05), while RAGE knockdown obviously suppressed TDI?HSA-induced upregulation of p-ERK and MUC5AC mRNA (P<0.05). Treatment with the ERK inhibitor U0126 also lowered TDI?HSA?induced up?regulation of MUC5AC mRNA in the cells (P<0.05). CONCLUSION: RAGE signaling induces MUC5AC expression via extracellular signal-regulated kinase pathway to promote mucus overproduction in mice with TDI-induced asthma.

Effects of serum of the rats ventilated with high tidal volume on endothelial cell permeability and therapeutic effects of ulinastatin.

BACKGROUND: With the widespread use of ventilators in treating critically ill patients, the morbidity of ventilator-induced lung injury (VILI) is increasing accordingly. VILI is characterized by a considerable increase in microvascular leakiness and activation of inflammatory processes. In this study we investigated the effects of inflammatory mediators in VILI rat serum on endothelial cytoskeleton and monolayer cellular permeability, as well as the therapeutic effect of ulinastatin, to explore the pathogenesis and the relationship between biotrauma and lung oedema induced by VILI. METHODS: Thirty healthy male Sprague-Dawley rats were randomly divided into three groups: group A (normal tidal volume ventilation), group B (high tidal volume ventilation) and group C (high tidal volume ventilation plus ulinastatin). The serum of each rat after ventilation was added to endothelial cell line ECV-304 medium for two hours to observe the effects of serum and/or ulinastatin on endothelial fibrous actin and permeability. RESULTS: Compared to rats ventilated with normal tidal volume, serum of rats ventilated with high tidal volume caused a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity at the peripheral filament bands and formation of the long and thick stress fibres in the centre resulting in endothelial contraction and higher permeability. Prior treatment with ulinastatin lessened the above changes significantly. The changes of permeability coefficient of endothelial permeability after group A, B or C rats serum stimulation were (6.95 +/- 1.66)%, (27.50 +/- 7.77)% and (17.71 +/- 4.66)% respectively with statistically significant differences (P < 0.05) among the three groups. CONCLUSIONS: The proinflammatory mediators in the serum of the rats given high tidal volume ventilation increases endothelial permeability by reorganizing actin cytoskeleton, and pretreatment with ulinastatin lessens the permeability by inhibiting of proinflammatory mediators.