Prenatal exposure to allergen, DNA methylation, and allergy in grandoffspring mice.

BACKGROUND: Prenatal allergen exposure has been linked to both induction and protection of allergic sensitization in offspring. We hypothesized that prenatal exposure of mice (F0) to Aspergillus fumigatus (A. fumigatus) would be associated with decreased immunoglobulin (Ig) E and airway eosinophilia and alterations in CpG methylation of T-helper genes in third-generation mice (F2). METHODS: Female BALB/c mice were sensitized to A. fumigatus (62.5, 125, 1250 µg, or saline) and re-exposed to the same dose on days 7 and 14 (early) or days 12 and 17 (late) gestation. Grandoffspring were treated with A. fumigatus (62.5 µg) at 9 weeks. IgE, IgG(1) , and IgG(2a) levels and cell counts from bronchoalveolar lavage fluid were determined. Lung DNA was pyrosequenced at multiple sites in the interferon (IFN)-γ and interleukin (IL)-4 promoters. RESULTS: Grandoffspring of mothers dosed with 1250 µg early during pregnancy developed increased airway eosinophilia (P < 0.05). Grandoffspring of mothers dosed late in pregnancy developed lower IgE (P < 0.05) and airway eosinophilia (P < 0.05). Grandoffspring of mothers dosed early had lower methylation at IL-4 CpG(-408) and CpG(-393) compared to late dosed mice (P < 0.005 across all doses). Few correlations were found between methylation levels and airway eosinophilia and IgE. CONCLUSION: Prenatal exposure to A. fumigatus late during pregnancy, but not early, was associated with lower IgE and airway eosinophilia in grandoffspring. Prenatal exposure to A. fumigatus was associated with changes in CpG methylation in the IFN-γ and IL-4 promoters that did not correlate consistently with indicators of allergic sensitization.

Alcohol feeding blocks methacholine-induced airway responsiveness in mice.

Historical accounts of alcohol administration to patients with breathing problems suggest that alcohol may have bronchodilating properties. We hypothesized that acute alcohol exposure will alter airway responsiveness (AR) in mice. To test this hypothesis, C57BL/6 mice were fed either 20% alcohol in drinking water (fed) or received a single intraperitoneal (ip) injection of alcohol (3 g/kg). Control groups received regular drinking water or ip saline. AR was assessed by means of ventilation or barometric plethysmography and reported as either total lung resistance or enhanced pause for each group of mice. To confirm alcohol exposure, elevated blood alcohol levels were documented. Alcohol feeding significantly blocked methacholine-triggered AR compared with water-fed controls. Comparable blunting of AR was also accomplished through a single ip injection of alcohol when compared with saline-injected controls. The alcohol response was slowly reversible in both routes of administration after withdrawal of alcohol: AR attenuation by alcohol persisted 12-20 h (ip) or up to 2 wk (fed) after blood alcohol cleared consistent with a sustained bronchodilator effect. These data demonstrate that brief alcohol exposure blunts AR in this murine model of alcohol exposure suggesting a role for alcohol in the modulation of bronchial motor tone.

Imaging the spatial distribution of transgene expression in the lungs with positron emission tomography.

This study was designed to evaluate the utility of positron emission tomography (PET) to quantify the magnitude and spatial distribution of transgene expression after different methods of adenoviral vector delivery (with surfactant- and saline-based vehicles) within rat lungs. In all, 17 animals (eight in the surfactant group, nine in the saline group) were studied 3 days after intratracheal administration of a replication-incompetent adenovirus encoding a mutant Herpes simplex virus-1 thymidine kinase (mHSV1-TK)-enhanced green fluorescent protein fusion gene driven by a Cytomegalovirus promoter (Ad-CMV-mNLS-HSV1sr39tk-egfp). PET images were obtained 1 h after i.v. administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), an imaging substrate for mHSV1-TK. Overall, the average lung concentration of [(18)F]-FHBG was significantly greater in the surfactant group than in the saline group (0.24+/-0.06 versus 0.17+/-0.03% injected dose/ml lung, P< or =0.05). Lung [(18)F]-FHBG distribution was more peripheral and more homogeneous in the surfactant group than in the saline group (mean coefficient of variation=31+/-4 versus 36+/-3%, respectively, P< or =0.05). Regions of increased tracer concentration in the surfactant group compared to the saline group were evenly distributed throughout the lungs. We conclude that PET imaging provides useful and meaningful information about the effectiveness of different gene transfer delivery strategies within the lungs, and that surfactant-based vehicles may be a superior strategy for pulmonary gene transfer.

Alveolar type 1 cells express the alpha2 Na,K-ATPase, which contributes to lung liquid clearance.

The alveolar epithelium is composed of alveolar type 1 (AT1) and alveolar type 2 (AT2) cells, which represent approximately 95% and approximately 5% of the alveolar surface area, respectively. Lung liquid clearance is driven by the osmotic gradient generated by the Na,K-ATPase. AT2 cells have been shown to express the alpha1 Na,K-ATPase. We postulated that AT1 cells, because of their larger surface area, should be important in the regulation of active Na+ transport. By immunofluorescence and electron microscopy, we determined that AT1 cells express both the alpha1 and alpha2 Na,K-ATPase isoforms. In isolated, ouabain-perfused rat lungs, the alpha2 Na,K-ATPase in AT1 cells mediated 60% of the basal lung liquid clearance. The beta-adrenergic agonist isoproterenol increased lung liquid clearance by preferentially upregulating the alpha2 Na,K-ATPase protein abundance in the plasma membrane and activity in alveolar epithelial cells (AECs). Rat AECs and human A549 cells were infected with an adenovirus containing the rat Na,K-ATPase alpha2 gene (Adalpha2), which resulted in the overexpression of the alpha2 Na,K-ATPase protein and caused a 2-fold increase in Na,K-ATPase activity. Spontaneously breathing rats were also infected with Adalpha2, which increased alpha2 protein abundance and resulted in a approximately 250% increase in lung liquid clearance. These studies provide the first evidence that alpha2 Na,K-ATPase in AT1 cells contributes to most of the active Na+ transport and lung liquid clearance, which can be further increased by stimulation of the beta-adrenergic receptor or by adenovirus-mediated overexpression of the alpha2 Na,K-ATPase.

Invited review: lung edema clearance: role of Na(+)-K(+)-ATPase.

Acute hypoxemic respiratory failure is a consequence of edema accumulation due to elevation of pulmonary capillary pressures and/or increases in permeability of the alveolocapillary barrier. It has been recognized that lung edema clearance is distinct from edema accumulation and is largely effected by active Na(+) transport out of the alveoli rather than reversal of the Starling forces, which control liquid flux from the pulmonary circulation into the alveolus. The alveolar epithelial Na(+)-K(+)-ATPase has an important role in regulating cell integrity and homeostasis. In the last 15 yr, Na(+)-K(+)-ATPase has been localized to the alveolar epithelium and its contribution to lung edema clearance has been appreciated. The importance of the alveolar epithelial Na(+)-K(+)-ATPase function is reflected in the changes in the lung's ability to clear edema when the Na(+)-K(+)-ATPase is inhibited or increased. An important focus of the ongoing research is the study of the mechanisms of Na(+)-K(+)-ATPase regulation in the alveolar epithelium during lung injury and how to accelerate lung edema clearance by modulating Na(+)-K(+)-ATPase activity.

Gene therapy for acute diseases.

The use of gene transfer systems to study cell function makes it apparent that overexpression of a transgene can restore or improve the function of a protein and positively influence cell function in a predetermined manner for purposes of counterbalancing cellular pathophysiology. The ability of some gene transfer vehicles to produce transgene product within hours of delivery positions gene transfer as a unique pharmaceutical administration system that can quickly affect production of biologic response modifiers in a highly compartmentalized fashion. This approach can be expected to overcome many of the adverse effects and high costs of systemic delivery of recombinant pharmaceuticals. This review highlights recent advances toward development of gene therapies for acute illnesses with particular emphasis on preclinical models of disease. In this context, a growing body of data suggests that gene therapies for polygenic and non-genetic diseases such as asthma, cardiogenic and non-cardiogenic pulmonary edema, stroke, subarachnoid hemorrhage, seizures, acute myocardial infarction, endovascular thrombosis, and infections may someday be options for the treatment of patients.

beta(2)-adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats.

beta-Adrenergic agonists accelerate the clearance of alveolar fluid by increasing the expression and activity of epithelial solute transport proteins such as amiloride-sensitive epithelial Na(+) channels (ENaC) and Na,K-ATPases. Here we report that adenoviral-mediated overexpression of a human beta(2)-adrenergic receptor (beta(2)AR) cDNA increases beta(2)AR mRNA, membrane-bound receptor protein expression, and receptor function (procaterol-induced cAMP production) in human lung epithelial cells (A549). Receptor overexpression was associated with increased catecholamine (procaterol)-responsive active Na(+) transport and increased abundance of Na,K-ATPases in the basolateral cell membrane. beta(2)AR gene transfer to the alveolar epithelium of normal rats improved membrane-bound beta(2)AR expression and function and increased levels of ENaC (alpha subunit) abundance and Na,K-ATPases activity in apical and basolateral cell membrane fractions isolated from the peripheral lung, respectively. Alveolar fluid clearance (AFC), an index of active Na(+) transport, in beta(2)AR overexpressing rats was up to 100% greater than sham-infected controls and rats infected with an adenovirus that expresses no cDNA. The addition of the beta(2)AR-specific agonist procaterol to beta(2)AR overexpressing lungs did not increase AFC further. AFC in beta(2)AR overexpressing lungs from adrenalectomized or propranolol-treated rats revealed clearance rates that were the same or less than normal, untreated, sham-infected controls. These experiments indicate that alveolar beta(2)AR overexpression improves beta(2)AR function and maximally upregulates beta-agonist-responsive active Na(+) transport by improving responsiveness to endogenous catecholamines. These studies suggest that upregulation of beta(2)AR function may someday prove useful for the treatment of pulmonary edema.

Role and regulation of lung Na,K-ATPase.

The recognition that pulmonary edema is cleared from the alveolar airspace by active Na+ transport has led to studies of the role and regulation of alveolar epithelial Na,K-ATPases. In the lung these heterodimers are predominantly composed of alpha1 and beta1-subunits and are located on the basolateral aspect of alveolar type 2 epithelial cells (AT2). Working with apically positioned epithelial Na+ channels they generate a transepithelial osmotic gradient which causes the movement of fluid out of the alveolar airspace. Accumulating data indicates that in some forms of pulmonary edema alveolar Na,K-ATPases function is reduced suggesting that pulmonary edema may be due, in part, to impairment of edema clearance mechanisms. Other studies suggest that Na,K-ATPase dysfunction or inhibition may contribute to airway reactivity. It is now recognized that lung Na,K-ATPases are positively regulated by glucocorticoids, aldosterone, catecholamines and growth hormones. These findings have led to investigations that show that enhancement of Na,K-ATPase function can accelerate pulmonary edema clearance in vitro, in normal and injured animal lungs in vivo, and in human lung explants. This review focuses on Na,K-ATPase data from lung and lung cell experiments that highlight the importance of Na,K-ATPases in airway reactivity and in maintaining a dry alveolar airspace. Review of data that suggests that there may be a role for therapeutic modulation of alveolar Na,K-ATPases for the purpose of treating patients with respiratory failure are also included.

GI complications in patients receiving mechanical ventilation.

Mechanical ventilation (MV) can be lifesaving by maintaining gas exchange until the underlying disorders are corrected, but it is associated with numerous organ-system complications, which can significantly affect the outcome of critically ill patients. Like other organ systems, GI complications may be directly attributable to MV, but most are a reflection of the severity of the underlying disease that required intensive care. The interactions of the underlying critical illness and MV with the GI tract are complex and can manifest in a variety of clinical pictures. Incorporated in this review are discussions of the most prevalent GI complications associated with MV, and current diagnosis and management of these problems.

Na,K-ATPase gene transfer mitigates an oxidant-induced decrease of active sodium transport in rat fetal ATII cells.

We investigated whether adenovirus-mediated transfer of genes encoding for subunits of the Na,K-ATPase increases transepithelial Na(+) transport in rat fetal distal lung epithelial (FDLE) monolayers and renders them more resistant to hydrogen peroxide injury. FDLE cells, isolated from rat fetuses at a gestational age of 19 to 20 d (22 d = term), were seeded on filters and infected with replication-incompetent human type 5 adenoviruses containing complementary DNAs encoding for rat Na,K-ATPase alpha(1) or beta(1) subunits (ad alpha(1) and ad beta(1), respectively). Once confluent monolayers were formed, the filters were mounted in Ussing chambers and short circuit currents (I(SC)) were measured. Increased levels of alpha(1) or beta(1) subunit proteins after infection with ad alpha(1) and ad beta(1), respectively, were confirmed by Western blot analysis. Baseline I(SC) increased after transfection with 2 plaque-forming units (pfu) of ad beta(1) from 5.1 +/- 0.3 to 6.1 +/- 0.3 microA/cm(2) (mean +/- SEM; P < 0.05). Permeabilization of the apical membrane with amphotericin B caused a large increase in I(SC); the ouabain-sensitive component of the amphotericin B-elicited I(SC) (ouab(max)) was increased from 4.0 +/- 0.2 (n = 69) in controls to 4.8 +/- 0.2 (n = 15), 5.9 +/- 0.3 (n = 53), 6.9 +/- 0.4 (n = 25), 7.7 +/- 0.9 (n = 16) in monolayers infected with 1, 2, 11, and 22 pfu of ad beta(1), respectively; transfection with ad alpha(1) had no effect on any measured variables. Further, transfection with ad beta(1) in comparison to noninfected monolayers resulted in higher baseline and ouab(max) I(SC) after injury with 500 microM H(2)O(2). We conclude that overexpression of the beta(1) subunit of the Na,K-ATPase may help maintain normal levels of vectorial Na(+) transport across ATII cell monolayers in pathologic conditions.

Adenovirus-mediated transfer of an Na+/K+-ATPase beta1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats.

Pulmonary edema is cleared via active Na(+) transport by alveolar epithelial Na(+)/K(+)-ATPases and Na(+) channels. Rats exposed to acute hyperoxia have a high mortality rate, decreased Na(+)/K(+)-ATPase function, and decreased alveolar fluid clearance (AFC). We hypothesized that Na(+)/K(+)-ATPase subunit gene overexpression could improve AFC in rats exposed to hyperoxia. We delivered 4 x 10(9) PFU of recombinant adenoviruses containing rat alpha(1) and beta(1) Na(+)/K(+)-ATPase subunit cDNAs (adalpha(1) and adbeta(1), respectively) to rat lungs 7 days prior to exposure to 100% O(2) for 64 hr. As compared with controls and ad alpha(1), AFC in the adbeta(1) rats was increased by >300%. Permeability for large solutes was less in the ad beta(1) than in the other hyperoxia groups. Glutathione oxidation, but not superoxide dismutase activity, was increased only in the adbeta(1) group. Survival through 14 days of hyperoxia was 100% in the adbeta(1) group but was not different from hyperoxic controls in animals given adalpha(1). Our data show that overexpression of a beta(1) Na(+)/K(+)-ATPase subunit augments AFC and improves survival in this model of acute lung injury via antioxidant-independent mechanisms. Conceivably, restoration of AFC via gene transfer of Na(+)/K(+)-ATPase subunit genes may prove useful for the treatment of acute lung injury and pulmonary edema.

Continuous enteral nutrition attenuates pulmonary edema in rats exposed to 100% oxygen.

Adult rats exposed to hyperoxia develop anorexia, weight loss, and a lung injury characterized by pulmonary edema and decreased lung liquid clearance. We hypothesized that maintenance of nutrition during hyperoxia could attenuate hyperoxia-induced pulmonary edema. To test this hypothesis, we enterally fed adult male Sprague-Dawley rats via gastrostomy tubes and exposed them to oxygen (inspired O(2) fraction >0.95) for 64 h. In contrast to controls, enterally fed hyperoxic animals did not lose weight and had smaller pleural effusions and wet-to-dry weight ratios (a measure of lung edema) that were not different from room air controls. Enterally fed rats exposed to hyperoxia had increased levels of mRNA for the Na(+)-K(+)-ATPase alpha(1)- and beta(1)-subunits and glutathione peroxidase. These findings suggest that maintenance of nutrition during an oxidative lung injury reduces lung edema, perhaps by allowing for continued expression and function of protective proteins such as the Na(+)-K(+)-ATPase.

Complications of mechanical ventilation.

Outcomes of critically ill patients are affected by the severity of the acute illnesses and by complications that may result from treatments. This article reviews the major complications associated with mechanical ventilation. Special emphasis is placed on recent advances in the understanding of ventilator-induced lung injury and strategies to avoid this life-threatening complication. In this update, the incidence, diagnosis, prevention, and management of complications associated with mechanical ventilation are discussed for each major organ system.

Tidal volume reduction for prevention of ventilator-induced lung injury in acute respiratory distress syndrome. The Multicenter Trail Group on Tidal Volume reduction in ARDS.

Because animal studies have demonstrated that mechanical ventilation at high volume and pressure can be deleterious to the lungs, limitation of airway pressure, allowing hypercapnia if necessary, is already used for ventilation of acute respiratory distress syndrome (ARDS). Whether a systematic and more drastic reduction is necessary is debatable. A multicenter randomized study was undertaken to compare a strategy aimed at limiting the end-inspiratory plateau pressure to 25 cm H2O, using tidal volume (VT) below 10 ml/kg of body weight, versus a more conventional ventilatory approach (with regard to current practice) using VT at 10 ml/kg or above and close to normal PaCO2. Both arms used a similar level of positive end-expiratory pressure. A total of 116 patients with ARDS and no organ failure other than the lung were enrolled over 32 mo in 25 centers. The two groups were similar at inclusion. Patients in the two arms were ventilated with different VT (7.1 +/- 1.3 versus 10.3 +/- 1.7 ml/kg at Day 1, p < 0.001) and plateau pressures (25.7 +/- 5. 0 versus 31.7 +/- 6.6 cm H2O at Day 1, p < 0.001), resulting in different PaCO2 (59.5 +/- 15.0 versus 41.3 +/- 7.6 mm Hg, p < 0.001) and pH (7.28 +/- 0.09 versus 7.4 +/- 0.09, p < 0.001), but a similar level of oxygenation. The new approach did not reduce mortality at Day 60 (46.6% versus 37.9% in control subjects, p = 0.38), the duration of mechanical ventilation (23.1 +/- 20.2 versus 21.4 +/- 16. 3 d, p = 0.85), the incidence of pneumothorax (14% versus 12%, p = 0. 78), or the secondary occurrence of multiple organ failure (41% versus 41%, p = 1). We conclude that no benefit could be observed with reduced VT titrated to reach plateau pressures around 25 cm H2O compared with a more conventional approach in which normocapnia was achieved with plateau pressures already below 35 cm H2O.

Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase beta1 subunit gene.

Previous studies have suggested that alveolar Na,K-ATPases play an important role in active Na+ transport and lung edema clearance. We reasoned that overexpression of Na,K-ATPase subunit genes could increase Na,K-ATPase function in lung epithelial cells and edema clearance in rat lungs. To test this hypothesis we produced replication deficient human type 5 adenoviruses containing cDNAs for the rat alpha1 and beta1 Na,K-ATPase subunits (adMRCMValpha1 and adMRCMVbeta1, respectively). As compared to controls, adMRCMVbeta1 increased beta1 subunit expression and Na,K-ATPase function by 2. 5-fold in alveolar type 2 epithelial cells and rat airway epithelial cell monolayers. No change in Na,K-ATPase function was noted after infection with adMRCMValpha1. Rat lungs infected with adMRCMVbeta1, but not adMRCMValpha1, had increased beta1 protein levels and lung liquid clearance 7 d after tracheal instillation. Alveolar epithelial permeability to Na+ and mannitol was mildly increased in animals infected with adMRCMVbeta1 and a similar Escherichia coli lacZ-expressing virus. Our data shows, for the first time, that transfer of the beta1 Na,K-ATPase subunit gene augments Na,K-ATPase function in epithelial cells and liquid clearance in rat lungs. Conceivably, overexpression of Na,K-ATPases could be used as a strategy to augment lung liquid clearance in patients with pulmonary edema.

Overexpression of the Na+,K+-ATPase alpha1 subunit increases Na+,K+-ATPase function in A549 cells.

We hypothesized that viral mediated transfer of Na+,K+-ATPase subunit genes to alveolar epithelial cells to overexpress Na+, K+-ATPase could increase Na+,K+-ATPase function. We produced replication-deficient human type 5 adenoviruses that contained cytomegalovirus (CMV)-driven cDNAs for the rat alpha1 and beta1 subunits of Na+,K+-ATPase (AdMRCMValpha1 and AdMRCMVbeta1, respectively). These viruses were used to transduce human adenocarcinoma cells (A549) in culture. Na+,K+-ATPase function was increased by 2.5-fold in the AdMRCMValpha1-infected cells. Sham and AdMRCMVbeta1-infected cells, and cells infected by a CMV-driven beta-galactosidase-expressing adenovirus, had no increases in Na+, K+-ATPase activity. A549 cells infected with multiplicities of infection of 10-200 of AdMRCMValpha1 demonstrated expression of a rat alpha1 mRNA and increased alpha1 protein; no change in beta1 message or protein was noted. Ouabain sensitivity was measured in A549 cells following infection with AdMRCMValpha1. In contrast to controls, AdMRCMValpha1-infected cells demonstrated two IC50s. The first was similar to the IC50s of the controls; the second IC50 was 2 logs greater than the first, consistent with the presence of both the rat and human alpha1 isozymes. These results demonstrate for the first time that adenoviruses can be used to augment Na+,K+-ATPase function.

Differential expression of Na-K-ATPase isoforms in rat alveolar epithelial cells.

Lung Na-K-ATPase has been shown to contribute to vectorial Na+ transport and edema clearance. The alpha 1- and beta 1-Na-K-ATPase subunits have been localized to alveolar type II (ATII) cells, and the alpha 2-Na-K-ATPase has been reported in rat lung homogenates. Expression of Na-K-ATPase alpha 1-, alpha 2-, and beta 1-subunits was investigated in rat ATII cells cultured for 7 days, a period during which they lose their phenotypic markers and differentiate to an alveolar type I (ATI)-like cell phenotype. Differentiation of ATII cells to an ATI-like phenotype resulted in a decrease of alpha 1- and an increase of alpha 2-mRNA and protein abundance without changes in the beta 1-subunit. Thus ATI-like cells exhibited a mixture of alpha 1- and alpha 2-isoforms. Nuclear run-on analysis suggests that these changes were transcriptionally regulated. The existence of the distinct functional classes of Na-K-ATPase in ATII and ATI-like cells was confirmed by ouabain inhibition of Na-K-ATPase activity. Ouabain inhibition of ATII cells was consistent with expression of the alpha 1-isozyme [50% inhibitory concentration (IC50) = 4 x 10(-5) M], whereas, in ATI-like cells, it was consistent with the presence of both alpha 1- and alpha 2-isozymes (IC50 = 9.0 x 10(-5) and 1.5 x 10(-7) M, respectively); [3H]ouabain binding studies corroborated these findings. Our results indicate that, during ATII cell cytodifferentiation with time in culture, there is a shift in isoform composition that may reflect physiological functions of alveolar epithelial cells.