James R. Sheller

Release of Free F2-isoprostanes from Esterified Phospholipids Is Catalyzed by Intracellular and Plasma Platelet-activating Factor Acetylhydrolases

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.

Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension

Free radicals have been implicated in the pathogenesis of an increasing number of diseases. Lipids, which undergo peroxidation, are major targets of free radical attack. We report the discovery of a pathway of lipid peroxidation that forms a series of isomers in vivo that are characterized by a substituted tetrahydrofuran ring structure, termed isofurans (IsoFs). We have proposed two distinct pathways by which IsoFs can be formed based on 18O2 and H218O labeling studies. Measurement of F2-isoprostanes (IsoPs), prostaglandin F2-like compounds formed nonenzymatically as products of lipid peroxidation, is considered one of the most reliable approaches for assessing oxidative stress status in vivo. However, one limitation with this approach is that the formation of IsoPs becomes limited at high oxygen tension. In contrast, the formation of IsoFs becomes increasingly favored as oxygen tension increases. IsoFs are present at readily detectable levels in normal fluids and tissues, and levels increase dramatically in CCl4-treated rats, an animal model of oxidant injury. The ratio of IsoFs to IsoPs in major organs varies according to normal steady-state tissue oxygenation. In addition, IsoFs show a marked increase early in the course of hyperoxia-induced lung injury, whereas IsoPs do not significantly increase. We propose that combined measurement of IsoFs and IsoPs should provide a more reliable index of oxidant stress severity than quantification of either alone because of the opposing modulation of the two pathways by oxygen tension, which can vary widely in different organs and disease states.

Airway Epithelium Controls Lung Inflammation and Injury through the NF-κB Pathway

Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-κB pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-κB activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IκB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-κB activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-κB in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IκB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-κB pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.

A controlled trial of the effect of the 5-lipoxygenase inhibitor, zileuton, on lung inflammation produced by segmental antigen challenge in human beings

BACKGROUND Segmental antigen challenge (SAC) and bronchoalveolar lavage (BAL) have been proven useful for investigating IgE-mediated lung inflammation in volunteers with allergies. OBJECTIVE This model was used to evaluate the pulmonary antiinflammatory effects of an experimental 5-lipoxygenase inhibitor (zileuton) in subjects allergic to ragweed. We hypothesized that decreased generation of leukotrienes by inhibition of the 5-lipoxygenase pathway of arachidonic acid metabolism would diminish the subsequent inflammatory response resulting from antigen challenge. METHODS Ten subjects with allergies received zileuton or placebo, 600 mg administered orally four times a day for 8 days, and then underwent bronchoscopy, BAL of a control segment, and SAC in the contralateral lung followed by BAL of the challenged segment 24 hours later in a double-blind, placebo-controlled, crossover protocol. Urinary excretion of leukotriene E4 induced by antigen challenge plus total and differential cell counts and the amount of total protein, albumin, urea, and eosinophil cationic protein in BAL fluid were determined. RESULTS A significant inhibition of leukotriene production (approximately 86%) was observed in subjects receiving zileuton. In addition, there was a statistically significant increase in eosinophils after antigen challenge (0.6 +/- 0.2 x 10(4) eosinophils/ml increasing to 49.0 +/- 25.0 x 10(4) in subjects receiving placebo, whereas the influx of eosinophils in subjects receiving zileuton was not statistically different from baseline (1.1 +/- 0.7 x 10(4) eosinophils/ml increasing to 16.5 +/- 4.1 x 10(4); analysis of variance for repeated measures with post hoc comparisons). CONCLUSIONS Treatment with zileuton altered the inflammatory response after antigen challenge. Products of the 5-lipoxygenase pathway appear to be important in recruiting eosinophils to the lung after SAC.

Constrictive bronchiolitis in soldiers returning from Iraq and Afghanistan.

BACKGROUND In this descriptive case series, 80 soldiers from Fort Campbell, Kentucky, with inhalational exposures during service in Iraq and Afghanistan were evaluated for dyspnea on exertion that prevented them from meeting the U.S. Armys standards for physical fitness. METHODS The soldiers underwent extensive evaluation of their medical and exposure history, physical examination, pulmonary-function testing, and high-resolution computed tomography (CT). A total of 49 soldiers underwent thoracoscopic lung biopsy after noninvasive evaluation did not provide an explanation for their symptoms. Data on cardiopulmonary-exercise and pulmonary-function testing were compared with data obtained from historical military control subjects. RESULTS Among the soldiers who were referred for evaluation, a history of inhalational exposure to a 2003 sulfur-mine fire in Iraq was common but not universal. Of the 49 soldiers who underwent lung biopsy, all biopsy samples were abnormal, with 38 soldiers having changes that were diagnostic of constrictive bronchiolitis. In the remaining 11 soldiers, diagnoses other than constrictive bronchiolitis that could explain the presenting dyspnea were established. All soldiers with constrictive bronchiolitis had normal results on chest radiography, but about one quarter were found to have mosaic air trapping or centrilobular nodules on chest CT. The results of pulmonary-function and cardiopulmonary-exercise testing were generally within normal population limits but were inferior to those of the military control subjects. CONCLUSIONS In 49 previously healthy soldiers with unexplained exertional dyspnea and diminished exercise tolerance after deployment, an analysis of biopsy samples showed diffuse constrictive bronchiolitis, which was possibly associated with inhalational exposure, in 38 soldiers.

Asthma outcomes: composite scores of asthma control.

BACKGROUND Current asthma guidelines recommend assessing the level of a patients asthma control. Consequently, there is increasing use of asthma control as an outcome measure in clinical research studies. Several composite assessment instruments have been developed to measure asthma control. OBJECTIVE National Institutes of Health institutes and federal agencies convened an expert group to propose the most appropriate standardized composite score of asthma control instruments to be used in future asthma studies. METHODS We conducted a comprehensive search of PubMed using both the National Library of Medicines Medical Subject Headings and key terms to identify studies that attempted to develop and/or test composite score instruments for asthma control. We classified instruments as core (required in future studies), supplemental (used according to study aims and standardized), or emerging (requiring validation and standardization). This work was discussed at a National Institutes of Health-organized workshop convened in March 2010 and finalized in September 2011. RESULTS We identified 17 composite score instruments with published validation information; all had comparable content. Eight instruments demonstrated responsiveness over time; 3 demonstrated responsiveness to treatment. A minimal clinically important difference has been established for 3 instruments. The instruments have demographic limitations; some are proprietary, and their use could be limited by cost. CONCLUSION Two asthma composite score instruments are sufficiently validated for use in adult populations, but additional research is necessary to validate their use in nonwhite populations. Gaps also exist in validating instruments for pediatric populations.

Respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice

Severe respiratory syncytial virus (RSV)‐induced disease is associated with childhood asthma and atopy. We combined models of allergen sensitization and RSV infection to begin exploring the immunologic interactions between allergic and virus‐induced airway inflammation and its impact on airway hypersensitivity. Airway resistance was measured after methacholine challenge in tracheally intubated mice by whole body plethysmography. Lung inflammation was assessed by bronchoalveolar lavage (BAL) and histopathology. RSV infection alone did not cause significant airway hyperresponsiveness (AHR) to methacholine. Ovalbumin (OVA)‐induced AHR lasted only a few days past the discontinuance of OVA aerosol in mice that were ovalbumin sensitized and mock infected. In contrast, OVA‐sensitized mice infected with RSV during the OVA aerosol treatments (OVA/RSV) had AHR for more than 2 weeks after infection. However, 2 weeks after either RSV or mock infection, OVA/RSV mice had significantly more lymphocytes found during BAL than OVA mice, whereas the OVA and OVA/RSV groups had the same number of eosinophils. Histopathologic analysis confirmed an increased inflammation in the lungs of OVA/RSV mice compared with OVA mice. In addition, OVA/RSV mice had a more widespread distribution of mucus in their airways with increased amounts of intraluminal mucus pools compared with the other groups. Thus, prolonged AHR in RSV‐infected mice during ovalbumin‐sensitization correlates with increased numbers of lymphocytes in BAL fluid, increased lung inflammation, and mucus deposition in the airways, but not with airway eosinophilia. A further understanding of the immunologic consequences of combined allergic and virus‐induced airway inflammation will impact the management of diseases associated with airway hyperreactivity. J. Med. Virol. 57:186–192, 1999.

Assessment of oxidant stress in allergic asthma by measurement of the major urinary metabolite of F2-isoprostane, 15-F2t-IsoP (8-iso-PGF2alpha).

Asthma is a chronic inflammatory disease of the airways which may involve an oxidant injury to the lung. Assessment of oxidant stress is difficult in vivo, but measurement of F2‐isoprostanes (F2‐IsoPs), free radical‐catalysed products of arachidonic acid, appears to offer a reliable approach for quantitative measurement of oxidative stress status in vivo. We have recently developed a mass spectrometric assay for 2,3‐dinor‐5,6‐dihydro‐15‐F2t‐IsoP (15‐F2t‐IsoP‐M), the major urinary metabolite of the F2‐IsoP, 15‐F2t‐IsoP (8‐iso‐PGF2a). Measurement of the urinary excretion of this metabolite offers a reliable index of oxidative stress status in vivo that has advantages over measuring unmetabolized F2‐IsoPs in urine and plasma.

Ethnic differences in response to morphine

Only recently has attention been focused on the importance of interethnic differences as determinants of interindividual variability in drug response. We compared the pharmacokinetics and pharmacodynamics of morphine in eight Chinese and eight white healthy men after 0.15 mg/kg of morphine intravenously. The clearance of morphine was significantly higher in the Chinese subjects than in the white subjects because of an increase in the partial metabolic clearance by glucuronidation. There was no interethnic difference in the metabolism to normorphine. Morphine depressed the respiratory response to rebreathing carbon dioxide more in white subjects than in Chinese subjects, resulting in a greater reduction in resting ventilation and resting end‐tidal Pco2. The slope of the ventilation/Pco2 response curve, a measure of carbon dioxide sensitivity, was reduced more in white subjects than Chinese subjects. As a result, white subjects had a greater depression in ventilation at a Pco2 of 55 mm Hg. The morphine‐induced reduction in blood pressure was also greater in white subjects than in Chinese subjects. Thus this study has shown ethnicity to be an important determinant of the disposition and effects of morphine.

Respiratory syncytial virus infection does not increase allergen-induced type 2 cytokine production, yet increases airway hyperresponsiveness in mice

Severe respiratory syncytial virus (RSV)‐induced disease is associated with childhood asthma and atopy. We combined murine models of allergen‐sensitization and RSV infection to explore the interaction of allergic and virus‐induced airway inflammation and its impact on airway hyperresponsiveness (AHR). We found that RSV infection during ova‐sensitization (OVA/RSV) increasedtlsb and prolonged AHR compared to mice only RSV‐infected (RSV) or ova‐sensitized (OVA). AHR is known to be associated with an increase in Type 2 cytokines (IL‐4, IL‐5, and IL‐13) in allergen‐sensitized mice. Therefore, we hypothesized that RSV‐induced enhancement of AHR was a result of potentiating the Type 2 cytokine profile promoted by ova‐sensitization. Surprisingly, we found that Type 2 cytokines induced by ova‐sensitization were not increased by RSV infection despite the increase in AHR, and in some cases were diminished. RNAse protection assay revealed no difference in IL‐4 and IL‐5 mRNA levels between the OVA and OVA/RSV groups, and IL‐13 mRNA was significantly decreased in the OVA/RSV mice compared to the OVA group. Flow cytometric analysis of Type 2 cytokines demonstrated the same frequency of IL‐4 and IL‐5 production in lung‐derived T lymphocytes from the OVA/RSV and OVA groups. Direct cytokine ELISA measurements of lung supernatant showed the level of IL‐13 was significantly decreased in the OVA/RSV group compared to OVA mice, while there was no difference in either IL‐4 or IL‐5 between these two groups. These data indicate that the enhanced and prolonged AHR caused by the interaction of allergic airway inflammation and virus‐induced immune responses is a complex process that can not be explained simply by aug‐mented production of Type 2 cytokines. J. Med. Virol. 63:178–188, 2001.