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Introduction:Neutrophil transmigration is multifactorial and primarily driven by selectins and β2-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.

In the past few decades, the frequency and severity of wildfires in California has increased. The majority of studies on wildfire-related human health effects have focused on the criteria pollutants in smoke. However, there are other minor constituents of smoke that may impact human health, particularly in specific geographic regions in California. This review summarizes what we currently know about the contribution of pesticides and wildfire suppression chemicals to wildfire-induced human health effects. In California there is heavy use of pesticides in agricultural settings and at the urban interface. When wildfires burn land treated with pesticide, these chemicals and their combustion products are volatilized and can be inhaled by humans. These constituents can be transported long distances in smoke, although those at the highest risk for exposure are near the wildfire source. Toxicity of some pesticides by inhalation has been demonstrated, although the health effects of pesticide combustion products in smoke have not been characterized. In order to effectively fight wildfires, large volumes of wildfire suppression chemicals are used. Wildfire suppression chemicals include retardants, for long-term application, and foams, for short term application. Based on the available data, foams are more likely to have an impact on human health than retardants. Moreover, foams tend to be applied at the urban interface, while the retardants are generally applied in remote areas. Understanding the health effects of the compounds we choose to introduce into our environment and how they alter and are altered by extreme events like wildfires is an important consideration for fire and land management. Our investigation has uncovered that there are significant data gaps in this area.

This article reviews the acute and chronic toxicity of the three oxidant air pollutants ozone, nitric oxide (NO), and nitrogen dioxide (NO2). The toxicity of binary mixtures of NO2 with other inhaled agents is also discussed. Newer studies are emphasized, especially those published in the last 5 years or still in press. Very recent data from our laboratory that suggest a new cellular mechanism of importance in lung injury in animals exposed to mixtures of ozone and NO2 that may have general relevance with regard to the effects of oxidant air pollutants on the lung are also presented.

Inhalation toxicology experiments in whole animals have demonstrated a remarkable lack of toxicity of sulfuric acid in the form of respirable aerosols, especially in rats and nonhuman primates. Thus, much of the current experimental emphasis has shifted to the evaluation of the potential health effects of acid aerosols as components of mixtures. Rats have been concurrently exposed to mixtures of ozone or nitrogen dioxide with respirable-sized aerosols of sulfuric acid, ammonium sulfate, or sodium chloride, or to each pollutant individually. Their responses to such exposures have been evaluated by various quantitative biochemical analysis of lung tissue or wash fluids ("lavage fluid") or by quantitative morphological methods ("morphometry"). Such studies have mainly been performed in the acute time frame due to the inherent limitations of the most sensitive assays available and have generally involved exposures for 1 to 9 days, depending on the assays used. Good correlations were found between the most sensitive biochemical indicators of lung damage (protein content of lung lavage fluid or whole lung tissue and lung collagen synthesis rate) and the exposure concentration of oxidant gas present alone or in mixtures with acidic aerosols showing interactive effects. Synergistic interaction between ozone and sulfuric acid aerosol was demonstrated to occur at environmentally relevant concentrations of both pollutants by several of the analytical methods used in this study. Such interactions were demonstrated at concentrations of ozone as low as 0.12 ppm and of sulfuric acid aerosol at concentrations as low as 5 to 20 micrograms/m3.(ABSTRACT TRUNCATED AT 250 WORDS)

Tracheal slices incubated with radioactive precursors in tissue culture medium secrete labeled mucus glycoproteins into the culture medium. We have used an in vivtro approach, a combined method utilizing exposure to pneumotoxins in vivo coupled with quantitation of mucus secretion rates in vitro, to study the effects of inhaled pollutants on mucus biosynthesis by rat airways. In addition, we have purified the mucus glycoproteins secreted by rat tracheal explants in order to determine putative structural changes that might by the basis for the observed augmented secretion rates after exposure of rats to H2SO4 aerosols in combination with high ambient levels of ozone. After digestion with papain, mucus glycoproteins secreted by tracheal explants may be separated into five fractions by ion-exchange chromatography, with recovery in high yield, on columns of DEAE-cellulose. Each of these five fractions, one neutral and four acidic, migrates as a single unique spot upon cellulose acetate electrophoresis at pH values of 8.6 and 1.2. The neutral fraction, which is labeled with [3H] glucosamine, does not contain radioactivity when Na2 35SO4 is used as the precursor. Acidic fractions I-IV are all labeled with either 3H-glucosamine or Na2 35SO4 as precursor. Acidic fraction II contains sialic acid as the terminal sugar on its oligosaccharide side chains, based upon its chromatographic behavior on columns of wheat-germ agglutinin-Agarose. Treatment of this fraction with neuraminidase shifts its elution position in the gradient to a lower salt concentration, coincident with acidic fraction I. After removal of terminal sialic acid residues with either neuraminidase or low pH treatment, the resultant terminal sugar on the oligosaccharide side chains is fucose. These results are identical with those observed with mucus glycoproteins secreted by cultured human tracheal explants and purified by these same techniques.

This presentation reports on newer animal models and techniques that we and others have developed. These models attempt to predict in a reasonable experimental time frame the occurrence of structural changes in the lungs that are the basis of various chronic diseases. Since the major chronic diseases of the lung parenchyma involving what we think of as well-understood structural changes are pulmonary fibrosis and emphysema, I will discuss in detail ways of looking at effects of air pollutants on lung collagen and lung elastin metabolism.

Effects of respirable aerosols of sulfuric acid, ammonium sulfate, sodium sulfite, and ammonium persulfate on lungs of rats are reviewed. The literature regarding interactions between ozone or nitrogen dioxide and acidic aerosols (ammonium sulfate, sulfuric acid) is discussed. An unexpected interaction between nitrogen dioxide and sodium chloride aerosol is also discussed. An attempt is made to identify bases for prediction of how and when acid aerosols might potentiate effects of inhaled gases.

Collagen is the major structural protein of the lung. At least five genetically distinct collagen types have been identified in lung tissue. However, the precise role of collagen in nonrespiratory lung function is not well understood, in part because of the difficulties inherent in studying lung collagen, regardless of the type of assay used. A major problem is the insolubility of lung collagen; generally less than 20% of total lung collagen can be solubilized as intact chains, even with harsh extraction procedures. Since such collagen may not be representative of total lung collagen, errors in quantitating collagen types, for example, may arise from using such material. Measurement of total lung collagen content may also pose problems, unless appropriate parameters of normalization are chosen. Biopsy dry weight, protein content, and DNA content, for example, may all change in certain disease states. Despite these difficulties, a number of changes in lung collagen have been documented in experimental pulmonary fibrosis, including increased collagen content, increased collagen synthesis rates, and changes in collagen type ratios. Many questions remain. For example, why do diverse toxic substances appear to cause essentially the same fibrotic response, even though initial sites of damage may vary? Conversely, why do similar toxic substances, such as ozone and NO2, cause diverse responses (fibrosis and emphysema, respectively)? Much work remains to be done to elucidate the mechanisms underlying the lung's choice of response.

Elemental sulfur, a natural substance, is used as a fungicide. Elemental sulfur is the most heavily used agricultural chemical in California. In 2003, annual sulfur usage in California was about 34% of the total weight of pesticide active ingredient used in production agriculture. Even though sulfur is mostly used in dust form, the respiratory health effects of elemental sulfur are not well documented. The purpose of this paper is to address the possible respiratory effect of elemental sulfur using the California Pesticide Illness Database and laboratory experiments with mice. We analyzed the California Pesticide Illness Database between 1991 and 2001. Among 127 reports of definite, probable, and possible illness involving sulfur, 21 cases (16%) were identified as respiratory related. A mouse model was used to examine whether there was an inflammatory or fibrotic response to elemental sulfur. Dust solutions were injected intratracheally into ovalbumin sensitized mice and lung damage was evaluated. Lung inflammatory response was analyzed via total lavage cell counts and differentials, and airway collagen content was analyzed histologically and biochemically. No significant differences from controls were seen in animals exposed to sulfur particles. The findings suggest that acute exposure of elemental sulfur itself may not cause an inflammatory reaction. However, further studies are needed to understand the possible health effects of chronic sulfur exposure and environmental weathering of sulfur dust. [ABSTRACT FROM AUTHOR]

Introduction: Neutrophil transmigration is multifactorial and primarily driven by selectins and β 2 -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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cagle, Linderholm, Franzi, Last, Simon, Kenyon and Harper.)

Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo . Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

Ras, a small GTPase protein, is thought to mediate Th2-dependent eosinophilic inflammation in asthma. Ras requires cell membrane association for its biological activity, and this requires the posttranslational modification of Ras with an isoprenyl group by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). We hypothesized that inhibition of FTase using FTase inhibitor (FTI)-277 would attenuate allergic asthma by depleting membrane-associated Ras. We used the OVA mouse model of allergic inflammation and human airway epithelial (HBE1) cells to determine the role of FTase in inflammatory cell recruitment. BALB/c mice were first sensitized then exposed to 1% OVA aerosol or filtered air, and half were injected daily with FTI-277 (20 mg/kg per day). Treatment of mice with FTI-277 had no significant effect on lung membrane-anchored Ras, Ras protein levels, or Ras GTPase activity. In OVA-exposed mice, FTI-277 treatment increased eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity. Human bronchial epithelial (HBE1) cells were pretreated with 5, 10, or 20 μM FTI-277 prior to and during 12 h IL-13 (20 ng/ml) stimulation. In HBE1 cells, FTase inhibition with FTI-277 had no significant effect on IL-13-induced STAT6 phosphorylation, eotaxin-3 peptide secretion, or Ras translocation. However, addition of exogenous FPP unexpectedly augmented IL-13-induced STAT6 phosphorylation and eotaxin-3 secretion from HBE1 cells without affecting Ras translocation. Pharmacological inhibition of FTase exacerbates allergic asthma, suggesting a protective role for FTase or possibly Ras farnesylation. FPP synergistically augments epithelial eotaxin-3 secretion, indicating a novel Ras-independent farnesylation mechanism or direct FPP effect that promotes epithelial eotaxin-3 production in allergic asthma., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Background: Positive-pressure mechanical ventilation is an essential therapeutic intervention, yet it causes the clinical syndrome known as ventilator-induced lung injury. Various lung protective mechanical ventilation strategies have attempted to reduce or prevent ventilator-induced lung injury but few modalities have proven effective. A model that isolates the contribution of mechanical ventilation on the development of acute lung injury is needed to better understand biologic mechanisms that lead to ventilator-induced lung injury., Objectives: To evaluate the effects of positive end-expiratory pressure and recruitment maneuvers in reducing lung injury in a ventilator-induced lung injury murine model in short- and longer-term ventilation., Methods: 5-12 week-old female BALB/c mice (n = 85) were anesthetized, placed on mechanical ventilation for either 2 hrs or 4 hrs with either low tidal volume (8 ml/kg) or high tidal volume (15 ml/kg) with or without positive end-expiratory pressure and recruitment maneuvers., Results: Alteration of the alveolar-capillary barrier was noted at 2 hrs of high tidal volume ventilation. Standardized histology scores, influx of bronchoalveolar lavage albumin, proinflammatory cytokines, and absolute neutrophils were significantly higher in the high-tidal volume ventilation group at 4 hours of ventilation. Application of positive end-expiratory pressure resulted in significantly decreased standardized histology scores and bronchoalveolar absolute neutrophil counts at low- and high-tidal volume ventilation, respectively. Recruitment maneuvers were essential to maintain pulmonary compliance at both 2 and 4 hrs of ventilation., Conclusions: Signs of ventilator-induced lung injury are evident soon after high tidal volume ventilation (as early as 2 hours) and lung injury worsens with longer-term ventilation (4 hrs). Application of positive end-expiratory pressure and recruitment maneuvers are protective against worsening VILI across all time points. Dynamic compliance can be used guide the frequency of recruitment maneuvers to help ameloriate ventilator-induced lung injury.

Proposition 2, which requires that egg-laying hens be confined only in ways that allow these animals to lie down, stand up, fully extend their limbs and turn around freely, was passed by the voters of California in 2008. These new housing requirements were introduced in the USA and European Union without considering the potential impact of changes in layer hen housing on the health of poultry workers in the new facilities. Particles were collected from ambient air inside a large layer hen complex featuring separate barns with conventional battery caging, enriched caging, or 'free range' (aviary) housing during winter, spring, and summer seasons over one year. Toxicity of the particles was evaluated by analysis of inflammatory cell influx into lung lavage fluid after intratracheal instillation into mice. Capacity of the particles to elicit oxidative stress was evaluated using a macrophage cell line engineered with a reporter gene sensitive to nuclear factor κB activation. We observed similar pro-inflammatory and pro-oxidant effects of the particles collected from different types of barns and over different seasons, suggesting that standard industrial hygiene techniques for evaluating respirable particles in ambient air can adequately monitor worker risk. Based on particle concentrations found in ambient air in the barns, we can rank the facilities for worker exposure to particles as conventional caging (now banned) approximately equal to enriched caging (permitted under Proposition 2). Aviary housing is associated with increased exposure of workers to particulate matter and, therefore, to greater risk of allergic reactions and/or decreased respiratory function.

Anesthetic protocols for murine models are varied within the literature and medetomidine has been implicated in the development of urethral plugs in male mice. Our objective was to evaluate the combination of butorphanol, dexmedetomidine, and tiletamine-zolazepam. A secondary objective was to identify which class of agent was associated with urethral obstructions in male mice. BALB/c male ( n = 13) and female ( n = 23) mice were assigned to dexmedetomidine and tiletamine-zolazepam with or without butorphanol or to single agent dexmedetomidine or tiletamine-zolazepam. Anesthesia was achieved in 58% (14/24) of mice without butorphanol and in 100% (24/24) of mice with butorphanol. The combination of dexmedetomidine (0.2 mg/kg), tiletamine-zolazepam (40 mg/kg), and butorphanol (3 mg/kg) resulted in an induction and anesthetic duration of 12 and 143 minutes, respectively. Urethral obstructions occurred in 66% (25/38) of trials in male mice that received dexmedetomidine with a mortality rate of 38% (5/13). Tiletamine-zolazepam, when used alone, resulted in a 0% (0/21) incidence of urethral obstructions. Combination use of dexmedetomidine, tiletamine-zolazepam, and butorphanol results in a longer and more reliable duration of anesthesia than the use of dexmedetomidine and tiletamine-zolazepam alone. Dexmedetomidine is not recommended for use in nonterminal procedures in male mice due to the high incidence of urethral obstructions and resultant high mortality rate., Competing Interests: The authors declare that there are no competing interests regarding the publication of this paper.

Systemic treatment with statins mitigates allergic airway inflammation, TH2 cytokine production, epithelial mucus production, and airway hyperreactivity (AHR) in murine models of asthma. We hypothesized that pravastatin delivered intratracheally would be quantifiable in lung tissues using mass spectrometry, achieve high drug concentrations in the lung with minimal systemic absorption, and mitigate airway inflammation and structural changes induced by ovalbumin. Male BALB/c mice were sensitized to ovalbumin (OVA) over 4 weeks, then exposed to 1% OVA aerosol or filtered air (FA) over 2 weeks. Mice received intratracheal instillations of pravastatin before and after each OVA exposure (30 mg/kg). Ultra performance liquid chromatography - mass spectrometry was used to quantify plasma, lung, and bronchoalveolar lavage fluid (BALF) pravastatin concentration. Pravastatin was quantifiable in mouse plasma, lung tissue, and BALF (BALF > lung > plasma for OVA and FA groups). At these concentrations pravastatin inhibited airway goblet cell hyperplasia/metaplasia, and reduced BALF levels of cytokines TNFα and KC, but did not reduce BALF total leukocyte or eosinophil cell counts. While pravastatin did not mitigate AHR, it did inhibit airway hypersensitivity (AHS). In this proof-of-principle study, using novel mass spectrometry methods we show that pravastatin is quantifiable in tissues, achieves high levels in mouse lungs with minimal systemic absorption, and mitigates some pathological features of allergic asthma. Inhaled pravastatin may be beneficial for the treatment of asthma by having direct airway effects independent of a potent anti-inflammatory effect. Statins with greater lipophilicity may achieve better anti-inflammatory effects warranting further research., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Objectives: We hypothesised that severe asthmatics taking a statin drug, in addition to inhaled corticosteroids/long-acting β-agonist inhaler therapy, would have better asthma symptom control and improved lung function compared to their controls., Study Design: A retrospective, cross-sectional study of 165 patients with severe asthma seen from 2001-2008. Hierarchical linear and logistic regression models were used for modelling fitting., Setting: University of California, Davis Medical Center (Sacramento, California, USA). Academic, single-centre, severe asthma subspecialty clinic., Participants: 612 screened, 223 eligible and 165 adult patients were included in the final study (N=165; 31 statin users and 134 non-users)., Primary and Secondary Outcome Measures: The primary endpoint was asthma control as measured by the Asthma Control Test (ACT). The secondary endpoints included lung function, symptoms and the need for corticosteroid burst and peripheral eosinophil count., Results: At baseline, statin users compared to non-users were older, had lower lung function (FEV1% predicted, FEV1, forced vital capacity and FEF25-75%) and had a higher prevalence of comorbid conditions. Statin use was associated with more aspirin and ipratropium inhaler use than in non-users. Patients in both groups were obese (body mass index ≥ 30). Statin users had better asthma symptom control compared to non-users (higher adjusted mean ACT score by 2.2±0.94 points, p<0.02). Median statin use was for 1 year. There were no statistically significant differences in lung function, corticosteroid or rescue bronchodilator use or peripheral eosinophilia between the two groups., Conclusions: In our severe asthma referral population, statin users already taking inhaled controller therapy achieved better asthma control compared to non-users. The implications of this study is that patients with severe asthma could potentially benefit from added statin treatment. Because our study population was on average obese, the obese severe asthmatic may be a viable asthma subphenotype for further studies. Prospective randomised clinical trials evaluating the safety and efficacy of statins in severe asthma are warranted.

In recent years, the international demand for commodities has prompted enormous growth in agriculture in most South American countries. Due to intensive use of fertilizers, cyanobacterial blooms have become a recurrent phenomenon throughout the continent, but their potential health risk remains largely unknown due to the lack of analytical capacity. In this paper we report the main results and conclusions of more than five years of systematic monitoring of cyanobacterial blooms in 20 beaches of Montevideo, Uruguay, on the Rio de la Plata, the fifth largest basin in the world. A locally developed microcystin ELISA was used to establish a sustainable monitoring program that revealed seasonal peaks of extremely high toxicity, more than one-thousand-fold greater than the WHO limit for recreational water. Comparison with cyanobacterial cell counts and chlorophyll-a determination, two commonly used parameters for indirect estimation of toxicity, showed that such indicators can be highly misleading. On the other hand, the accumulated experience led to the definition of a simple criterion for visual classification of blooms, that can be used by trained lifeguards and technicians to take rapid on-site decisions on beach management. The simple and low cost approach is broadly applicable to risk assessment and risk management in developing countries., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The Fogarty International Center (FIC) of the United States National Institutes of Health includes the International Training and Research in Environmental and Occupational Health (ITREOH) Program. The "International Training Program in Environmental Toxicology and Public Health" Center, funded in 2002 is based at the University of California, Davis, and is part of the ITREOH group of Centers. It has major efforts focused at the public universities in Montevideo, Uruguay, and Salta, Argentina., Results: Training and research efforts in Salta begun in 2005 in the College of Engineering. A donated used real-time PCR machine was the starting point and the initial FIC support was instrumental to face other problems including physical space, research projects and grants, trainees, training, networking, and distractions/opportunities in order to develop local capacities in Environmental Engineering using modern methodology. After 6 years of successful work, the Salta center has become a reference Center in the field, and is still growing and consolidating., Conclusions: This program has had a significant impact locally and regionally. The model used in Argentina could be easily adapted to other fields or types of projects in Argentina and in other developing countries., (Copyright © 2012 Wiley Periodicals, Inc.)

Our previous work has shown that coarse particulate matter (PM(10-2.5)) from wildfire smoke is more toxic to lung macrophages on an equal dose (by mass) basis than coarse PM isolated from normal ambient air, as evidenced by decreased numbers of macrophages in lung lavage fluid 6 and 24hours after PM instillation into mouse lungs in vivo and by cytotoxicity to a macrophage cell line observed directly in vitro. We hypothesized that pulmonary macrophages from mice instilled with wildfire coarse PM would undergo more cytotoxicity than macrophages from controls, and that there would be an increase in oxidative stress in their lungs. Cytotoxicity was quantified as decreased viable macrophages and increased percentages of dead macrophages in the bronchoalveolar lavage fluid (BALF) of mice instilled with wildfire coarse PM. At 1hour after PM instillation, we observed both decreased numbers of viable macrophages and increased dead macrophage percentages as compared to controls. An increase in free isoprostanes, an indicator of oxidative stress, from control values of 28.1±3.2pg/mL to 83.9±12.2pg/mL was observed a half-hour after PM instillation. By 1hour after PM instillation, isoprostane values had returned to 30.4±7.6pg/mL, not significantly different from control concentrations. Lung sections from mice instilled with wildfire coarse PM showed rapid Clara cell responses, with decreased intracellular staining for the Clara cell secretory protein CCSP 1hour after wildfire PM instillation. In conclusion, very rapid cytotoxicity occurs in pulmonary macrophages and oxidative stress responses are seen 0.5-1hour after wildfire coarse PM instillation. These results define early cellular and biochemical events occurring in vivo and support the hypothesis that oxidative stress-mediated macrophage toxicity plays a key role in the initial response of the mouse lung to wildfire PM exposure., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cystic echinococcosis is still a major concern in South America. While some regions show advances in the control of the disease, others have among the highest incidence in the world. To reverse this situation the Pan American Health Organization (PAHO) has launched a regional project on cystic echinococcosis control and surveillance. An early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. Under this premise, we have developed a new copro-ELISA test after extensive screening of a large panel of monoclonal antibodies (MAbs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. The key component of the test, MAbEg9 has a convenient IgG isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. Time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. The test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. This characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in Peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion.

The ability to accurately determine the elastic modulus of each layer of the human cornea is a crucial step in the design of better corneal prosthetics. In addition, knowledge of the elastic modulus will allow design of substrates with relevant mechanical properties for in vitro investigations of cellular behavior. Previously, we have reported elastic modulus values for the anterior basement membrane and Descemet's membrane of the human cornea, the surfaces in contact with the epithelial and endothelial cells, respectively. We have completed the compliance profile of the stromal elements of the human cornea by obtaining elastic modulus values for Bowman's layer and the anterior stroma. Atomic force microscopy (AFM) was used to determine the elastic modulus, which is a measure of the tissue stiffness and is inversely proportional to the compliance. The elastic response of the tissue allows analysis with the Hertz equation, a model that provides a relationship between the indentation force and depth and is a function of the tip radius and the modulus of the substrate. The elastic modulus values for each layer of the cornea are: 7.5±4.2 kPa (anterior basement membrane), 109.8±13.2 kPa (Bowman's layer), 33.1±6.1 kPa (anterior stroma), and 50±17.8 kPa (Descemet's membrane). These results indicate that the biophysical properties, including elastic modulus, of each layer of the human cornea are unique and may play a role in the maintenance of homeostasis as well as in the response to therapeutic agents and disease states. The data will also inform the design and fabrication of improved corneal prosthetics., (Copyright © 2012. Published by Elsevier Ltd.)

The mechanistic basis of the high toxicity to lung macrophages of coarse PM from the California wildfires of 2008 was examined in cell culture experiments with mouse macrophages. Wildfire PM directly killed macrophages very rapidly in cell culture at relatively low doses. The wildfire coarse PM is about four times more toxic to macrophages on an equal weight basis than the same sized PM collected from normal ambient air (no wildfires) from the same region and season. There was a good correlation between the extent of cytotoxicity and the amount of oxidative stress observed at a given dose of wildfire PM in vitro. Our data implicate NF-κB signaling in the response of macrophages to wildfire PM, and suggest that most, if not all, of the cytotoxicity of wildfire PM to lung macrophages is the result of oxidative stress. The relative ratio of toxicity and of expression of biomarkers of oxidant stress between wildfire PM and "normal" PM collected from ambient air is consistent with our previous results in mice in vivo, also suggesting that most, if not all, of the cytotoxicity of wildfire PM to lung macrophages is the result of oxidative stress. Our findings from this and earlier studies suggest that the active components of coarse PM from the wildfire are heat-labile organic compounds. While we cannot rule out a minor role for endotoxin in coarse PM preparations from the collected wildfire PM in our observed results both in vitro and in vivo, based on experiments using the inhibitor Polymyxin B most of the oxidant stress and pro-inflammatory activity observed was not due to endotoxin., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Single-domain antibodies (sdAbs) found in camelids lack a light chain, and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. Although this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low-affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC(50) 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, K(D) 0.98-1.37 nM (SPR), which allowed development of a competitive assay for TCC with an IC(50) = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC(50) attained with other antihapten VHHs reported thus far. Despite the modest overall antihapten sdAbs response in llamas, a small subpopulation of high-affinity VHHs is generated that can be isolated by careful design of the selection process.

Exhaled breath nitric oxide (NO) is an accepted asthma biomarker. Lung concentrations of NO and its amino acid precursor, L-arginine, are regulated by the relative expressions of the NO synthase (NOS) and arginase isoforms. Increased expression of arginase I and NOS2 occurs in murine models of allergic asthma and in biopsies of asthmatic airways. Although clinical trials involving the inhibition of NO-producing enzymes have shown mixed results, small molecule arginase inhibitors have shown potential as a therapeutic intervention in animal and cell culture models. Their transition to clinical trials is hampered by concerns regarding their safety and potential toxicity. In this review, we discuss the paradigm of arginase and NOS competition for their substrate L-arginine in the asthmatic airway. We address the functional role of L-arginine in inflammation and the potential role of arginase inhibitors as therapeutics.

In this review, we compare the reported values of Young's modulus (YM) obtained from indentation and tensile deformations of soft biological tissues. When the method of deformation is ignored, YM values for any given tissue typically span several orders of magnitude. If the method of deformation is considered, then a consistent and less ambiguous result emerges. On average, YM values for soft tissues are consistently lower when obtained by indentation deformations. We discuss the implications and potential impact of this finding.

Purpose: Elevated intraocular pressure (IOP) is a risk factor for glaucoma. The principal outflow pathway for aqueous humor in the human eye is through the trabecular meshwork (HTM) and Schlemm's canal (SC). The junction between the HTM and SC is thought to have a significant role in the regulation of IOP. A possible mechanism for the increased resistance to flow in glaucomatous eyes is an increase in stiffness (increased elastic modulus) of the HTM. In this study, the stiffness of the HTM in normal and glaucomatous tissue was compared, and a mathematical model was developed to predict the impact of changes in stiffness of the juxtacanalicular layer of HTM on flow dynamics through this region., Methods: Atomic force microscopy (AFM) was used to measure the elastic modulus of normal and glaucomatous HTM. According to these results, a model was developed that simulated the juxtacanalicular layer of the HTM as a flexible membrane with embedded pores., Results: The mean elastic modulus increased substantially in the glaucomatous HTM (mean = 80.8 kPa) compared with that in the normal HTM (mean = 4.0 kPa). Regional variation was identified across the glaucomatous HTM, possibly corresponding to the disease state. Mathematical modeling suggested an increased flow resistance with increasing HTM modulus., Conclusions: The data indicate that the stiffness of glaucomatous HTM is significantly increased compared with that of normal HTM. Modeling exercises support substantial impairment in outflow facility with increased HTM stiffness. Alterations in the biophysical attributes of the HTM may participate directly in the onset and progression of glaucoma.

Airway remodeling in asthma contributes to airway hyperreactivity, loss of lung function, and persistent symptoms. Current therapies do not adequately treat the structural airway changes associated with asthma. The statins are cholesterol-lowering drugs that inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase, which is the rate-limiting step of cholesterol biosynthesis in the mevalonate (MA) pathway. These drugs have been associated with improved respiratory health, and ongoing clinical trials are testing their therapeutic potential in asthma. We hypothesized that simvastatin treatment of ovalbumin (OVA)-exposed mice would attenuate early features of airway remodeling by a mevalonate-dependent mechanism. BALB/c mice initially were sensitized to OVA and then exposed to 1% OVA aerosol for 2 weeks after sensitization for 6 exposures. Simvastatin (40 mg/kg) or simvastatin plus MA (20 mg/kg) were injected intraperitoneally before each OVA exposure. Treatment with simvastatin attenuated goblet cell hyperplasia, arginase-1 protein expression, and total arginase enzyme activity, but it did not alter airway hydroxyproline content or transforming growth factor-β1. Inhibition of goblet cell hyperplasia by simvastatin was mevalonate-dependent. No appreciable changes to airway smooth muscle cells were observed in any control or treatment groups. In conclusion, in an acute mouse model of allergic asthma, simvastatin inhibited early hallmarks of airway remodeling, which are indicators that can lead to airway thickening and fibrosis. Statins are potentially novel treatments for airway remodeling in asthma. Additional studies using subchronic or chronic allergen exposure models are needed to extend these initial findings., (Copyright © 2010 Mosby, Inc. All rights reserved.)

The atomic force microscope (AFM) is widely used in materials science and has found many applications in biological sciences but has been limited in use in vision science. The AFM can be used to image the topography of soft biological materials in their native environments. It can also be used to probe the mechanical properties of cells and extracellular matrices, including their intrinsic elastic modulus and receptor-ligand interactions. In this review, the operation of the AFM is described along with a review of how it has been thus far used in vision science. It is hoped that this review will serve to stimulate vision scientists to consider incorporating AFM as part of their research toolkit.

The authors have previously demonstrated that wildfire-derived coarse or fine particulate matter (PM) intratracheally instilled into lungs of mice induce a strong inflammatory response. In the current study, the authors demonstrate that wildfire PM simultaneously cause major increases in oxidative stress in the mouse lungs as measured by decreased antioxidant content of the lung lavage supernatant fluid 6 and 24 h after PM administration. Concentrations of neutrophil chemokines/cytokines and of tumor necrosis factor (TNF)-alpha were elevated in the lung lavage fluid obtained 6 and 24 h after PM instillation, consistent with the strong neutrophilic inflammatory response observed in the lungs 24 h after PM administration, suggesting a relationship between the proinflammatory activity of the PM and the measured level of antioxidant capacity in the lung lavage fluid. Chemical analysis shows relatively low levels of polycyclic aromatic hydrocarbons compared to published results from typical urban PM. Coarse PM fraction is more active (proinflammatory activity and oxidative stress) on an equal-dose basis than the fine PM despite its lower content of polycyclic aromatic hydrocarbons. There does not seem to be any correlation between the content of any specific polycyclic aromatic hydrocarbon (or of total polycyclic aromatic hydrocarbon content) in the PM fraction and its toxicity. However, the concentrations of the oxidation products of phenanthrene and anthracene, phenanthraquinone and anthraquinone, were several-fold higher in the coarse PM than the fine fraction, suggesting a significant role for atmospheric photochemistry in the formation of secondary pollutants in the wildfire PM and the possibility that such secondary pollutants could be significant sources of toxicity in the wildfire PM.

The environmental impact of rice agriculture is poorly studied in developing countries, mainly due to limitations of the analytical capacity. Here, we report the development of a clomazone enzyme-linked immunosorbent assay as a fast and cost-effective tool to monitor the dissipation of this herbicide along the harvest. Antibodies were prepared using different strategies of hapten conjugation, and the best hapten/antibody pair was selected. It proved to be a reliable tool to measure the herbicide in the 2.0-20 ng/mL range in field samples, with excellent correlation with high-performance liquid chromatography results. The assay was used to study the dissipation of the herbicide in the floodwater of experimental rice paddies in Uruguay. Large differences in the residual amounts of herbicide were observed depending on the flooding practices. Because of its robustness and simplicity, the assay may be useful to delineate and monitor management practices that can contribute to minimizing the release of the herbicide in the environment.

Objectives and Design: The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism., Materials or Subjects: Mice from a C57BL/6 wild-type, NOS1(-/-), NOS2(-/-), and NOS3(-/-) genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation., Methods: We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content., Results: Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3(-/-) strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1(-/-) animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2(-/-), and NOS3(-/-) allergen-exposed mice., Conclusion: The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This "homeostatic" mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia.

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.

Arginase1 and nitric oxide synthase2 (NOS2) utilize l-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N(omega)-hydroxy-nor-l-arginine (nor-NOHA) significantly increased total l-arginine content in the airway compartment. We hypothesized that such an increase in l-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyperreactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2-/-) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyperreactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyperreactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2-/- mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyperreactivity in all treatment groups. NOS2-/- mice had significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2-/- mice. We conclude that l-arginine metabolism plays an important role in the development of airway hyperreactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be attributable to NOS2, which is a major source of NO production in the inflamed airway, although arginase inhibition may also be affecting the turnover of arginine by the other NOS isoforms, NOS1 and NOS3. The increased l-arginine content in the airway compartment of mice treated with nor-NOHA may directly or indirectly, through NOS2, control arginase expression both in response to OVA exposure and at a basal level.

Recent studies indicate that the biophysical properties of the cellular microenvironment strongly influence a variety of fundamental cell behaviors. The extracellular matrix's (ECM) response to mechanical force, described mathematically as the elastic modulus, is believed to play a particularly critical role in regulatory and pathological cell behaviors. The basement membrane (BM) is a specialization of the ECM that serves as the immediate interface for many cell types (e.g. all epithelial cells) and through which cells are connected to the underlying stroma. Matrigel is a commercially available BM-like complex and serves as an easily accessible experimental simulant of native BMs. However, the local elastic modulus of Matrigel has not been defined under physiological conditions. Here we present the procedures and results of indentation tests performed on Matrigel with atomic force microscopy (AFM) in an aqueous, temperature controlled environment. The average modulus value was found to be approximately 450 Pa. However, this result is considerably higher than macroscopic shear storage moduli reported in the scientific literature. The reason for this discrepancy is believed to result from differences in test methods and the tendency of Matrigel to soften at temperatures below 37 degrees C.

Biophysical cues such as substrate modulus have been shown to influence a variety of cell behaviors. We have determined the elastic modulus of the anterior basement membrane and Descemet's membrane of the human cornea with atomic force microscopy (AFM). A spherical probe was used with a radius approximating that of a typical cell focal adhesion. Values obtained for the elastic modulus of the anterior basement membrane range from 2 to 15 kPa, with a mean of 7.5+/-4.2 kPa. The elastic modulus of Descemet's membrane was found to be slightly higher than those observed for the anterior basement membrane, with a mean of 50+/-17.8 kPa and a range of 20-80 kPa. The topography of Descemet's membrane has been shown to be similar to that of the anterior basement, but with smaller pore sizes resulting in a more tightly packed structure. This structural difference may account for the observed modulus differences. The determination of these values will allow for the design of a better model of the cellular environment as well as aid in the design and fabrication of artificial corneas.

Background: During the last week of June 2008, central and northern California experienced thousands of forest and brush fires, giving rise to a week of severe fire-related particulate air pollution throughout the region. California experienced PM(10-2.5) (particulate matter with mass median aerodynamic diameter > 2.5 mum to < 10 mum; coarse ) and PM(2.5) (particulate matter with mass median aerodynamic diameter < 2.5 mum; fine) concentrations greatly in excess of the air quality standards and among the highest values reported at these stations since data have been collected., Objectives: These observations prompt a number of questions about the health impact of exposure to elevated levels of PM(10-2.5) and PM(2.5) and about the specific toxicity of PM arising from wildfires in this region., Methods: Toxicity of PM(10-2.5) and PM(2.5) obtained during the time of peak concentrations of smoke in the air was determined with a mouse bioassay and compared with PM samples collected under normal conditions from the region during the month of June 2007., Results: Concentrations of PM were not only higher during the wildfire episodes, but the PM was much more toxic to the lung on an equal weight basis than was PM collected from normal ambient air in the region. Toxicity was manifested as increased neutrophils and protein in lung lavage and by histologic indicators of increased cell influx and edema in the lung., Conclusions: We conclude that the wildfire PM contains chemical components toxic to the lung, especially to alveolar macrophages, and they are more toxic to the lung than equal doses of PM collected from ambient air from the same region during a comparable season.

Coarse and fine particulate matter (PM(2.5-10) and PM(2.5), respectively) are regulated ambient air pollutants thought to have major adverse health effects in exposed humans. The role of endotoxin and other bioaerosol components in the toxicity of PM from ambient air is controversial. This study evaluated the inflammatory lung response in mice instilled intratracheally with PM(2.5-10) and PM(2.5) emitted from a working dairy barn, a source presumed to have elevated concentrations of endotoxin. PM(2.5-10) was more pro-inflammatory on an equal weight basis than was PM(2.5); both fractions elicited a predominantly neutrophilic response. The inflammatory response was reversible, with a peak response to PM(2.5-10) observed at 24 h after instillation, and a return to control values by 72 h after instillation. The major active pro-inflammatory component in whole PM(2.5-10), but not in whole PM(2.5), is heat-labile, consistent with it being endotoxin. A heat treatment protocol for the gradual inactivation of biological materials in the PM fractions over a measurable time course was developed and optimized in this study using pure lipopolysaccharide (LPS) as a model system. The time course of heat inactivation of pure LPS and of endotoxin activity in PM(2.5-10) as measured by Limulus bioassay is identical. The active material in both PM(2.5-10) and PM(2.5) remained in the insoluble fraction when the whole PM samples were extracted with physiological saline solution. Histological analysis of lung sections from mice instilled with PM(2.5-10) or PM(2.5) showed evidence of inflammation consistent with the cellular responses observed in lung lavage fluid. The major pro-inflammatory components present in endotoxin-rich PM were found in the insoluble fraction of PM(2.5-10); however, in contrast with PM(2.5-10) isolated from ambient air in the Central Valley of California, the active components in the insoluble fraction were heat-labile.

Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, l-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses - inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration - were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nomega-hydroxy-nor-l-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2 knockout mice exposed to ovalbumin.

Uncontrolled combustion due to garbage recycling is a widespread activity among slum dwellers in distressed economy countries and has been indicated as a major source of dioxin contamination. However, because of the high cost and complexity of gas chromatography/high-resolution mass spectrometry (GC-HRMS) analysis, the magnitude of the problem remains largely unknown. The present study describes a first approach toward the use of a dioxin antibody-based enzyme-linked immunosorbent assay (ELISA) as the basis for a sustainable, simple, and low-cost monitoring program to assess the toxicological impact of uncontrolled combustion in slums. A panel of 16 samples was analyzed by GC-HRMS and ELISA on split extracts. Close to 20% of the analyzed samples showed dioxin concentrations up to almost twice the guidance level for residential soil in several countries, pointing out the need for performing a large-scale monitoring program. Despite the potential for variations in dioxin congener distribution due to the mixed nature of the incinerated material, there was a good correlation between the toxic equivalents as determined by GC-HRMS and ELISA. Furthermore, an interlaboratory ELISA validation showed that the capacity to perform the dioxin ELISA was successfully transferred between laboratories. It was concluded that the ELISA method performed very well as a screening tool to prioritize samples for instrumental analysis, which allows cutting down costs significantly.

Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans(1) (PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content-effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, and had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.

Arginase gene expression in the lung has been linked to asthma both in clinical studies of human patients and in the well-studied mouse model of ovalbumin-induced airway inflammation. Arginase is thought to regulate NO levels in the lung by its ability to divert arginine, the substrate for nitric oxide synthases that produce citrulline and NO, into an alternative metabolic pathway producing ornithine and urea. In the present study arginase I and arginase II concentrations were measured in isolated microdissected airway preparations from sensitized Balb/c mice exposed to ovalbumin aerosol. We found that arginase II was constitutively expressed in the airways of normal mice, whereas arginase I was undetectable in normal airways, while its expression was increased in airways of mice exposed to ovalbumin. The expression of arginase I strongly correlated with the presence of lung inflammation, as quantified by differential cell counts in lung lavage, suggesting that most, or all, of the arginase I in lungs of mice exposed to ovalbumin is present in the inflammatory cells rather than in the airway epithelium. There was also a significant correlation between increased expression of arginase I in the isolated airways and decreased lung compliance. On the other hand, while we found arginase II expression to also be significantly increased in airways from mice exposed to ovalbumin as compared with normal airways, the relative increase was much less than that observed for arginase I, suggesting that there was a smaller contribution of inflammatory cells to the arginase II content of the airways in mice exposed to ovalbumin. There was no apparent correlation between the content of arginase in isolated airways and exhaled NO concentration in the expired air from mice exposed to ovalbumin. However, there was a correlation between exhaled NO concentration from mice exposed to ovalbumin and the lymphocyte content of the lung lavage. The concentration of arginine found in isolated airways from Balb/c mice exposed for 2 weeks to ovalbumin was about half of the value found in isolated microdissected airways from normal mice. Treatment of mice systemically with an arginase inhibitor significantly increased the amount of NO produced, as measured as the amount of nitrite+nitrate (NOx) in lung lavage supernatant prepared from mice exposed to ovalbumin. Our results are consistent with the hypothesis that the response of the lung to ovalbumin challenge includes an adaptive response in the large airways regulating the concentration of arginine within cells of the airway epithelium and subepithelial layer, by shunting of arginine into the metabolic pathway for increased synthesis of NO.

Particulate matter (PM) elicits inflammatory and toxic responses in the lung specific to its constituents, which can vary by region, time, and particle size. To identify the mechanism of toxicity in PM collected in a rural area in the San Joaquin Valley of Central California, we studied coarse particles of 2.5-10 mum diameter (PM(2.5)-PM(10)). Potential pro-inflammatory and toxic effects of PM(2.5)-PM(10) in the lung were investigated using intratracheally instilled mice. We determined total and differential cell profiles and inflammatory chemokines in lung lavage fluid, and biomarkers of toxicity resulting from coarse PM exposure. Responses of the mice were readily observed with total doses of 25-50 mug of PM per mouse. Changes in pro-inflammatory cellular profiles and chemokines showed both dose and time responses; peak responses were observed 24 h after PM instillation, with recovery as early as 48 h. Furthermore, macrophage inflammatory protein (MIP-2) profiles following PM exposures were correlated to levels of measured macrophages and neutrophils recovered from lung lavage fluid of PM-treated animals. Our data suggest that pro-inflammatory effects observed from coarse PM collected during the summer months from California's hot and dry Central Valley are driven largely by the insoluble components of the PM mixture, and are not caused by endotoxin.

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.

Vitellogenin (VTG) and choriogenin (CHO) are valuable biomarkers of endocrine-disrupting compound (EDC) exposure in fish. Existing immunoassays are limited to a few species, which restricts their use for the analysis of local wildlife sentinels. Using C. facetum as a relevant South American model fish, this work presents a new strategy for the preparation of antibodies to VTG and CHO, with zero cross-reactivity with fish serum components. Recombinant fragments of Cichlasoma facetum VTG (280-mer) and CHO (223-mer) were prepared by degenerate primer RT-PCR and expression in E. coli. Polyclonal and monoclonal antibodies prepared with these antigens were used to develop rapid dotblot assays for VTG and CHO. Both the polyclonal and monoclonal antibodies prepared with the recombinant antigens reacted against the native proteins adsorbed on to nitrocellulose allowing the set up of sensitive dotblot assays. The VTG assay was further validated with spiked samples and purified native VTG. Exposure experiments with several estrogenic compounds revealed the potential of C. facetum as a sensitive biomonitor that produced measurable responses at concentrations of 100 ng L(-1) of 17-beta-estradiol, 100 ng L(-1) of ethynylestradiol, and 6.6 microg L(-1) of nonylphenol. The approach described here may be applied to other native species to produce highly specific and sensitive rapid tests. It may be particularly advantageous for species that cannot be kept in captivity or when homogeneous purification of the immunizing proteins is particularly challenging. In conclusion, we present a novel approach to develop a strategy for the generation of immunoassay reagents for vitellogenin (VTG) and choriogenin (CHO), which will facilitate regional studies on the impact of endocrine-disrupting chemicals on local wildlife.

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.