Altered Expression of Substance P and NK1R in CCR3+ and CD123+HLA-DR- Basophils Under Airway Allergic Conditions.

PURPOSE: To explore expression of SP and NK1R in basophils of allergic asthma (AA), allergic rhinitis (AR) and AR combined with AA (ARA), and influence of allergens and immunoglobulin E (IgE) mediated mechanisms on SP and NK1R expression. METHODS: Expression of SP and NK1R was detected by flow cytometry, NK1R mRNA expression was detected by real time quantitative polymerase chain reaction (qPCR), and mouse AR and AA models were employed for in vivo study. RESULTS: SP+ and NK1R+ cells increased in CCR3+ and CD123+HLA-DR- granulocytes of AA. PPE elevated proportions of SP+ cells in CCR3+ and CD123+HLA-DR- granulocytes, whereas ASWE and HDME augmented SP+ cells in CD123+HLA-DR- granulocytes of AR and ARA patients. ASWE, HDME and PPE increased proportions of NK1R+ cells in CCR3+ PBMC and CD123+HLA-DR- granulocytes of AR patients. OVA, Der p1, IL-33, IL-37, IgE and SP enhanced NK1R expression on KU812 cells. NK1R expressing basophils were increased in blood of OVA sensitized and challenged AR and AA mice. FcεRI-KO AA mice seemed to have less NK1R+ basophils than WT AA mice in their blood. CONCLUSION: CCR3+ and CD123+HLA-DR- cells are likely involved in AA and AR via SP and NK1R. IgE-related mechanism may participate in upregulation of NK1R expression.

Long non-coding RNA NKILA alleviates airway inflammation in asthmatic mice by promoting M2 macrophage polarization and inhibiting the NF-κB pathway.

Asthma remains a severe public health problem. Long non-coding RNAs (lncRNAs) are potent regulators in various diseases including asthma. This study investigated the mechanism of lncRNA NF-κB interacting lncRNA (NKILA) in asthma. The model of asthma in mice was induced by ovalbum (OVA). LncRNA NKILA expression, serum total IgE level and expressions of inflammatory cytokines (IL-4, IL-5, IL-13, and TNF-α) in OVA-induced asthmatic mice were detected. NKILA was overexpressed to evaluate the airway inflammation and airway hyperresponsiveness (AHR) in asthmatic mice. Macrophage abundance, M1/M2-polarized macrophage numbers, and expressions of macrophage polarization-related genes were detected. Levels of the NF-κB pathway-related proteins were determined. Downregulated NKILA and upregulated total IgE level and expressions of inflammatory cytokines were observed in asthmatic mice. NKILA overexpression alleviated AHR and airway inflammation in asthmatic mice. NKILA reduced macrophage abundance and promoted M2 macrophage polarization in asthmatic mice. NKILA inhibited the NF-κB pathway in asthmatic mice. We highlighted that lncRNA NKILA limited the asthmatic airway inflammation via promoting M2 macrophage polarization and inhibiting the NF-κB pathway.

lncRNA H19 promotes viability and epithelial-mesenchymal transition of lung adenocarcinoma cells by targeting miR-29b-3p and modifying STAT3.

Considering the joint contribution of long non­coding RNAs (lncRNAs) and microRNAs (miRNAs/miRs) to tumorigenesis, the aim of the present study was to investigate whether and how lncRNA H19 targets miR­29b­3p to affect the progression of lung adenocarcinoma by the modulation of signal transducer and activator of transcription 3 (STAT3). A total of 305 lung adenocarcinoma tissues and four human lung adenocarcinoma cell lines (i.e. Calu­3, NCI­H1975, A549 and NCI­H23) were used. pcDNA3.1­H19, short interfering RNA (si­)H19, miR­29b­3p mimic, miR­29b­3p inhibitor and negative control (NC) were transfected into the cells, and the proliferation, viability and apoptosis of the cells were determined using a Cell Counting Kit­8 assay, colony formation assay and flow cytometry, respectively. The results indicated that highly expressed H19 and poorly expressed miR­29b­3p could serve as predictors for the poor prognosis of lung adenocarcinoma patients. Additionally, si­H19 and miR­29b­3p mimic significantly increased the apoptosis of lung adenocarcinoma cells, and decreased the survival rate and viability of cells. Simultaneously, expression of epithelial­mesenchymal transition (EMT)­specific proteins was significantly altered, i.e. increased epithelial cadherin expression, as well as decreased vimentin, Snail and Slug expression. Furthermore, miR­29b­3p was verified to be targeted and regulated by H19, and STAT3 was targeted and modified by miR­29b­3p. Ultimately, STAT3 was identified to decrease lung adenocarcinoma cell viability, survival, apoptosis and EMT imposed by miR­29b­3p. In conclusion, the results of the present study indicated that lncRNA H19/miR­29b­3p/STAT3 signaling was involved in the development of lung adenocarcinoma, which may be critical for developing effective diagnostic and treatment strategies for lung adenocarcinoma.

Silencing of PINK1 represses cell growth, migration and induces apoptosis of lung cancer cells.

PTEN induced putative kinase 1 (PINK1) has been found to be up-regulated, which promotes the proliferation and chemoresistance in lung cancer. Nevertheless, the role and detailed mechnisms of PINK1 in lung cancer have not been fully understood, which need to be further clarified. In this study, the resluts showed that silencing of PINK1 inhibited proliferation and blocked cell cycle of lung cancer cells. Furthermore, the apoptosis rate was enhanced by PINK1 suppression, as evidenced by increased protein levels of Bax, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP), and decreased level of Bcl-2. The migration and invasion abilities were also restrained by PINK1 silencing. Silencing of PINK1 also resulted in oxygen species (ROS) overproduction and decreased mitochondrial membrane potential. Finally, suppression of PINK1 repressed the growth of xenograft tumor and induced apoptosis in tumor tissues in vivo. This study might lead to PINK1 kinase as a novel therapeutic target for lung cancer treatment.

Naringin attenuates diabetic retinopathy by inhibiting inflammation, oxidative stress and NF-κB activation in vivo and in vitro.

OBJECTIVES: Naringin, an essential flavonoid, inhibits inflammatory response and oxidative stress in diabetes. However, whether naringin has beneficial effects on diabetic retinopathy (DR) remains unknown. MATERIALS AND METHODS: Streptozotocin (STZ, 65 mg/kg) was intraperitoneally injected into male rats (8 weeks old weighting 200-250 g) to establish diabetic model, then naringin (20, 40 or 80 mg/kg/day) was intraperitoneally injected into the diabetic rats for twelve weeks. Glial fibrillary acidic protein (GFAP) level, thickness of ganglion cell layer (GCL) and ganglion cell counts were assessed in diabetic retina in vivo. Naringin (50 µM) that significantly inhibited high glucose (HG, 25 mM)-induced cell proliferation was used to treat rat Muller cell line (rMC1) in vitro. Inflammatory response, oxidative stress and activation of nuclear factor kappa B (NF-κB) p65 were evaluated in retina in vivo and in rMC1 cells in vitro. RESULTS: Naringin alleviated DR symptoms as evidenced by the increased retinal ganglion cells and decreased GFAP level in rat retina. Naringin exhibited anti-inflammatory and antioxidative effects as confirmed by the down-regulated pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), and the up-regulated antioxidants, glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) in DR rats. Moreover, we found that naringin inhibited HG-induced proliferation, abnormal inflammatory response and oxidative stress in rMC1 cells. In addition, the enhanced nuclear translocation of NF-κB p65 in diabetic rat retina and HG-induced rMC1 cells was suppressed by naringin. CONCLUSION: Naringin attenuates inflammatory response, oxidative stress and NF-κB activation in experimental models of DR.

[Elevated IL-18, IL-18 binding protein and IL-18 receptor in eosinophil-enriched blood cells in patients with asthma].

Objective To investigate the changes of interleukin-18 (IL-18), interleukin 18 binding protein (IL-18BP) and interleukin 18 receptor (IL-18R) expressions in eosinophil-enriched cells from asthmatic patients, and study the correlations among them. Methods Peripheral venous blood from asthmatic patients and normal subjects were collected and stimulated with the extracts of Artemisia pollen, dust mite, and Platanus pollen. The expressions of IL-18, IL-18BP and IL-18R in eosinophil-enriched blood cells were detected by flow cytometry, and the correlations among them were analyzed. Results The proportion of IL-18+ cells in eosinophil-enriched cells from asthmatic patients increased 15-fold compared with normal subjects. The proportion of IL-18+ cells increased about 1.3-fold and 1.5-fold in eosinophil-enriched cells from asthmatic patients whose blood was stimulated with dust mite and Platanus pollen extracts, respectively. The average fluorescence intensity (MFI) of IL-18BP+ cells increased about 1.5-fold and the proportion of IL-18R+ cells increased about 2-fold in eosinophil-enriched cells from asthmatic patients' blood stimulated with Platanus pollen extracts. In addition, the expressions of IL-18BP+ cells and IL-18R+ cells showed a positive correlation (r=0.639) in eosinophil-enriched cells from asthmatic patients stimulated with allergens. However, the expressions of IL-18, IL-18BP and IL-18R in eosinophil-enriched cells did not obviously change in normal subjects. Conclusion IL-18, IL-18R and IL-18BP expressed in eosinophils may be involved in the inflammatory reaction of asthma.

The significant effect of chronic intermittent hypoxia on prostaglandin D2 biosynthesis in rat brain.

Obstructive sleep apnea (OSA) is a common disorder characterized by chronic intermittent hypoxia (CIH). Excessive daytime sleepiness (EDS) is one of severe complications frequently associated with OSA. Lipocalin-type prostaglandin synthase (L-PGDS) is potentially responsible for the production of prostaglandin D2 (PGD2) which is an endogenous sleep inducer. To date, whether the content of PGD2 and PGDS is related to intermittent hypoxia has never been reported. The aim of this study was to compare the content of PGD2 and L-PGDS in rats' brains with and without intermittent hypoxia. Adult male Wistar rats (n = 48; 8-10 weeks) were averagely divided into two groups. One was control group, and the other group was exposed to IH (12 h/day for 6 weeks). In each group there are four time-points including 0, 2, 4 and 6 weeks, and six rats were killed and studied at each time-point. At the end of 0, 2, 4 and 6 weeks, the concentrations of PGD2 in brains were measured by LC-MS/MS. In addition, the expressions of L-PGDS protein and mRNA in brains were investigated by western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed the concentrations of PGD2 in CIH rat brains were higher than those in control groups from the second week. At the end of 6 weeks, the concentrations of PGD2 in CIH and control groups were 11.1 and 5.9 ng/g, respectively. The levels of L-PGDS protein and mRNA followed the same trend during the whole 6 weeks. The results will provide a new idea to explore that patients with OSA are always accompanied by excessive daytime sleepiness.

Early Growth Response Protein-1 Involves in Transforming Growth factor-ß1 Induced Epithelial-Mesenchymal Transition and Inhibits Migration of Non-Small-Cell Lung Cancer Cells.

The zinc finger transcription factor EGR1 has a role in controlling synaptic plasticity, wound repair, female reproductive capacity, inflammation, growth control, apoptosis and tumor progression. Recent studies mainly focused on its role in growth control and apoptosis, however, little is known about its role in epithelial-mesenchymal transition (EMT). Here, we aim to explore whether EGR 1 is involved in TGF-ß1-induced EMT in non-small- cell lung cancer cells. Transforming growth factor (TGF)-ß1 was utilized to induce EMT in this study. Western blotting, RT-PCR, and transwell chambers were used to identify phenotype changes. Western blotting was also used to observe changes of the expression of EGR 1. The lentivirus-mediated EGR 1 vector was used to increase EGR1 expression. We investigated the change of migration to evaluate the effect of EGR 1 on non-small-cell lung cancer cells migration by transwell chambers. After stimulating with TGF-ß1, almost all A549 cells and Luca 1 cells (Non-small-cell lung cancer primary cells) changed to mesenchymal phenotype and acquired more migration capabilities. These cells also had lower EGR 1 protein expression. Overexpression of EGR 1 gene with EGR 1 vector could decrease tumor cell migration capabilities significantly after adding TGF-ß1. These data showed an important role of EGR 1 in the EMT of non-small-cell lung cancer cells, as well as migration.

Identification and characterization of a serious multidrug resistant Stenotrophomonas maltophilia strain in China.

An S. maltophilia strain named WJ66 was isolated from a patient; WJ66 showed resistance to more antibiotics than the other S. maltophilia strains. This bacteraemia is resistant to sulphonamides, or fluoroquinolones, while the representative strain of S. maltophilia, K279a, is sensitive to both. To explore drug resistance determinants of this strain, the draft genome sequence of WJ66 was determined and compared to other S. maltophilia sequences. Genome sequencing and genome-wide evolutionary analysis revealed that WJ66 was highly homologous with the strain K279a, but strain WJ66 contained additional antibiotic resistance genes. Further analysis confirmed that strain WJ66 contained an amino acid substitution (Q83L) in fluoroquinolone target GyrA and carried a class 1 integron, with an aadA2 gene in the resistance gene cassette. Homology analysis from the pathogen-host interaction database showed that strain WJ66 lacks raxST and raxA, which is consistent with K279a. Comparative genomic analyses revealed that subtle nucleotide differences contribute to various significant phenotypes in close genetic relationship strains.

Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-ß-lactamase blaL1 in Beijing, China.

Intrinsic ß-lactam resistance in Stenotrophomonas maltophilia is caused by bla L1 and/or bla L2, a kind of metallo-ß-lactamase with a broad substrate spectrum including carbapenems. A rapid and sensitive molecular method for the detection of bla L1 in clinical samples is needed to guide therapeutic treatment. In present study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of bla L1 in clinical samples by using two methods including a chromogenic method using calcein/Mn(2+) complex and the real-time turbidity monitoring to assess the reaction. Then dissemination of L1-producing S. maltophilia was investigated from ICU patients in three top hospital in Beijing, China. The results showed that both methods detected the target DNA within 60 min under isothermal conditions (65°C). The detection limit of LAMP was 3.79 pg/µl DNA, and its sensitivity 100-fold greater than that of conventional PCR. All 21 test strains except for S. maltophilia were negative for bla L1, indicative of the high-specificity of the primers for the bla L1. A total of 22 L1-positive isolates were identified for LAMP-based surveillance of bla L1 from 105 ICU patients with clinically suspected multi-resistant infections. The sequences of these bla L1 genes were conservative with only a few sites mutated, and the strains had highly resistant to ß-lactam antibiotics. The MLST recovered that 22 strains belonged to seven different S. maltophilia sequence types (STs). Furthermore, co-occurrence of bla L1 and bla L2 genes were detected in all of isolates. Strikingly, S. maltophilia DCPS-01 was recovered to contain bla L1, bla L2, and bla NDM-1 genes, possessing an ability to hydrolyse all ß-lactams antibiotics. Our data showed the diversity types of S. maltophilia carrying bla L1 and co-occurrence of many resistant genes in the clinical strains signal an ongoing and fast evolution of S. maltophilia resulting from their wide spread in the respiratory infections, and therefore will be difficult to control.

Association between genetic polymorphisms in the ADAM33 gene and asthma risk: a meta-analysis.

The aim of this study was to evaluate the associations between the rs3918396 G>A and rs528557 C>G polymorphisms in the disinterring and metalloproteinase domain 33 (ADAM33) gene and asthma risk. We searched CISCOM, CINAHL, Web of Science, PubMed, Google Scholar, EBSCO, Cochrane Library, and CBM databases from inception through August 1st, 2013 without language restrictions. Meta-analysis was performed using the STATA 12.0 software. Crude odds ratios (ORs) with their 95% confidence intervals (95% CI) were calculated. Thirteen case-control studies were included with a total of 7104 asthma patients and 8172 healthy controls. Our meta-analysis results revealed that ADAM33 rs528557 C>G polymorphism was associated with an increased risk of asthma (all p<0.05). However, we found no correlation between the ADAM33 rs3918396 G>A polymorphism and asthma risk (all p>0.05). Subgroup analysis by ethnicity indicated that the ADAM33 rs528557 C>G polymorphism might be strongly associated with an increased risk of asthma among both Caucasian and Asian populations (All p<0.05). No significant association was found between the ADAM33 rs3918396 G>A polymorphism and the risk of asthma among the studied ethnicities (All p>0.05). The present meta-analysis suggests that the ADAM33 rs528557 C>G polymorphism may contribute to susceptibility to asthma. Thus, the ADAM33 rs528557 C>G polymorphism may be utilized as a biomarker for early diagnosis of asthma.

[Expression of high-level cephalosporinase due to mutation in the AmpC attenuator of a clinical Escherichia coli strain].

OBJECTIVE: To study the resistant phenotype of a clinical strain of Escherichia coli and to explore the effect of its attenuator mutation on AmpC expression. METHODS: A clinical strain of Escherichia coli 20022 (ECO20022) resistant to cefoxitin was isolated clinically. The phenotype was examined by three-dimensional methods, isoelectric focusing (IEF), and microdilution method. The regulator genes of ECO20022 were amplified and sequenced, and the difference between them was analyzed by BLAST method. Then the regulator genes were cloned into pCAT3-basic vector (a promoterless reporter gene vector). Microdilution method was used to detect the minimal inhibitory concentration (MIC) of chloramphenicol and ampicillin to this strain with E. coli ATCC25922 as quality control bacterium. ELISA was used to detect the content of chloramphenicol acetyl transferase (CAT). RESULTS: Compared to the standard E. coli K-12, there were four base substitutions, i.e., 22C-T, 26, 27TA-GT, and 32G-A in the attenuator region of ECO20022. Three-dimensional method showed that this strain was high AmpC-producing. IEF found that it produced three beta-lactamases with the values of PI of 5.4, 8.2, and 9.0 respectively. The beta-lactamase with the PI of 9.0 could be inhibited by cloxacillin but not by clavulanate. The strain was resistant to not only most of third generation cephalosporins, but also to cefepime; however it was still susceptible to carbapenem. The secondary structure of the attenuator RNA of ECO20022 was different from the traditional structure of E. coli K-12. The regulator gene was successfully cloned into pCAT3-basic vector and direct and indirect tests indicated that this regulator gene enhanced the CAT expressing level as much as 10 times that of Escherichia coli K-12. CONCLUSION: AmpC attenuator mutation leads to high AmpC expression in Escherichia coli, resulting in a significant rise of resistance level to beta-lactamase and a great menace to clinical antibiotic therapy.