Physical and chemical characteristics of PM2.5 and its toxicity to human bronchial cells BEAS-2B in the winter and summer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

With the increasing occurrence of haze during the summer, the physicochemical characteristics and toxicity differences in PM2.5 in different seasons are of great concern. Hangzhou is located in an area that has a subtropical monsoon climate where the humidity is very high during both the summer and winter. However, there are limited studies on the seasonal differences in PM2.5 in these weather conditions. In this test, PM2.5 samples were collected in the winter and summer, the morphology and chemical composition of PM2.5 were analyzed, the toxicity of PM2.5 to human bronchial cells BEAS-2B was compared, and the correlation between PM2.5 toxicity and the chemical composition was discussed. The results showed that during both the winter and summer, the main compounds in the PM2.5 samples were water-soluble ions, particularly SO42-, NO3-, and NH4+, followed by organic components, while heavy metals were present at lower levels. The higher the mass concentration of PM2.5, the greater its impact on cell viability and ROS levels. However, when the mass concentration of PM2.5 was similar, the water extraction from the summer samples showed a greater impact on BEAS-2B than that from the winter samples. The cytotoxicity of PM2.5 was closely associated with heavy metals and organic pollutants but less related to water-soluble ions.

Detection of bronchial function of NHBD lung following one-h warm ischemia by organ bath model / 华中科技大学学报（医学）（英德文版）

This study investigated the feasibility and effects of organ bath to be used for detection of bronchial function of non-heart-beating donor (NHBD) lung after 1-h warm ischemia. Sixteen Swedish pigs were divided into two groups randomly: heart-beating donor (HBD) group and NHBD with 1-h warm ischemia (NHBD-1 h) group. The bronchial rings whose lengths and inner diameters were both 1.5 mm were obtained from isolated left lungs of all the pigs. Acetylcholine, arachidonic acid natrium and papaverine were used to test and compare the contractile and relaxant function of bronchial smooth muscles and epithelium-dependent relaxation (EpiDR) response between HBD and NHBD-1 h groups. The results showed that there was no significant difference in the values of bronchial precontraction between HBD and NHBD-1 h groups (5.18+/-0.07 vs 5.10+/-0.11 mN, P>0.05). No significant difference in the values of EpiDR responses between HBD and NHBD-1 h groups (1.26+/-0.05 vs 1.23+/-0.07 mN, P>0.05) was observed either. During the process of EpiDR induction, the rings had no spontaneous relaxation in two groups. In addition, papaverine solution completely relaxed the bronchial smooth muscles of all bronchial rings. It was concluded that after warm ischemia for 1 h, the contractile and relaxant abilities of bronchial smooth muscles, and the epithelium-dependent adjustment both kept intact. Organ bath model could be a liable and scientific way to evaluate the bronchial function of NHBD lung.

The Effect of Interleukin-4 and Amphiregulin on the Proliferation of Human Airway Smooth Muscle Cells and Cytokine Release

Airway smooth muscle (ASM) hyperplasia and angiogenesis are important features associated with airway remodeling. We investigated the effect of IL-4 and amphiregulin, an epidermal growth factor family member, on the proliferation of human ASM cells and on the release of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1 from human ASM cells. Human ASM cells were growth-arrested for 48 hr and incubated with platelet-derived growth factor (PDGF)- BB, interleukin (IL)-4, amphiregulin, and VEGF to evaluate cell proliferation. The cells were treated with PDGF, IL-4 and amphiregulin to evaluate the release of VEGF, MCP-1. IL-4 suppressed unstimulated and PDGF-stimulated ASM cell proliferation. Amphiregulin stimulated ASM cell proliferation in a dose-dependent manner. VEGF did not have any influence on ASM cell proliferation. IL-4 stimulated VEGF secretion by the ASM cells in a dose-dependent manner and showed added stimulatory effects when co-incubated with PDGF. Amphiregulin did not promote VEGF secretion. IL-4 and amphiregulin showed no stimulatory effects on MCP-1 secretion. The results of this study showed that IL-4 had bifunctional effects on airway remodeling, one was the suppression of the proliferation of the ASM cells and the other was the promotion of VEGF release by the ASM cells, and amphiregulin can promote human ASM cell proliferation.

Korean Ginseng-Induced Occupational Asthma and Determination of IgE Binding Components

A number of case reports on occupational asthma caused by herbal medicines have been issued, for example, on Sanyak, Chunkung, Banha, and Brazilian ginseng. Recently, cases of occupational asthma induced by Sanyak and Korean ginseng have been reported, but the pathogenic mechanisms involved are unknown. This study was carried out to evaluate the immunologic mechanism underlying Korean ginseng-induced occupational asthma. A patient engaged in Korean ginseng wholesale was referred for recurrent dyspnea, wheezing, and nasal symptoms, which were aggravated at work. Allergen bronchial provocation testing to Korean ginseng extract showed a typical immediate response, and skin prick testing to Korean ginseng extract also showed a strong positive response. Moreover, serum-specific IgE levels to Korean ginseng extract were significantly higher than in controls. Enzymelinked immunosorbent assay (ELISA) inhibition tests showed a dose-dependent inhibition by Korean ginseng, but not by Dermatophagoides farinae, wheat flour, or Chinese balloon flower. Sodium dodecylsulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and immunoblotting revealed four specific Immunoglobulin E (IgE) binding components at 26, 30, 47, and 60 kDa, which were not bound by control sera. These results strongly suggest that occupation asthma induced by Korean ginseng is induced via an IgE-mediated mechanism.

Handling of ferric iron by branchial and intestinal epithelia of climbing perch (Anabas testudineus Bloch).

With a view to test how the branchial and intestinal tissues of fish, the two sites of metal acquisition, utilize the water-borne ferric [Fe(III)] iron and whether the accumulation of this form of iron influences cellular Na/K gradient in these tissues, the gills and intestines of climbing perch adapted to freshwater (FW) and acclimated to dilute seawater (20 ppt; SW) were analyzed for ouabain-sensitive Na+, K+-ATPase activity, Fe and electrolyte contents after loading a low (8.95 microM) or high dose (89.5 microM) of Fe(III) iron in the water. The SW gills showed higher levels of total Fe after treating with 8.95 microM of Fe(III) iron which was not seen in the FW gills. Na+, K+-ATPase activity, reflecting Na/K pump activity, showed an increase in the FW gills and not in the SW gills. Substantial increase in the branchial Na and K content was observed in the SW gills, but the FW gills failed to show such effects after Fe(III) loading. The total Fe content was declined in the FW intestine but not in the SW intestine. Water-borne Fe(III) iron decreased the activity of Na+, K+-ATPase in the SW intestine while not changing its activity in the FW intestine. The Na and K content in the FW intestine did not respond to Fe(III) iron exposure but showed a reduction in its Na levels in the SW intestine. The moisture content in the gills and intestines of both the FW and SW perch remained unaffected after Fe(III) loading. In FW fish, the plasma Na levels were decreased by a low dose of Fe(III) iron, though a high dose of Fe(III) iron was required in the SW fish for such an effect. Overall, the results for the first time provide evidence that gills act as a major site for Fe(III) iron absorption and accumulation during salinity acclimation which depends on a high cellular Na/K gradient.

TACI:Fc scavenging B cell activating factor (BAFF) alleviates ovalbumin-induced bronchial asthma in mice

Asthma was induced by the sensitization and challenge with ovalbumin (OVA) in mice. B-cell activating factor (BAFF) plays a role in mature B cell generation and maintenance. Here, we investigated whether, BAFF expression was changed in OVA-induced mice and whether the control of BAFF expression level alleviates the symptom of bronchial asthma. BAFF expression was detected in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues. BAFF protein was also increased in OVA-induced mouse serum. The increased BAFF transcripts was detected in OVA-induced mouse splenocytes. OVA-induced asthma was associated with the increased number of eosinophils in bronchoalveolar lavage fluid (BALF). When TACI:Fc scavenging soluble BAFF was injected to OVA-induced mice, a significant inhibition was detected in the thickness of airway smooth muscle and glycol-containing cellular elements in airway that were visualized by hematoxylin/eosin Y and periodic acid-Schiff staining, respectively. In addition, when mice were treated with TACI:Fc protein, BAFF protein level was decreased in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues compared to control mice. When compared to OVA-induced control, TACI:Fc treatment reduced the percentage of non-lymphoid cells and no changes were detected in lymphoid cell population. Hypodiploid cell formation in BALF was decreased by OVA-challenge but it was recovered by TACI:Fc treatment. Collectively, data suggest that asthmatic symptom could be alleviated by scavenging BAFF and then BAFF could be a novel target for the develpoment of anti-asthmatic agents.

Nitric oxide metabolites in induced sputum: a noninvasive marker of airway inflammation in asthma.

OBJECTIVE: This study was designed to examine for nitric oxide (NO) metabolites in induced sputum as a marker of airway inflammation in asthmatic children. DESIGN. Prospective interventional SETTING: Pediatric Allergy and Asthma Clinic of a tertiary care referral hospital in Northern India. SUBJECTS: Twenty-one children with asthma who were not receiving corticosteroids for the preceding 3 months and 10 healthy controls were enrolled. METHODS: Hypertonic saline-induced sputum was obtained at study entry in controls, and at study entry and after 6 weeks of inhaled corticosteroid (ICS) therapy in asthmatic children. Fresh expectorated sputum was treated with dithiothreitol and cytospinned for cell count. NO metabolites were measured in the supernatant by the modified Griess reaction. RESULTS: Asthmatic children, compared with controls, had significantly higher concentration of NO metabolites (22.4 +/- 209.69 vs 39.2 +/- 15.9 (moL/L, P <0.01) and a higher percentage of eosinophils (15.3 +/- 12.0 vs 0.8 +/- 1.1%, P <0.01) in induced sputum. Both NO metabolites and eosinophil percentage declined following treatment with ICS for 6 weeks (P <0.01). CONCLUSION: The study confirms that the level of NO metabolites is increased in the tracheobronchial secretions of asthmatic children and decreases following ICS therapy. Measurement of NO metabolites in induced sputum may be useful for monitoring airway inflammation in children with asthma.

Effect of cigarette smoke extract on the role of protein kinase C in the proliferation of passively sensitized human airway smooth muscle cells / 华中科技大学学报（医学）（英德文版）

To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A1, A2 and A3 and the group B was sub-divided into the group of B1, B2, B3, B4 and B5. No other agents were added to the group A1 and B1. The cells of group A2 and B2 were stimulated with 5% CSE for 24 h. HASMCs from group A3 and B3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5%) for 24 h. PKC inhibitor Ro-31-8220 (5 micromol/L) was added to the HASMCs of group B4 for 24 h. The cells from group B5 were stimulated with Ro-31-8220 (5 micromol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-a in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-alpha mRNA and the A value of PKC-alpha protein in HASMCs from group B1, B2 and B3 were significantly increased compared to those of group A1, A2 and A3 correspondingly and respectively (P< 0.01). The proliferation of HASMCs of group A2 and B2 stimulated with CSE and group A3 and B3 stimulated with CSE and PMA were also significantly enhanced when group A1, A2 and A3 and group B1, B2 and B3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-alpha mRNA and the A value of PKC-alpha protein in HASMCs from group B4 treated with Ro-31-8220 and group B5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B1 and B2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.

Effect of cigarette smoke extract on the proliferation of human airway epithelial cells and expression and activation of FAK / 华中科技大学学报（医学）（英德文版）

The effect of cigarette smoke extract (CSE) on the proliferation of human airway epithelial cells and the possible mechanism was studied. After airway epithelial cells were treated with different concentrations of CSE for 24 h, the cell proliferation was measured by MTT and the distribution of different cell cycles by flow cytometry. The FAK expression level was detected by Western blot and the degree of tyrosine phosphorylation by immunoprecipitation. The results showed that CSE could inhibit the proliferation of human airway epithelial cells, arrest the epithelial cells in G1 phase of cell cycle, dramatically decrease the number of epithelial cells in S and G2 phases; Meanwhile CSE could decrease the expression level of FAK and the degree of its tyrosine phosphorylation. The above effects of CSE were concentration-dependent. The expression of FAK and the degree of its phosphorylation was positively correlated to the increased number of epithelial cells in G1 phase, and negatively to the number of epithelial cells in S and G2 phases. It was concluded that the mechanism by which CSE could inhibit the proliferation of human epithelial cells was contributed to the increased expression and activation of FAK.

Expression of heat stress protein 70 mRNA in patients with chronic obstructive pulmonary disease and its significance / 华中科技大学学报（医学）（英德文版）

The effects of cigarette smoke extract (CSE) on the expression of heat stress protein 70 (Hsp70) in human bronchi smooth muscle cells were investigated in vitro, and the changes in Hsp70 mRNA in the patients with chronic obstructive pulmonary disease and their significance were explored. Human bronchi smooth muscle cells were cultured with CSE at the different concentrations. The expression of Hsp70 mRNA and Hsp70 was detected by reverse translation-polymerase chain reaction (RT-PCR) and Western blotting respectively. Levels of Hsp70 mRNA and Hsp70 in lymphocytes from 20 patients with COPD and 20 healthy smoking control subjects were measured by RT-PCR and Western blotting. The results showed the expression of both Hsp70 mRNA and Hsp70 was decreased conformably in human bronchi smooth muscle cells treated with CSE at certain concentration in vitro. The A values of the Hsp70 mRNA expression were 0.24 +/- 0.11 and 0. 42 +/- 0.13 respectively in COPD patients and healthy smoking controls with the difference being significant (P < 0.01). There was also significant difference in the A values of the Hsp70 expression between COPD patients and healthy smoking controls (20.9 +/- 9.9 vs 44.8 +/- 15.3, P < 0.01). The levels of Hsp70 mRNA had strongly positive correlation with Hsp70 protein (r = 0.85, P < 0.01). It was suggested that the expression of Hsp70 mRNA was in concordance with the expression of Hsp70, which could provide a basis on the study of Hsp70 gene regulation and Hsp70 gene in the development of COPD.

Cap-independent protein translation is initially responsible for 4-(N-methylnitrosamino)-1-(3-pyridyl)-butanone (NNK)-induced apoptosis in normal human bronchial pithelial cells

Evidences show that eukaryotic mRNAs can perform protein translation through internal ribosome entry sites (IRES). 5'-Untranslated region of the mRNA encoding apoptotic protease-activating factor 1 (Apaf-1) contains IRES, and, thus, can be translated in a cap-independent manner. Effects of changes in protein translation pattern through rapamycin pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-butanone(NNK, tobacco-specific lung carcinogen)-induced apoptosis in human bronchial epithelial cells were examined by caspase assay, FACS analysis, Western blotting, and transient transfection. Results showed that NNK induced apoptosis in concentration- and time-dependent manners. NNK-induced apoptosis occurred initially through cap-independent protein translation, which during later stage was replaced by cap-dependent protein translation. Our data may be pplicable as the mechanical basis of lung cancer treatment.

Retinoic Acid Receptor-beta Expression in Stage I Non-Small Cell Lung Cancer and Adjacent Normal Appearing Bronchial Epithelium

Retinoic acid receptor- (RAR-beta) is induced by and mediates the growth-inhibitory and apoptotic effects of retinoic acid (RA), suggesting that loss of RAR-betaexpression may be one of the critical events involved in the carcinogenesis/ progression of non-small cell lung cancer (NSCLC) and in the responsiveness to retinoid chemotherapy. However, recent contradictory reports that the expression of RAR-beta is associated with poor clinical outcome, and the fact that treatment of serum-deprived type 2 alveolar cells with RA leads to a stimulation of cell proliferation, require the verification of RAR-beta as a biomarker of chemoprevention or prognosis. The expression status of RAR-beta in cancer cells and adjacent normal appearing bronchial epithelium from 39 patients, diagnosed as stage I NSCLC and undergone a curative lung resection, was analyzed in paraffin-embedded tissue sections by IHC staining. The normal appearing bronchial epithelium of 14 out of 33 (42.4%) specimens expressed RAR-beta, whereas 22 out of the 39 (56.4%) stage I NSCLC specimens expressed RAR-beta. RAR-beta was more frequently expressed in the adenocarcinoma (72.7%) than in the squamous cell carcinoma (31.3%) (p=0.026). Neither the expression status in normal appearing adjacent tissue nor that in the tumor tissue had prognostic implications. The higher expression of RAR-beta in cancer tissue, the focal and uneven distribution in normal appearing adjacent bronchial epithelium, and inconsistency with the corresponding tumor tissue, suggest that the expression status of RAR-beta as a biomarker for chemoprevention/early diagnosis or the prognosis of NSCLC requires further consideration.

Relationship between apoptosis and E-cadherin expression in bronchial epithelium of smoking mouse / 华中科技大学学报（医学）（英德文版）

To investigate whether apoptosis is associated with cell adhesion in bronchial epithelium, and whether it contributes to the kinetics of injury and repair of surface epithelia, this study was performed for E-cadherin expression by using immunohistochemistry technique and for apoptosis by TUNEL method. An animal model of smoking was used for this study. The results showed that epithelial cells with membrane anchored E-cadherin decreased remarkably at several time points during 6 months of exposure to smoke (P 0.05). All these suggested that apoptosis is associated with E-cadherin expression in bronchial epithelium of smoking mouse.

Açäo broncodilatadora do salbutamol e formoterol em asmáticos adultos / The bronchodilator agents action of salbutamol and formoterol in asthmathics adults

O trabalho apresenta uma revisäo bibliográfica sobre dois agonistas ß2 seletivos, Salbutamol e Formoterol, no tratamento da asma leve e moderada, incluindo um breve estudo a respeito da classificaçäo e manifestaçöes clínicas da asma.

Bronchopulmonary absorption of aerosolised insulin.

Seven non-insulin dependent diabetes mellitus (NIDDM) patients aged 48 to 78 years, inhaled approximately 10 to 20 units of aerosolised insulin (human actrapid) by nebuliser. In 5 cases, approximately 10 units of insulin aerosol inhalation was given 4 hours after breakfast. Blood glucose level started falling after 15 minutes with a maximum fall at 45 minutes to 1 hour. In 2 cases insulin aerosol inhalation was given 2 hours after lunch. The first dose of 10 units of insulin did not show any significant change in blood glucose level at 30 minutes. A second dose of 10 units of insulin aerosol was inhaled after 30 minutes and blood glucose level started falling in a pattern similar to the first group of patients. The first dose appears to withhold the rise of blood glucose in the postprandial phase and the second dose lowers the blood glucose level. The result is compared with placebo inhalation and there is a significant fall of blood glucose level with insulin aerosol inhalation. It admits of little doubt that insulin is absorbed from the bronchopulmonary mucosa.

Bacteriology of bronchial secretions in non-tubercular lower respiratory tract infections.

Fifty samples of bronchial secretions collected from patients of non-tubercular lower respiratory tract infections through fiberoptic bronchoscopy (FOB) were cultured both for aerobic and anaerobic organisms. Thirty-three (66%) samples yielded bacteria. Out of these, thirty were isolated in pure culture and from three, a mixed growth of aerobic and anaerobic micro-organisms was obtained. Aerobic bacteria were the predominant isolates. Stephylococcus aureus (10), pseudomonas (9) and streptococcus pneumoniae (8) were the major aerobic isolates. Ciprofloxacin was found to be the most effective drug against aerobes and metronidazole against anaerobes in vitro susceptibility tests.

Upregulation of the tracheobronchial mucin gene involves cyclic AMP response elements.

The cAMP response element (CRE)-binding transcription factor CREB can mediate induction of gene transcription in response to cAMP. Since the tracheobronchial mucin gene (TBM) 5'-flanking region contains CREs (located between residues -289 and -376) with an octamer-like motif (TGACGTCC), the cAMP responsiveness of the TBM CREs was investigated in human tracheal epithelial cells HBE1. These cells were isolated from non-cystic fibrosis subjects and immortalized with HPV18 genes E6 and E7 (ref. 1). HBE1 cells express a homolog of canine TBM (as demonstrated by TBM expression at the transcription and translation level). Electrophoretic mobility shift assay (EMSA) indicated that CREs provide a binding site for nuclear proteins. Transient transfection analysis [using the chloramphenicol acetyl transferase (CAT) reporter gene] and nuclear run on analysis indicated cAMP induced transcription of the TBM gene. The transcriptional activity of the HBE1 transfected cells containing CRE was selectively modulated by extracellular 8Br-cAMP in a dose-dependent manner; a 6-fold increase in activity was detected when cells were incubated for 12 hr in the presence of 2 microM vs 1 nM 8BrcAMP. Since mucin gene is over-expressed in diseases such as cystic fibrosis (CF) and asthma, the information presented here will help us understand the mechanisms involved in transcriptional regulation of mucin gene expression in disease states.

Colheita do fluido brônquio-alveolar de bezerros através da traqueocentese transcutânea / Colection of bronchoalveolar fluid in calves by transcutaneous tracheocenthesis

Foi estudada uma técnica da traqueocentese transcutânea, praticada na regiäo cervical ventro-medial, para colheita de secreçäo brônquica de bezerros acometidos de broncopneumonia. Foram colhidas amostras de secreçäo nasal, através de "swabs" para comparaçäo da microflora da secreçäo brônquica, empregando-se a traqueocentese em 52 bezerros com diagnóstico clínico de broncopneumonia. A traqueocentese foi realizada em nível de campo, com material de fácil aquisiçäo e manipulaçäo, mostrando-se eficiente para colheita de secreçäo brônquica, facilmente exequível com o animal em estaçäo, sob contençäo mínima e sem necessidade de anestesia. A quantidade de secreçäo brônquica colhida foi suficiente para execuçäo dos exames bacteriológicos. Os exames demonstraram que a traqueocentese reduziu significativamente (P<0,01) o número de enterobactérias, possivelmente contaminantes, em relaçäo ao "swab" nasal

Peptide mapping reveals differences in the non-glycosylated domains of cystic fibrosis and normal tracheobronchial mucins.

Tracheobronchial mucins from lung mucus secretions of healthy individuals and from patients with cystic fibrosis (CF) were purified according to a protocol established in our laboratory. Following digestion of the purified, reduced-alkylated mucin (free of 118 kDa and 70 kDa components) with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2 and TR-3) were observed upon chromatography on a Superose 6 column using FPLC. TR-1 (glycosylated fraction) contained all of the carbohydrate, while TR-2 and TR-3 fractions had no detectable sugars. Comparison of the amino acid composition of TR-1 fractions from normal and CF individuals revealed no significant differences, while the TR-2 fractions from these mucins showed noticeable differences. Peptide mapping of TR-2 fractions from normal and CF mucins was performed on a C18 reverse phase column using FPLC. The peptide maps of normal mucins were markedly different from CF mucins. A greater number of peptides were seen in the TR-2 fractions of normal mucins when compared to CF mucin TR-2 fractions. In addition, normal TR-2 fractions appeared to be comprised of more hydrophobic peptides when compared to CF TR-2 fractions. These data provide evidence of possible structural differences in the non-glycosylated regions of CF and non-CF mucins, since the TR-2 fractions are essentially derived from the T-domains in the "naked" stretches of the mucin polypeptide backbone.