Early mechanisms of neutrophil activation and transmigration in acute lung injury.

Introduction:Neutrophil transmigration is multifactorial and primarily driven by selectins and ß2-integrins (CD11b/CD18), whose expression are dependent on the underlying stimulus. Ventilator-induced lung injury (VILI) results in a predominantly CD18-independent mechanism of neutrophil recruitment, while direct endotoxin-induced lung injury results from a CD18-dependent mechanism. We previously observed that lack of NADPH oxidases DUOX1 and DUOX2 resulted in reduced neutrophil influx in a VILI model of lung injury but had no influence on neutrophil influx after LPS exposure. Based on these observations, we hypothesized that DUOX1/DUOX2 are an important component of CD18-independent mechanisms of neutrophil recruitment in the lung. Methods:We exposed Duoxa -/- (KO) mice and Duoxa +/+ (WT) mice to either an intratracheal exposure of lipopolysaccharide (LPS/endotoxin)-or high tidal volume ventilation and compared expression of neutrophil markers between groups. WT mice (129S6/SvEvTac) were obtained from Taconic Biosciences (One Discovery Drive Suite 304; Rensselaer, NY 1244) and were allowed to acclimatize for one week prior to study enrollment. KO mice were generated as previously described [Grasberger 2012] and bred in-house on a 129S6 background. We provided positive-pressure ventilation at a tidal volume of 10 ml/kg with 2 cmH20 positive end-expiratory pressure (PEEP). Mice were assigned to groups consisting of KO (n = 5) and WT (n = 5) in each group and divided into non-ventilated, positive-pressure ventilation, or LPS IT exposure groups. Positive-pressure ventilation was instituted for 4-h using a FlexiVent (Flexiware 8.1, Scireq, Montreal, QC, Canada). Lipopolysaccharide (Salmonella enterica serotype tryphimurium L6143, Millipore Sigma) was administered via an intratracheal (IT) route at a dose of 0.1 mg/kg. Mice were humanely euthanized at 4-h post-injection consistent with the UC Davis IAUCAC-approved protocol. Results:As previously observed, neutrophilic influx into the airways was significantly impaired in the Duoxa -/- (KO) mice after VILI, but not after LPS exposure. LPS-induced lung injury resulted in upregulation of CD11b+ neutrophils and shedding of CD62L and CD162 regardless of DUOX expression, whereas VILI resulted in upregulation of CD49+ neutrophils in the Duoxa +/+ (WT) mice but not the Duoxa -/- (KO) mice. Conclusion:Our data suggest DUOX is required for CD18-independent mechanisms of neutrophil recruitment in the lung induced by acute lung injury, but not for canonical CD18depedent mechanisms after LPS exposure.

Water quality issues and infant diarrhoea in a South American province.

In the province of Salta, in the Northwest region of Argentina, almost two-thirds of the population live in absolute poverty, and diseases associated with poverty are rampant. Almost 12% of the total population of the province are children below 5 years of age; almost half of these infants are living in situations where the basic necessities are not available. Primitive sanitary conditions, including widespread contamination of available water supplies with pathogens, contribute to a major public health problem. Infant mortality was 17% higher for Salta than for Argentina as a whole in 2001. A major cause of death for these children is infectious disease, especially respiratory and intestinal diseases. In Salta, more than half of the total population of infants is affected by diarrhoea annually. The infectious pathogens are diverse: bacteria (predominantly in spring and summer), viruses (especially in the winter) and parasites (endemic in some situations). This paper evaluates current methods used to test for the presence of pathogens in drinking water; discusses why these methods are less than adequate; documents an episode of contamination in a local water supply source; and suggests appropriate methods that can be used to better address this major public health issue effectively.

High-throughput method for ranking the affinity of peptide ligands selected from phage display libraries.

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major "bottleneck" of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred "en masse" from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide-BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide-BAP protein can find direct application as a tracer reagent.

Polyclonal antibody-based noncompetitive immunoassay for small analytes developed with short peptide loops isolated from phage libraries.

To date, there are a few technologies for the development of noncompetitive immunoassays for small molecules, the most common of which relies on the use of anti-immunocomplex antibodies. This approach is laborious, case specific, and relies upon monoclonal antibody technology for its implementation. We recently demonstrated that, in the case of monoclonal antibody-based immunoassays, short peptide loops isolated from phage display libraries can be used as substitutes of the anti-immunocomplex antibodies for noncompetitive immunodetection of small molecules. The aim of this work was to demonstrate that such phage ligands can be isolated even when the selector antibodies are polyclonal in nature. Using phenoxybenzoic acid (PBA), a major pyrethroid metabolite, as a model system, we isolated the CFNGKDWLYC peptide after panning a cyclic peptide library on the PBA/anti-PBA immunocomplex. The sensitivity of the noncompetitive enzyme-linked immunosorbent assay (ELISA) setup with this peptide was 5-fold (heterologous) or 400-fold (homologous) higher than that of the competitive assay setup with the same antibody. Phage anti-immunocomplex assay (PHAIA) was also easily adapted into a rapid and highly sensitive dipstick assay. The method not only provides a positive readout but also constitutes a major shortcut in the development of sensitive polyclonal-based assays, avoiding the need of synthesizing heterologous competing haptens.

Phage anti-immune complex assay: general strategy for noncompetitive immunodetection of small molecules.

Due to their size, small molecules cannot be simultaneously bound by two antibodies, precluding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. This has prompted the development of anti-immune complex antibodies, but these are difficult to produce, and often exhibit high cross-reactivity with the unliganded primary antibody. This work demonstrates that anti-immune complex antibodies can be substituted by phage particles isolated from phage display peptide libraries. Phages bearing specific small peptide loops allowed to focus the recognition to changes in the binding area of the immune complex. The concept was tested using environmental and drug analytes; with improved sensitivity and ready adaptation into on-site formats. Peptides specific for different immune complexes can be isolated from different peptide libraries in a simple and systematic fashion allowing the rapid development of noncompetitive assays for small molecules.

Workshop summary: phosgene-induced pulmonary toxicity revisited: appraisal of early and late markers of pulmonary injury from animal models with emphasis on human significance.

A workshop was held February 14, 2007, in Arlington, VA, under the auspices of the Phosgene Panel of the American Chemistry Council. The objective of this workshop was to convene inhalation toxicologists and medical experts from academia, industry and regulatory authorities to critically discuss past and recent inhalation studies of phosgene in controlled animal models. This included presentations addressing the benefits and limitations of rodent (mice, rats) and nonrodent (dogs) species to study concentration x time (C x t) relationships of acute and chronic types of pulmonary changes. Toxicological endpoints focused on the primary pulmonary effects associated with the acute inhalation exposure to phosgene gas and responses secondary to injury. A consensus was reached that the phosgene-induced increased pulmonary extravasation of fluid and protein can suitably be probed by bronchoalveolar lavage (BAL) techniques. BAL fluid analyses rank among the most sensitive methods to detect phosgene-induced noncardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum protein concentrations in BAL fluid occurred within 1 day after exposure, typically followed by a latency period up to about 15 h, which is reciprocal to the C x t exposure relationship. The C x t relationship was constant over a wide range of concentrations and single exposure durations. Following intermittent, repeated exposures of fixed duration, increased tolerance to recurrent exposures occurred. For such exposure regimens, chronic effects appear to be clearly dependent on the concentration rather than the cumulative concentration x time relationship. The threshold C x t product based on an increased BAL fluid protein following single exposure was essentially identical to the respective C x t product following subchronic exposure of rats based on increased pulmonary collagen and influx of inflammatory cells. Thus, the chronic outcome appears to be contingent upon the acute pulmonary threshold dose. Exposure concentrations high enough to elicit an increased acute extravasation of plasma constituents into the alveolus may also be associated with surfactant dysfunction, intra-alveolar accumulation of fibrin and collagen, and increased recruitment and activation of inflammatory cells. Although the exact mechanisms of toxicity have not yet been completely elucidated, consensus was reached that the acute pulmonary toxicity of phosgene gas is consistent with a simple, irritant mode of action at the site of its initial deposition/retention. The acute concentration x time mortality relationship of phosgene gas in rats is extremely steep, which is typical for a local, directly acting pulmonary irritant gas. Due to the high lipophilicity of phosgene gas, it efficiently penetrates the lower respiratory tract. Indeed, more recent published evidence from animals or humans has not revealed appreciable irritant responses in central and upper airways, unless exposure was to almost lethal concentrations. The comparison of acute inhalation studies in rats and dogs with focus on changes in BAL fluid constituents demonstrates that dogs are approximately three to four times less susceptible to phosgene than rats under methodologically similar conditions. There are data to suggest that the dog may be useful particularly for the study of mechanisms associated with the acute extravasation of plasma constituents because of its size and general morphology and physiology of the lung as well as its oronasal breathing patterns. However, the study of the long-term sequelae of acute effects is experimentally markedly more demanding in dogs as compared to rats, precluding the dog model to be applied on a routine base. The striking similarity of threshold concentrations from single exposure (increased protein in BAL fluid) and repeated-exposure 3-mo inhalation studies (increased pulmonary collagen deposition) in rats supports the notion that chronic changes depend on acute threshold mechanisms.

Mouse models of asthma: can they give us mechanistic insights into the role of nitric oxide?

New clinical practice guidelines for patients with asthma include the recommendation to monitor exhaled breath nitric oxide (NO) levels. NO concentrations in exhaled breath are increased in asthmatics and increased NO levels correlate with worsening airway inflammation and asthma symptoms. The multiple roles of NO in the lung have not been delineated clearly. Clinical trials are being performed presently that test the apparently conflicting hypotheses that either donors or inhibitors of NO in the lung are effective strategies for treating asthma. These strategies evolved, in part, from results of pre-clinical studies performed in mice and other animal models. This review evaluates the existing literature with regard to mouse models of asthma and explores the often conflicting data on the role of NO, the nitric oxide synthase (NOS) enzymes, and the arginase enzymes in allergic airway inflammation. While we will emphasize the ovalbumin exposure mouse model, we will also examine other models. Where inconsistencies are identified among the studies, we attempt to determine whether such inconsistencies arise from methodological differences or alternative mechanisms. Ultimately, we address whether the allergen-exposed mouse is a suitable model for identifying promising new drugs for the treatment of human asthma. While a consensus is building that NO is beneficial or protective in subsets of asthmatics, results from studies using mouse models to investigate the individual roles of NO and the NOS enzymes in airway inflammation are often contradictory. Further research efforts with this model will allow us to distinguish which asthma patients may benefit best from NO donors and which may benefit from NO inhibitors.

The immunogenicity of Echinococcus granulosus antigen 5 is determined by its post-translational modifications.

Since its early introduction as a marker for the immunodiagnosis of hydatid disease, antigen 5 (Ag5) has been regarded as one of the more relevant antigens of Echinococcus granulosus, and it is still widely used in different confirmation techniques. In this work we prepared 2 recombinant forms of the antigen, namely, rAg5 (corresponding to the unprocessed polypeptide chain of the antigen) and rAg5-38s (corresponding to its 38 kDa subunit). Their antigenicities were compared to that of the native antigen using a human serum collection. There was a major drop in the reactivity of the sera, particularly against rAg5-38s, which was confirmed by analysis of the cross-reactivity of 2 panels of monoclonal antibodies specific for rAg5-38s and the native antigen. Using the chemically deglycosylated native antigen, we demonstrated that the reduced antigenicity of the recombinants is due to the loss of the sugar determinants, and not to their misfolding. Inhibition experiments using phosphorylcholine confirmed that this moiety also contributes to the reactivity of the antigen, but to a much lesser extent. The presence of immunodominant highly cross-reactive glycan moieties in the Ag5 molecule may involve a parasite evasion mechanism.

Reversible and irreversible airway inflammation and fibrosis in mice exposed to inhaled ovalbumin.

OBJECTIVE AND DESIGN: We examined the reversibility of several changes in the lungs and airways of mice immediately after exposure to ovalbumin aerosol and after a period of recovery breathing clean air. METHODS: Mice were exposed for 1, 2, 4, 6, 8, or 10 weeks, with recovery in clean air for 1-3 weeks. RESULTS: Airway collagen content, exhaled NO, airway mucous cell hyperplasia, and lung lavage inflammatory cell content increased upon exposure to ovalbumin aerosol. All parameters except airway fibrosis decreased partially or completely to control values with recovery in clean air. CONCLUSIONS: Airway mucous cell hypertrophy and hyperplasia appear to be completely reversible after recovery in clean air, while exhaled NO and airway inflammation appear to be mostly reversible, except for persistence of lymphocytes in the lung lavage fluid. Airway fibrosis appears to be reversible when mice are exposed to ovalbumin aerosol for periods of up to 4 weeks of exposure, but becomes irreversible after 6 or more weeks of exposure.

TGF-beta1 causes airway fibrosis and increased collagen I and III mRNA in mice.

BACKGROUND: Subepithelial collagen and extracellular matrix protein deposition are important pathophysiological components of airway remodelling in chronic asthma. Animal models based on the local reaction to antigens show structural alterations in the airway submucosal region and provide important information regarding disease pathophysiology. We describe a murine model of peribronchial fibrosis using intratracheally instilled transforming growth factor (TGF)-beta(1) in BALB/C mice that facilitates a mechanistic approach to understanding the cellular and molecular pathways leading to airway fibrosis. METHODS: BALB/C mice were intratracheally instilled with either TGF-beta(1) or buffered saline. Airway fibrosis was assessed by light microscopy, hydroxyproline content, and polymerase chain reaction (PCR) for collagen I and III on microdissected airway samples. The lysyl oxidase inhibitor beta-aminoproprionitrile (BAPN) was administered to TGF-beta(1) treated mice to block airway collagen deposition. Airway hyperresponsiveness was also measured after treatment with TGF-beta(1). RESULTS: During the 7 days after administration of TGF-beta(1) the mice developed increased subepithelial collagen which could be blocked by BAPN. Increased mRNAs for collagen types I and III were seen in microdissected airways 1 week after TGF-beta(1), and significantly increased total collagen was found in the airways 4 weeks after TGF-beta(1). A detectable increase in airway hyperreactivity occurred. CONCLUSIONS: This new model should facilitate detailed study of airway remodelling that occurs in the absence of detectable cellular inflammation, and allow examination of the functional consequences of a major structural alteration in the conducting airways uncomplicated by inflammatory cell influx.

Mechanism of inhibition of lysyl hydroxylase activity by the organophosphates malathion and malaoxon.

Direct inhibition of lysyl hydroxylase by malathion and malaoxon was observed in an in vitro enzyme assay with recombinant lysyl hydroxylase expressed via a baculoviral system. The IC50 values for malathion and malaoxon were estimated to be approximately 60 and 45 mM, respectively. Additional kinetic studies showed this inhibition to be competitive or partially competitive with respect to the synthetic (collagen) peptide, partially uncompetitive with respect to Fe(2+), and partially noncompetitive with respect to ascorbic acid. The calculated values for the K(i) were consistent with the IC50 values. Allosteric effects were not found for any of the cofactors tested, the peptide substrate, or the inhibitors. Interactions were found to be unimolecular for lysyl hydroxylase and its substrate and cofactors as well as for the inhibitors malathion and malaoxon. A computer search of a protein structure database showed an unexpected region of partial homology between the active site sequence of acetylcholinesterase and a segment of lysyl hydroxylase, suggesting a possible molecular basis for these observations. These results suggest the possibility of a novel and hitherto unexpected class of inhibitors of lysyl hydroxylase, based on the organophosphate structure, that might be of value for testing as antifibrotic drugs.

Lipid membrane reorganization induced by chemical recognition.

Nanoscale structural reorganization of a lipid bilayer membrane induced by a chemical recognition event has been imaged using in situ atomic force microscopy (AFM). Supported lipid bilayers, composed of distearylphosphatidylcholine (DSPC) and a synthetic lipid functionalized with a Cu(2+) receptor, phase-separate into nanoscale domains that are distinguishable by the 9 A height difference between the two molecules. Upon binding of Cu(2+) the electrostatic nature of the receptor changes, causing a dispersion of the receptor molecules and subsequent shrinking of the structural features defined by the receptors in the membrane. Complete reversibility of the process was demonstrated through the removal of metal ions with EDTA.

Nanostructures. Self-assembled domain patterns.

The ordered domain patterns that form spontaneously in a wide variety of chemical and physical systems as a result of competing interatomic interactions can be used as templates for fabricating nanostructures. Here we describe a new self-assembling domain pattern on a solid surface that involves two surface structures of lead on copper. The evolution of the system agrees with theoretical predictions, enabling us to probe the interatomic force parameters that are crucial to the process.

Inhibition of lysyl hydroxylase by malathion and malaoxon.

The inhibition of lysyl hydroxylation in newly synthesized collagen by malathion and its oxidation product malaoxon were studied with cultured rat fetal lung fibroblasts. Exposure of these cells to 125 microM malathion or malaoxon for 96 h resulted in a 25 and a 30% decrease in the ratio of hydroxylated lysine/lysine residues, respectively, in acid hydrolyzed cell lysates compared to control values. This relative decrease in hydroxylysine was not caused by cytotoxicity or changes in total collagen content, which were found to remain constant as measured by Alamar Blue metabolism and Sircol dye binding assays. Direct inhibition of lysyl hydroxylase by malathion and malaoxon was observed using an in vitro enzyme assay with recombinant lysyl hydroxylase with a baculoviral system. The IC50 values for malathion and malaoxon were estimated to be approximately 60 and 45 microM, respectively. These observed IC50 values are consistent with calculated values for the intracellular concentration of malathion in the cell culture experiments. These results support a significant role for inhibition of lysyl hydroxylase activity as causing, or contributing to, the teratogenic effects of malathion and malaoxon.

Early phase collagen synthesis in lungs of rats exposed to bleomycin.

Skin wound healing exhibits type III collagen synthesis occurring transiently as early as 10 h after injury, with subsequent synthesis of type I to form a scar. We hypothesized that similar collagen type switching also occurred in the bleomycin model of lung fibrosis in the rat. We could measure elevated lung collagen synthesis rates as early as 4 days after administration of bleomycin. Collagen type I:III ratios in whole lung remained constant for the first 7 days at the control level of 2:1, then increased to as high as 5:1 at day 21. Procollagen mRNA content, expressed as a ratio of type I:III mRNAs, was consistent with the protein synthesis data and the observed ratio of collagen types being made by the lungs at the various time points evaluated. We conclude that a transient increase in type III relative to type I collagen does not occur in the bleomycin rat lung model. Therefore, the sequence of type-specific collagen expression and deposition in the skin wound healing model is not entirely analogous to this widely used animal model of pulmonary fibrosis.

Differential expression of stress proteins in nonhuman primate lung and conducting airway after ozone exposure.

The presence of seven stress proteins including various heat shock proteins [27-kDa (HSP27), 60-kDa (HSP60), 70-kDa (HSP70) and its constitutive form HSC70, and 90-kDa (HSP90) HSPs] and two glucose-regulated proteins [75-kDa (GRP75) and 78-kDa (GRP78) GRPs] in ozone-exposed lungs of nonhuman primates and in cultured tracheobronchial epithelial cells was examined immunohistochemically by various monoclonal antibodies. Heat treatment (42 degrees C) resulted in increased HSP70, HSP60, and HSP27 and slightly increased HSC70 and GRP75 but no increase in GRP78 in primary cultures of monkey tracheobronchial epithelial cells. Ozone exposure did not elevate the expression of these HSPs and GRPs. All of these HSPs including HSP90, which was undetectable in vitro, were suppressed in vivo in monkey respiratory epithelial cells after ozone exposure. Both GRP75 and GRP78 were very low in control cells, and ozone exposure in vivo significantly elevated these proteins. These results suggest that the stress mechanism exerted on pulmonary epithelial cells by ozone is quite different from that induced by heat. Furthermore, differences between in vitro and in vivo with regard to activation of HSPs and GRPs suggest a secondary mechanism in vivo, perhaps related to inflammatory response after ozone exposure.

Centriacinar remodeling and sustained procollagen gene expression after exposure to ozone and nitrogen dioxide.

Sprague-Dawley rats were exposed to 0.8 ppm ozone (O3), to 14.4 ppm nitrogen dioxide (NO2), or to both gases simultaneously for 6 h per day for up to 90 d. The extent of histopathologic changes within the central acinus of the lungs was compared after 7 or 78 to 90 d of exposure using morphometric analysis by placement of concentric arcs radiating outward from a single reference point at the level of the bronchiole- alveolar duct junction. Lesions in the lungs of rats exposed to the mixture of gases extended approximately twice as far into the acinus as in those exposed to each individual gas. The extent of tissue involvement was the same at 78 to 90 d as noted at 7 d in all exposure groups. At the end of exposure, in situ hybridization for procollagen types I and III demonstrated high levels of messenger RNA within central acini in the lungs of animals exposed to the combination of O3 and NO2. In contrast, animals exposed to each individual gas had a similar pattern of message expression compared with that seen in control animals, although centriacinar histologic changes were still significantly different from control animals. We conclude that the progressive pulmonary fibrosis that occurs in rats exposed to the combination of O3 and NO2 is due to sustained, elevated expression of the genes for procollagen types I and III. This effect at the gene level is correlated with the more severe histologic lesions seen in animals exposed to both O3 and NO2 compared with those exposed to each individual gas. In contrast, the sustained expression of the procollagen genes is not associated with a shift in the distribution of the lesions because the area of change in each group after 7 d of exposure was the same as after 78 to 90 d of exposure.

Chronic exposure of rats to ozone and sulfuric acid aerosol: biochemical and structural responses.

Groups of rats were exposed to either 0.12 or 0.20 ppm of ozone, 20, 100, or 150 ppm of sulfuric acid aerosol (0.4-0.8 microm diameter), or their mixtures in whole body exposure chambers for up to 90 days. Matched control animals were exposed to filtered air in comparable chambers. The rats were examined biochemically and morphometrically for centriacinar fibrosis or other indicators of pollutant-induced changes in the terminal bronchiole-alveolar duct junction region of the lung at the end of the exposures. By evaluating different markers of lung injury, we had previously demonstrated a synergistic interaction between ozone and sulfuric acid aerosol after acute exposures to these same concentrations of the pollutants. The present experiments were designed to answer the question of whether there was any interaction between ozone and respirable sized aerosols of sulfuric acid, synergistic or antagonistic, after chronic exposures. Exposure of rats to 0.12 or 0.20 ppm of ozone elicited tissue and cellular changes at the bronchiole-alveolar duct junction. Concurrent exposure to sulfuric acid aerosol did not affect the extent or magnitude of these changes. Intermittent exposure (12 h per day) to ozone, with or without the acid aerosol, elicited a greater response than did continuous exposure (24 h per day). No consistent effects of exposure to sulfuric acid aerosol alone were observed, either morphometrically or biochemically. The biochemical data were consistent with the morphometric analyses, showing trends towards or significantly increased lung 4-hydroxyproline content in the rats exposed to ozone, with or without sulfuric acid aerosol, in the intermittent exposure experiment, but not after continuous exposure. No interactive effects between ozone and sulfuric acid aerosol were observed with any of the biochemical parameters examined. We conclude that ozone and sulfuric acid aerosols do not exhibit synergistic interactions after chronic exposures (90 days) of rats to the concentrations tested in this study, which correspond to concentrations showing synergistic interactions in previously performed acute studies. We also observed that exposure of rats to ozone for 12 h per day elicited greater lung changes, which we interpret to indicate a mild fibrotic response, than did exposure of rats for 24 h per day, whether or not there was accompanying exposure to the acid aerosol.