Postexposure Liponucleotide Prophylaxis and Treatment Attenuates Acute Respiratory Distress Syndrome in Influenza-infected Mice.

There is an urgent need for new drugs for patients with acute respiratory distress syndrome (ARDS), including those with coronavirus disease (COVID-19). ARDS in influenza-infected mice is associated with reduced concentrations of liponucleotides (essential precursors for de novo phospholipid synthesis) in alveolar type II (ATII) epithelial cells. Because surfactant phospholipid synthesis is a primary function of ATII cells, we hypothesized that disrupting this process could contribute significantly to the pathogenesis of influenza-induced ARDS. The goal of this study was to determine whether parenteral liponucleotide supplementation can attenuate ARDS. C57BL/6 mice inoculated intranasally with 10,000 plaque-forming units/mouse of H1N1 influenza A/WSN/33 virus were treated with CDP (cytidine 5'-diphospho)-choline (100 µg/mouse i.p.) ± CDP -diacylglycerol 16:0/16:0 (10 µg/mouse i.p.) once daily from 1 to 5 days after inoculation (to model postexposure influenza prophylaxis) or as a single dose on Day 5 (to model treatment of patients with ongoing influenza-induced ARDS). Daily postexposure prophylaxis with CDP-choline attenuated influenza-induced hypoxemia, pulmonary edema, alterations in lung mechanics, impairment of alveolar fluid clearance, and pulmonary inflammation without altering viral replication. These effects were not recapitulated by the daily administration of CTP (cytidine triphosphate) and/or choline. Daily coadministration of CDP-diacylglycerol significantly enhanced the beneficial effects of CDP-choline and also modified the ATII cell lipidome, reversing the infection-induced decrease in phosphatidylcholine and increasing concentrations of most other lipid classes in ATII cells. Single-dose treatment with both liponucleotides at 5 days after inoculation also attenuated hypoxemia, altered lung mechanics, and inflammation. Overall, our data show that liponucleotides act rapidly to reduce disease severity in mice with severe influenza-induced ARDS.

ATP catabolism by tissue nonspecific alkaline phosphatase contributes to development of ARDS in influenza-infected mice.

Influenza A viruses are highly contagious respiratory pathogens that are responsible for significant morbidity and mortality worldwide on an annual basis. We have shown previously that influenza infection of mice leads to increased ATP and adenosine accumulation in the airway lumen. Moreover, we demonstrated that A1-adenosine receptor activation contributes significantly to influenza-induced acute respiratory distress syndrome (ARDS). However, we found that development of ARDS in influenza-infected mice does not require catabolism of ATP to adenosine by ecto-5'-nucleotidase (CD73). Hence, we hypothesized that increased adenosine generation in response to infection is mediated by tissue nonspecific alkaline phosphatase (TNAP), which is a low-affinity, high-capacity enzyme that catabolizes nucleotides in a nonspecific manner. In the current study, we found that whole lung and BALF TNAP expression and alkaline phosphatase enzymatic activity increased as early as 2 days postinfection (dpi) of C57BL/6 mice with 10,000 pfu/mouse of influenza A/WSN/33 (H1N1). Treatment at 2 and 4 dpi with a highly specific quinolinyl-benzenesulfonamide TNAP inhibitor (TNAPi) significantly reduced whole lung alkaline phosphatase activity at 6 dpi but did not alter TNAP gene or protein expression. TNAPi treatment attenuated hypoxemia, lung dysfunction, histopathology, and pulmonary edema at 6 dpi without impacting viral replication or BALF adenosine. Treatment also improved epithelial barrier function and attenuated cellular and humoral immune responses to influenza infection. These data indicate that TNAP inhibition can attenuate influenza-induced ARDS by reducing inflammation and fluid accumulation within the lung. They also further emphasize the importance of adenosine generation for development of ARDS in influenza-infected mice.

Heterozygosity for the F508del mutation in the cystic fibrosis transmembrane conductance regulator anion channel attenuates influenza severity.

BACKGROUND: Seasonal and pandemic influenza are significant public health concerns. Influenza stimulates respiratory epithelial Cl(-) secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). The purpose of this study was to determine the contribution of this effect to influenza pathogenesis in mice with reduced CFTR activity. METHODS: C57BL/6-congenic mice heterozygous for the F508del CFTR mutation (HET) and wild-type (WT) controls were infected intranasally with 10 000 focus-forming units of influenza A/WSN/33 (H1N1) per mouse. Body weight, arterial O2 saturation, and heart rate were monitored daily. Pulmonary edema and lung function parameters were derived from ratios of wet weight to dry weight and the forced-oscillation technique, respectively. Levels of cytokines and chemokines in bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay. RESULTS: Relative to WT mice, influenza virus-infected HET mice showed significantly delayed mortality, which was accompanied by attenuated hypoxemia, cardiopulmonary dysfunction, and pulmonary edema. However, viral replication and weight loss did not differ. The protective HET phenotype was correlated with exaggerated alveolar macrophage and interleukin 6 responses to infection and was abrogated by alveolar macrophage depletion, using clodronate liposomes. CONCLUSIONS: Reduced CFTR expression modulates the innate immune response to influenza and alters disease pathogenesis. CFTR-mediated Cl(-) secretion is therefore an important host determinant of disease, and CFTR inhibition may be of therapeutic benefit in influenza.

Reuse of Disposable Isolation Gowns in Rodent Facilities during a Pandemic.

Reuse of disposable personal protective equipment is traditionally discouraged, yet in times of heightened medical applications such as the SARS CoV-2 pandemic, it can be difficult to obtain. In this article we examine the reuse of disposable gowns with respect to still providing personnel protection. XR7, a fluorescent powder, was used to track contamination of gowns after manipulation of rodent cages. Mouse cages were treated with XR7 prior to manipulations. Disposable gowns were labeled for single person use and hung in common procedure spaces within the vivarium between usages. A simulated rack change of 140 cages was completed using XR7-treated cages. One individual changed all cages with a break occurring after the first 70 cages, requiring the gown to be removed and reused once. To simulate research activities, 5 individuals accessed 3 XR7-treated cages daily for 5 d. Each mouse in the XR7-treated cages was manipulated at least once before returning cages to the housing room. Disposable gowns were reused 5 times per individual. Gowns, gloves, clothing, bare arms, and hands were scanned for fluorescence before and after removing PPE. Fluorescence was localized to gloves and gown sleeves in closest contact with animals and caging. No fluorescence was detected on underlying clothing, or bare arms and hands after removing PPE. Fluorescence was not detected in procedure spaces where gowns were hung. The lack of fluorescence on personnel or surfaces indicate that gowns can be reused 1 time for routine husbandry tasks and up to 5 times for research personnel. A method for decontamination of used gowns using Vaporized Hydrogen Peroxide (VHP) was also validated for use in areas where animals are considered high risk such as quarantine, or for fragile immunocompromised rodent colonies.

Metabolic shifts modulate lung injury caused by infection with H1N1 influenza A virus.

Influenza A virus (IAV) infection alters lung epithelial cell metabolism in vitro by promoting a glycolytic shift. We hypothesized that this shift benefits the virus rather than the host and that inhibition of glycolysis would improve infection outcomes. A/WSN/33 IAV-inoculated C57BL/6 mice were treated daily from 1 day post-inoculation (d.p.i.) with 2-deoxy-d-glucose (2-DG) to inhibit glycolysis and with the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) to promote flux through the TCA cycle. To block OXPHOS, mice were treated every other day from 1 d.p.i. with the Complex I inhibitor rotenone (ROT). 2-DG significantly decreased nocturnal activity, reduced respiratory exchange ratios, worsened hypoxemia, exacerbated lung dysfunction, and increased humoral inflammation at 6 d.p.i. DCA and ROT treatment normalized oxygenation and airway resistance and attenuated IAV-induced pulmonary edema, histopathology, and nitrotyrosine formation. None of the treatments altered viral replication. These data suggest that a shift to glycolysis is host-protective in influenza.

Comparison of methods for detection of pinworms in mice and rats.

Though pinworm infestation remains common in laboratory rodent colonies, there is little information regarding current practices for pinworm detection and their relative efficacy. The authors surveyed research institutions to find out the prevalence of pinworm infestations and the detection methods they used. They also tested mice and rats from colonies that were known to be infested with Syphacia sp. and compared the following detection methods: perianal tape test, fecal flotation, fecal concentration, cecal content examination, cecal flotation and histological examination. Though the different methods yielded comparable efficacy overall, the authors recommend using more than one type of test to increase detection potential.

Institutional Animal Care and Use Committee Considerations Regarding the Use of Virus-Induced Carcinogenesis and Oncolytic Viral Models.

The use of virus-induced carcinogenesis and oncologic experimental animal models is essential in understanding the mechanisms of cancer development to advance prevention, diagnosis, and treatment methods. The Institutional Animal Care and Use Committee (IACUC) is responsible for both the complex philosophical and practical considerations associated with animal models of cancer. Animal models of cancer carry their own unique issues that require special consideration from the IACUC. Many of the considerations to be discussed apply to cancer models in general; specific issues related to viral carcinogenesis or oncolytic viruses will be specifically discussed as they arise. Responsible animal use integrates good science, humane care, and regulatory compliance. To meet those standards, the IACUC, in conjunction with the research investigator and attending veterinarian, must address a wide range of issues, including animal model selection, cancer model selection, humane end point considerations, experimental considerations, postapproval monitoring, reporting requirements, and animal management and personnel safety considerations.

Effect of Ventilated Caging on Water Intake and Loss in 4 Strains of Laboratory Mice.

Food availability, temperature, humidity, strain, and caging type all affect water consumption by mice. Measurement of transepidermal water loss (TEWL) is a new technique for the quantification of water turnover in mice. To understand water turnover in common strains of adult mice, male and female SCID, SKH, C57BL/6, and FVB mice were housed in same-sex groups of 5 animals in static cages or IVC. Body weight, TEWL, urine osmolality, and water consumption of mice and intracage temperature and humidity were measured every 48 h for comparison. Static cages were monitored for 7 d and IVC for 14 d before cage change. Female SCID, FVB, and C57 mice drank less water than did their male counterparts. Male and female SCID, SKH, and FVB mice in IVC drank less water and had higher urine osmolality than did those in static cages. In SCID and SKH mice, TEWL paralleled water consumption. C57 mice in static cages drank less water, had lower urine osmolality, and had less TEWL than did those in IVC. Temperature and humidity within the cage was higher than the macroenvironmental levels for all housing conditions, mouse strains, and sexes. Temperatures within IVC ranged from 76.6 to 81.4 °F compared with 69±0.4 °F in the room. Humidity within IVC ranged from 68% to 79% compared with 27.o%±2.7% within the room. These data demonstrate that mouse strain and housing conditions significantly influence water balance and indicate that macroenvironmental measurements do not always reflect the intracage environment.

Role of innate immunity in respiratory mycoplasma infection.

Mycoplasmas are unique among respiratory pathogens. They possess very small genomes, lack cell walls and are strictly dependent on the host for survival. These pathogens have developed the ability to quickly adapt to the host environment through attachment to target cells within the host. Mycoplasmas have been identified as commensal microbial flora of healthy persons yet, infection of the upper and lower respiratory tracts can result in acute cough, fever and headache, and even chronic disease involving multiple organs. The lung contains a complex system of defense mechanisms with which to combat these pathogens, including innate (nonspecific) and acquired (specific) immune responses. Innate defenses include mechanical clearance, cellular responses provided by host phagocytes and molecular protection in the form of antimicrobial peptides. The interaction of mycoplasmas with different components of the innate immune system and mechanisms by which they incite pathology has proved elusive. The mechanisms by which pathogenic mycoplasmas evade the innate immune system are unknown. The purpose of this review is to summarize current knowledge of these interactions in the hope of identifying new avenues for research and therapy.

Implications of natural occlusion of ventilated racks on ammonia and sanitation practices.

Examination of ventilated rat racks prior to semiannual sanitation revealed silicone nozzles and ventilation ports that were partially or completely occluded with granular debris. We subsequently sought to document performance standards for rack sanitation and investigate the effect of ventilation port occlusion on rack function and animal husbandry practices. We hypothesized that individually ventilated cages with occluded airflow would require more frequent cage changes, comparable to those for static cages (that is, every 3 to 4 d). Sprague-Dawley rats were housed under one of 4 conditions: no airflow occlusion, occluded air-supply inlet, occluded air-exhaust outlet, and occlusion of both inlet and outlet. Cages were changed when daily ammonia concentration exceeded 20 ppm or after 14 d had elapsed. Most cages with unoccluded or partial airflow occlusion remained below the 20 ppm limit until day 12 or 13. Cages with occlusion of both inlet and outlet exceeded 20 ppm ammonia by as early as day 5. Airflow was significantly lower in cages with occlusion of both inlet and outlet airflow. Weekly inspection revealed that occlusion of ventilation ports was detectable by 3 mo after semiannual sanitation. This study demonstrates that silicone nozzles should be removed prior to rack sanitation to improve the effectiveness of cleaning ventilation ports and nozzles. While the rack is in use, silicone nozzles and ventilation ports should be inspected regularly to identify occlusion that is likely to diminish environmental quality in the cage. Intracage ammonia levels are significantly higher when both inlet and outlet airflow are occluded.

Processing and treatment of corncob bedding affects cage-change frequency for C57BL/6 mice.

The goal of this study was to evaluate the effectiveness of a new proprietary processed corncob bedding material (PCC)compared with standard corncob in ventilated and static mouse housing systems. Intracage ammonia levels, bacterial growth, and absorptive capacity of bedding were measured for cages of C57BL/6 mice under nonautoclaved and autoclaved conditions on static and ventilated racks in a barrier facility. Ammonia concentration was measured daily, and cages were removed from the study when measurements reached or exceeded 25 ppm. Bacterial growth in bedding was quantified and speciated before exposure to mice and at the time of cage removal. The absorptive capacity of all bedding material was determined under autoclaved and nonautoclaved conditions. Ventilated cages with PCC or autoclaved corncob took longer to reach ammonia concentrations of 25 ppm than did those with corncob or autoclaved PCC; PCC-filled cages remained below 25 ppm NH3 for at least 3 wk. The type of bedding material did not affect the number of days required to reach 25 ppm in static cages. Compared with other bedding types in the absence of mice, 1/4-in. PCC had a lower and 1/8-in. corncob a higher bacterial load. Autoclaving altered the absorptive capacity of 1/4-in. bedding materials, and for 1/8-in. bedding, corncob was more absorptive than PCC regardless of autoclaving. The results of this study indicate that PCC is comparable to autoclaved corncob in controlling intracage ammonia levels, and a cage-change interval of 3 wk is possible when ventilated cages are used with this bedding.

Effectiveness of shoe covers for bioexclusion within an animal facility.

The personal protective equipment (PPE) required for entry into rodent barrier rooms often includes a hair bonnet, face mask, disposable gown, gloves, and shoe covers. Traditionally, shoe covers have been considered essential PPE for maintaining a 'clean' animal room. The introduction of microisolation caging and ventilated rack housing prompted us to reevaluate the contribution of shoe covers to bioexclusion. Contamination powder that fluoresces under black light was to track particle dispersal on the floor and personnel. The test mouse room contained a ventilated microisolation rack and biosafety cabinet. Powder was applied directly inside or outside the animal room doorway. PPE with or without shoe covers was donned outside of the animal room doorway and discarded on exiting. Participants either were scanned on entry into the room for the presence of florescence or asked to complete a simulated standard animal room activity while wearing full PPE. Animal rooms were scanned for florescence after exit of participants. All participants donning shoe covers fluoresced in multiple areas, primarily on gloves and gowns. Shoe covers had no effect on the spread of powder in normal traffic patterns, with no powder detected within caging. Powder also was used to determine the distance substances could be carried on the floor from building entry points. Results indicate that shoe covers do not improve (and actually may compromise) bioexclusion. Donning of shoe covers offers a potential for contamination of personnel from contact with shoe bottoms.

Post-infection A77-1726 blocks pathophysiologic sequelae of respiratory syncytial virus infection.

Despite respiratory syncytial virus (RSV) bronchiolitis remaining the most common cause of lower respiratory tract disease in infants worldwide, treatment has progressed little in the past 30 years. The aim of our study was to determine whether post-infection administration of de novo pyrimidine synthesis inhibitors could prevent the reduction in alveolar fluid clearance (AFC) and hypoxemia that occurs at Day 2 after intranasal infection of BALB/c mice with RSV. BALB/c mice were infected intranasally with RSV strain A2. AFC was measured in anesthetized, ventilated mice after instillation of 5% bovine serum albumin into the dependent lung. Post-infection systemic treatment with leflunomide has no effect on AFC. However, when added to the AFC instillate, leflunomide's active metabolite, A77-1726, blocks RSV-mediated inhibition of AFC at Day 2. This block is reversed by uridine (which allows pyrimidine synthesis via the scavenger pathway) and not recapitulated by genistein (which mimics the tyrosine kinase inhibitor effects of A77-1726), indicating that the effect is specific for the de novo pyrimidine synthesis pathway. More importantly, when administered intranasally at Day 1, A77-1726, but not its vehicle dimethyl sulfoxide, maintains its beneficial effect on AFC and lung water content until Day 2. Intranasal instillation of A77-1726 at Day 1 also reduces bronchoalveolar lavage nucleotide levels, lung inflammation, and hypoxemia at Day 2 without impairing viral replication at Day 2 or viral clearance at Day 8. Post-infection intranasal or aerosolized treatment with pyrimidine synthesis inhibitors may provide symptomatic relief from the pathophysiologic sequelae of impaired AFC in children with RSV bronchiolitis.

Respiratory syncytial virus induces insensitivity to beta-adrenergic agonists in mouse lung epithelium in vivo.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and children worldwide. We wished to determine whether intratracheal administration of beta-agonists improved alveolar fluid clearance (AFC) across the distal respiratory epithelium of RSV-infected mice. Following intranasal infection with RSV strain A2, AFC was measured in anesthetized, ventilated BALB/c mice by instillation of 5% BSA into the dependent lung. We found that direct activation of protein kinase A by forskolin or 8-bromo-cAMP increased AFC at day 2 after infection with RSV. In contrast, short- and long-acting beta-agonists had no effect at either day 2 or day 4. Insensitivity to beta-agonists was not a result of elevated plasma catecholamines or lung epithelial cell beta-adrenergic receptor degradation. Instead, RSV-infected mice had significantly higher levels of phosphorylated PKCzeta in the membrane fractions of their lung epithelial cells. In addition, insensitivity to beta-agonists was mediated in a paracrine fashion by KC (the murine homolog of CXCL8) and reversed by inhibition of either PKCzeta or G protein-coupled receptor kinase 2 (GRK2). These results indicate that insufficient response to beta-agonists in RSV may be caused, at least in part, by impaired beta-adrenergic receptor signaling, as a consequence of GRK2-mediated uncoupling of beta-adrenergic receptors from adenylyl cyclase.

Surfactant dysfunction in SP-A-/- and iNOS-/- mice with mycoplasma infection.

Surfactant dysfunction was studied in C57BL/6 (B6), B6.SP-A(-/-), and B6.iNOS(-/-) mice with pulmonary mycoplasma infection (10(7) colony-forming units). Cell-free bronchoalveolar lavage (BAL) from uninfected B6.SP-A(-/-) versus B6 mice had a reduced content of very large aggregates (VLA) and an increase in intermediate large aggregates (ILA), with no difference in total large aggregates (LA = VLA + ILA). However, LA from uninfected B6.SP-A(-/-) versus B6 mice contained less protein and were more sensitive to inhibition by serum albumin and lysophosphatidylcholine in pulsating bubble studies in vitro. Infection with Mycoplasma pulmonis caused significant lung injury and surfactant abnormalities in B6.SP-A(-/-), B6.iNOS(-/-), and B6 mice at 24, 48, 72 h after infection compared with uninfected mice of the same strain. Analyses of time-pooled data indicated that mycoplasma-infected B6.SP-A(-/-) and B6.iNOS(-/-) mice had significantly lower levels of LA and higher protein/phospholipid ratios in BAL compared with infected B6 mice. Infected B6.iNOS(-/-) versus B6 mice also had increased minimum surface tensions on the pulsating bubble and decreased levels of surfactant protein (SP)-B in BAL. These results indicate that pulmonary mycoplasma infection in vivo causes lung injury and surfactant abnormalities that are dependent in part on iNOS and SP-A. In addition, SP-A deficiency modifies surfactant aggregate content and lowers the inhibition resistance of LA surfactant in vitro compared with congenic normal mice.

Transgenic mice.

For the past 20 years researchers have used transgenic mice to help understand the basic mechanisms associated with inherited human and animal diseases. The ability to integrate exogenous genetic information into the mouse genome has revolutionised the analysis of gene function. Both gene addition and gene replacement can be performed and the capability exists to create 'conditional' mutations and to study gene dosage effects. The aim of the present review is to provide a framework of information on transgenic mouse methodologies that can be applied to any area of research. A basic understanding of transgenic technology, recognising its advantages and disadvantages, is essential knowledge for the scientist in the 21st century.

Bactericidal function of alveolar macrophages in mechanically ventilated rabbits.

Protective ventilation strategies have been universally embraced because of reduced mortality. We tested the hypothesis that tidal volume (VT) in an in vivo model of mechanical ventilation would modulate bactericidal function of alveolar macrophages (AMs). Adult New Zealand White rabbits were mechanically ventilated for 4 h with a VT of 6 ml/kg (low) or a VT of 12 ml/kg (traditional), with each group receiving 3 cm H2O positive end-expiratory pressure with and without intratracheal lipopolysaccharide (LPS) instillation (20 mg/kg). AMs were isolated from bronchoalveolar lavage fluid taken from the whole left lung and used for bacterial killing assays. There were no significant differences in steady-state levels of nitrite or AM phagocytosis and killing of Klebsiella pneumoniae, although these values trended to be slightly higher in the traditional VT group. However, bronchoalveolar lavage fluid protein concentrations were significantly increased in traditional VT groups receiving LPS compared with animals ventilated with a low VT (1,407.8 +/- 121.4 versus 934.7 +/- 118.2; P < 0.001). Lung wet:dry weight ratio in the traditional VT group was increased when compared with the low VT group without LPS (7.3 +/- 0.4 versus 6.1 +/- 0.3, respectively; P < 0.05). Additionally, IL-8 expression was significantly greater under conditions of LPS treatment and mechanical ventilation at VT of 12 ml/kg. These results suggest that the traditional ventilator approach (12 ml/kg VT) in a model of in vivo mechanical ventilation results in lung pathology without affecting AM antibacterial function.

Leflunomide prevents alveolar fluid clearance inhibition by respiratory syncytial virus.

RATIONALE: Previously, we demonstrated that intranasal infection of BALB/c mice with respiratory syncytial virus (RSV) resulted in an early 40% reduction in alveolar fluid clearance (AFC), an effect mediated via P2Y purinergic receptors. OBJECTIVES: To confirm that RSV-induced inhibition of AFC is mediated by uridine triphosphate (UTP), and to demonstrate that inhibition of de novo pyrimidine synthesis with leflunomide prevents increased UTP release after RSV infection, and thereby also prevents inhibition of AFC by RSV. METHODS: BALB/c mice were infected intranasally with RSV strain A2. AFC was measured in anesthetized, ventilated mice by instillation of 5% bovine serum albumin into the dependent lung. Some mice were pretreated with leflunomide or 6-mercaptopurine. MEASUREMENTS AND MAIN RESULTS: RSV-mediated inhibition of AFC is associated temporally with a 20-nM increase in UTP and ATP content of bronchoalveolar lavage fluid, hypoxemia, and altered nasal potential difference. RSV-mediated nucleotide release, AFC inhibition, and physiologic sequelae thereof can be prevented by pretreatment of mice with the de novo pyrimidine synthesis inhibitor leflunomide, which is not toxic to the mice, and which does not affect RSV replication in the lungs. In contrast, pretreatment of mice with 6-mercaptopurine, an inhibitor of de novo purine synthesis, has no beneficial effect on AFC or other indicators of disease progression. Finally, RSV-mediated inhibition of AFC is prevented by volume-regulated anion channel inhibitors. CONCLUSION: Pyrimidine synthesis or release pathways may provide novel therapeutic targets to counter the pathophysiologic sequelae of impaired AFC in RSV disease.

Reactive species mediate inhibition of alveolar type II sodium transport during mycoplasma infection.

RATIONALE: Mycoplasma pneumoniae is a significant cause of pneumonia in humans. OBJECTIVES: To determine the impact of mycoplasma infection and the host inflammatory response on alveolar type II (ATII) cell ion transport in vivo and in vitro. METHODS: Mice were infected with M. pulmonis for measurements of alveolar fluid clearance (AFC) in vivo and isolation of ATII cells. ATII cells were infected in vivo for determination of epithelial Na+ channel (ENaC) total and cell surface protein levels by biotinylation and Western blot and in vitro for whole cell patch clamp recording and measurement of nitric oxide (NO) production by chemiluminescence. RESULTS: Mycoplasma infection significantly inhibited AFC at 24 h and total and amiloride-sensitive AFC by 48 h postinfection (pi). In contrast, infected myeloperoxidase-deficient mice had similar basal and amiloride-sensitive AFC values to uninfected control mice at 48 h pi. Addition of forskolin restored total and amiloride-sensitive AFC to control values at 48 h pi. ATII cells isolated from infected mice demonstrated normal alpha, beta, and gamma ENaC total protein levels; however, infected whole-lung cell-surface levels of gamma ENaC were significantly decreased. Patch-clamp recordings demonstrated a significant decrease in total and amiloride-sensitive Na+ currents at 24 h pi. ATII cells demonstrated a significant increase in the production of NO at 24 h pi and inhibition of NO by ATII cells before infection reversed the decrease in total Na+ currents. CONCLUSIONS: These data indicate that mycoplasma infection results in decreased AFC and functional ENaC via the production of reactive oxygen nitrogen intermediates.

Hyperoxia impairs antibacterial function of macrophages through effects on actin.

Oxidative stress may impair alveolar macrophage function in patients with inflammatory lung diseases or those exposed to high concentrations of oxygen. We investigated putative mechanisms of injury to macrophages by oxidative stress, using RAW 264.7 cells exposed to 95% oxygen for 48 h. Hyperoxia-exposed macrophages were less able to phagocytose and kill Klebsiella pneumoniae than normoxic controls, despite increased production of nitric oxide, a free radical important in pathogen killing. Exposure of macrophages to hyperoxia had marked effects on the actin cytoskeleton, including increased actin polymerization, loss of cortical actin, formation of stress fibers, de novo synthesis of actin, and actin oxidation. Hyperoxia induced changes in cell morphology, with increased cell size and pseudopod formation. Exposure of macrophages to jasplakinolide, an agent that increases actin polymerization, also impaired their ability to phagocytose Klebsiella. Alveolar macrophages isolated from mice exposed to 100% oxygen for 84 h also demonstrated impaired phagocytic function, as well as similar effects on the actin cytoskeleton and cell morphology to macrophages exposed to hyperoxia in vitro. We conclude that oxidative stress in vitro and in vivo impairs macrophage antibacterial function through effects on actin.