Deep-Masker: A Deep Learning-based Tool to Assess Chord Length from Murine Lung Images.

Chord length is an indirect measure of alveolar size and a critical endpoint in animal models of chronic obstructive pulmonary disease (COPD). In assessing chord length, the lumens of nonalveolar structures are eliminated from measurement by various methods, including manual masking. However, manual masking is resource intensive and can introduce variability and bias. We created a fully automated deep learning-based tool to mask murine lung images and assess chord length to facilitate mechanistic and therapeutic discovery in COPD called Deep-Masker (available at http://47.93.0.75:8110/login). We trained the deep learning algorithm for Deep-Masker using 1,217 images from 137 mice from 12 strains exposed to room air or cigarette smoke for 6 months. We validated this algorithm against manual masking. Deep-Masker demonstrated high accuracy with an average difference in chord length compared with manual masking of -0.3 ± 1.4% (rs = 0.99) for room-air-exposed mice and 0.7 ± 1.9% (rs = 0.99) for cigarette-smoke-exposed mice. The difference between Deep-Masker and manually masked images for change in chord length because of cigarette smoke exposure was 6.0 ± 9.2% (rs = 0.95). These values exceed published estimates for interobserver variability for manual masking (rs = 0.65) and the accuracy of published algorithms by a significant margin. We validated the performance of Deep-Masker using an independent set of images. Deep-Masker can be an accurate, precise, fully automated method to standardize chord length measurement in murine models of lung disease.

ADAMTS5 Deficiency Protects Mice From Chronic Tobacco Smoking-induced Intervertebral Disc Degeneration.

STUDY DESIGN: ADAMTS5-deficient and wild type (WT) mice were chronically exposed to tobacco smoke to investigate effects on intervertebral disc degeneration (IDD). OBJECTIVE: The aim of this study was to demonstrate a role for ADAMTS5 in mediating tobacco smoking-induced IDD. SUMMARY OF BACKGROUND DATA: We previously demonstrated that chronic tobacco smoking causes IDD in mice because, in part, of proteolytic destruction of disc aggrecan. However, it was unknown which matrix proteinase(s) drive these detrimental effects. METHODS: Three-month-old WT (C57BL/6) and ADAMTS5 mice were chronically exposed to tobacco smoke (four cigarettes/day, 5 day/week for 6 months). ADAMTS-mediated cleavage of disc aggrecan was analyzed by Western blot. Disc total glycosaminoglycan (GAG) content was assessed by dimethyl methylene blue assay and safranin O/fast green histology. Vertebral osteoporosity was measured by microcomputed tomography. Human nucleus pulposus (hNP) cell cultures were also exposed directly to tobacco smoke extract (TSE), a condensate containing the water-soluble compounds inhaled by smokers, to measure ADAMTS5 expression and ADAMTS-mediated cleavage of aggrecan. Activation of nuclear factor (NF)-κB, a family of transcription factors essential for modulating the cellular response to stress, was measured by immunofluorescence assay. RESULTS: Genetic depletion of ADAMTS5 prevented vertebral bone loss, substantially reduced loss of disc GAG content, and completely obviated ADAMTS-mediated proteolysis of disc aggrecan within its interglobular domain (IGD) in mice following exposure to tobacco smoke. hNP cell cultures exposed to TSE also resulted in upregulation of ADAMTS5 protein expression and a concomitant increase in ADAMTS-mediated cleavage within aggrecan IGD. Activation of NF-κB, known to be required for ADAMTS5 gene expression, was observed in both TSE-treated hNP cell cultures and disc tissue of tobacco smoke-exposed mice. CONCLUSION: The findings demonstrate that ADAMTS5 is the primary aggrecanase mediating smoking-induced disc aggrecanolysis and IDD. Mouse models of chronic tobacco smoking are important and useful for probing the mechanisms of disc aggrecan catabolism and IDD. LEVEL OF EVIDENCE: N/A.

Variable Susceptibility to Cigarette Smoke-Induced Emphysema in 34 Inbred Strains of Mice Implicates Abi3bp in Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is caused by a complex interaction of environmental exposures, most commonly cigarette smoke, and genetic factors. Chronic cigarette smoke exposure in the mouse is a commonly used animal model of COPD. We aimed to expand our knowledge about the variable susceptibility of inbred strains to this model and test for genetic variants associated with this trait. To that end, we sought to measure differential susceptibility to cigarette smoke-induced emphysema in the mouse, identify genetic loci associated with this quantitative trait, and find homologous human genes associated with COPD. Alveolar chord length (CL) in 34 inbred strains of mice was measured after 6 months of exposure to cigarette smoke. After testing for association, we connected a murine candidate locus to a published meta-analysis of moderate-to-severe COPD. We identified deleterious mutations in a candidate gene in silico and measured gene expression in extreme strains. A/J was the most susceptible strain in our survey (Δ CL 7.0 ± 2.2 µm) and CBA/J was the least susceptible (Δ CL -0.3 ± 1.2 µm). By integrating mouse and human genome-wide scans, we identified the candidate gene Abi3bp. CBA/J mice harbor predicted deleterious variants in Abi3bp, and expression of the gene differs significantly between CBA/J and A/J mice. This is the first report of susceptibility to cigarette smoke-induced emphysema in 34 inbred strains of mice, and Abi3bp is identified as a potential contributor to this phenotype.

Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain.

RATIONALE: Macrophage elastase (matrix metalloproteinase [MMP]-12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C-terminal domain (CTD). OBJECTIVES: To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer. METHODS: We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non-small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and KrasLSL/G12D mouse models of lung cancer. MEASUREMENTS AND MAIN RESULTS: We show that SR20 triggers tumor apoptosis by up-regulation of gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL-mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer. CONCLUSIONS: The MMP12 CTD initiates TRAIL-mediated tumor cell death through its conserved SR20 peptide.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor.

Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high-fat diet (HFD) in some inbred mouse strains. We conducted a genome-wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD-induced PH. Mice from 36 inbred and wild-derived strains were fed with regular diet or HFD for 20 weeks beginning at 6-12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end-systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest-path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH-related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high-fat feeding. Network-based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest-path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high-fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network-based approach adds evidence for a role for Egfr in murine PH.

Automated Measurement of Blood Vessels in Tissues from Microscopy Images.

The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual measurements of discrete wall regions along the vessel periphery have become common practice for wall thickness quantification. The aim of this study is to introduce, validate, and facilitate the use of an improved method for medial wall thickness quantification. We describe a novel method of wall thickness calculation based on image skeletonization and compare its results to those of common techniques. Using both theoretical and empirical approaches, we demonstrate the accuracy and superiority of the skeleton-based method for measuring wall thickness while discussing its interpretation and limitations. Finally, we present a new freely available software tool, the VMI Calculator, to facilitate wall thickness measurements using our novel method. © 2016 by John Wiley & Sons, Inc.

Enhancing Autophagy with Drugs or Lung-directed Gene Therapy Reverses the Pathological Effects of Respiratory Epithelial Cell Proteinopathy.

Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy.

The receptor for advanced glycation end products (RAGE) contributes to the progression of emphysema in mice.

Several recent clinical studies have implied a role for the receptor for advanced glycation end products (RAGE) and its variants in chronic obstructive pulmonary disease (COPD). In this study we have defined a role for RAGE in the pathogenesis of emphysema in mice. RAGE deficient mice (RAGE-/-) exposed to chronic cigarette smoke were significantly protected from smoke induced emphysema as determined by airspace enlargement and had no significant reduction in lung tissue elastance when compared to their air exposed controls contrary to their wild type littermates. The progression of emphysema has been largely attributed to an increased inflammatory cell-mediated elastolysis. Acute cigarette smoke exposure in RAGE-/- mice revealed an impaired early recruitment of neutrophils, approximately a 6-fold decrease compared to wild type mice. Hence, impaired neutrophil recruitment with continued cigarette smoke exposure reduces elastolysis and consequent emphysema.

Macrophage elastase suppresses white adipose tissue expansion with cigarette smoking.

Macrophage elastase (MMP12) is a key mediator of cigarette smoke (CS)-induced emphysema, yet its role in other smoking related pathologies remains unclear. The weight suppressing effects of smoking are a major hindrance to cessation efforts, and MMP12 is known to suppress the vascularization on which adipose tissue growth depends by catalyzing the formation of antiangiogenic peptides endostatin and angiostatin. The goal of this study was to determine the role of MMP12 in adipose tissue growth and smoking-related suppression of weight gain. Whole body weights and white adipose depots from wild-type and Mmp12-deficient mice were collected during early postnatal development and after chronic CS exposure. Adipose tissue specimens were analyzed for angiogenic and adipocytic markers and for content of the antiangiogenic peptides endostatin and angiostatin. Cultured 3T3-L1 adipocytes were treated with adipose tissue homogenate to examine its effects on vascular endothelial growth factor (VEGF) expression and secretion. MMP12 content and activity were increased in the adipose tissue of wild-type mice at 2 weeks of age, leading to elevated endostatin production, inhibition of VEGF secretion, and decreased adipose tissue vascularity. By 8 weeks of age, adipose MMP12 levels subsided, and the protein was no longer detectable. However, chronic CS exposure led to macrophage accumulation and restored adipose MMP12 activity, thereby suppressing adipose tissue mass and vascularity. Our results reveal a novel systemic role for MMP12 in postnatal adipose tissue expansion and smoking-associated weight loss by suppressing vascularity within the white adipose tissue depots.

Investigating the role of DNA damage in tobacco smoking-induced spine degeneration.

BACKGROUND CONTEXT: Tobacco smoking is a key risk factor for spine degeneration. However, the underlying mechanism by which smoking induces degeneration is not known. Recent studies implicate DNA damage as a cause of spine and intervertebral disc degeneration. Because tobacco smoke contains many genotoxins, we hypothesized that tobacco smoking promotes spine degeneration by inducing cellular DNA damage. PURPOSE: To determine if DNA damage plays a causal role in smoking-induced spine degeneration. STUDY DESIGN: To compare the effect of chronic tobacco smoke inhalation on intervertebral disc and vertebral bone in normal and DNA repair-deficient mice to determine the contribution of DNA damage to degenerative changes. METHODS: Two-month-old wild-type (C57BL/6) and DNA repair-deficient Ercc1(-/Δ) mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 7 weeks) to model first-hand smoking in humans. Total disc proteoglycan (PG) content (1,9-dimethylmethylene blue assay), PG synthesis ((35)S-sulfate incorporation assay), aggrecan proteolysis (immunoblotting analysis), and vertebral bone morphology (microcomputed tomography) were measured. RESULTS: Exposure of wild-type mice to tobacco smoke led to a 19% increase in vertebral porosity and a 61% decrease in trabecular bone volume. Intervertebral discs of smoke-exposed animals also showed a 2.6-fold decrease in GAG content and an 8.1-fold decrease in new PG synthesis. These smoking-induced degenerative changes were similar but not worse in Ercc1(-/Δ) mice. CONCLUSIONS: Short-term exposure to high levels of primary tobacco smoke inhalation promotes degeneration of vertebral bone and discs. Disc degeneration is primarily driven by reduced synthesis of proteoglycans needed for vertebral cushioning. Degeneration was not exacerbated in congenic DNA repair-deficient mice, indicating that DNA damage per se does not have a significant causal role in driving smoke-induced spine degeneration.

Integration of mouse and human genome-wide association data identifies KCNIP4 as an asthma gene.

Asthma is a common chronic respiratory disease characterized by airway hyperresponsiveness (AHR). The genetics of asthma have been widely studied in mouse and human, and homologous genomic regions have been associated with mouse AHR and human asthma-related phenotypes. Our goal was to identify asthma-related genes by integrating AHR associations in mouse with human genome-wide association study (GWAS) data. We used Efficient Mixed Model Association (EMMA) analysis to conduct a GWAS of baseline AHR measures from males and females of 31 mouse strains. Genes near or containing SNPs with EMMA p-values <0.001 were selected for further study in human GWAS. The results of the previously reported EVE consortium asthma GWAS meta-analysis consisting of 12,958 diverse North American subjects from 9 study centers were used to select a subset of homologous genes with evidence of association with asthma in humans. Following validation attempts in three human asthma GWAS (i.e., Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG) and two human AHR GWAS (i.e., SHARP, DAG), the Kv channel interacting protein 4 (KCNIP4) gene was identified as nominally associated with both asthma and AHR at a gene- and SNP-level. In EVE, the smallest KCNIP4 association was at rs6833065 (P-value 2.9e-04), while the strongest associations for Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG were 1.5e-03, 1.0e-03, 3.1e-03 at rs7664617, rs4697177, rs4696975, respectively. At a SNP level, the strongest association across all asthma GWAS was at rs4697177 (P-value 1.1e-04). The smallest P-values for association with AHR were 2.3e-03 at rs11947661 in SHARP and 2.1e-03 at rs402802 in DAG. Functional studies are required to validate the potential involvement of KCNIP4 in modulating asthma susceptibility and/or AHR. Our results suggest that a useful approach to identify genes associated with human asthma is to leverage mouse AHR association data.

Emerging genetics of COPD.

Since the discovery of alpha-1 antitrypsin in the early 1960s, several new genes have been suggested to play a role in chronic obstructive pulmonary disease (COPD) pathogenesis. Yet, in spite of those advances, much about the genetic basis of COPD still remains to be discovered. Unbiased approaches, such as genome-wide association (GWA) studies, are critical to identify genes and pathways and to verify suggested genetic variants. Indeed, most of our current understanding about COPD candidate genes originates from GWA studies. Experiments in form of cross-study replications and advanced meta-analyses have propelled the field towards unravelling details about COPD's pathogenesis. Here, we review the discovery of genetic variants in association with COPD phenotypes by discussing the available approaches and current findings. Limitations of current studies are considered and future directions provided.

CX3CR1+ lung mononuclear phagocytes spatially confined to the interstitium produce TNF-α and IL-6 and promote cigarette smoke-induced emphysema.

Increased numbers of macrophages are found in the lungs of smokers and those with chronic obstructive pulmonary disease. Experimental evidence shows the central role of macrophages in elaboration of inflammatory mediators such as TNF-α and the progression toward cigarette smoke-induced emphysema. We investigated the role of CX3CR1 in recruitment of mononuclear phagocytes, inflammatory cytokine responses, and tissue destruction in the lungs after cigarette smoke exposure. Using mice in which egfp is expressed at the locus of the cx3cr1 gene, we show that alveolar macrophages increased transmembrane ligand CX3CL1 expression and soluble CX3CL1 was detectable in the airspaces, but cx3cr1(GFP/GFP) and cx3cr1(GFP/+) mice failed to show recruitment of CX3CR1(+) cells into the airspaces with cigarette smoke. In contrast, cigarette smoke increased the accumulation of CX3CR1(+)CD11b(+) mononuclear phagocytes that were spatially confined to the lung interstitium and heterogenous in their expression of CD11c, MHC class II, and autofluorescent property. Although an intact CX3CL1-CX3CR1 pathway amplified the percentage of CX3CR1(+)CD11b(+) mononuclear phagocytes in the lungs, it was not essential for recruitment. Rather, functional CX3CR1 was required for a subset of tissue-bound mononuclear phagocytes to produce TNF-α and IL-6 in response to cigarette smoke, and the absence of functional CX3CR1 protected mice from developing tissue-destructive emphysema. Thus, CX3CR1(+) "tissue resident" mononuclear phagocytes initiate an innate immune response to cigarette smoke by producing TNF-α and IL-6 and are capable of promoting emphysema.

Comparison of unrestrained plethysmography and forced oscillation for identifying genetic variability of airway responsiveness in inbred mice.

Lung function detection in mice is currently most accurately measured by invasive techniques, which are costly, labor intensive, and terminal. This limits their use for large-scale or longitudinal studies. Noninvasive assays are often used instead, but their accuracy for measuring lung function parameters such as resistance and elastance has been questioned in studies involving small numbers of mouse strains. Here we compared parameters detected by two different methods using 29 inbred mouse strains: enhanced pause (Penh), detected by unrestrained plethysmography, and central airway resistance and lung elastance, detected by a forced oscillation technique. We further tested whether the phenotypic variations were determined by the same genomic location in genome-wide association studies using a linear mixed model algorithm. Penh, resistance, and elastance were measured in nonexposed mice or mice exposed to saline and increasing doses of aerosolized methacholine. Because Penh differed from airway resistance in several strains and because the peak genetic associations found for Penh, resistance, or elastance were located at different genomic regions, we conclude that using Penh as an indicator for lung function changes in high-throughput genetic studies (i.e., genome-wide association studies or quantitative trait locus studies) measures something fundamentally different than airway resistance and lung elastance.

The role of the receptor for advanced glycation end-products in a murine model of silicosis.

BACKGROUND: The role of the receptor for advanced glycation end-products (RAGE) has been shown to differ in two different mouse models of asbestos and bleomycin induced pulmonary fibrosis. RAGE knockout (KO) mice get worse fibrosis when challenged with asbestos, whereas in the bleomycin model they are largely protected against fibrosis. In the current study the role of RAGE in a mouse model of silica induced pulmonary fibrosis was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Wild type (WT) and RAGE KO mice received a single intratracheal (i.t.) instillation of silica in saline or saline alone as vehicle control. Fourteen days after treatment mice were subjected to a lung mechanistic study and the lungs were lavaged and inflammatory cells, protein and TGF-beta levels in lavage fluid determined. Lungs were subsequently either fixed for histology or excised for biochemical assessment of fibrosis and determination of RAGE protein- and mRNA levels. There was no difference in the inflammatory response or degree of fibrosis (hydroxyproline levels) in the lungs between WT and RAGE KO mice after silica injury. However, histologically the fibrotic lesions in the RAGE KO mice had a more diffuse alveolar septal fibrosis compared to the nodular fibrosis in WT mice. Furthermore, RAGE KO mice had a significantly higher histologic score, a measure of affected areas of the lung, compared to WT silica treated mice. A lung mechanistic study revealed a significant decrease in lung function after silica compared to control, but no difference between WT and RAGE KO. While a dose response study showed similar degrees of fibrosis after silica treatment in the two strains, the RAGE KO mice had some differences in the inflammatory response compared to WT mice. CONCLUSIONS/SIGNIFICANCE: Aside from the difference in the fibrotic pattern, these studies showed no indicators of RAGE having an effect on the severity of pulmonary fibrosis following silica injury.

A survey of airway responsiveness in 36 inbred mouse strains facilitates gene mapping studies and identification of quantitative trait loci.

Airway hyper-responsiveness (AHR) is a critical phenotype of human asthma and animal models of asthma. Other studies have measured AHR in nine mouse strains, but only six strains have been used to identify genetic loci underlying AHR. Our goals were to increase the genetic diversity of available strains by surveying 27 additional strains, to apply haplotype association mapping to the 36-strain survey, and to identify new genetic determinants for AHR. We derived AHR from the increase in airway resistance in females subjected to increasing levels of methacholine concentrations. We used haplotype association mapping to identify associations between AHR and haplotypes on chromosomes 3, 5, 8, 12, 13, and 14. And we used bioinformatics techniques to narrow the identified region on chromosome 13, reducing the region to 29 candidate genes, with 11 of considerable interest. Our combined use of haplotype association mapping with bioinformatics tools is the first study of its kind for AHR on these 36 strains of mice. Our analyses have narrowed the possible QTL genes and will facilitate the discovery of novel genes that regulate AHR in mice.

Duration of allergic susceptibility in maternal transmission of asthma risk.

PROBLEM: Asthma has its origins in early-life. Maternal, not paternal asthma is an important risk factor, but the mechanisms and the duration of the maternal effect are unknown. METHOD OF STUDY: Offspring of asthmatic and normal mice were sensitized with ovalbumin (OVA) at 1, 3, 6 and 10 weeks of age, challenged with aerosolized OVA 2 weeks later and tested for airway hyperresponsiveness (AHR) and allergic airway inflammation (AI). RESULTS: Offspring of asthmatic, but not normal, mothers showed AHR and AI after OVA sensitization at week 1. Similarly increased susceptibility to OVA was observed when sensitized at 3 or 6 weeks, although the magnitude diminished. Offspring sensitized at 10 and challenged at 12 weeks showed some AI but no AHR. CONCLUSION: In offspring from asthmatic mothers increased allergic susceptibility persisted through the young adulthood, albeit with gradual decline, which suggests a long-lived but reversible skewing of the developing immune system.

Effects of positive end-expiratory pressure in an experimental model of acute myocardial infarct in wistar rats.

UNLABELLED: Our purpose in this study was to access the pulmonary effects of mechanical ventilation with positive end-expiratory pressure (PEEP; 10 cmH2O) or without PEEP (zero PEEP-ZEEP) in a rat model of acute myocardial infarction that resulted in hypotension but not in pulmonary congestion. METHODS: Wistar rats were anesthetized (1.5% isoflurane) and myocardial infarct was induced by ligature of the anterior interventricular coronary artery. Rats with myocardial infarct were compared with sham-operated (Sham) and closed thorax groups. RESULTS AND CONCLUSION: There was a significant decrease in MAP in the acute myocardial infarct group (92.5 +/- 4.2 mmHg) when compared with closed chest group (113.0 +/- 4.4 mmHg). There was no significant difference between acute myocardial infarct and Sham groups in PEEP or ZEEP. Mechanical ventilation for 120 min resulted in a significant increase in respiratory system elastance in the groups ventilated with ZEEP (2.59 +/- 0.17 and 2.32 +/- 0.17 cmH2O.mL, Sham and acute myocardial infarct groups, respectively). This effect of mechanical ventilation was not observed in the presence of PEEP in both groups. There was no significant increase in the amount of perivascular pulmonary edema measured in all groups studied. Mean airspace linear intercept and lung tissue distortion index also did not show statistically significant difference between Sham and acute myocardial infarct groups. We conclude that in this experimental model of acute myocardial infarct (12.4 +/- 4.1% area of necrotic tissue and 26.4 +/- 4.0% area of ischemic tissue), there was a protective pulmonary effect of PEEP.

Effects of nitric oxide synthases in chronic allergic airway inflammation and remodeling.

The precise role of each nitric oxide (NO) synthase (NOS) isoform in the pathobiology of asthma is not well established. Our objective was to investigate the contribution of constitutive NO synthase (cNOS) and inducible NOS (iNOS) isoforms to lung mechanics and inflammatory and remodeling responses in an experimental model of chronic allergic pulmonary inflammation. Guinea pigs were submitted to seven ovalbumin exposures with increasing doses (1 approximately 5 mg/ml) for 4 wk. The animals received either chronic L-NAME (N-nitro-L-arginine methyl ester, in drinking water) or 1,400 W (iNOS-specific inhibitor, intraperitoneal) treatments. At 72 h after the seventh inhalation of ovalbumin solution, animals were anesthetized, mechanically ventilated, exhaled NO was collected, and lung mechanical responses were evaluated before and after antigen challenge. Both L-NAME and 1,400 W treatments increased baseline resistance and decreased elastance of the respiratory system in nonsensitized animals. After challenge, L-NAME increased resistance of the respiratory system and collagen deposition on airways, and decreased peribronchial edema and mononuclear cell recruitment. Administration of 1,400 W reduced resistance of the respiratory system response, eosinophilic and mononuclear cell recruitment, and collagen and elastic fibers content in airways. L-NAME treatment reduced both iNOS- and neuronal NOS-positive eosinophils, and 1,400 W diminished only the number of eosinophils expressing iNOS. In this experimental model, inhibition of NOS-derived NO by L-NAME treatment amplifies bronchoconstriction and increases collagen deposition. However, blockage of only iNOS attenuates bronchoconstriction and inflammatory and remodeling processes.

Role of breast milk in a mouse model of maternal transmission of asthma susceptibility.

Epidemiologic data suggest a link between nursing by asthmatic mothers and increased risk of allergy in babies. We sought to experimentally test the potential contribution of breast milk mediator(s) in a mouse model of maternal transmission of asthma risk by evaluating the effect of adoptive nursing on asthma susceptibility in the offspring. We measured airway hyperresponsiveness (AHR) and allergic airway inflammation (AI) after an intentionally suboptimal OVA Ag sensitization, tested the allergen independence of the maternal effect by using a second allergen, casein, for sensitization of the baby mice, and tested the potential role of cytokines by measuring their levels in breast milk. Offspring of asthmatic, but not normal, mothers showed AHR and AI, indicating a maternal transfer of asthma risk. After adoptive nursing, both groups (litters born to asthmatic mothers and nursed by normal mothers, and normal babies nursed by asthmatic mothers) showed AHR (enhanced pause after methacholine aerosol, 50 mg/ml, 3.7 +/- 0.7, 4.2 +/- 0.5, respectively, vs 1.1 +/- 0.1 normal controls, n = 25, p < 0.01) and AI, seen as eosinophilia on histology and bronchoalveolar lavage (40.7 +/- 4.5%, 28.7 +/- 3.7%, vs 1.0 +/- 0.5% normals, n = 25, p < 0.01) after OVA sensitization. Similar results using casein allergen were observed. Multiplex assays for cytokines (IFN-gamma, IL-2, IL-4, IL-5, TNF-alpha, and IL-13) in breast milk were negative. Breast milk is sufficient, but not necessary, to mediate allergen-independent maternal transmission of asthma risk to offspring.