Linkage analysis of susceptibility to ozone-induced lung inflammation in inbred mice

Through a survey of all departments of pediatrics, neurology and neuropathology in Germany, we calculated the incidence of all major forms of leukodystrophy. Only diagnoses based on specific biochemical tests in association with typical findings and/or neuroradiologically proven white matter involvement were accepted. In accordance with these strict criteria, 617 cases of leukodystrophy were found (incidence of all forms: app. 2.0/100,000). Minimal incidence was estimated at 0.8/100,000 for adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN), 0.6/100,000 for metachromatic leukodystrophy (MLD), and 0.6/100,000 for Krabbe disease. Thus ALD/AMN is apparently underdiagnosed in Germany. A considerable proportion of leukodystrophies could not be classified in spite of adequate diagnostic procedures in experienced centers.     

Regional clearance of solute from peripheral airway epithelia: recovery after sublobar exposure to ozone

The influence of local exposure to ozone (O3) on respiratory epithelial permeability of sublobar lung segments was studied by using aerosolized 99mTc-diethylenetriamine pentaacetic acid (DTPA; mol wt, 492). Two bronchoscopes were inserted through an endotracheal tube in anesthetized, mechanically ventilated, mixed breed dogs and were wedged into sublobar bronchi located in the right and left lower lobes, respectively. Segments were ventilated via the bronchoscope with 5% CO2 in air delivered at 200 ml/min, and an aerosol of 99mTc-DTPA was generated and delivered through the scope and into the sublobar segment over a 30-s period. Clearance of 99mTc-DTPA was measured simultaneously from right and left lower lung segments at baseline and 1, 7, and 14 days after a 6-h sublobar exposure to filtered air or 400 parts per billion O3. O3 treatment significantly decreased the clearance halftime (t50) of 99mTc-DTPA by 50% from the baseline mean of 32.3 to 16.0 min at 1 day postexposure. After 7 days of recovery, t50 was still reduced by 28. 8%; however, by 14 days postexposure, clearance of 99mTc-DTPA had recovered, and the t50 had a mean value of 30.0 min. 99mTc-DTPA clearance was not altered by exposure to filtered air, and t50 values were comparable to baseline at 1, 7, and 14 days postexposure. These results reveal that a single local exposure to O3 increases transepithelial clearance, but only for epithelia directly exposed to O3, and that 7-14 days of recovery are required before permeability to small-molecular-weight solutes returns to normal.     

Effects of ozone and airway inflammation on glutathione status and iron homeostasis in the lungs of horses

OBJECTIVE: To investigate the effects of ozone and airway inflammation on indices of oxidant injury in horses. ANIMALS: 5 clinically normal horses and 25 horses referred for poor performance. PROCEDURE: Blood, tracheal wash, and bronchoalveolar lavage fluid samples were collected before and after ozone exposure (n = 5) or from clinical cases (n = 25), and were analyzed for reduced glutathione (GSH), glutathione disulfide (GSSG), and free and total iron (Fe) values. A scoring system (0 to 5) was used to assess airway inflammation on the basis of clinical signs and cytologic analysis of the tracheal wash and bronchoalveolar lavage fluid samples. RESULTS: Ozone induced significant (P < 0.05) increases in GSH (195.4 +/- 68.5 microM), GSSG (19.4 +/- 6.4 microM), and free (25.5 +/- 16.1 microM) and total (93.1 +/- 13.4 microM) Fe values in the pulmonary epithelial lining fluid, compared with preozone samples (49.2 +/- 18.6, 2.4 +/- 1.2, 0.0, and 33.1 +/- 5.9 microM, respectively). The presence of airway inflammation (19/25) was associated with high GSSG and free and total Fe, but not GSH, values in epithelial lining fluid, compared with values for clinically normal horses (6/25). There were no differences in the systemic values of GSH, GSSG, and free and total Fe between any of the groups. A strong correlation (r = 0.84; P < 0.001) existed between inflammation score and the glutathione redox ratio (GSSG/[GSH + GSSG]) in the 25 horses admitted for clinical examination. CONCLUSIONS: Oxidant injury in the lung will induce changes in the glutathione status and Fe homeostasis that could affect pathogenesis of the disease. CLINICAL RELEVANCE: Measurement of indices of oxidant injury may be useful in the diagnosis of airway inflammation and the response to inhaled oxidants.     

Breathing and pulmonary surfactant function in mice 24 h after ozone exposure

Systemic administration of pilocarpine, which results in status epilepticus followed by recurrent seizures in rats, is a widely used experimental model of chronic limbic epilepsy. Marked structural alterations have been documented in pilocarpine-induced epilepsy, and these include cell loss in the hippocampus and other brain areas, and sprouting of mossy and cholinergic fibers in the hippocampus. Evidence is accumulating that neurotrophins and neurotrophin receptors are involved in the cascade of these events. Two and 4 months after pilocarpine-induced epilepsy, neurons containing the 75-kDa low affinity neurotrophin receptor (p75NTR) were investigated with immunohistochemistry in the medial septal and diagonal band nuclei. No significant differences in the distribution and number of immunoreactive neurons were found in the epileptic rats compared to control saline-treated animals. However, in the epileptic animals, a significant decrease in the perikaryal size of p75NTR-immunoreactive neurons of the septal/diagonal band region was found by 60 days, and such atrophic changes were more marked in the diagonal band nuclei by 120 days. These findings indicate that the p75NTR-containing cell bodies, which include the neurons projecting to the hippocampal formation and are cholinergic in the normal brain, survive after months of spontaneous recurrent seizures, during which, therefore, a supply of p75NTR to target regions is maintained in the chronic epileptic brain. However, the present data point out that these p75NTR-containing neurons undergo a significant shrinkage in pilocarpine-induced chronic epilepsy, thus indicating that they are involved in the brain pathology of temporal lobe epilepsy.     

Interleukin-9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice

Human atopic asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and airway hyperresponsiveness. Recent studies demonstrate that the degree of airway responsiveness is strongly associated with interleukin (IL)-9 expression in murine lung. To investigate the contribution of IL-9 to airway hyperresponsiveness, and to explore directly its relationship to airway inflammation, we studied transgenic mice overexpressing IL-9. In this report we show that IL-9 transgenic mice (FVB/N-TG5), in comparison with FVB/NJ mice, display significantly enhanced eosinophilic airway inflammation, elevated serum total immunoglobulin E, and airway hyperresponsiveness following lung challenge with a natural antigen (Aspergillus fumigatus). These data support a central role for IL-9 in the complex pathogenesis of allergic inflammation.     

Induction of microsomal epoxide hydrolase activity in inbred mice by chronic phenytoin exposure

A lower microsomal epoxide hydrolase (mEH) activity has been associated with increased likelihood of fetal hydantoin syndrome. While phenytoin anticonvulsive regimens are long-term, there are no data regarding induction of mEH by chronic phenytoin exposure. Two inbred mouse strains which differ in their susceptibility (A/J > C57BL/6J) to phenytoin-induced oral clefting were treated with an oral gavage of phenytoin for 14 consecutive days. The mice were sacrificed on the 15th day, and hepatic microsomes were prepared. mEH activity was determined using benzo[a]pyrene-4,5-oxide. The dihydrodiol product was separated by HPLC and quantified. There was no significant difference (P = 0.15) in the phenytoin plasma level between the two strains on Day 15. There was no significant difference (P = 0.07) between control and sham control groups within each strain, so they were combined for further analysis. There was a significant strain difference (P = 0.0001) between the control and phenytoin-exposed group means, with the C57BL/6J strain having the greater activity before and after phenytoin exposure. The A/J phenytoin-exposed group activity was 51% higher (P = 0.01) than the A/J control, while the C57BL/6J phenytoin-exposed group activity was 78% higher (P = 0.001) than the C57BL/6J control. The greater mEH activity in the phenytoin-induced clefting resistant strain (C57BL/6J) before and after phenytoin exposure is consistent with a putative oxidative metabolism mechanism of phenytoin teratogenecity. Chronic phenytoin exposure induced mEH activity in both strains, although the strain with the greater enzyme activity prior to the exposure continued to have the greater activity following induction.     

Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice

The earliest contact between antigen and the innate immune system is thought to direct the subsequent antigen-specific T cell response. We hypothesized that cells of the innate immune system, such as natural killer (NK) cells, NK1.1(+) T cells (NKT cells), and gamma/delta T cells, may regulate the development of allergic airway disease. We demonstrate here that depletion of NK1.1(+) cells (NK cells and NKT cells) before immunization inhibits pulmonary eosinophil and CD3(+) T cell infiltration as well as increased levels of interleukin (IL)-4, IL-5, and IL-12 in bronchoalveolar lavage fluid in a murine model of allergic asthma. Moreover, systemic allergen-specific immunoglobulin (Ig)E and IgG2a levels and the number of IL-4 and interferon gamma-producing splenic cells were diminished in mice depleted of NK1.1(+) cells before the priming regime. Depletion of NK1.1(+) cells during the challenge period only did not influence pulmonary eosinophilic inflammation. CD1d1 mutant mice, deficient in NKT cells but with normal NK cells, developed lung tissue eosinophilia and allergen-specific IgE levels not different from those observed in wild-type mice. Mice deficient in gamma/delta T cells showed a mild attenuation of lung tissue eosinophilia in this model. Taken together, these findings suggest a critical role of NK cells, but not of NKT cells, for the development of allergen-induced airway inflammation, and that this effect of NK cells is exerted during the immunization. If translatable to humans, these data suggest that NK cells may be critically important for deciding whether allergic eosinophilic airway disease will develop. These observations are also compatible with a pathogenic role for the increased NK cell activity observed in human asthma.     

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

Exposure to ambient ozone (O3) is associated with increased exacerbations of asthma. We sought to determine whether mast cell degranulation is induced by in vivo exposure to O3 in mice and whether mast cells play an essential role in the development of pulmonary pathophysiological alterations induced by O3. For this we exposed mast cell-deficient WBB6F1-kitW/kitW-v (kitW/kitW-v) mice and the congenic normal WBB6F1 (+/+) mice to air or to 1 or 3 parts/million O3 for 4 h and studied them at different intervals from 4 to 72 h later. We found evidence of O3-induced cutaneous, as well as bronchial, mast cell degranulation. Polymorphonuclear cell influx into the pulmonary parenchyma was observed after exposure to 1 part/milllion O3 only in mice that possessed mast cells. Airway hyperresponsiveness to intravenous methacholine measured in vivo under pentobarbital anesthesia was observed in both kitW/kitW-v and +/+ mice after exposure to O3. Thus, although mast cells are activated in vivo by O3 and participate in O3-induced polymorphonuclear cell infiltration into the pulmonary parenchyma, they do not participate detectably in the development of O3-induced airway hyperresponsiveness in mice.     

Oral administration of L-arginine potentiates allergen-induced airway inflammation and expression of interleukin-5 in mice

The role of nitric oxide in the airway hyperresponsiveness and inflammation of bronchial asthma has not yet been established. However, L-arginine, the substrate for nitric oxide synthases, reportedly alleviates airway hyperresponsiveness caused by parainfluenza virus and reduces granulocytic inflammation induced by ischemia-reperfusion. We investigated the effects of L-arginine on a murine model of allergic asthma that included airway hyperresponsiveness, eosinophilic inflammation and expression of interleukin (IL)-5 in the lung. The mice received drinking water with or without L-arginine for 9 weeks. Histologic evaluation and cellular profiles in bronchoalveolar lavage fluid showed that p.o. administration of L-arginine (72 micromol/kg/day) significantly enhanced eosinophilic airway inflammation and goblet cell proliferation that were associated with intratracheal instillation of ovalbumin. L-Arginine also increased protein levels of IL-5 and IL-2 in supernatants from the lung exposed to ovalbumin. The number of eosinophils in bronchoalveolar lavage fluid correlated significantly with the expression of IL-5. L-Arginine did not reverse ovalbumin-associated airway hyperresponsiveness to inhaled ACh. These results suggest that p.o. administration of L-arginine aggravates allergen-induced eosinophilic airway inflammation via expression of IL-5, and in this model it does not show therapeutic efficacy against airway hyperresponsiveness associated with allergen exposure. Oral administration of L-arginine, the precursor of nitric oxide, may not be an effective intervention in allergic asthma.     

Methods of evaluating airway inflammation

Airway inflammation is a feature of bronchial asthma and can be quantified invasively with bronchial mucosal biopsy and bronchoalveolar lavage. The induction of sputum by the inhalation of hypertonic saline however is safer and more noninvasive when compared with such methods. Evidence of airway inflammation may be revealed by examining hypertonic saline-induced sputum for eosinophils, cytokines and eosinophil cationic protein. There is a clear need however to develop further noninvasive discriminant measurement of airway inflammation.     

Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice

We have used a mouse model of allergen-induced airway hyperresponsiveness to demonstrate that immunostimulatory DNA sequences (ISS) containing a CpG DNA motif significantly inhibit airway eosinophilia and reduce responsiveness to inhaled methacholine. ISS not only inhibited eosinophilia of the airway (by 93%) and lung parenchyma (91%), but also significantly inhibited blood eosinophilia (86%), suggesting that ISS was exerting a significant effect on the bone marrow production of eosinophils. The inhibition of the bone marrow production of eosinophils by 58% was associated with a significant inhibition of T cell-derived cytokine generation (IL-5, granulocyte-macrophage CSF, and IL-3). ISS exerted this inhibitory effect on T cell cytokine production indirectly by stimulating monocytes/macrophages and NK cells to generate IL-12 and IFNs. The onset of the ISS effect on reducing the number of tissue eosinophils was both immediate (within 1 day of administration) and sustained (lasted 6 days), and was not due to ISS directly inducing eosinophil apoptosis. ISS was effective in inhibiting eosinophilic airway inflammation when administered either systemically (i.p.), or mucosally (i.e., intranasally or intratracheally). Interestingly, a single dose of ISS inhibited airway eosinophilia as effectively as daily injections of corticosteroids for 7 days. Moreover, while both ISS and corticosteroids inhibited IL-5 generation, only ISS was able to induce allergen-specific IFN-gamma production and redirect the immune system toward a Th1 response. Thus, systemic or mucosal administration of ISS before allergen exposure could provide a novel form of active immunotherapy in allergic diseases.     

The laryngeal mask airway and positive-pressure ventilation

BACKGROUND: The utility of the laryngeal mask airway during positive-pressure ventilation has yet to be determined. Our study was designed to assess whether significant leaks occurred with positive-pressure ventilation and if leaks were associated with gastroesophageal insufflation. METHODS: Forty-eight patients undergoing elective surgery were studied. After induction of anesthesia and paralysis, controlled ventilation was used with four different peak pressure settings in each patient (15, 20, 25, and 30 cmH2O). The order of ventilator pressure settings was assigned from a randomized block schedule. Data collected included inspiratory and expiratory volumes, qualitative assessments of gastroesophageal insufflation, and leak at the neck. After data collection during laryngeal mask use, the anesthesiologist intubated the trachea and measurements were repeated for tracheal tube ventilation. Leak was calculated by subtracting the expiratory from the inspiratory volume and expressed as a fraction of the inspiratory volume. RESULTS: Ventilation with the laryngeal mask airway was adequate at all ventilation pressures and comparable with tracheal tube ventilation. Leak fraction (mean +/- SD) at 15, 20, 25, and 30 cmH2O for laryngeal mask ventilation were 0.13 +/- 0.15, 0.21 +/- 0.18, 0.25 +/- 0.16 and 0.27 +/- 0.17, respectively, and 0.03 +/- 0.03, 0.05 +/- 0.03, 0.05 +/- 0.03 and 0.04 +/- 0.03, respectively, for tracheal tube ventilation. Leak fractions for ventilation with the laryngeal mask were consistently greater than those measured for tracheal tube ventilation at similar ventilation pressures. Leak fraction with laryngeal mask ventilation increased with increasing airway pressures, whereas leak with tracheal tube ventilation remained unchanged. The frequency of gastroesophageal insufflation ranged from 2.1% at a ventilation pressure of 15 cmH2O to 35.4% at 30 cmH2O. CONCLUSIONS: Ventilation using the laryngeal mask appears to be adequate if airway resistance and pulmonary compliance are normal. Gastroesophageal insufflation of air will become a problem in the presence increased ventilation pressure.     

Asthma and airway inflammation: potential anti-inflammatory activities of phosphodiesterase inhibitors

Although mental imagery has historically been ignored by the scientific community, it is one of the six most commonly used alternative therapies chosen by cancer patients. Investigations of immune response to psychosocial interventions have been largely anecdotal or quasi-experimental. Although reports of increased survival predominate among cancer patients, few investigators have measured psychological or immunological outcomes to explain the mechanisms that contribute to improved survival. The efficacy of imagery or support groups in patient adaptation (emotionally and physically) to the stresses of cancer, heightened immune function, and improved morbidity and mortality remain uncertain in the absence of objective evidence from randomized, controlled clinical trials. The imagery and group emotional support study pilot (IMAGES-P) is a 12-month feasibility study designed to examine the effect of group support and imagery/relaxation in a randomized, controlled clinical trial, differentiating the effects of these therapy modalities on immune function, quality of life, and the emotional well-being of women who have completed treatment for breast cancer. Secondary aims are to explore the quantitative relationships among emotional well-being, quality of life, and immune function. For this study established clinical trial methodology was used to examine functional immune responses associated with emotional states induced by the therapy interventions. As a pilot study, IMAGES-P will provide direction for future longitudinal studies evaluating the effectiveness of these interventions on emotional well-being and survival.     

Involvement of superoxide and nitric oxide on airway inflammation and hyperresponsiveness induced by diesel exhaust particles in mice

We previously demonstrated that chronic intratracheal instillation of diesel exhaust particles (DEP) induces airway inflammation and hyperresponsiveness in the mouse, and that these effects were partially reversed by the administration of superoxide dismutase (SOD). In the present study, we have investigated the involvement of superoxide in DEP-induced airway response by analyzing the localization and activity of two enzymes: (1) a superoxide producer, NADPH cytochrome P-450 reductase (P-450 reductase), and (2) a superoxide scavenger, SOD, in the lungs of the exposed mice and controls. P-450 reductase was detected mainly in ciliated cells and clara cells: its activity was increased by the repeated intratracheal instillation of DEP. While CuZn-SOD and Mn-SOD were also present in the airway epithelium, their activity was significantly decreased following DEP instillation. Exposure to DEP doubled the level of nitric oxide (NO) in the exhaled air. DEP exposure also increased the level of constitutive NO synthase (cNOS) in the airway epithelium and inducible NO synthase (iNOS) in the macrophages. Pretreatment with N-G-monomethyl L-arginine, a nonspecific inhibitor of NO synthase, significantly reduced the airway hyperresponsiveness induced by DEP. These results indicate that superoxide and NO may each contribute to the airway inflammation and hyperresponsiveness induced by the repeated intratracheal instillation of DEP in mice.     

Treatment of airway inflammation in cystic fibrosis

Airway inflammation is now recognized as a major factor in the pathogenesis of cystic fibrosis (CF) lung disease. Therapies aimed at decreasing the inflammatory response represent a new strategy for treatment, and attention has focused primarily on the therapeutic potential of corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs). Alternate-day prednisone (1 mg/kg) may be beneficial; however, unacceptable adverse effects limit long-term use. Inhaled corticosteroids are under investigation as a safer alternative. High-dose ibuprofen (approximately 20-30 mg/kg twice daily) has been shown to decrease the progression of CF lung disease, particularly in children with mild lung disease, and it is without significant toxicity. Other NSAIDs (piroxicam) are under consideration, as well as pentoxifylline and fish oil. The rationale for all of these agents lies in their potential to decrease neutrophil influx into the lung. Because of the large burden and deleterious effects of uninhibited neutrophil elastase and oxidants in the CF airway, antiproteases and antioxidants are also being studied. To optimize anti-inflammatory therapy, it is necessary to understand the mechanism of action of these agents in the CF lung, to determine which of these agents would provide the most benefit to patients with CF, and to determine which therapies should be initiated at what age or stage of lung disease. It is hoped that adding anti-inflammatory therapy to an already comprehensive treatment program will decrease morbidity and improve the quality of life for patients with CF.     

Epithelia cells as regulators of airway inflammation

The past decade has seen major advances in our understanding of the metabolic and biochemical functions of the epithelial cell. As a result, the traditional view of the epithelium as a relatively passive physical barrier to the internal environment has been superseded by the concept that the epithelial plays a key role in regulating airway inflammation. The epithelial cell experts this modulatory effect in several ways. Epithelial cells can function maintain mucosal integrity and to modulate local immune responses. They can also limit inflammatory processes by degrading, or inhibiting, proinflammatory mediators and proteins. However, the epithelium also responds to a range of stimuli by producing biologically active mediators that can influence airway inflammation. These include, but are not limited to a broad range of cytokines and chemokines that can exert profound effects on inflammatory cells, as well as lipid and peptide mediators. This review will highlight some of these aspects of the role of the epithelium in regulating airway inflammation.     

Time-course of antigen-induced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness

The causative relationship between airway inflammation and hyperreactivity is unclear, since inflammatory changes have been examined at one or, at most, a few time-points after antigen challenge in both human asthma and animal models. We have made a detailed investigation of inflammatory and functional changes in the airways up to 8 days after antigen challenge in guinea-pigs. In particular, we examined the hypothesis that eosinophil-derived mediators contribute to tissue damage and the development of airway hyperresponsiveness. Following antigen challenge, the influx of inflammatory cells and mediator release in airway tissue and bronchoalveolar lavage fluid were correlated temporally with histopathological changes in airway tissue and airway responsiveness. Eosinophil influx was demonstrable at 4 h. Eosinophilia peaked after 24 h and persisted for at least 8 days. Parallel increases in the concentrations of major basic protein and eosinophil cationic protein in bronchoalveolar lavage fluid indicated that the eosinophils were activated. Eosinophilia was accompanied by subepithelial oedema and epithelial damage co-localized with major basic protein immunoreactivity. A transient neutrophilia (< 48 h duration) and an increase in neutrophil elastase in bronchoalveolar lavage fluid peaked at 14 h. The proportion of airway macrophages with an activated morphology increased at 8 h and remained markedly elevated until 72 h. Airways were hyperresponsive to histamine at 4 h and for at least 8 days. The antigen-induced airway inflammation resemble in time-course and histopathology that seen in antigen-challenged asthmatics, and indicate that the eosinophil and its cytotoxic proteins may be major mediators of airway mucosal damage and airway hyperresponsiveness.     

Radionuclide imaging of airway obstruction following assisted ventilation

Radioaerosol lung scintigraphy was performed in five infants in whom obstructive airway complications developed following assisted ventilation. These studies suggest that a primary functional defect was obstruction to airflow at the level of the major airways, which occurred during expiration. Perfusion lung scintigraphy showed areas of diminished or redistributed pulmonary blood flow, which, like the results of the radioaerosol scintigraphic studies, are findings often associated with adult chronic obstructive pulmonary disease.     

Cardiovascular effects of ozone exposure in human volunteers

We hypothesized that ozone (O3) exposure acutely affects cardiovascular hemodynamics in humans and, in particular, in subjects with essential hypertension. We studied 10 nonmedicated hypertensive and six healthy male adults. Each subject, after catheterization of the right heart and a radial artery, was exposed in an environmentally controlled chamber to filtered air (FA) on one day and to 0.3 ppm O3 on the following day for 3 h with intermittent exercise. Relative to FA exposure, O3 exposure induced no statistically significant changes in cardiac index, ventricular performance, pulmonary artery pressure, pulmonary and systemic vascular resistances, ECG, serum cardiac enzymes, plasma catecholamines and atrial natriuretic factor, and SaO2. The overall results did not indicate major acute cardiovascular effects of O3 in either the hypertensive or the control subjects. However, mean preexposure to postexposure changes were significantly (p < 0.02) larger with O3 than with FA for rate-pressure product (1,353 beats/min/mm Hg) and for heart rate (8 beats/min); these responses were not significantly different between the hypertensive and the control subjects. Significant O3 effects were also observed for mean FEV1 (-6%), and AaPO2 (> 10 mm Hg increase), which were not significantly different between the two groups. These results suggest that O3 exposure can increase myocardial work and impair pulmonary gas exchange to a degree that might be clinically important in persons with significant preexisting cardiovascular impairment, with or without concomitant lung disease.