Extracellular glutathione inhibits oxygen-induced permeability changes in alveolar epithelial monolayers.

Exposure to high fractional inspired oxygen for 24 h increases permeability of the alveolar epithelium, contributing to the clinical manifestations of oxygen toxicity. Utilizing a model of the alveolar epithelium in which isolated rat type II cells form polarized monolayers on polycarbonate filters [transepithelial resistance (R(t)) > 1 k Omega x cm(2) by day 4], we evaluated the ability of reduced glutathione (GSH) to ameliorate these changes. On day 4, apical fluid was replaced with culture medium containing 1) no additives, 2) GSH (500 microM), or 3) GSH (500 microM) + glutathione reductase (0.5 U/ml) + nicotinamide adenine dinucleotide phosphate (250 microM). Monolayers were exposed (for 24 h) to room air (control) or 95% O(2), each containing 5% CO(2). After 24 h of hyperoxia, R(t) for condition 1 decreased by 45% compared with control (P < 0.001). In conditions 2 and 3, R(t) did not decrease significantly (P = not significant). Hyperoxia-induced decreases in active ion transport were observed for conditions 1 and 2 (P < 0.05), but not for condition 3 (P = not significant). These findings indicate that extracellular GSH may protect the alveolar epithelium against hyperoxia-induced injury. Addition of glutathione reductase and nicotinamide adenine dinucleotide phosphate may further augment these protective effects of GSH.

Successful nonoperative management of delayed spontaneous esophageal perforation in patients with human immunodeficiency virus.

OBJECTIVE: To describe the clinical outcome of esophageal stenting for repair of distal esophageal perforation in one patient with septic shock and human immunodeficiency virus. DESIGN: Case report. SETTING: Medical-surgical intensive care units of one university teaching hospital. PATIENT: One patient with human immunodeficiency virus infection and septic shock in whom there was a delay in diagnosis of spontaneous perforation at the distal thoracic esophagus. INTERVENTION: A 10 cm x 2 cm silicone lined, partially coated, expandable metal stent was fluoroscopically placed in the distal esophagus at the perforation. Other treatment included chest tube thoracostomy, sump drainage of proximal esophagus, percutaneous gastrostomy, and antibiotics. MEASUREMENT AND MAIN RESULTS: Septic shock and the distal esophageal perforation were successfully treated with combined esophageal stenting, thoracostomy pleural drainage and antibiotics. Esophageal stenting was accomplished fluoroscopically with a partially coated, silicone-lined, expandable metal stent. CONCLUSION: Esophageal stenting, tube thoracostomy drainage, and antibiotics may be a management option for gravely ill patients with human immunodeficiency virus, esophageal perforation, and a delay in diagnosis. An optimal outcome requires a thoughtful, individualized approach and adherence to basic principles.

Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis.

Cystic fibrosis (CF) is characterized by accumulation of activated neutrophils and macrophages on the respiratory epithelial surface (RES); these cells release toxic oxidants, which contribute to the marked epithelial derangements seen in CF. These deleterious consequences are magnified, since reduced glutathione (GSH), an antioxidant present in high concentrations in normal respiratory epithelial lining fluid (ELF), is deficient in CF ELF. To evaluate the feasibility of increasing ELF GSH levels and enhancing RES antioxidant protection, GSH aerosol was delivered (600 mg twice daily for 3 days) to seven patients with CF. ELF total, reduced, and oxidized GSH increased (P < 0.05, all compared with before GSH therapy), suggesting adequate RES delivery and utilization of GSH. Phorbol 12-myristate 13-acetate-stimulated superoxide anion (O2-.) release by ELF inflammatory cells decreased after GSH therapy (P < 0.002). This paralleled observations that GSH added in vitro to CF ELF inflammatory cells suppressed O2-. release (P < 0.001). No adverse effects were noted during treatment. Together, these observations demonstrate the feasibility of using GSH aerosol to restore RES oxidant-antioxidant balance in CF and support the rationale for further clinical evaluation.

Systemic deficiency of glutathione in cystic fibrosis.

Cystic fibrosis (CF), a disorder characterized by mutations of the CF transmembrane regulator gene, is characterized in the lung by chronic inflammation, leading to progressive damage to the airway epithelium, bronchiectasis, and chronic obstructive lung disease. One process contributing to the airway derangement is the chronic burden of oxidants released by inflammatory cells on the respiratory epithelial surface. With this background, we hypothesized that glutathione in respiratory epithelial lining fluid (ELF) in CF patients might be oxidized and/or diminished in amount compared with that in normal subjects. Recovery of ELF by bronchoalveolar lavage from young adults with CF (n = 21) and normal subjects (n = 25) demonstrated marked neutrophil-dominated inflammation in ELF in CF patients. As predicted, ELF in CF patients was characterized by a deficiency of glutathione (P < 0.001), but this was secondary to a reduction in reduced glutathione (P < 0.001), inasmuch as there were no differences in ELF levels of oxidized glutathione (P > 0.2). Unexpectedly, there was also a marked deficiency of reduced glutathione in plasma (P < 0.02); i.e., the glutathione "deficiency" observed in ELF in CF patients is not limited to the site of the inflammation but is systemic. Although the etiology of this generalized deficiency of extracellular glutathione is unknown, it is important in considering options for treating the concomitant and devastating lung pathology in this disorder.

Correction of glutathione deficiency in the lower respiratory tract of HIV seropositive individuals by glutathione aerosol treatment.

BACKGROUND: Concentrations of glutathione, a ubiquitous tripeptide with immune enhancing and antioxidant properties, are decreased in the blood and lung epithelial lining fluid of human immunodeficiency virus (HIV) seropositive individuals. Since the lung is the most common site of infection in those who progress to AIDS it is rational to consider whether it is possible to safely augment glutathione levels in the epithelial lining fluid of HIV seropositive individuals, thus potentially improving local host defence. METHODS: Purified reduced glutathione was delivered by aerosol to HIV seropositive individuals (n = 14) and the glutathione levels in lung epithelial lining fluid were compared before and at one, two, and three hours after aerosol administration. RESULTS: Before treatment total glutathione concentrations in the epithelial lining fluid were approximately 60% of controls. After three days of twice daily doses each of 600 mg reduced glutathione, total glutathione levels in the epithelial lining fluid increased and remained in the normal range for at least three hours after treatment. Strikingly, even though > 95% of the glutathione in the aerosol was in its reduced form, the percentage of oxidised glutathione in epithelial lining fluid increased from 5% before treatment to about 40% three hours after treatment, probably reflecting the use of glutathione as an antioxidant in vivo. No adverse effects were observed. CONCLUSIONS: It is feasible and safe to use aerosolised reduced glutathione to augment the deficient glutathione levels of the lower respiratory tract of HIV seropositive individuals. It is rational to evaluate further the efficacy of this tripeptide in improving host defence in HIV seropositive individuals.

Pulmonary Langerhans cell granulomatosis (histiocytosis X). A clinicopathologic study of 48 cases.

We report the clinical and histologic findings of lung biopsies from 48 patients with pulmonary Langerhans cell granulomatosis (PLCG) and show how special techniques such as immunohistochemistry, electron microscopy (EM), and high resolution computerized tomography (HRCT) of the lungs can be useful in diagnostically challenging cases. Nineteen patients were men and 29 were women. The median age was 33 years (range 15-54 years). Two had pituitary involvement. Bone lesions were observed in four patients and biopsy proven in two. All patients were cigarette smokers. In six patients HRCT revealed a combination of thin-walled cystic and nodular lesions. The pathologic diagnosis was established on the basis of open lung biopsy specimens in 44 cases and transbronchial biopsy specimens in 4 of 10 cases. In two transbronchial biopsies, diagnostic PLCG infiltrates were seen in toluidine blue-stained thick sections in the tissue processed for EM but not on the tissue processed for histology. EM in both of these cases revealed Birbeck granules within LCs. The diagnosis was supported by a positive bone biopsy in one of these patients and characteristic HRCT findings in the other. The antibody to S100 protein stained the LC infiltrates in the five cases studied. This staining and the characteristic findings on HRCT confirmed the diagnosis in one case in which the PLCG lesions were obscured by atelectasis. The frequent finding of intraluminal fibrosis (78% of open lung biopsies) supports the recent suggestion that this alteration plays an important role in the pathogenesis of fibrotic remodeling in PLCG. The strong association of PLCG with cigarette smoking and the frequent peribronchiolar location of PLCG lesions (87% of open lung biopsies) in our cases are consistent with the concept that in adults this disorder is associated with an abnormal response to cigarette smoke.

Aerosolization of superoxide dismutase. Augmentation of respiratory epithelial lining fluid antioxidant screen by aerosolization of recombinant human Cu++/Zn++ superoxide dismutase.

Various human pulmonary diseases are characterized by an increased oxidant burden on the respiratory epithelial surface. As a step toward developing a therapy to augment the antioxidant defenses of respiratory epithelial lining fluid (ELF) of the human lung, we have evaluated the feasibility of aerosolizing a human protein antioxidant to the respiratory epithelial surface of an experimental animal sufficiently large to permit repetitive sampling of ELF. To accomplish this, recombinant human Cu++/Zn++ superoxide dismutase (rSOD) was aerosolized to sheep, and the levels of human superoxide dismutase (SOD) and antisuperoxide anion (O2.-) capacity were quantified in ELF over time. In vitro aerosolization did not alter the specific activity of rSOD (p > 0.5). In vivo aerosolization of rSOD (100 mg) to sheep (n = 7) resulted in peak amounts of human Cu++/Zn++ SOD in ELF of 3.1 +/- 0.6 mumol/L, with a parallel increase in the anti-O2.- capacity of ELF. For the duration of the study (5 h), levels of SOD and anti-O2.- in ELF remained elevated, with a value 50 percent of the peak at 5 h. Aerosolization of phosphate-buffered saline (n = 5) had no effect on SOD or anti-O2.- levels in ELF. In animals receiving rSOD, there was no change in the specific activity of SOD recovered in ELF compared to the starting material (p > 0.4). We conclude that rSOD can be delivered by aerosol to the ELF of a large animal with preservation of specific activity and that a substantial increase in both the amount of SOD and the anti-O2.- capacity can be achieved for a period of time applicable to human therapy, supporting the rationale for evaluation of rSOD aerosol as an antioxidant in human pulmonary disease.

Activation of alveolar macrophages in asymptomatic HIV-infected individuals.

After the initial infection with HIV, there is evidence of immune dysfunction despite an apparent normal clinical state. In the context that the lung is a major site affected by opportunistic infection during the progression of this immune dysfunction, and that some components of the immune system are activated during early HIV infection, we hypothesized that there may be activation of alveolar macrophages (AM), a key component of the pulmonary host defense system, during the asymptomatic phase of HIV infection. Compared to normals, in HIV-infected individuals the class II MHC molecules DR, DQ, and DP were all expressed more frequently and in greater cell surface density on AM (p < 0.03, all comparisons), and there was increased spontaneous release of superoxide anion (O2-.) by AM (p < 0.002). To gain insight into whether the activation of the AM was an inherent property of the cells or dependent on the in vivo milieu, AM were evaluated after 24 h in culture for O2-. release. In contrast to the findings in fresh AM, after 24 h in culture, O2-. release by HIV AM was not different from normals (p > 0.7), suggesting that these AM had been activated in vivo. To assess whether IFN-gamma could be mediating these effects, mRNA levels of the IP-10 gene (a gene specifically induced by increased concentrations of IFN-gamma) were quantified in AM. Strikingly, the IP-10 gene was expressed only in AM of HIV-seropositive individuals, suggesting the AM had been exposed to IFN-gamma in vivo. Overall, these observations are consistent with the concept that the HIV-seropositive state is associated with activation of AM, in part due to local exposure to IFN-gamma.

Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterised by alveolar inflammation, exaggerated release of oxidants, and subnormal concentrations of the antioxidant glutathione in respiratory epithelial lining fluid (ELF). Glutathione (600 mg twice daily for 3 days) was given by aerosol to 10 patients with IPF. Total ELF glutathione rose transiently, ELF oxidised glutathione concentrations increased, and there was a decrease in spontaneous superoxide anion release by alveolar macrophages. Thus, glutathione by aerosol could be a means of reversing the oxidant-antioxidant imbalance in IPF.

Effects of propranolol and indomethacin on muscarinic airway reactivity in unanesthetized guinea pigs.

To investigate whether endogenous beta-adrenergic stimulation or cyclooxygenase products normally affect muscarinic reactivity in conscious, spontaneously breathing guinea pigs, we measured specific airway resistance (SRaw) during acetylcholine (ACh) infusion before and after treatment with propranolol (10 mg/kg ip) or indomethacin (30 mg/kg ip). Airway reactivity was assessed by measuring changes in SRaw upon increasing ACh infusion. We found that propranolol treatment increased reactivity to parenteral ACh, but did not change baseline SRaw. Furthermore, propranolol reduced the range in muscarinic reactivity for the group, and it enhanced thr reproducibility of measurements in individual animals. In contrast, indomethacin had no effect on either baseline SRaw or muscarinic reactivity. Our results suggest that beta-blockade of endogenous adrenergic stimulation increases the muscarinic reactivity of guinea pig airways, but does not influence resting airway tone. It appears that propranolol treatment allows a more reproducible assessment of muscarinic reactivity in the guinea pig. In contrast, cyclooxygenase products do not seem to significantly affect baseline airway resistance, reactivity, or reproducibility in the guinea pig.

Indomethacin increases bronchial reactivity after exposure to subthreshold ozone levels.

We investigated the effects of indomethacin on bronchial reactivity after ozone exposure. Guinea pigs in groups of six were treated with indomethacin (30 mg/kg IP) and studied before and 2 h after a 2 h exposure to either 1.5 or 3.0 ppm ozone. These animals were compared to similarly exposed groups that were untreated. Reactivity was determined by measuring specific airway resistance (SRaw) upon intravenous acetylcholine infusion. Prior to ozone exposure, indomethacin had no effect on either SRaw or muscarinic reactivity. In all untreated guinea pigs (n = 12) exposed to 1.5 ppm ozone, there was no significant change in SRaw or muscarinic reactivity. In contrast, all treated animals exposed to 1.5 ppm showed a substantial increase in reactivity. Those treated animals exposed to 3.0 ppm showed significant elevations in SRaw, making interpretations of changes in their reactivity difficult. We conclude that indomethacin treatment increases bronchial reactivity in guinea pigs exposed to subthreshold ozone levels.

Bronchial hyperreactivity occurs in steroid-treated guinea pigs depleted of leukocytes by cyclophosphamide.

The effects of cyclophosphamide and cortisone acetate treatment on O3-induced changes in airway mucosal morphology and bronchial reactivity were assessed in guinea pigs. Animals in groups of four were studied at 2 or 6 h after O3 (3.0 ppm, 2 h) and in one control group. Reactivity was determined by measuring specific airway resistance during intravenous acetylcholine infusion in intact, unanesthetized, spontaneously breathing animals. After testing, tracheal tissue was obtained from all animals for light microscopic examination. Another group of 10 drug-treated and 10 normal animals were tested at 2 h, 6 h, 1 day, and 4 days after O3. Drug treatment resulted in substantial decreases in both circulating and airway mucosal granulocytes. Two hours after O3, a marked decrease in airway mucosal goblet cells as well as ciliated cell damage occurred in both normal and treated animals. However, only in normal animals did neutrophilic infiltration develop thereafter. Nonetheless, hyperreactivity postozone occurred and progressed similarly in both groups. Our results indicate that acute O3-induced bronchial hyperreactivity at 2 h is associated with signs of airway mucosal injury but appears independent of granulocyte changes. Airway neutrophilic infiltration and eosinophil depletion seem to be consequences of mucosal injury from O3 and not causes of the bronchial hyperreactivity that results.

Sequence of pathologic changes in the airway mucosa of guinea pigs during ozone-induced bronchial hyperreactivity.

We assessed the nature and progression of airway mucosal disease and muscarinic bronchial reactivity in guinea pigs studied in groups of 4 at 2 h, 6 h, 14 h, 1 day, 2 days, or 4 days after ozone exposure (3.0 ppm for 2 h), and in 1 control group. Muscarinic reactivity was determined by measuring specific airway resistance as a function of increasing doses of intravenous acetylcholine in 31 intact, unanesthetized, spontaneously breathing animals. After testing, each group was killed to obtain tracheal tissue for light microscopic examination. We found that airway hyperreactivity to acetylcholine occurred in 96% of the animals exposed to ozone. Its degree at 2 h was substantial. Complete remission was not observed until the fourth day. In association with the acute bronchial hyperreactivity found at 2 h, a marked decrease in airway mucosal goblet cells and an increase in mucosal mast cells occurred. Neutrophilic infiltration occurred later and lasted longer, despite remission of the hyperreactivity. Our results indicate that acute, ozone-induced bronchial hyperreactivity is related to signs of airway mucosal injury and mast cell infiltration. After this early phase of airway damage, neutrophilic infiltration occurs and persists, suggesting that it is a consequence of the damage rather than a cause of the increased airway reactivity after ozone exposure.

Ozone-induced changes in muscarinic bronchial reactivity by different testing methods.

We examined the effect of ozone (O3) on muscarinic bronchial reactivity in the guinea pig and compared reactivity determined by two different routes of agonist delivery. Reactivity before and from 4 h to 2 days after O3 exposure (3.0 ppm, 2 h) was determined by measuring specific airway resistance upon administration of intravenous acetylcholine and/or aerosolized methacholine challenge in 34 unanesthetized, spontaneously breathing animals. Before exposure, we observed more gradual and reproducible results to intravenous agonist. After exposure, hyperreactivity to parenteral agonist occurred consistently, but not to inhaled agonist. Hyperreactivity demonstrable by either route was similar in magnitude and time course within 14 h of exposure. Two days later, hyperreactivity to inhaled agonist had remitted; that to intravenous drug persisted. Our results indicate that variability in the occurrence and time course of O3-induced hyperreactivity to inhaled agonist may be a consequence of the technique employed. The consistent occurrence of hyperreactivity after O3 to parenteral agonist suggests mechanisms other than airway mucosal hyperpermeability are responsible for this hyperreactivity.