表皮生长因子对培养的气道上皮细胞抗臭氧损伤的保护作用

本研究观察到臭氧（Ｏ３）对体外培养经３Ｈ－ＵｄＲ标记的免气道上皮细胞有明显细胞毒性作用,且损伤程度与Ｏ３作用时间呈正相关。Ｏ３暴露组细胞内丙二醛（ＭＤＡ）产生增多（Ｐ＜０．０１）,提示Ｏ３损伤细胞的机制与胞膜脂质过氧化有关。表皮生长因子（ＥＧＦ）可明显降低Ｏ３所致的３Ｈ释放率（Ｐ＜０．０１）、降低Ｏ３的细胞毒指数及细胞内ＭＤＡ含量（Ｐ＜０．０１）,证明ＥＧＦ对气道上皮细胞有保护作用。进一步还观察到浓度为５ｎｇ／ｍｌ的ＥＯＦ可以取消Ｏ３所引起的细胞内还原型谷胱甘肽（ＧＳＨ）含量降低（Ｐ＜０．０１）,并增加细胞内谷胱甘肽总含量（Ｐ＜０．０５）,但不能改变Ｏ３所致的氧化型谷胱甘肽（ＧＳＳＧ）含量的增加（Ｐ＞０．０５）,对ＧＳＨ／ＧＳＳＧ比值也无明显提高,这些都提示ＥＧＦ的细胞保护机理可能与其促进细胞内谷胱甘肽合成有关,而对ＧＳＳＧ转化为ＧＳＨ的还原过程影响不明显。     

Effect of ultrasonically nebulized distilled water on airway epithelial cell swelling in guinea pigs.

To investigate the pathogenesis of ultrasonically nebulized distilled water-induced airway narrowing, we studied the role of airway epithelial cells during a distilled water-inhalation challenge in an animal model of airway inflammation. Guinea pigs were divided into four groups: 1) a sham/saline (S/S) group: sham ozone followed by saline inhalation; 2) a sham/water (S/W) group: sham ozone followed by water inhalation; 3) an ozone/saline (O/S) group: ozone followed by saline inhalation; and 4) an ozone/water (O/W) group: ozone followed by water inhalation. After exposure to either 3.0 parts/million ozone or air at the same flow rate for 2 h, guinea pigs were anesthetized and tracheostomized, and then lung resistance (RL) was measured. For morphometric assessment, tissues were fixed with formaldehyde, stained with hematoxylin and eosin, and cut into transverse sections. Airway dimensions were either measured directly or calculated from the internal perimeter, the external perimeter, and airway wall area. There were no statistical differences in the values of RL before distilled water inhalation between the sham groups and the ozone groups. RL increased significantly after 10 min of distilled water inhalation in both the S/W group and the O/W group. In the S/W group, epithelial cells were swollen, and intercellular spaces were wider, resulting in significant increase in epithelial wall thickness, but there was no significant infiltration by inflammatory cells. In the O/S group, the epithelium showed infiltration by inflammatory cells without change in cell volume. In the O/W group, the epithelium showed both infiltration and a greater increase in epithelial wall thickness compared with the S/W group. These results suggest that airway epithelial cell swelling, induced by inhaled distilled water, increases with RL in guinea pigs and that this reaction may be accelerated by airway inflammation.     

Inflammatory and Repair Pathways Induced in Human Bronchoalveolar Lavage Cells with Ozone Inhalation

Background

Inhalation of ambient levels of ozone causes airway inflammation and epithelial injury.

Methods

To examine the responses of airway cells to ozone-induced oxidative injury, 19 subjects (7 with asthma) were exposed to clean air (0ppb), medium (100ppb), and high (200ppb) ambient levels of ozone for 4h on three separate occasions in a climate-controlled chamber followed by bronchoscopy with bronchoalveolar lavage (BAL) 24h later. BAL cell mRNA expression was examined using Affymetrix GeneChip Microarray. The role of a differentially expressed gene (DEG) in epithelial injury was evaluated in an in vitro model of injury [16HBE14o- cell line scratch assay].

Results

Ozone exposure caused a dose-dependent up-regulation of several biologic pathways involved in inflammation and repair including chemokine and cytokine secretion, activity, and receptor binding; metalloproteinase and endopeptidase activity; adhesion, locomotion, and migration; and cell growth and tumorigenesis regulation. Asthmatic subjects had 1.7- to 3.8-fold higher expression of many DEGs suggestive of increased proinflammatory and matrix degradation and remodeling signals. The most highly up-regulated gene was osteopontin, the protein level of which in BAL fluid increased in a dose-dependent manner after ozone exposure. Asthmatic subjects had a disproportionate increase in non-polymerized osteopontin with increasing exposure to ozone. Treatment with polymeric, but not monomeric, osteopontin enhanced the migration of epithelial cells and wound closure in an α9β1 integrin-dependent manner.

Conclusions

Expression profiling of BAL cells after ozone exposure reveals potential regulatory genes and pathways activated by oxidative stress. One DEG, osteopontin, promotes epithelial wound healing in an in vitro model of injury.     

Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.     

Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys

Children chronically exposed to high levels of ozone (O(3)), the principal oxidant pollutant in photochemical smog, are more vulnerable to respiratory illness and infections. The specific factors underlying this differential susceptibility are unknown but may be related to air pollutant-induced nasal alterations during postnatal development that impair the normal physiological functions (e.g., filtration and mucociliary clearance) serving to protect the more distal airways from inhaled xenobiotics. In adult animal models, chronic ozone exposure is associated with adaptations leading to a decrease in airway injury. The purpose of our study was to determine whether cyclic ozone exposure induces persistent morphological and biochemical effects on the developing nasal airways of infant monkeys early in life. Infant (180-day-old) rhesus macaques were exposed to 5 consecutive days of O(3) [0.5 parts per million (ppm), 8 h/day; "1-cycle"] or filtered air (FA) or 11 biweekly cycles of O(3) (FA days 1-9; 0.5 ppm, 8 h/day on days 10-14; "11-cycle"). The left nasal passage was processed for light microscopy and morphometric analysis. Mucosal samples from the right nasal passage were processed for GSH, GSSG, ascorbate (AH(2)), and uric acid (UA) concentration. Eleven-cycle O(3) induced persistent rhinitis, squamous metaplasia, and epithelial hyperplasia in the anterior nasal airways of infant monkeys, resulting in a 39% increase in the numeric density of epithelial cells. Eleven-cycle O(3) also induced a 65% increase in GSH concentrations at this site. The persistence of epithelial hyperplasia was positively correlated with changes in GSH. These results indicate that early life ozone exposure causes persistent nasal epithelial alterations in infant monkeys and provide a potential mechanism for the increased susceptibility to respiratory illness exhibited by children in polluted environments.     

Prolonged ozone exposure in an allergic airway disease model: Adaptation of airway responsiveness and airway remodeling

Background

Short-term exposure to high concentrations of ozone has been shown to increase airway hyper-responsiveness (AHR). Because the changes in AHR and airway inflammation and structure after chronic ozone exposure need to be determined, the goal of this study was to investigate these effects in a murine model of allergic airway disease.

Methods

We exposed BALB/c mice to 2 ppm ozone for 4, 8, and 12 weeks. We measured the enhanced pause (Penh) to methacholine and performed cell differentials in bronchoalveolar lavage fluid. We quantified the levels of IL-4 and IFN-γ in the supernatants of the bronchoalveolar lavage fluids using enzyme immunoassays, and examined the airway architecture under light and electron microscopy.

Results

The groups exposed to ozone for 4, 8, and 12 weeks demonstrated decreased Penh at methacholine concentrations of 12.5, 25, and 50 mg/ml, with a dose-response curve to the right of that for the filtered-air group. Neutrophils and eosinophils increased in the group exposed to ozone for 4 weeks compared to those in the filtered-air group. The ratio of IL-4 to INF-γ increased significantly after exposure to ozone for 8 and 12 weeks compared to the ratio for the filtered-air group. The numbers of goblet cells, myofibroblasts, and smooth muscle cells showed time-dependent increases in lung tissue sections from the groups exposed to ozone for 4, 8, and 12 weeks.

Conclusion

These findings demonstrate that the increase in AHR associated with the allergic airway does not persist during chronic ozone exposure, indicating that airway remodeling and adaptation following repeated exposure to air pollutants can provide protection against AHR.

     

Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection

Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA) is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI). Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs) to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.     

Effects of N-Acetylcysteine in Ozone-Induced Chronic Obstructive Pulmonary Disease Model

Introduction

Chronic exposure to high levels of ozone induces emphysema and chronic inflammation in mice. We determined the recovery from ozone-induced injury and whether an antioxidant, N-acetylcysteine (NAC), could prevent or reverse the lung damage.

Methods

Mice were exposed to ozone (2.5 ppm, 3 hours/12 exposures, over 6 weeks) and studied 24 hours (24h) or 6 weeks (6W) later. Nac (100 mg/kg, intraperitoneally) was administered either before each exposure (preventive) or after completion of exposure (therapeutic) for 6 weeks.

Results

After ozone exposure, there was an increase in functional residual capacity, total lung volume, and lung compliance, and a reduction in the ratio of forced expiratory volume at 25 and 50 milliseconds to forced vital capacity (FEV₂₅/FVC, FEV₅₀/FVC). Mean linear intercept (L_m) and airway hyperresponsiveness (AHR) to acetylcholine increased, and remained unchanged at 6W after cessation of exposure. Preventive NAC reduced the number of BAL macrophages and airway smooth muscle (ASM) mass. Therapeutic NAC reversed AHR, and reduced ASM mass and apoptotic cells.

Conclusion

Emphysema and lung function changes were irreversible up to 6W after cessation of ozone exposure, and were not reversed by NAC. The beneficial effects of therapeutic NAC may be restricted to the ASM.     

Effect of acute ozone induced airway inflammation on human sympathetic nerve traffic: a randomized, placebo controlled, crossover study

Background

Ozone concentrations in ambient air are related to cardiopulmonary perturbations in the aging population. Increased central sympathetic nerve activity induced by local airway inflammation may be one possible mechanism.

Methodology/Principal Findings

To elucidate this issue further, we performed a randomized, double-blind, cross-over study, including 14 healthy subjects (3 females, age 22–47 years), who underwent a 3 h exposure with intermittent exercise to either ozone (250 ppb) or clean air. Induced sputum was collected 3 h after exposure. Nineteen to 22 hours after exposure, we recorded ECG, finger blood pressure, brachial blood pressure, respiration, cardiac output, and muscle sympathetic nerve activity (MSNA) at rest, during deep breathing, maximum-inspiratory breath hold, and a Valsalva maneuver. While the ozone exposure induced the expected airway inflammation, as indicated by a significant increase in sputum neutrophils, we did not detect a significant estimated treatment effect adjusted for period on cardiovascular measurements. Resting heart rate (clean air: 59±2, ozone 60±2 bpm), blood pressure (clean air: 121±3/71±2 mmHg; ozone: 121±2/71±2 mmHg), cardiac output (clean air: 7.42±0.29 mmHg; ozone: 7.98±0.60 l/min), and plasma norepinephrine levels (clean air: 213±21 pg/ml; ozone: 202±16 pg/ml), were similar on both study days. No difference of resting MSNA was observed between ozone and air exposure (air: 23±2, ozone: 23±2 bursts/min). Maximum MSNA obtained at the end of apnea (air: 44±4, ozone: 48±4 bursts/min) and during the phase II of the Valsalva maneuver (air: 64±5, ozone: 57±6 bursts/min) was similar.

Conclusions/Significance

Our study suggests that acute ozone-induced airway inflammation does not increase resting sympathetic nerve traffic in healthy subjects, an observation that is relevant for environmental health. However, we can not exclude that chronic airway inflammation may contribute to sympathetic activation.     

Ozone inhalation promotes CX3CR1-dependent maturation of resident lung macrophages that limit oxidative stress and inflammation

Inhalation of ambient ozone alters populations of lung macrophages. However, the impact of altered lung macrophage populations on the pathobiology of ozone is poorly understood. We hypothesized that subpopulations of macrophages modulate the response to ozone. We exposed C57BL/6 mice to ozone (2 ppm × 3 h) or filtered air. At 24 h after exposure, the lungs were harvested and digested and the cells underwent flow cytometry. Analysis revealed a novel macrophage subset present in ozone-exposed mice, which were distinct from resident alveolar macrophages and identified by enhanced Gr-1(+) expression [Gr-1 macrophages (Gr-1 Macs)]. Further analysis showed that Gr-1(+) Macs exhibited high expression of MARCO, CX3CR1, and NAD(P)H:quinone oxioreductase 1. Gr-1(+) Macs were present in the absence of CCR2, suggesting that they were not derived from a CCR2-dependent circulating intermediate. Using PKH26-PCL to label resident phagocytic cells, we demonstrated that Gr-1 Macs were derived from resident lung cells. This new subset was diminished in the absence of CX3CR1. Interestingly, CX3CR1-null mice exhibited enhanced responses to ozone, including increased airway hyperresponsiveness, exacerbated neutrophil influx, accumulation of 8-isoprostanes and protein carbonyls, and increased expression of cytokines (CXCL2, IL-1β, IL-6, CCL2, and TNF-α). Our results identify a novel subset of lung macrophages, which are derived from a resident intermediate, are dependent upon CX3CR1, and appear to protect the host from the biological response to ozone.     

Mitochondrial ROS and NLRP3 inflammasome in acute ozone-induced murine model of airway inflammation and bronchial hyperresponsiveness

Oxidative stress is a key mechanism underlying ozone-induced lung injury. Mitochondria can release mitochondrial reactive oxidative species (mtROS), which may lead to the activation of NLRP3 inflammasome. The goal of this study was to examine the roles of mtROS and NLRP3 inflammasome in acute ozone-induced airway inflammation and bronchial hyperresponsiveness (BHR). C57/BL6 mice (n?=?8/group) were intraperitoneally treated with vehicle (phosphate buffered saline, PBS) or mitoTEMPO (mtROS inhibitor, 20?mg/kg), or orally treated with VX-765 (caspse-1 inhibitor, 100?mg/kg) 1?h before the ozone exposure (2.5?ppm, 3?h). Compared to the PBS-treated ozone-exposed mice, mitoTEMPO reduced the level of total malondialdehyde in bronchoalveolar lavage (BAL) fluid and increased the expression of mitochondrial complexes II and IV in the lung 24?h after single ozone exposure. VX-765 inhibited ozone-induced BHR, BAL total cells including neutrophils and eosinophils, and BAL inflammatory cytokines including IL-1α, IL-1β, KC, and IL-6. Both mitoTEMPO and VX-765 reduced ozone-induced mtROS and inhibited capase-1 activity in lung tissue whilst VX-765 further inhibited DRP1 and MFF expression, increased MFN2 expression, and down-regulated caspase-1 expression in the lung tissue. These results indicate that acute ozone exposure induces mitochondrial dysfunction and NLRP3 inflammasome activation, while the latter has a critical role in the pathogenesis of ozone-induced airway inflammation and BHR.     

20-HETE Mediates Ozone-Induced,Neutrophil-Independent Airway Hyper-Responsiveness in Mice

Background

Ozone, a pollutant known to induce airway hyper-responsiveness (AHR), increases morbidity and mortality in patients with obstructive airway diseases and asthma. We postulate oxidized lipids mediate in vivo ozone-induced AHR in murine airways.

Methodology/Principal Findings

Male BALB/c mice were exposed to ozone (3 or 6 ppm) or filtered air (controls) for 2 h. Precision cut lung slices (PCLS; 250 µm thickness) containing an intrapulmonary airway (∼0.01 mm² lumen area) were prepared immediately after exposure or 16 h later. After 24 h, airways were contracted to carbachol (CCh). Log EC₅₀ and E_max values were then calculated by measuring the airway lumen area with respect to baseline. In parallel studies, dexamethasone (2.5 mg/kg), or 1-aminobenzotriazol (ABT) (50 mg/kg) were given intraperitoneal injection to naïve mice 18 h prior to ozone exposure. Indomethacin (10 mg/kg) was administered 2 h prior. Cell counts, cytokine levels and liquid chromatography-mass spectrometry (LC-MS) for lipid analysis were assessed in bronchoalveolar lavage (BAL) fluid from ozone exposed and control mice. Ozone acutely induced AHR to CCh. Dexamethasone or indomethacin had little effect on the ozone-induced AHR; while, ABT, a cytochrome P450 inhibitor, markedly attenuated airway sensitivity. BAL fluid from ozone exposed animals, which did not contain an increase in neutrophils or interleukin (IL)-6 levels, increased airway sensitivity following in vitro incubation with a naïve PCLS. In parallel, significant increases in oxidized lipids were also identified using LC-MS with increases of 20-HETE that were decreased following ABT treatment.

Conclusions/Significance

These data show that ozone acutely induces AHR to CCh independent of inflammation and is insensitive to steroid treatment or cyclooxygenase (COX) inhibition. BAL fluid from ozone exposed mice mimicked the effects of in vivo ozone exposure that were associated with marked increases in oxidized lipids. 20-HETE plays a pivotal role in mediating acute ozone-induced AHR.     

Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows

Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to examine the biochemical composition of follicular fluid harvested from different-sized follicles and its relationship with that of blood serum in dairy cattle. Following slaughter, blood samples were collected from dairy cows (n=30) and follicular fluid aspirated from three size classes of non-atretic follicles (<4 mm, 6–8 mm and >10 mm diameter). Samples remained independent between cows and between size classes within cows. Serum and follicular fluid samples were assayed using commercial clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and metabolites (glucose, β-hydroxybutyrate (β-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, β-OHB and total cholesterol increased from small to large follicles and decreased for potassium, chloride, lactate, urea and triglycerides. There was a significant concentration gradient for all variables between their levels in serum and follicular fluid (P<0.05). Significant correlations were observed for chloride (r=0.40), glucose (r=0.56), β-OHB (r=0.85), urea (r=0.95) and total protein (r=0.60) for all three follicle size classes and for triglycerides (r=0.43), NEFA (r=0.50) and total cholesterol (r=0.42) for large follicles (P<0.05). The results from the present study suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical environment that changes from small to large follicles. Furthermore, the significant correlation between the composition of serum and follicular fluid for the above-mentioned metabolites suggests that metabolic changes in serum levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.     

Biomarkers of oxidative stress study V: Ozone exposure of rats and its effect on lipids,proteins, and DNA in plasma and urine

Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins, and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F₂-isoprostanes, protein carbonyls, methionine oxidation, and tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F₂-isoprostanes were investigated with various techniques. The criterion used to recognize a marker in the model of ozone exposure was that a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and the control groups at either ozone dose and time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criteria of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies.     

Effect of inhaled indomethacin on distilled water-induced airway epithelial cell swelling.

We evaluated the mechanism of the anti-asthmatic effect of inhaled indomethacin (Indo) by using an animal model (guinea pigs) of airway inflammation. After being exposed to either ozone or room air at identical flow rates (5 l/min) for 2 h, guinea pigs were anesthetized, tracheostomized, and lung resistance (RL) was subsequently measured. Guinea pigs inhaled either saline or Indo (1.5 mg/ml) for 1 min before undergoing an ultrasonically nebulized distilled water (UNDW) inhalation test. RL increased significantly after 10 min of UNDW inhalation in the room air and ozone groups but more so in the ozone group. This increase in RL was significantly suppressed by pretreatment with Indo. In the morphometric assessment of airway mucosa, a significant swelling of the epithelial cells after UNDW inhalation was observed in both the room air and ozone groups but especially so in the ozone group. This increase was also suppressed with Indo pretreatment. These results suggest that the increase in RL and the swelling of airway epithelial cells induced by inhaled UNDW in ozone-exposed guinea pigs was suppressed by pretreatment of inhaled Indo and that this suppression may be one of the reasons for the anti-asthmatic effect of inhaled Indo.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

Wound repair and anti-oxidative capacity is regulated by ITGB4 in airway epithelial cells

Integrin beta 4 (ITGB4) is a structural adhesion molecule which engages in maintaining the integrity of airway epithelial cells. Its specific cytomembrane structural feature strongly indicates that ITGB4 may engage in many signaling pathways and physiologic processes. However, in addition to adhesion, the specific biologic significance of ITGB4 in airway epithelial cells is almost unknown. In this article, we investigated the expression and functional properties of ITGB4 in airway epithelial cells in vivo and in vitro. Human bronchial epithelial cell line (16HBE14O-cells) and primary rat tracheal epithelial cells (RTE cells) were used to determine ITGB4 expression under ozone tress or mechanical damage, respectively. An ovalbumin (OVA)-challenged asthma model was used to investigate ITGB4 expression after antigen exposure in vivo. In addition, an ITGB4 overexpression vector and ITGB4 silence virus vector were constructed and transfected into RTE cells. Then, wound repair ability and anti-oxidation capacity was evaluated. Our results demonstrated that, on the edge of mechanically wounded cell areas, ITGB4 expression was increased after mechanical injury. After ozone stress, upregulation expression of ITGB4 was also detected. In the OVA-challenged asthma model, ITGB4 expression was decreased on airway epithelial cells accompanying with structural disruption and damage of anti-oxidation capacity. Besides, our study revealed that upregulation of ITGB4 promotes wound repair ability and anti-oxidative ability, while such abilities were blocked when ITGB4 was silenced. Taken together, these results showed that ITGB4 was a new interesting molecule involved in the regulation of wound repair and anti-oxidation processes for airway epithelial cells.     

Airway responses to chronic ozone exposure are partially mediated through mast cells.

Airways inflammation and epithelial injury induced by chronic ozone (O(3)) in genetically mast cell-deficient mice (Kit(W)/Kit(W-v)) were compared with those in mast cell-sufficient mice (+/+) and Kit(W)/Kit(W-v) mice repleted of mast cells (Kit(W)/Kit(W-v)-BMT). Mice were exposed to 0.26 ppm O(3) 8 h/day, 5 days/wk, for 1-90 days. Background was 0.06 ppm O(3). Age-matched mice were exposed to filtered air for O(3) controls. Reversibility of lesions was evaluated 35 days after exposure. Compared with Kit(W)/Kit(W-v), O(3) caused greater increases in lavageable macrophages, epithelial cells, and polymorphonuclear leukocytes in +/+ and Kit(W)/Kit(W-v)-BMT mice. O(3) also caused lung hyperpermeability, but the genotypic groups were not different. Cells and permeability returned to air control levels after O(3). O(3) induced lung cell proliferation only in +/+ and Kit(W)/Kit(W-v)-BMT mice; proliferation remained elevated or increased in +/+ and Kit(W)/Kit(W-v)-BMT mice after O(3). Greater O(3)-induced cell proliferation was found in nasal epithelium of +/+ and Kit(W)/Kit(W-v)-BMT mice compared with Kit(W)/Kit(W-v) mice. Results are consistent with the hypothesis that mast cells affect airway responses induced by chronic O(3) exposure.     

Hog barn dust extract augments lymphocyte adhesion to human airway epithelial cells.

The dust of hog confinement facilities induces airway inflammation. Mechanisms by which this dust modulates inflammation are not completely defined, although it is clear that exposure to dust can modulate both epithelial cell and inflammatory cell function. In this work, we demonstrate that airway epithelial cell (BEAS-2B) treatment with hog barn dust extract (HDE) results in augmentation of peripheral blood lymphocyte adhesion to epithelial cell cultures in vitro. The augmentation of lymphocyte adhesion to epithelial cells is dependent on the concentration of HDE and time of HDE exposure, with twofold increases observed by 3 h and maintained at 24 h. Similar results are seen with primary human bronchial epithelial cells in culture. Lymphocyte adhesion to epithelial cells is inhibited in a concentration-dependent fashion by the treatment of epithelial cells with antibody to intercellular adhesion molecule-1 (ICAM-1). In addition, HDE exposure of epithelial cells results in an approximate twofold increase in ICAM-1 expression as determined by flow cytometry analysis. Pretreatment of epithelial cells with a protein kinase C-alpha (PKC-alpha) inhibitor, G?-6976, also inhibited subsequent lymphocyte adhesion to HDE-exposed epithelial cells. These data suggest that airway epithelial cell HDE exposure enhances subsequent lymphocyte adhesion to epithelial cells that is mediated in part by HDE modulation of ICAM-1 expression and PKC-alpha.