Publications by authors named "Jerold Last"

45 Publications

Role of Dual Oxidases in Ventilator-induced Lung Injury.

Am J Respir Cell Mol Biol 2021 02;64(2):208-215

University of California Lung Center, University of California, Davis, Davis, California.

Positive-pressure ventilation results in ventilator-induced lung injury, and few therapeutic modalities have been successful at limiting the degree of injury to the lungs. Understanding the primary drivers of ventilator-induced lung injury will aid in the development of specific treatments to ameliorate the progression of this syndrome. There are conflicting data for the role of neutrophils in acute respiratory distress syndrome pathogenesis. Here, we specifically examined the importance of neutrophils as a primary driver of ventilator-induced lung injury in a mouse model known to have impaired ability to recruit neutrophils in previous models of inflammation. We exposed and mice to low- or high-tidal volume ventilation with or without positive end-expiratory pressure (PEEP) and recruitment maneuvers for 4 hours. Absolute neutrophils in BAL fluid were significantly reduced in mice compared with mice (6.7 cells/μl; 16.4 cells/μl;  = 0.003), consistent with our hypothesis that neutrophil translocation across the capillary endothelium is reduced in the absence of DUOX1 or DUOX2 in response to ventilator-induced lung injury. Reduced lung neutrophilia was not associated with a reduction in overall lung injury in this study, suggesting that neutrophils do not play an important role in early features of acute lung injury. Surprisingly, mice exhibited significant hypoxemia, as measured by the arterial oxygen tension/fraction of inspired oxygen ratio and arterial oxygen content, which was out of proportion with that seen in the mice (141, 257,  = 0.012). These findings suggest a role for dual oxidases to limit physiologic impairment during early ventilator-induced lung injury.
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http://dx.doi.org/10.1165/rcmb.2020-0197OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874397PMC
February 2021

Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

FASEB J 2019 12 26;33(12):14354-14369. Epub 2019 Oct 26.

Division of Nephrology, Department of Internal Medicine, University of California-Davis, Davis, California, USA.

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 as well as to reduce pulmonary fibrosis in bleomycin-treated mice . 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.
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http://dx.doi.org/10.1096/fj.201901705RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894092PMC
December 2019

Farnesyltransferase Inhibition Exacerbates Eosinophilic Inflammation and Airway Hyperreactivity in Mice with Experimental Asthma: The Complex Roles of Ras GTPase and Farnesylpyrophosphate in Type 2 Allergic Inflammation.

J Immunol 2018 06 27;200(11):3840-3856. Epub 2018 Apr 27.

Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, University of California, Davis, Davis, CA 95817;

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.
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http://dx.doi.org/10.4049/jimmunol.1601317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964018PMC
June 2018

Effects of positive end-expiratory pressure and recruitment maneuvers in a ventilator-induced injury mouse model.

PLoS One 2017 7;12(11):e0187419. Epub 2017 Nov 7.

Center for Comparative Respiratory Biology and Medicine, University of California, Davis, Davis, CA, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187419PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675408PMC
November 2017

Injectable Anesthesia for Mice: Combined Effects of Dexmedetomidine, Tiletamine-Zolazepam, and Butorphanol.

Anesthesiol Res Pract 2017 22;2017:9161040. Epub 2017 Jan 22.

Division of Pulmonary, Critical Care, and Sleep Medicine, School of Medicine, University of California, Davis, CA, USA.

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 ( = 13) and female ( = 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.
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http://dx.doi.org/10.1155/2017/9161040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5292157PMC
January 2017

Lung toxicity in mice of airborne particulate matter from a modern layer hen facility containing Proposition 2-compliant animal caging.

Toxicol Ind Health 2017 Mar 9;33(3):211-221. Epub 2016 Jul 9.

Department of Internal Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, School of Medicine, University of California, Davis, CA, USA.

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.
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http://dx.doi.org/10.1177/0748233716630490DOI Listing
March 2017

Intratracheal instillation of pravastatin for the treatment of murine allergic asthma: a lung-targeted approach to deliver statins.

Physiol Rep 2015 May;3(5)

University of California, Davis, California Department of Internal Medicine, University of California, Davis, California Division of Pulmonary, Critical Care and Sleep Medicine, University of California, Davis, California Center for Comparative Respiratory Biology and Medicine (CCRBM) University of California, Davis, California.

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.
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http://dx.doi.org/10.14814/phy2.12352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4463814PMC
May 2015

Statin use and asthma control in patients with severe asthma.

BMJ Open 2013 Aug 13;3(8). Epub 2013 Aug 13.

Department of Internal Medicine, University of California, Davis School of Medicine, Sacramento, California, USA.

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.
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http://dx.doi.org/10.1136/bmjopen-2013-003314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752054PMC
August 2013

A monoclonal antibody-based copro-ELISA kit for canine echinococcosis to support the PAHO effort for hydatid disease control in South America.

PLoS Negl Trop Dis 2013 10;7(1):e1967. Epub 2013 Jan 10.

Comisión Nacional de Zoonosis, Ministerio de Salud Pública, Montevideo, Uruguay.

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.
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http://dx.doi.org/10.1371/journal.pntd.0001967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542170PMC
June 2013

Limited analytical capacity for cyanotoxins in developing countries may hide serious environmental health problems: simple and affordable methods may be the answer.

J Environ Manage 2013 Jan 4;114:63-71. Epub 2012 Dec 4.

Cátedra de Bioquímica, Departamento de Biociencias, Facultad de Quimíca, Universidad de la Republica, Montevideo, Uruguay.

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.
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http://dx.doi.org/10.1016/j.jenvman.2012.10.052DOI Listing
January 2013

Cell-specific oxidative stress and cytotoxicity after wildfire coarse particulate matter instillation into mouse lung.

Toxicol Appl Pharmacol 2013 Jan 7;266(1):48-55. Epub 2012 Nov 7.

Pulmonary, Critical Care, and Sleep Medicine, School of Medicine, University of California, Davis, CA, USA.

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.
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http://dx.doi.org/10.1016/j.taap.2012.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546532PMC
January 2013

Increasing capacity for environmental engineering in Salta, Argentina.

Am J Ind Med 2013 Jan 29;56(1):11-9. Epub 2012 Mar 29.

Instituto de Investigaciones para la Industria Química, INIQUI-CONICET, UNSa, Salta, Argentina.

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.
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http://dx.doi.org/10.1002/ajim.22044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410034PMC
January 2013

Why is particulate matter produced by wildfires toxic to lung macrophages?

Toxicol Appl Pharmacol 2011 Dec 16;257(2):182-8. Epub 2011 Sep 16.

Center for Comparative Respiratory Biochemistry and Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, School of Medicine, University of California-Davis, 451 Health Sciences Drive, Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.1016/j.taap.2011.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221783PMC
December 2011

Competitive metabolism of L-arginine: arginase as a therapeutic target in asthma.

J Biomed Res 2011 Sep;25(5):299-308

Department of Internal Medicine, Division of Pulmonary and Critical Care and Sleep Medicine, University of California, Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.1016/S1674-8301(11)60041-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596726PMC
September 2011

Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response.

Anal Chem 2011 Sep 29;83(18):7213-20. Epub 2011 Aug 29.

Cátedra de Inmunología, Facultad de Química, Instituto de Higiene, Universidad de la República, Montevideo, Uruguay.

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.
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http://dx.doi.org/10.1021/ac201824zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3193053PMC
September 2011

Simvastatin inhibits goblet cell hyperplasia and lung arginase in a mouse model of allergic asthma: a novel treatment for airway remodeling?

Transl Res 2010 Dec;156(6):335-49

Division of Pulmonary & Critical Care Medicine, Department of Internal Medicine, Center for Comparative Respiratory Biology & Medicine, Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.1016/j.trsl.2010.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2990975PMC
December 2010

Nitric oxide synthase enzymes in the airways of mice exposed to ovalbumin: NOS2 expression is NOS3 dependent.

Mediators Inflamm 2010 5;2010. Epub 2010 Oct 5.

Pulmonary and Critical Care Medicine, School of Medicine, Genome and Biomedical Sciences Facility, University of California, Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.1155/2010/321061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952819PMC
February 2011

Neutral sphingomyelinase 2: a novel target in cigarette smoke-induced apoptosis and lung injury.

Am J Respir Cell Mol Biol 2011 Mar 6;44(3):350-60. Epub 2010 May 6.

Genome and Biomedical Sciences Facility, Division of Pulmonary and Critical Care Medicine, University of California Davis, School of Medicine, USA.

Chronic obstructive pulmonary disease (COPD) is caused by exposure to cigarette smoke (CS). One mechanism of CS-induced lung injury is aberrant generation of ceramide, which leads to elevated apoptosis of epithelial and endothelial cells in the alveolar spaces. Recently, we discovered that CS-induced ceramide generation and apoptosis in pulmonary cells is governed by neutral sphingomyelinase (nSMase) 2. In the current experiments, we expanded our studies to investigate whether nSMase2 governs ceramide generation and apoptosis in vivo using rodent and human models of CS-induced lung injury. We found that exposure of mice or rats to CS leads to colocalizing elevations of ceramide levels and terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling-positive cells in lung tissues. These increases are nSMase2 dependent, and are abrogated by treatment with N-acetyl cysteine or anti-nSMase2 small interfering RNA (siRNA). We further showed that mice that are heterozygous for nSMase2 demonstrate significant decrease in ceramide generation after CS exposure, whereas acidic sphingomyelinase (aSMase) knockout mice maintain wild-type ceramide levels, confirming our previous findings (in human airway epithelial cells) that only nSMase2, and not aSMase, is activated by CS exposure. Lastly, we found that lung tissues from patients with emphysema (smokers) display significantly higher levels of nSMase2 expression compared with lung tissues from healthy control subjects. Taken together, these data establish the central in vivo role of nSMase2 in ceramide generation, aberrant apoptosis, and lung injury under CS exposure, underscoring its promise as a novel target for the prevention of CS-induced airspace destruction.
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http://dx.doi.org/10.1165/rcmb.2009-0422OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095936PMC
March 2011

Lung antioxidant and cytokine responses to coarse and fine particulate matter from the great California wildfires of 2008.

Inhal Toxicol 2010 Jun;22(7):561-70

Departments of Pulmonary and Critical Care Medicine, University of California-Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.3109/08958370903571849DOI Listing
June 2010

A clomazone immunoassay to study the environmental fate of the herbicide in rice (Oryza sativa) agriculture.

J Agric Food Chem 2010 Apr;58(7):4367-71

Catedra de Immunologia, Instituto de Higiene, DEPBIO, Facultad de Quimica, UdelarR, Montevideo, Uruguay.

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.
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http://dx.doi.org/10.1021/jf9043259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878771PMC
April 2010

Arginase inhibition in airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin.

Toxicol Appl Pharmacol 2010 Jan 2;242(1):1-8. Epub 2009 Oct 2.

Department of Pulmonary and Critical Care Medicine, CCRBM, School of Medicine, University of California, Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.1016/j.taap.2009.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221650PMC
January 2010

Simvastatin inhibits airway hyperreactivity: implications for the mevalonate pathway and beyond.

Am J Respir Crit Care Med 2009 Oct 16;180(8):731-40. Epub 2009 Jul 16.

Center for Comparative Respiratory Biology and Medicine, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of California Davis, Davis, California 95616, USA.

Rationale: Statin use has been linked to improved lung health in asthma and chronic obstructive pulmonary disease. We hypothesize that statins inhibit allergic airway inflammation and reduce airway hyperreactivity via a mevalonate-dependent mechanism.

Objectives: To determine whether simvastatin attenuates airway inflammation and improves lung physiology by mevalonate pathway inhibition.

Methods: BALB/c mice were sensitized to ovalbumin over 4 weeks and exposed to 1% ovalbumin aerosol over 2 weeks. Simvastatin (40 mg/kg) or simvastatin plus mevalonate (20 mg/kg) was injected intraperitoneally before each ovalbumin exposure.

Measurements And Main Results: Simvastatin reduced total lung lavage leukocytes, eosinophils, and macrophages (P < 0.05) in the ovalbumin-exposed mice. Cotreatment with mevalonate, in addition to simvastatin, reversed the antiinflammatory effects seen with simvastatin alone (P < 0.05). Lung lavage IL-4, IL-13, and tumor necrosis factor-alpha levels were all reduced by treatment with simvastatin (P < 0.05). Simvastatin treatment before methacholine bronchial challenge increased lung compliance and reduced airway hyperreactivity (P = 0.0001).

Conclusions: Simvastatin attenuates allergic airway inflammation, inhibits key helper T cell type 1 and 2 chemokines, and improves lung physiology in a mouse model of asthma. The mevalonate pathway appears to modulate allergic airway inflammation, while the beneficial effects of simvastatin on lung compliance and airway hyperreactivity may be independent of the mevalonate pathway. Simvastatin and similar agents that modulate the mevalonate pathway may prove to be treatments for inflammatory airway diseases, such as asthma.
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http://dx.doi.org/10.1164/rccm.200901-0018OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2778150PMC
October 2009

California wildfires of 2008: coarse and fine particulate matter toxicity.

Environ Health Perspect 2009 Jun 2;117(6):893-7. Epub 2009 Feb 2.

Department of Pulmonary and Critical Care Medicine, University of California, Davis, California, USA.

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.
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http://dx.doi.org/10.1289/ehp.0800166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2702402PMC
June 2009

Mouse lung inflammation after instillation of particulate matter collected from a working dairy barn.

Toxicol Appl Pharmacol 2009 May 9;236(3):348-57. Epub 2009 Mar 9.

Pulmonary and Critical Care Medicine, University of California, Davis, Genome and Biomedical Sciences Facility, Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.1016/j.taap.2009.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680696PMC
May 2009

Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin.

Toxicol Appl Pharmacol 2009 Feb 5;234(3):273-80. Epub 2008 Nov 5.

Department of Pulmonary and Critical Care Medicine, CCRBM, School of Medicine, University of California, Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.1016/j.taap.2008.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2666129PMC
February 2009

Enzyme-linked immunosorbent assay for screening dioxin soil contamination by uncontrolled combustion during informal recycling in slums.

Environ Toxicol Chem 2008 Nov;27(11):2224-32

Cátedra de Bioquímica, Department of Biosciences, Faculty of Chemistry, University de la República, Montevideo, Uruguay.

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.
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http://dx.doi.org/10.1897/07-660.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2858572PMC
November 2008

Arginases I and II in lungs of ovalbumin-sensitized mice exposed to ovalbumin: sources and consequences.

Toxicol Appl Pharmacol 2008 Aug 15;230(3):269-75. Epub 2008 Mar 15.

Pulmonary and Critical Care Medicine, CCRBM, 6519 GBSF, School of Medicine, University of California, Davis, CA 95616-8685, USA.

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.
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http://dx.doi.org/10.1016/j.taap.2008.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2578817PMC
August 2008

Lung response to coarse PM: bioassay in mice.

Toxicol Appl Pharmacol 2008 Jul 26;230(2):159-66. Epub 2008 Feb 26.

Pulmonary and Critical Care Medicine Division, School of Medicine, University of California, 6519 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616, USA.

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.
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http://dx.doi.org/10.1016/j.taap.2008.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2517986PMC
July 2008

Non-enzymatic glycation of type I collagen diminishes collagen-proteoglycan binding and weakens cell adhesion.

J Cell Biochem 2008 Aug;104(5):1684-98

Department of Medicine, Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5099, USA.

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.
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http://dx.doi.org/10.1002/jcb.21735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746388PMC
August 2008

Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR.

Anal Bioanal Chem 2007 Dec 6;389(7-8):2195-202. Epub 2007 Oct 6.

Facultad de Química, Instituto de Higiene, Universidad de la República, Av. A. Navarro 3051, piso 2, Montevideo, 11600, Uruguay.

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.
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http://dx.doi.org/10.1007/s00216-007-1630-3DOI Listing
December 2007
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