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Biomedical subjects

Ian C Davis

Publications and source records attributed to Ian C Davis.

10 recordsLinked to original sources
PubMed2005

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.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

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.

Animals↗
PubMed2003

Nucleotide-mediated inhibition of alveolar fluid clearance in BALB/c mice after respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in infants and children worldwide. Intranasal infection of BALB/c mice with RSV strain A2, but not ultraviolet-inactivated RSV, for 2 or 4 days reduced basal alveolar fluid clearance (AFC), a seminal function of bronchoalveolar epithelium, and caused loss of AFC sensitivity to amiloride inhibition. Reduced AFC was temporally associated with increased lung water content but was not a consequence of increased epithelial permeability or cell death. Reduced AFC was also not due to decreased transcription of epithelial Na+ channel subunit genes in lung tissue. RSV-mediated inhibition of AFC 2 days after infection was rapidly prevented by addition to the instillate of P2Y receptor antagonists (suramin and XAMR-0721) or enzymes that degrade UTP, but not those that degrade ATP. After UTP degradation, AFC returned to control levels but was no longer sensitive to amiloride. UTP at nanomolar concentrations recapitulated the AFC inhibitory effect of RSV in normal mice and mice infected with RSV for 6 days, indicating that normalization of AFC at this time point is a consequence of cessation of UTP release, rather than P2Y receptor desensitization. We conclude that RSV infection of the bronchoalveolar epithelium results in reduced AFC as a consequence of autocrine feedback inhibition mediated by UTP. These studies are the first to demonstrate AFC inhibition by an important pulmonary viral pathogen. Reduced AFC may result in formation of an increased volume of fluid mucus, airway congestion, and rhinorrhea, all features of severe RSV disease.

Animals↗
PubMed2003

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.

Actins↗
PubMed2002

Inhibition of human surfactant protein A function by oxidation intermediates of nitrite.

NitraNitration of protein tyrosine residues by peroxynitrite (ONOO - ) has been implicated in a variety of inflammatory diseases such as acute respiratory distress syndrome (ARDS). Pulmonary surfactant protein A (SP-A) has multiple functions including host defense. We report here that a mixture of hypochlorous acid (HOCl) and nitrite (NO 2 - ) induces nitration, oxidation, and chlorination of tyrosine residues in human SP-A and inhibits SP-A's ability to aggregate lipids and bind mannose. Nitration and oxidation of SP-A was not altered by the presence of lipids, suggesting that proteins are preferred targets in lipid-rich mixtures such as pulmonary surfactant. Moreover, both horseradish peroxidase and myeloperoxidase (MPO) can utilize NO 2 - and hydrogen peroxide (H 2 O 2 ) as substrates to catalyze tyrosine nitration in SP-A and inhibit its lipid aggregation function. SP-A nitration and oxidation by MPO is markedly enhanced in the presence of physiological concentrations of Cl - and the lipid aggregation function of SP-A is completely abolished. Collectively, our results suggest that MPO released by activated neutrophils during inflammation utilizes physiological or pathological levels of NO 2 - to nitrate proteins, and may provide an additional mechanism in addition to ONOO - formation, for tissue injury in ARDS and other inflammatory diseases associated with upregulated *NO and oxidant production.

Bronchoalveolar Lavage Fluid↗
PubMed2002

Modulation of sodium transport in fetal alveolar epithelial cells by oxygen and corticosterone.

Regulation of active Na(+) transport across fetal distal lung epithelial cells (FDLE) by corticosterone (CST), corticotropin-releasing hormone (CRH), and oxygen tension may be crucial for postnatal adaptation. FDLE isolated from 19-day rat fetuses (term: 22 days) were grown on permeable supports to confluent monolayers (duration 3 days) in 2.5, 5, 12, or 20% O(2) with 5% CO(2)-balance N(2) and mounted in Ussing chambers for measurement of short-circuit currents (I(sc)). FDLE monolayers grown in 20% O(2) had significantly higher levels of total I(sc) and of their amiloride-sensitive (I(amil)) and ouabain-sensitive (I(ouab)) components than hypoxic cells. Values (microA/cm(2) +/- SE) for 2.5-5% O(2) and 20% O(2) were, respectively, I(sc) 5.3 +/- 0.2 vs. 8.4 +/- 0.3 (P < 0.001), I(amil) 3.4 +/- 0.2 vs. 4.3 +/- 0.2 (P < 0.01), and I(ouab) 3.4 +/- 0.6 vs. 9.1 +/- 0.6 (P < 0.001). Addition of CST but not CRH to the culture medium at any O(2) concentration increased I(amil). FDLE cells grown at 5% O(2) expressed significantly lower levels of alpha-, beta-, and gamma-epithelial Na(+) channel (ENaC), and of the alpha(1)-Na(+)-K(+)-ATPase, as determined by Western blotting. We conclude that higher O(2) concentrations increased total vectorial Na(+) transport, and the function of Na(+)-K(+)-ATPase and apical amiloride-sensitive Na(+) conductance, whereas CST only increased ENaC function.

Amiloride↗
PubMed2002

Elevated generation of reactive oxygen/nitrogen species in hantavirus cardiopulmonary syndrome.

Hantavirus cardiopulmonary syndrome (HCPS) is a life-threatening respiratory disease characterized by profound pulmonary edema and myocardial depression. Most cases of HCPS in North America are caused by Sin Nombre virus (SNV), which is carried asymptomatically by deer mice (Peromyscus maniculatus). The underlying pathophysiology of HCPS is poorly understood. We hypothesized that pathogenic SNV infection results in increased generation of reactive oxygen/nitrogen species (RONS), which contribute to the morbidity and mortality of HCPS. Human disease following infection with SNV or Andes virus was associated with increased nitrotyrosine (NT) adduct formation in the lungs, heart, and plasma and increased expression of inducible nitric oxide synthase (iNOS) in the lungs compared to the results obtained for normal human volunteers. In contrast, NT formation was not increased in the lungs or cardiac tissue from SNV-infected deer mice, even at the time of peak viral antigen expression. In a murine (Mus musculus) model of HCPS (infection of NZB/BLNJ mice with lymphocytic choriomeningitis virus clone 13), HCPS-like disease was associated with elevated expression of iNOS in the lungs and NT formation in plasma, cardiac tissue, and the lungs. In this model, intraperitoneal injection of 1400W, a specific iNOS inhibitor, every 12 h during infection significantly improved survival without affecting intrapulmonary fluid accumulation or viral replication, suggesting that cardiac damage may instead be the cause of mortality. These data indicate that elevated production of RONS is a feature of pathogenic New World hantavirus infection and that pharmacologic blockade of iNOS activity may be of therapeutic benefit in HCPS cases, possibly by ameliorating the myocardial suppressant effects of RONS.

Adolescent↗
PubMed2002

Killing of Klebsiella pneumoniae by human alveolar macrophages.

We investigated putative mechanisms by which human surfactant protein A (SP-A) effects killing of Klebsiella pneumoniae by human alveolar macrophages (AMs) isolated from bronchoalveolar lavagates of patients with transplanted lungs. Coincubation of AMs with human SP-A (25 microg/ml) and Klebsiella resulted in a 68% decrease in total colony forming units by 120 min compared with AMs infected with Klebsiella in the absence of SP-A, and this SP-A-mediated effect was abolished by preincubation with N(G)-monomethyl-L-arginine. Incubation of transplant AMs with SP-A increased intracellular Ca(2+) concentration ([Ca(2+)](i)) by 70% and nitrite and nitrate (NO(x)) production by 45% (from 0.24 +/- 0.02 to 1.3 +/- 0.21 nmol small middle dot 10(6) AMs(-1).h(-1)). Preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester inhibited the increase in [Ca(2+)](i) and abrogated the SP-A-mediated Klebsiella phagocytosis and killing. In contrast, incubation of AMs from normal volunteers with SP-A decreased both [Ca(2+)](i) and NO(x) production and did not result in killing of Klebsiella. Significant killing of Klebsiella was also seen in a cell-free system by sustained production of peroxynitrite (>1 microM/min) at pH 5 but not at pH 7.4. These findings indicate that SP-A mediates pathogen killing by AMs from transplant lungs by stimulating phagocytosis and production of reactive oxygen-nitrogen intermediates.

Bronchoalveolar Lavage Fluid↗