IL-17A Modulates Oxidant Stress-Induced Airway Hyperresponsiveness but Not Emphysema

IL-17A induces the release of pro-inflammatory cytokines and of reactive oxygen species which could lead to neutrophilic inflammation. We determined the role of IL-17 receptor (IL-17R) signalling in oxidant-induced lung emphysema and airway hyperresponsiveness. IL-17R^−/− and wild-type C57/BL6 mice were exposed to ozone (3 ppm; 3 hours) for 12 times over 6 weeks. Bronchial responsiveness to acetylcholine was measured, and lungs were retrieved. Mean linear intercept (L_m) and isometric contractile responses of intrapulmonary airways to acetylcholine were determined. In wild-type mice but not in IL-17R^−/−, chronic ozone exposure caused airway hyperresponsiveness. The increase in L_m after chronic ozone exposure of wild-type mice was also observed in IL-17R^−/− mice. The increased maximal contractile response to acetylcholine seen in airways of wild-type mice exposed to ozone was abolished in IL-17R^−/− mice. p38-mitogen-activated protein kinase (MAPK) and dexamethasone-dependent increase in contractile response was reduced in airways from IL-17R^−/− ozone-exposed mice. Lung inflammation scores were not altered in IL-17R^−/− mice exposed to ozone compared to wild-type mice. The increased release of IL-17 and IL-1β, and the activation of p38 MAPK in the lungs of ozone-exposed mice was reduced in IL-17R^−/− mice. IL-17R signalling underlies the increase in airway hyperresponsiveness seen after ozone exposure, mediated by the increased contractility of airway smooth muscle. The emphysema and lung inflammation induced by ozone is not dependent on IL-17.     

Ozone-induced pulmonary inflammation and epithelial proliferation are partially mediated by PAF

Ozone(O₃) exposure stimulates airwayinflammation and epithelial sloughing in a number of species, includingmice. Platelet-activating factor (PAF) is a lipid mediator released byactivated mast cells, macrophages, and epithelial cells and causespulmonary inflammation and hyperpermeability. We hypothesized that theactivation of PAF receptors is central to the development ofinflammation and epithelial injury induced by acuteO₃ exposure in mice. To test thishypothesis, O₃-susceptibleC57BL/6J mice were treated with a PAF-receptor antagonist, UK-74505, orvehicle either before or immediately after 3-h exposure toO₃ (2 parts/million) or filtered air. Bronchoalveolar lavage (BAL) fluids were collected 6 and 24 hafter exposure. Differential cell counts and protein content of thelavage were used as indicators of inflammation in the airways. O₃-induced epithelial injury wasassessed by light microscopy, and DNA synthesis in epithelium ofterminal bronchioles was estimated by using abromodeoxyuridine-labeling index. Intercellular adhesion molecule 1 (ICAM-1) expression was also examined in the lung by immunohistochemical localization.O₃ caused significant increases inpolymorphonuclear leukocytes and protein in the BAL fluid, increasedpulmonary epithelial proliferation, and increased epithelial expressionof ICAM-1 compared with air-exposed, vehicle-treated control mice.Relative to O₃-exposed,vehicle-treated control mice, UK-74505 before exposure significantly(P < 0.05) decreased BAL protein,polymorphonuclear leukocytes, and epithelial cells. O₃-induced inflammation wassimilarly attenuated in mice treated with UK-74505 after exposure.These experiments thus support the hypothesis thatO₃-induced airways inflammationand epithelial damage in mice are partially mediated by activation ofPAF receptors, possibly through modulation of ICAM-1 expression.

     

Proinflammatory role of inducible nitric oxide synthase in acute hyperoxic lung injury

BackgroundHyperoxic exposures are often found in clinical settings of respiratory insufficient patients, although oxygen therapy (>50% O₂) can result in the development of acute hyperoxic lung injury within a few days. Upon hyperoxic exposure, the inducible nitric oxide synthase (iNOS) is activated by a variety of proinflammatory cytokines both in vitro and in vivo. In the present study, we used a murine hyperoxic model to evaluate the effects of iNOS deficiency on the inflammatory response.MethodsWild-type and iNOS-deficient mice were exposed to normoxia, 60% O₂ or >95% O₂ for 72 h.ResultsExposure to >95% O₂ resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. No significant effects of iNOS deficiency on cell differential in bronchoalveolar lavage fluid were observed. However, hyperoxia induced a significant increase in total cell count, protein concentration, LDH activity, lipid peroxidation, and TNF-α concentration in the bronchoalveolar lavage fluid compared to iNOS knockout mice. Moreover, binding activity of NF-κB and AP-1 appeared to be higher in wild-type than in iNOS-deficient mice.ConclusionTaken together, our results provide evidence to suggest that iNOS plays a proinflammatory role in acute hyperoxic lung injury.     

Respiratory syncytial virus infection reduces lung inflammation and fibrosis in mice exposed to vanadium pentoxide

Background

Vanadium pentoxide (V₂O₅) exposure is a cause of occupational bronchitis and airway fibrosis. Respiratory syncytial virus (RSV) is a ubiquitous pathogen that causes airway inflammation. It is unknown whether individuals with pre-existing respiratory viral infection are susceptible to V₂O₅-induced bronchitis. We hypothesized that respiratory viral infection will exacerbate vanadium-induced lung fibrosis.

Methods

In this study we investigated the effect of RSV pre- or post-exposure to V₂O₅ in male AKR mice. Mice were pre-exposed by intranasal aspiration to RSV or media vehicle prior to intranasal aspiration of V₂O₅ or saline vehicle at day 1 or day 7. A parallel group of mice were treated first with V₂O₅ or saline vehicle at day 1 and day 7 then post-exposed to RSV or media vehicle at day 8.

Results

V₂O₅-induced airway inflammation and fibrosis were decreased by RSV pre- or post-exposure. Real time quantitative RT-PCR showed that V₂O₅ significantly increased lung mRNAs encoding pro-fibrogenic growth factors (TGF-β1, CTGF, PDGF-C) and collagen (Col1A2), but also increased mRNAs encoding anti-fibrogenic type I interferons (IFN-α, -β) and IFN-inducible chemokines (CXCL9 and CXCL10). RSV pre- or post-exposure caused a significantly reduced mRNAs of pro-fibrogenic growth factors and collagen, yet reduced RNA levels of anti-fibrogenic interferons and CXC chemokines.

Conclusions

Collectively these data suggest that RSV infection reduces the severity of V₂O₅-induced fibrosis by suppressing growth factors and collagen genes. However, RSV suppression of V₂O₅-induced IFNs and IFN-inducible chemokines suggests that viral infection also suppresses the innate immune response that normally serves to resolve V₂O₅-induced fibrosis.     

Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells

Although it is understood that hydrogen peroxide (H₂O₂) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H₂O₂ in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H₂O₂. The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0  ±  4.7 h versus 28.6  ±  0.8 h, p < 0.05), consistent with a diminished growth rate and H₂O₂ release. Incubation with aminotriazole, a catalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G₀/G₁ phase in hCatTg MAECs compared to the wild-type cells (25.0  ±  0.9 h versus 15.9  ±  1.4 h, p  <  0.05). The hCatTg MAECs also exhibited decreased activities of the cyclin-dependent kinase (Cdk) complexes responsible for G₀/G₁- to S-phase transition in the cell cycle, including the cyclin D–Cdk4 and cyclin E–Cdk2 complexes. Moreover, the reduction in cyclin–Cdk activities in hCatTg MAECs was accompanied by increased protein levels of two Cdk inhibitors, p21 and p27, which inhibit the Cdk activity required for the G₀/G₁- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H₂O₂ scavenger, suggest that endogenously produced H₂O₂ mediates MAEC proliferation by fostering the transition from G₀/G₁ to S phase.     

Behavioral characterization of mice lacking the A3 adenosine receptor: sensitivity to hypoxic neurodegeneration

1. The potential neuroprotective actions of the A₃ adenosine receptor (A₃AR) were investigated using mice with functional deletions of the A₃AR (A₃AR^–/–) in behavioral assessments of analgesia, locomotion, tests predictive of depression and anxiety, and the effects of mild hypoxia on cognition and neuronal survival.2. Untreated A₃AR^–/– mice were tested in standard behavioral paradigms, including activity in the open field, performance in the hot-plate, tail-flick, tail-suspension, and swim tests, and in the elevated plus maze. In addition, mice were exposed repeatedly to a hypoxic environment containing carbon monoxide (CO). The cognitive effects of this treatment were assessed using the contextual fear conditioning test. After testing, the density of pyramidal neurons in the CA1, 2, and 3 subfields of the hippocampus was determined using standard histological and morphometric techniques.3. A₃AR^–/– mice showed increased locomotion in the open field test, elevated plus maze (number of arm entries) and light/dark box (number of transitions). However, they spent more time immobile in two different tests of antidepressant activity (Swim and tail suspension tests). A₃AR^–/– mice also showed evidence of decreased nociception in the hot-plate, but not tail-flick tests. Further, A₃AR^–/– mice were more vulnerable to hippocampal pyramidal neuron damage following episodes of carbon monoxide (CO)-induced hypoxia. One week after exposure to CO a moderate loss of pyramidal neurons was observed in all hippocampal subfields of both wild-type (A₃AR^+/+) and A₃AR^–/– mice. However, the extent of neuronal death in the CA2–3 subfields was less pronounced in A₃AR^+/+ than A₃AR^–/– mice. This neuronal loss was accompanied by a decline in cognitive function as determined using contextual fear conditioning. These histological and cognitive changes were reproduced in wild-type mice by repeatedly administering the A₃AR-selective antagonist MRS 1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate 1 mg/kg i.p.).4. These results indicate that pharmacologic or genetic suppression of A₃AR function enhances some aspects of motor function and suppresses pain processing at supraspinal levels, while acting as a depressant in tests predictive of antidepressant action. Consistent with previous reports of the neuroprotective actions of A₃AR agonists, A₃AR^–/– mice show an increase in neurodegeneration in response to repeated episodes of hypoxia.     

Developmental changes in the cochlear hair cell mechanotransducer channel and their regulation by transmembrane channel–like proteins

Vibration of the stereociliary bundles activates calcium-permeable mechanotransducer (MT) channels to initiate sound detection in cochlear hair cells. Different regions of the cochlea respond preferentially to different acoustic frequencies, with variation in the unitary conductance of the MT channels contributing to this tonotopic organization. Although the molecular identity of the MT channel remains uncertain, two members of the transmembrane channel–like family, Tmc1 and Tmc2, are crucial to hair cell mechanotransduction. We measured MT channel current amplitude and Ca²⁺ permeability along the cochlea’s longitudinal (tonotopic) axis during postnatal development of wild-type mice and mice lacking Tmc1 (Tmc1−/−) or Tmc2 (Tmc2−/−). In wild-type mice older than postnatal day (P) 4, MT current amplitude increased ∼1.5-fold from cochlear apex to base in outer hair cells (OHCs) but showed little change in inner hair cells (IHCs), a pattern apparent in mutant mice during the first postnatal week. After P7, the OHC MT current in Tmc1−/− (dn) mice declined to zero, consistent with their deafness phenotype. In wild-type mice before P6, the relative Ca²⁺ permeability, P_Ca, of the OHC MT channel decreased from cochlear apex to base. This gradient in P_Ca was not apparent in IHCs and disappeared after P7 in OHCs. In Tmc1−/− mice, P_Ca in basal OHCs was larger than that in wild-type mice (to equal that of apical OHCs), whereas in Tmc2−/−, P_Ca in apical and basal OHCs and IHCs was decreased compared with that in wild-type mice. We postulate that differences in Ca²⁺ permeability reflect different subunit compositions of the MT channel determined by expression of Tmc1 and Tmc2, with the latter conferring higher P_Ca in IHCs and immature apical OHCs. Changes in P_Ca with maturation are consistent with a developmental decrease in abundance of Tmc2 in OHCs but not in IHCs.     

Effects of rapid transfer from sea water to fresh water on respiratory variables,blood acid-base status and O2 affinity of haemoglobin in Atlantic salmon (Salmo salar L.)

Summary Oxygen consumption, gill ventilation, blood acid-base/ionic status and haemoglobin oxygen affinity were studied in seawater-adapted adult salmon (Salmo salar) during five weeks after transfer into fresh water. Freshwater exposure induced the following changes: Standard oxygen consumption ( ) and ventilatory flow ( ) decreased markedly during the first days after transfer, then decreased more gradually until a new steady-state was achieved at which and were about 80% and 56% of the control values, respectively. The marked increase in oxygen extraction coefficient (Ew _{O 2}) and the marked decrease in the oxygen convection requirement ( ) were associated with a reduction in the partial pressure of carbon dioxide in arterial blood (Pa _{CO 2}), in spite of a decrease of both ventilatory flow and water CO₂ capacitance. These results suggested that transfer into fresh water induced an increase in branchial diffusive conductance. A biphasic pattern was observed in the time-course of the changes in both plasma ion concentration and acid-base status. During the first 10 days, plasma Na⁺, K⁺, and Cl^– concentrations fell abruptly, then more gradually. [Cl^–] decreased more than [Na⁺] resulting in a progressive increase in the [Na⁺]/[Cl^–] ratio. During the second phase of acclimation to fresh water plasma Na⁺, K⁺, and Cl^– concentrations progressively increased. [Cl^–] increased more than [Na⁺], so that [Na⁺]/[Cl^–] ratio decreased. Transfer into fresh water did not significantly change plasma lactate concentration. Upon exposure to fresh water, blood pH increased from 7.94±0.04 to 8.43±0.06 at day 10 and then decreased to 8.08±0.03 at day 34. The increase in blood pH induced by transfer to fresh water initially represented a mixed metabolic/respiratory alkalosis. However, after 15 days Pa _{CO 2} had returned to pretransfer values and the alkalosis was purely metabolic. The metabolic component of the alkalosis was associated with appropriate changes in the plasma strong ion difference (S.I.D.). Blood alkalosis moved the oxygen dissociation curve to the left, so that P₅₀ was decreased by 30% below the value in seawater for the maximal increase in blood pH. This rise in haemoglobin affinity for O₂, associated with a marked increase in blood buffer capacity, are regarded as adaptative processes allowing the salmon to cope with the markedly increased energy expenditure required for upstream migration.     

Maximal oxygen uptake, maximal voluntary isometric contraction and physical activity in young Danish adults

In a randomly selected sample of 88 men and 115 women, aged 23–27 years from Denmark, maximal oxygen uptake ( O_2max), maximal voluntary isometric contraction (MVC) in four muscle groups and physical activity were studied. The O_2max was 48.0 ml · min^–1 kg^–1 and 39.6 ml · min^–1 · kg^–1 for the men and the women, respectively. The MVC was 10% lower than in a comparable group of Danes of the same age and height studied 35 years ago. Only in men was sports activity directly related to O_2max (ml · min^–1 · kg^–1; r=0.31, P<0.01). The MVC of the knee extensors was related to O_2max in the men (r=0.31, P<0.01), but there was no relationship between the other measurements of MVC and O_2max. In the women O_2max (ml · min^–1 · kg^–1) was only related to body size, i.e. body mass index, percentage body fat and body mass [(r= –0.47, –0.48 (both P<0.001) and –0.34. (P<0.01), respectively)]. There were differences in O_2max in the men, according to education and occupation. Blue collar workers and subjects attending vocational or trade schools in 1983 had lower O_2max and more of them were physically inactive. In the women differences were also found, but there was no clear pattern among the groups. More of the women participated regularly in sports activity, but more of the men were very active compared to the women.     

Lung injury during LPS-induced inflammation occurs independently of the receptor P2Y<Subscript>1</Subscript>

Disruption of the lung endothelial and epithelial barriers during acute inflammation leads to excessive neutrophil migration. It is likely that activated platelets promote pulmonary recruitment of neutrophils during inflammation, and previous studies have found that anti-platelet therapy and depletion of circulating platelets have lung-protective effects in different models of inflammation. Because ADP signaling is important for platelet activation, I investigated the role of the ADP-receptor P2Y₁, a G protein-coupled receptor expressed on the surface of circulating platelets, during lipopolysaccharide (LPS)-induced inflammation and lung injury in P2Y₁-null and wild-type mice. Systemic inflammation was induced by a single intraperitoneal dose of LPS (3 mg/kg), and the mice were analyzed 24 h posttreatment. The data show that the LPS-induced inflammation levels were comparable in the P2Y₁-null and wild-type mice. Specifically, splenomegaly, counts of circulating platelets and white blood cells (lymphocytes and neutrophils), and assessments of lung injury (tissue architecture and cell infiltration) were similar in the P2Y₁-null and wild-type mice. Based on my results, I conclude that lung injury during LPS-induced inflammation in mice is independent of P2Y₁ signaling. I propose that if a blockade of purinergic signaling in platelets is a potential lung-protective strategy in the treatment of acute inflammation, then it is more likely to be a result of the disruption of the signaling pathway mediated by P2Y₁₂, another G protein-coupled receptor that mediates platelet responses to ADP.     

Cyclooxygenase-2 in newborn hyperoxic lung injury

Supraphysiological O₂ concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD).Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD.Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice.The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury.We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization.Newborn C3H/HeN micewere injected daily with vehicle, aspirin (nonselective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life.Additional studies utilized wild-type (C57Bl/6, COX-2^+/+), heterozygous (COX-2^+/-), and homozygous (COX-2^-/-) transgenic mice.Micewere exposed to room air (21% O₂) or hyperoxia (85% O₂) for 14 days.Aspirin-injected and COX-2^-/- pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL).Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and air spaces.Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D₂ metabolites.Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits.Our data suggest thatincreased COX-2 activity may contribute to proinflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia.Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD.     

Absence of the Common Gamma Chain (γc), a Critical Component of the Type I IL-4 Receptor,Increases the Severity of Allergic Lung Inflammation

The T_H2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and drive the alternative activation of macrophages (AAM). Although both cytokines share receptor subunits, IL-4 and IL-13 have differential roles in asthma pathogenesis: IL-4 regulates T_H2 cell differentiation, while IL-13 regulates airway hyperreactivity and mucus production. Aside from controlling T_H2 differentiation, the unique contribution of IL-4 signaling via the Type I receptor in airway inflammation remains unclear. Therefore, we analyzed responses in mice deficient in gamma c (γ_c) to elucidate the role of the Type I IL-4 receptor. OVA primed CD4⁺ OT-II T cells were adoptively transferred into RAG2^−/− and γ_c ^−/− mice and allergic lung disease was induced. Both γ_c ^−/− and γ_cxRAG2^−/− mice developed increased pulmonary inflammation and eosinophilia upon OVA challenge, compared to RAG2^−/− mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater numbers of FIZZ1+ or YM1+ airways were present in γ_c ^−/− mice. Absence of γ_c in macrophages, however, resulted in reduced YM1 expression. We observed higher T_H2 cytokine levels in the BAL and an altered DC phenotype in the γ_c ^−/− recipient mice suggesting the potential for dysregulated T cell and dendritic cell (DC) activation in the γ_c-deficient environment. These results demonstrate that in absence of the Type I IL-4R, the Type II R can mediate allergic responses in the presence of T_H2 effectors. However, the Type I R regulates AAM protein expression in macrophages.     

Airway Surface Dehydration Aggravates Cigarette Smoke-Induced Hallmarks of COPD in Mice

Introduction

Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown.

Objective

We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the β-subunit of the epithelial Na⁺ channel (βENaC).

Methods

βENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured.

Results

Airway surface dehydration in βENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in βENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements.

Conclusions

We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD.     

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice,but not in those from gp91-phox-deficient mice

Macrophages produce superoxide (O₂^–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O₂^– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H₂DCFDA), O₂^– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H₂DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O₂^–-producing cells, but not in O₂^–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H₂O₂ dismutated from O₂^–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.     

Synthesis, structural analysis and anticonvulsant activity of a ternary Cu(II) mononuclear complex containing 1,10-phenanthroline and the leading antiepileptic drug valproic acid

The synthesis and characterization of the binary complex of copper(II) with the antiepileptic drug valproic acid sodium salt (Valp) and the related ternary complex with 1,10-phenanthroline (phen) are reported, as well as the anticonvulsant properties of the latter. The characterization was carried out by means of elemental analyses, infrared (IR), UV–visible (UV–vis) spectrophotometry and Electron Paramagnetic Resonance (EPR). The X-ray crystal structure of the mononuclear complex bis(2-propylpentanoate)(1,10-phenanthroline)copper(II) [Cu(Valp)₂phen] is showed for the first time. It crystallized in C2/c space group with unit cell dimensions of a = 14.939(1) Å, b = 19.280(1) Å, c = 9.726(1) Å, β = 97.27(2)°, V = 2778.8(4) Å ³ and Z = 8. The carboxylates bond in an asymmetric chelating mode and the copper atom adopts a highly distorted octahedral coordination, characterized by the sum of the angles of 365.9° around Cu(II) and its nearest atoms in the CuN₂O₂ + O₂ chromophore instead of the expected 360° for a basal square planar geometry found in most Cu(II) complexes. Molecules assemble three by three through slipped π–π stacking of the aromatic phen with respectively 3.519 and 3.527 Å distances, in a head-to-tail arrangement. Studies of the anticonvulsant properties of this bioligand chelate evidenced its lack of efficacy in preventing MES-induced seizures. Interestingly, complex 4 protected mice against the Minimal Clonic seizures at doses that do not cause Rotorod toxicity, with an ED₅₀ documenting very potent anticonvulsant activity in this model of seizure, a particularly useful pharmacological profile of activity for the treatment of Petit Mal seizures.     

Molecular mechanisms of nitrogen dioxide induced epithelial injury in the lung

The lung can be exposed to a variety of reactive nitrogen intermediates through the inhalation of environmental oxidants and those produced during inflammation. Reactive nitrogen species (RNS) include, nitrogen dioxide (·NO₂) and peroxynitrite (ONOO^–). Classically known as a major component of both indoor and outdoor air pollution, ·NO₂ is a toxic free radical gas. ·NO₂ can also be formed during inflammation by the decomposition of ONOO^– or through peroxidase-catalyzed reactions. Due to their reactive nature, RNS may play an important role in disease pathology. Depending on the dose and the duration of administration, ·NO₂ has been documented to cause pulmonary injury in both animal and human studies. Injury to the lung epithelial cells following exposure to ·NO₂ is characterized by airway denudation followed by compensatory proliferation. The persistent injury and repair process may contribute to airway remodeling, including the development of fibrosis. To better understand the signaling pathways involved in epithelial cell death by ·NO₂ or other RNS, we routinely expose cells in culture to continuous gas-phase ·NO₂. Studies using the ·NO₂ exposure system revealed that lung epithelial cell death occurs in a density dependent manner. In wound healing experiments, ·NO₂ induced cell death is limited to cells localized in the leading edge of the wound. Importantly, ·NO₂-induced death does not appear to be dependent on oxidative stress per se. Potential cell signaling mechanisms will be discussed, which include the mitogen activated protein kinase, c-Jun N-terminal Kinase and the Fas/Fas ligand pathways. During periods of epithelial loss and regeneration that occur in diseases such as asthma or during lung development, epithelial cells in the lung may be uniquely susceptible to death. Understanding the molecular mechanisms of epithelial cell death associated with the exposure to ·NO₂ will be important in designing therapeutics aimed at protecting the lung from persistent injury and repair.     

Role of Tissue Factor in Mycobacterium tuberculosis-Induced Inflammation and Disease Pathogenesis

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.     

Dietary antioxidants and the biochemical response to oxidant inhalation

We fed female strain A/St mice selenium (Se) test diets containing either no Se (?Se) or 1 ppm Se (+Se) for 11 wk. Both diets contained 55 ppm vitamin E. We then exposed three groups of mice from each dietary regimen to either 0.8 ppm (1568 μg/m³) O₃ (low-level) continuously for 5 d, 10.0 ppm (19,600 μg/m³) O₃ (high-level) for 12 h, or filtered room air, where the latter served as a control for both O₃ exposures. After O₃ exposures we analyzed the lungs for various physical and biochemical parameters, and compared the results to those obtained from the air controls. The results showed that the difference in dietary Se intake produced an eightfold difference in Se content and a three-fold difference in glutathione peroxidase (GP) activity in the lung, but few changes in other lung parameters. With low-level O₃ exposure, NADPH production increased significantly in +Se mice, but did not change in ?Se mice. With high-level O₃ exposure we observed comparable effects for both dietary regimens, including animal mortality, which was 24% for ?Se and 14% for +Se mice. Thus, it seems that diminished GP activity resulting from Se deficiency and the ensuing lack of increase in NADPH production were poorly correlated with mouse tolerance to O₃. The lung Se content increased in both dietary regimens after O₃ exposure, but the increase was greater after high-level O₃ exposure. This suggests a “mobilization” of Se to the lung under O₃ stress. It is possible that such a mobilization contributes to the lung reserve of antioxidants, and hence the comparable mortality in both dietary Se regimens.     

Mast cell activation is not required for induction of airway hyperresponsiveness by ozone in mice

Exposure to ambient ozone(O₃) is associated withincreased exacerbations of asthma. We sought to determine whether mastcell degranulation is induced by in vivo exposure toO₃ in mice and whether mast cellsplay an essential role in the development of pulmonarypathophysiological alterations induced byO₃. For this we exposed mastcell-deficientWBB6F₁-kit^W/kit^W-v(kit^W/kit^W-v)mice and the congenic normalWBB6F₁ (+/+) mice to air or to 1 or 3 parts/million O₃ for 4 h andstudied them at different intervals from 4 to 72 h later. We foundevidence of O₃-induced cutaneous,as well as bronchial, mast cell degranulation. Polymorphonuclear cellinflux into the pulmonary parenchyma was observed after exposure to 1 part/milllion O₃ only in mice thatpossessed mast cells. Airway hyperresponsiveness to intravenousmethacholine measured in vivo under pentobarbital anesthesia wasobserved in bothkit^W/kit^W-vand +/+ mice after exposure to O₃.Thus, although mast cells are activated in vivo byO₃ and participate inO₃-induced polymorphonuclear cellinfiltration into the pulmonary parenchyma, they do not participate detectably in the development ofO₃-induced airwayhyperresponsiveness in mice.

     

Signal role for activation of caspase-3-like protease and burst of superoxide anions during Ce<Superscript>4+</Superscript>-induced apoptosis of cultured <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells

The signal events of 1 mM Ce⁴⁺ (Ce(NH₄)₂(NO₃)₆)-induced apoptosis of cultured Taxus cuspidata cells were investigated. The percentage of apoptotic cells increased from 0.82% to 51.32% within 6 days. Caspase-3-like protease activity became notable during the second day of Ce⁴⁺-treatment, and the maximum activity was 5-fold higher than that of control cells at the fourth day. When the experiment system was pretreated with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) at 100 M, caspase-3-like activity resulted in distinct inhibition by 70% and 77.3% after 3 and 4 days of induction. Furthermore, 100 M Ac-DEVD-CHO partially reduced the apoptotic cells by 58.6% and 60.8% at day 4 and 5 respectively. Ce⁴⁺ induced superoxide anions (O₂·–) transient burst, and the first peak appeared at around 3.7–4 h, the second appeared at about 7 h. Both O₂·– burst and cell apoptosis were effectively suppressed by application of diphenyl iodonium (NADPH oxidase inhibitor). Inhibition of O₂·– production attenuated caspase-3-like activation by 49% and 53.6% during day 3 and 4 respectively. In addition, a total of 15 protein spots changed in response to caspase-3-like protease activation were identified by two-dimensional gel electrophoresis. These results suggest that Ce⁴⁺ of 1 mM induces apoptosis in suspension cultures of T. cuspidata through O₂·– burst as well as caspase-3-like protease activation. The burst of O₂·– exerts its activity as an upstream of caspase-3-like activation. Our results also implicate that other signal pathways independent of an O₂·– burst possibly participate in mediating caspase-3-like protease activation.