BackgroundThough the possibility of using malignant pleural effusions (MPEs) as alternatives for metastatic pleural tumor tissues (MPTTs) in epidermal growth factor receptor (EGFR) mutation test has been examined, due to the lack of studies comparing the results in matching MPEs and MPTTs, the clinical value of MPEs for advanced adenocarcinoma patients with pleural effusions is not confirmed.MethodsEGFR mutation statuses in matching MPTTs, MPE supernatants and cell blocks, of 41 patients with advanced lung adenocarcinoma as diagnosed by thoracoscopy were analyzed using amplification refractory mutation system (ARMS).ResultsEGFR mutations were detected in 46.3% (19/41) of MPTTs, 43.9% (18/41) of MPE supernatants and 56.3% (18/32) of MPE cell blocks by ARMS analysis. Generally, the same EGFR statuses were identified in both MPTTs and matching MPE cell blocks of 81.3% patients (26/32), whereas MPTTs and matching MPE supernatants of 87.8% (36/41) patients shared the same EGFR status. Compared with EGFR mutation detection in MPTTs, the sensitivity of EGFR mutation detection in MPE-cell blocks was 87.5% (14/16), specificity was 75.0% (12/16), while the sensitivity of EGFR mutation detection in MPE-supernatants was 84.2% (16/19), specificity was 90.9% (20/22).ConclusionsThe high concordance of EGFR mutation statuses between MPEs and MPTTs in lung adenocarcinoma patients with pleural metastasis as determined by ARMS analysis suggests that MPEs, particularly MPE supernatants, may be substitutes for MPTTs in EGFR mutation test.

Intrahepatic cholangiocarcinoma (ICC) is a rare and highly aggressive malignancy. In this study, we identified the presence of gene deletion and missense mutation leading to inactivation or underexpression of liver kinase B1 (LKB1) tumor suppressor and excluded the involvement of LKB1 gene hypermethylation in ICC tissues. Immunohistochemical analysis showed that LKB1 was underexpressed in a portion of 326 ICC tissues compared to their adjacent normal tissues. By statistical analysis underexpression of LKB1 in ICC tissues significantly correlated with poor survival and malignant disease characteristics in ICC patients. Moreover, we showed that knockdown of LKB1 significantly enhanced growth, migration, and invasion of three LKB1-competent ICC cell lines. Global transcriptional profiling analysis identified multiple malignancy-promoting genes, such as HIF-1α, CD24, Talin1, Vinculin, Wnt5, and signaling pathways including Hedgehog, Wnt/β-catenin, and cell adhesion as novel targets of LKB1 underexpression in ICC cells. Furthermore, knockdown of LKB1 gene expression dramatically enhanced Wnt/β-catenin signaling in ICC cells, while an inverse correlation between LKB1 and nuclear β-catenin was observed in ICC tissues. Our findings suggest a novel mechanism for ICC carcinogenesis in which LKB1 underexpression enhances multiple signaling pathways including Wnt/β-catenin to promote disease progression.

Our study provides a historical perspective on the progress of research on asthma. Studies conducted in well-developed European countries and North America, published in high-impact journals had the highest citations.

Asthma is the most common chronic disease and is characterized by airway remodeling and chronic inflammation. Increasingly, studies have found that the activation and M1 phenotypic transformation of macrophages play important roles in asthma progress, including airway remodeling. However, the reversal of M1 macrophages to the M2 phenotype has been shown to attenuate airway remodeling. Exosomes are nano-sized extracellular vesicles derived from endosomes; they play direct roles in governing physiological and pathological conditions by the intracellular transfer of bioactive cargo, such as proteins, enzymes, nucleic acids (microRNA [miRNA], mRNA, DNA), and metabolites. However, transfer mechanisms are unclear. To uncover potential therapeutic mechanisms, we constructed an ovalbumin-induced asthma mouse model and lipopolysaccharide-induced RAW264.7 macrophages cells. Highthroughput sequencing showed that mmu_circ_0001359 was downregulated in asthmatic mice when compared with normal mice. Adipose-derived stem cell (ADSC)-exosome treatment suppressed inflammatory cytokine expression by the conversion of M1 macrophages to the M2 phenotype, under lipopolysaccharide-induced conditions. Exosomes from mmu_circ_0001359 overexpression in ADSCs increased therapeutic effects, in terms of cytokine expression, when compared with wild-type exosomes. Luciferase reporter assays confirmed that exosomes from mmu_circ_0001359-modified ADSCs attenuated airway remodeling by enhancing FoxO1 signalingmediated M2-like macrophage activation, via sponging miR-183-5p. In conclusion, mmu_circ_0001359-enriched exosomes attenuated airway remodeling by promoting M2-like macrophages.

Salmeterol is a long-acting b2-agonist that activates adenylate cyclase, causing long-lasting bronchodilation and has been used for many years to control asthma. However, little information is available about the immunoregulatory effects of salmeterol. We found that salmeterol decreases the production of pro-inflammatory cytokines in a model of allergen-challenged mice that expressed tumor-necrosis factor-alpha, interleukin-1 and interleukin-6. Dendritic cells (DCs) are antigen-presenting cells and act as sentinels in the airway. We found that salmeterol (10 25 mol/l) reduced the inflammation caused by lipopolysaccharide (0.1 mg/ml) in activated murine bone marrow-derived DCs. Moreover, western blots demonstrated that this protective effect was mediated partially by inhibiting signaling through the nuclear factor-kappa B (NF-kB), mitogen-activated protein kinase (MAPK) pathways and dramatically decreased levels of p-ERK. We suggest that salmeterol regulates the inflammation of allergen-induced asthma by modulating DCs. In conclusion, we provide evidence that DCs are the target immune cells responsible for the action of salmeterol against asthma.

Copper is an essential trace metal, but imbalance in copper homeostasis can induce oxidative damage. Inflammation is a fundamental element of various pulmonary diseases. Although a positive relationship between copper and chronic pulmonary diseases has been reported, the underlying reasons are still not clear. The copper level in the sputum of patients with various pulmonary diseases was measured. An inflammatory model was established to evaluate the impact of inflammation on copper uptake in the lung. We found that the level of sputum copper was increased in patients with various pulmonary diseases, especially chronic obstructive pulmonary disease and asthma. Then, we confirmed that mice with pulmonary inflammation were susceptible to copper‐mediated oxidative damage because of copper overload in lung tissue. Further investigation demonstrated that interleukin (IL)‐17 and tumor necrosis factor (TNF)‐α exerted synergistic effects in airway epithelial cells by upregulating the expression of six‐transmembrane epithelial antigens of prostate 4 (STEAP4), a metalloreductase that reduces extracellular copper ions from the cupric state to the cuprous state and facilitates copper uptake. Inhibition of STEAP4 decreased the copper uptake of cells and inhibited copper‐mediated oxidative damage. Moreover, we demonstrated that the upregulation of STEAP4 by IL‐17 and TNF‐α was largely dependent on TNF receptor‐associated factor 4 (TRAF4). Traf4−/− mice were resistant to copper‐mediated oxidative damage. Our data suggest a novel IL‐17/TNF‐α–TRAF4–STEAP4 axis that regulates copper homeostasis.

Abstract. Gallbladder cancer (GBC) is a rare disease associated with an extremely poor patient prognosis, and occasionally, aberrant expression of p53 is present. Considering that p53 is one of the most widely studied tumor-suppressor genes, we used a cell-penetrating peptide, 11R, to enhance the transferring efficiency of the oncolytic adenovirus carrying the p53 gene by constructing SG7605-11R-p53, a gene-viral therapy system which has higher specificity, enhanced safety, and efficacy. After infection with SG7605-11R-p53 at a multiplicity of infection (MOI) of 1 PFU/cell in vitro, the survival rate of EH-GB1 cells was lower than 50%, and that of EH-GB2 cells was lower than 40%, while the survival rate was higher than 90% for BJ human fibroblast cells, demonstrating that SG7605-11R-p53 has potent specific cytotoxicity against GBC cells. The tumor growth was greatly inhibited in nude mice bearing EH-GB2 xenografts when the total dose of SG7605-11R-p53 was 1x10 9 PFU, and terminal dUTP nick end-labeling (TUNEL) revealed that the apoptotic rate of cancer cells was 66.75±6.702%. Compared with existing gene therapy with longstanding shortcomings, our new system offers an additional option for patients with advanced GBC and other cancers who may not be suitable for chemotherapy, radiotherapy or who are not indicated for surgical treatment. IntroductionGallbladder cancer (GBC) is a rare but highly lethal disease, known to humans since 1777 (1,2). It is now recognized as one of the most common biliary tract malignancies (3). Up to 75% of such patients suffer from this unresectable disease at the time of surgical evaluation (4).Currently available treatments such as chemotherapy and radiotherapy have little effect on advanced GBC, and the 5-year survival rate is only ~5% in such cases (5). Therefore, development of new treatment modalities, such as gene therapy, merits high priority. Yet, few studies are available concerning GBCs.Gene therapy has been widely accepted as an important strategy for treating malignancies (6). Yet, the clinical application of tumor gene therapy still has some limitations, such as low gene transferring efficiency, poor transgene expression, and limited target specificity to tumors (7). Among the possible candidate genes is the gene that codes for the p53 protein, whose product is mainly enriched around the nucleoli of normal cells and can specifically bind with DNA. The protein activity is regulated by phosphorylation and is readily degraded (8,9). In contrast, the p53 protein loses its function quite readily due to its gene mutation, especially on its 393 amino acid residue (10).The wt-p53 gene has been a popular candidate for gene replacement therapy since it suppresses tumor growth in various types of solid tumors (11)(12)(13)(14)(15). Mutation in the p53 gene may cause various types of cancers. Researchers have also proven that the growth of tumors with p53 gene mutation may be inhibited even in the late stages, and the function of p53 may still be recovered by clinical therapy. As a resu...

BackgroundIntratumoral heterogeneity reflects subclonal diversity and accounts for a variety of clinically defined phenotypes including the development of drug resistance and recurrence. However, intratumoral heterogeneity of bile duct carcinoma (BDC) is rarely studied.MethodsTwo highly heterogeneous cell lines named EH-CA1a and EH-CA1b were established from a primary tumor tissue of a pathologically proven BDC. Distinct heterogeneity and underlying mechanisms of two cell lines in karyotype, colony formation, tumorgenicity, and sensitivity to chemoradiotherapy were intensively studied.ResultsBoth cell lines showed typical morphology of cancer cells. EH-CA1a cells grew as free-floating aggregates, while EH-CA1b cells grew adherently as a monolayer. EH-CA1a cells had higher cloning efficiencies and were able to keep proliferating under hypoxic condition. Coincidentally, hypoxia-induced factor-1α (HIF1α) and vascular endothelial growth factor (VEGF) mRNA were significantly higher in EH-CA1a cells than in EH-CA1b cells. Both cell lines were tumorigenic in nude mouse, however, EH-CA1a cells showed more aggressive characteristics. Most importantly, the EH-CA1a cells showed much more resistance against radiation and chemotherapy with gemcitabine. Metastasis-related genes including matrix metalloproteinase 2 (MMP-2), MMP-9, epithelial-mesenchymal transition (EMT) markers such as Vimentin, Snail, and Twist, are more highly expressed in EH-CA1a cells than in EH-CA1b cells. Moreover, the percentage of cells expressing cancer stem cell-like marker, CD133, in EH-CA1a cells is much higher than that in EH-CA1b cells. Moreover, knockdown of CD133 in both EH-CA1a and EH-CA1b cells significantly reduced their invasive potential and increased their sensitivities to radiation and gemcitabine, suggesting the differential expression of CD133 protein may partially account for the difference in malignancy between these two cancer cells.ConclusionEstablishment of these two cell lines will not only shed light on intratumoral heterogeneities of BDC, but also potentially facilitate the development of novel therapeutic approaches of BDC.