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1.  (Too) Great Expectations 
doi:10.1164/rccm.200903-0456ED
PMCID: PMC4263324  PMID: 19498061
2.  Dissection of Regenerating T-Cell Responses against Tuberculosis in HIV-infected Adults Sensitized by Mycobacterium tuberculosis 
Rationale
Combination antiretroviral treatment (cART) reduces the risk of tuberculosis in HIV-infected people. Therefore a novel approach to gain insight into protection against tuberculosis is to analyze the T cells that expand in people sensitized by Mycobacterium tuberculosis (MTB) during cART.
Objectives
To longitudinally analyze CD4 T-cell subsets during the first year of cART, from the time of starting cART (Day 0), in 19 HIV-infected, MTB-sensitized adults.
Methods
Peripheral blood mononuclear cells were obtained on Day 0, Weeks 2, 4, 12, 24, 36, and 48 of cART and were stimulated with purified protein derivative (PPD) followed by flow cytometry to analyze surface markers and intracellular cytokines.
Measurements and Main Results
CD4+ T cells significantly increased during follow-up and the viral load fell to undetectable levels in each patient, indicating successful immune restoration. Central memory CD27+CD45RA− and CD27+CCR5− CD4+ cells expanded by 12 weeks (P < 0.02) followed by naive CD27+CD45RA+ cells at 36 weeks (P = 0.02). Terminally differentiated effector CD4+CD27−CCR7− cells decreased by 12 weeks (P = 0.02), paralleled by a proportional decline of PPD-specific CD4+IFN-γ+ cells (P = 0.02). However, the absolute numbers of PPD-specific IFN-γ–producing cells, determined by enzyme-linked immunospot assay, increased (P = 0.02).
Conclusions
Rapid effector responses are often measured when evaluating immunity. We show that although cART is associated with an absolute increase in effector function, the proportional response decreased and the strongest correlate of increased cART-mediated immunity in this study was the central memory response.
doi:10.1164/rccm.200904-0568OC
PMCID: PMC4176738  PMID: 19628776
bacterial infections; RNA viruses; drug therapy
3.  C5a Mediates Peripheral Blood Neutrophil Dysfunction in Critically Ill Patients 
Rationale
Critically ill patients are highly susceptible to hospital-acquired infection. Neutrophil function in critical illness remains poorly understood.
Objectives
To characterize and define mechanisms of peripheral blood neutrophil (PBN) dysfunction in critically ill patients. To determine whether the inflamed lung contributes additional phagocytic impairment.
Methods
Prospective collection of blood and bronchoalveolar lavage fluid from patients with suspected ventilator-associated pneumonia and from age- and sex-matched volunteers; laboratory analysis of neutrophil functions.
Measurements and Main Results
Seventy-two patients and 21 volunteers were included. Phagocytic capacity of PBNs was 36% lower in patients than in volunteers (P < 0.0001). From several biologically plausible candidates only activated complement was significantly associated with impaired PBN phagocytosis (P < 0.0001). Phagocytosis was negatively correlated with serum C3a and positively correlated with expression of C5a receptor type 1 (CD88) on PBNs. C5a recapitulated impaired PBN phagocytosis and significantly down-regulated CD88 expression in vitro. C5a-mediated phagocytic impairment was prevented by blocking either CD88 or phosphoinositide 3-kinase, and completely reversed by granulocyte-macrophage colony-stimulating factor. C5a also impaired killing of Pseudomonas aeruginosa by, and migration of, PBNs, indicating that effects were not restricted to phagocytosis. Bronchoalveolar lavage fluid leukocytes from patients also demonstrated significantly impaired function, and lavage supernatant reduced phagocytosis in healthy neutrophils by 43% (P = 0.0001). However, lavage fluid did not affect CD88 expression and lavage-mediated impairment of phagocytosis was not blocked by anti-CD88 antibody.
Conclusions
Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs in the inflamed lung.
doi:10.1164/rccm.200812-1928OC
PMCID: PMC2948533  PMID: 19324972
complement; natural immunity; intensive care; phagocytosis
4.  TLR4 Is Necessary for Hyaluronan-mediated Airway Hyperresponsiveness after Ozone Inhalation 
Rationale: Ozone is a common environmental air pollutant that contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced airway hyperresponsiveness, remain poorly understood. We have previously reported that toll-like receptor 4 (TLR4)–deficient animals are protected against ozone-induced airway hyperresponsiveness (AHR) and that hyaluronan (HA) mediates ozone-induced AHR. However, the relation between TLR4 and hyaluronan in the airway response to ozone remains unexplored.
Objectives: We hypothesized that HA acts as an endogenous TLR4 ligand for the development of AHR after ozone-induced environmental airway injury.
Methods: TLR4-deficient and wild-type C57BL/6 mice were exposed to either inhaled ozone or intratracheal HA and the inflammatory and AHR response was measured.
Measurements and Main Results: TLR4-deficient mice have similar levels of cellular inflammation, lung injury, and soluble HA levels as those of C57BL/6 mice after inhaled ozone exposure. However, TLR4-deficient mice are partially protected from AHR after ozone exposure as well as after direct intratracheal instillation of endotoxin-free low molecular weight HA. Similar patterns of TLR4-dependent cytokines were observed in the bronchial alveolar lavage fluid after exposure to either ozone or HA. Exposure to ozone increased immunohistological staining of TLR4 on lung macrophages. Furthermore, in vitro HA exposure of bone marrow–derived macrophages induced NF-κB and production of a similar pattern of proinflammatory cytokines in a manner dependent on TLR4.
Conclusions: Our observations support the observation that extracellular matrix HA contributes to ozone-induced airways disease. Furthermore, our results support that TLR4 contributes to the biological response to HA by mediating both the production of proinflammatory cytokines and the development of ozone-induced AHR.
doi:10.1164/rccm.200903-0381OC
PMCID: PMC2868499  PMID: 20007931
environmental airways injury; asthma; toll-like receptor; macrophage; TNF-α
5.  Shared Treatment Decision Making Improves Adherence and Outcomes in Poorly Controlled Asthma 
Rationale: Poor adherence to asthma controller medications results in poor treatment outcomes.
Objectives: To compare controller medication adherence and clinical outcomes in 612 adults with poorly controlled asthma randomized to one of two different treatment decision-making models or to usual care.
Methods: In shared decision making (SDM), nonphysician clinicians and patients negotiated a treatment regimen that accommodated patient goals and preferences. In clinician decision making, treatment was prescribed without specifically eliciting patient goals/preferences. The otherwise identical intervention protocols both provided asthma education and involved two in-person and three brief phone encounters.
Measurements and Main Results: Refill adherence was measured using continuous medication acquisition (CMA) indices—the total days' supply acquired per year divided by 365 days. Cumulative controller medication dose was measured in beclomethasone canister equivalents. In follow-up Year 1, compared with usual care, SDM resulted in: significantly better controller adherence (CMA, 0.67 vs. 0.46; P < 0.0001) and long-acting β-agonist adherence (CMA, 0.51 vs. 0.40; P = 0.0225); higher cumulative controller medication dose (canister equivalent, 10.9 vs. 5.2; P < 0.0001); significantly better clinical outcomes (asthma-related quality of life, health care use, rescue medication use, asthma control, and lung function). In Year 2, compared with usual care, SDM resulted in significantly lower rescue medication use, the sole clinical outcome available for that year. Compared with clinician decision making, SDM resulted in: significantly better controller adherence (CMA, 0.67 vs. 0.59; P = 0.03) and long-acting β-agonist adherence (CMA, 0.51 vs. 0.41; P = 0.0143); higher cumulative controller dose (CMA, 10.9 vs. 9.1; P = 0.005); and quantitatively, but not significantly, better outcomes on all clinical measures.
Conclusions: Negotiating patients' treatment decisions significantly improves adherence to asthma pharmacotherapy and clinical outcomes.
Clinical trials registered with www.clinicaltrials.gov (NCT00217945 and NCT00149526).
doi:10.1164/rccm.200906-0907OC
PMCID: PMC2841026  PMID: 20019345
randomized controlled trial; asthma control; patient–clinician communication
6.  Latent Herpesvirus Infection Augments Experimental Pulmonary Fibrosis 
Rationale: No effective treatment exists for idiopathic pulmonary fibrosis, and its pathogenesis remains unclear. Accumulating evidence implicates herpesviruses as cofactors (either initiating or exacerbating agents) of fibrotic lung disease, but a role for latent herpesvirus infection has not been studied.
Objectives: To develop a murine model to determine whether latent herpesvirus infection can augment fibrotic responses and to gain insight into potential mechanisms of enhanced fibrogenesis.
Methods: Mice were infected with murine γherpesvirus 14 to 70 days before a fibrotic challenge with fluorescein isothiocyanate or bleomycin so that the virus was latent at the time of fibrotic challenge. Measurements were made after viral infection alone or after the establishment of fibrosis.
Measurements and Main Results: γHerpesvirus is latent by 14 days post infection, and infection 14 to 70 days before fibrotic challenge augmented fibrosis. Fibrotic augmentation was not dependent on reactivation of the latent virus to a lytic state. Total cell numbers and fibrocyte numbers were increased in the lungs of latently infected mice administered fibrotic challenge compared with mock-infected mice that received fibrotic challenge. Latent infection up-regulates expression of proinflammatory chemokines, transforming growth factor-β1, and cysteinyl leukotrienes in alveolar epithelial cells.
Conclusions: Latent γherpesvirus infection augments subsequent fibrotic responses in mice. Enhanced fibrosis is associated with the induction of profibrotic factors and the recruitment of fibrocytes. Our data complement existing human and animal data supporting the hypothesis that γherpesviruses can serve as initiating cofactors in the pathogenesis of pulmonary fibrosis.
doi:10.1164/rccm.200905-0798OC
PMCID: PMC2830399  PMID: 20185751
chemokine; epithelial cells; fibrocyte; interstitial lung disease
7.  Physiological and Computed Tomographic Predictors of Outcome from Lung Volume Reduction Surgery 
Rationale: Previous investigations have identified several potential predictors of outcomes from lung volume reduction surgery (LVRS). A concern regarding these studies has been their small sample size, which may limit generalizability. We therefore sought to examine radiographic and physiologic predictors of surgical outcomes in a large, multicenter clinical investigation, the National Emphysema Treatment Trial.
Objectives: To identify objective radiographic and physiological indices of lung disease that have prognostic value in subjects with chronic obstructive pulmonary disease being evaluated for LVRS.
Methods: A subset of the subjects undergoing LVRS in the National Emphysema Treatment Trial underwent preoperative high-resolution computed tomographic (CT) scanning of the chest and measures of static lung recoil at total lung capacity (SRtlc) and inspiratory resistance (Ri). The relationship between CT measures of emphysema, the ratio of upper to lower zone emphysema, CT measures of airway disease, SRtlc, Ri, the ratio of residual volume to total lung capacity (RV/TLC), and both 6-month postoperative changes in FEV1 and maximal exercise capacity were assessed.
Measurements and Main Results: Physiological measures of lung elastic recoil and inspiratory resistance were not correlated with improvement in either the FEV1 (R = −0.03, P = 0.78 and R = –0.17, P = 0.16, respectively) or maximal exercise capacity (R = –0.02, P = 0.83 and R = 0.08, P = 0.53, respectively). The RV/TLC ratio and CT measures of emphysema and its upper to lower zone ratio were only weakly predictive of postoperative changes in both the FEV1 (R = 0.11, P = 0.01; R = 0.2, P < 0.0001; and R = 0.23, P < 0.0001, respectively) and maximal exercise capacity (R = 0.17, P = 0.0001; R = 0.15, P = 0.002; and R = 0.15, P = 0.002, respectively). CT assessments of airway disease were not predictive of change in FEV1 or exercise capacity in this cohort.
Conclusions: The RV/TLC ratio and CT measures of emphysema and its distribution are weak but statistically significant predictors of outcome after LVRS.
doi:10.1164/rccm.200906-0911OC
PMCID: PMC2830400  PMID: 19965810
8.  Impact of Untreated Obstructive Sleep Apnea on Glucose Control in Type 2 Diabetes 
Rationale: Obstructive sleep apnea (OSA), a treatable sleep disorder that is associated with alterations in glucose metabolism in individuals without diabetes, is a highly prevalent comorbidity of type 2 diabetes. However, it is not known whether the severity of OSA is a predictor of glycemic control in patients with diabetes.
Objectives: To determine the impact of OSA on hemoglobin A1c (HbA1c), the major clinical indicator of glycemic control, in patients with type 2 diabetes.
Methods: We performed polysomnography studies and measured HbA1c in 60 consecutive patients with diabetes recruited from outpatient clinics between February 2007 and August 2009.
Measurements and Main Results: A total of 77% of patients with diabetes had OSA (apnea–hypopnea index [AHI] ≥5). Increasing OSA severity was associated with poorer glucose control, after controlling for age, sex, race, body mass index, number of diabetes medications, level of exercise, years of diabetes and total sleep time. Compared with patients without OSA, the adjusted mean HbA1c was increased by 1.49% (P = 0.0028) in patients with mild OSA, 1.93% (P = 0.0033) in patients with moderate OSA, and 3.69% (P < 0.0001) in patients with severe OSA (P < 0.0001 for linear trend). Measures of OSA severity, including total AHI (P = 0.004), rapid eye movement AHI (P = 0.005), and the oxygen desaturation index during total and rapid eye movement sleep (P = 0.005 and P = 0.008, respectively) were positively correlated with increasing HbA1c levels.
Conclusions: In patients with type 2 diabetes, increasing severity of OSA is associated with poorer glucose control, independent of adiposity and other confounders, with effect sizes comparable to those of widely used hypoglycemic drugs.
doi:10.1164/rccm.200909-1423OC
PMCID: PMC2830401  PMID: 20019340
sleep disordered breathing; glycemic control; diabetes
9.  The Ratio of FEV1 to FVC as a Basis for Establishing Chronic Obstructive Pulmonary Disease 
Rationale: The lambda-mu-sigma (LMS) method is a novel approach that defines the lower limit of normal (LLN) for the ratio of FEV1/FVC as the fifth percentile of the distribution of Z scores. The clinical validity of this threshold as a basis for establishing chronic obstructive pulmonary disease is unknown.
Objective: To evaluate the association between the LMS method of determining the LLN for the FEV1/FVC, set at successively higher thresholds, and clinically meaningful outcomes.
Methods: Using data from a nationally representative sample of 3,502 white Americans aged 40–80 years, we stratified the FEV1/FVC according to the LMS-LLN, with thresholds set at the 5th, 10th, 15th, 20th, and 25th percentiles (i.e., LMS-LLN5, LMS-LLN10, etc.). We then evaluated whether these thresholds were associated with an increased risk of death or prevalence of respiratory symptoms. Spirometry was not specifically completed after a bronchodilator.
Measurements and Main Results: Relative to an FEV1/FVC greater than or equal to LMS-LLN25 (reference group), the risk of death and the odds of having respiratory symptoms were elevated only in participants who had an FEV1/FVC less than LMS-LLN5, with an adjusted hazard ratio of 1.68 (95% confidence interval, 1.34–2.12) and an adjusted odds ratio of 2.46 (95% confidence interval, 2.01–3.02), respectively, representing 13.8% of the cohort. Results were similar for persons aged 40–64 years and those aged 65–80 years.
Conclusions: In white persons aged 40–80 years, an FEV1/FVC less than LMS-LLN5 identifies persons with an increased risk of death and prevalence of respiratory symptoms. These results support the use of the LMS-LLN5 threshold for establishing chronic obstructive pulmonary disease.
doi:10.1164/rccm.200909-1366OC
PMCID: PMC3159085  PMID: 20019341
chronic obstructive pulmonary disease; FEV1/FVC; lower limit of normal; mortality; respiratory symptoms
10.  Pulmonary Alveolar Proteinosis in Workers at an Indium Processing Facility 
Two cases of pulmonary alveolar proteinosis, including one death, occurred in workers at a facility producing indium-tin oxide (ITO), a compound used in recent years to make flat panel displays. Both workers were exposed to airborne ITO dust and had indium in lung tissue specimens. One worker was tested for autoantibodies to granulocytemacrophage–colonystimulating factor (GM-CSF) and found to have an elevated level. These cases suggest that inhalational exposure to ITO causes pulmonary alveolar proteinosis, which may occur via an autoimmune mechanism.
doi:10.1164/rccm.200907-1022CR
PMCID: PMC3159086  PMID: 20019344
pulmonary alveolar proteinosis; indium
11.  Identification of Asthma Phenotypes Using Cluster Analysis in the Severe Asthma Research Program 
Rationale: The Severe Asthma Research Program cohort includes subjects with persistent asthma who have undergone detailed phenotypic characterization. Previous univariate methods compared features of mild, moderate, and severe asthma.
Objectives: To identify novel asthma phenotypes using an unsupervised hierarchical cluster analysis.
Methods: Reduction of the initial 628 variables to 34 core variables was achieved by elimination of redundant data and transformation of categorical variables into ranked ordinal composite variables. Cluster analysis was performed on 726 subjects.
Measurements and Main Results: Five groups were identified. Subjects in Cluster 1 (n = 110) have early onset atopic asthma with normal lung function treated with two or fewer controller medications (82%) and minimal health care utilization. Cluster 2 (n = 321) consists of subjects with early-onset atopic asthma and preserved lung function but increased medication requirements (29% on three or more medications) and health care utilization. Cluster 3 (n = 59) is a unique group of mostly older obese women with late-onset nonatopic asthma, moderate reductions in FEV1, and frequent oral corticosteroid use to manage exacerbations. Subjects in Clusters 4 (n = 120) and 5 (n = 116) have severe airflow obstruction with bronchodilator responsiveness but differ in to their ability to attain normal lung function, age of asthma onset, atopic status, and use of oral corticosteroids.
Conclusions: Five distinct clinical phenotypes of asthma have been identified using unsupervised hierarchical cluster analysis. All clusters contain subjects who meet the American Thoracic Society definition of severe asthma, which supports clinical heterogeneity in asthma and the need for new approaches for the classification of disease severity in asthma.
doi:10.1164/rccm.200906-0896OC
PMCID: PMC2822971  PMID: 19892860
asthma phenotype; definition; cluster analysis; severe asthma
12.  A Role for Wnt Signaling Genes in the Pathogenesis of Impaired Lung Function in Asthma 
Rationale: Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes.
Objectives: We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma.
Methods: Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV1, FVC, and FEV1/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study.
Measurements and Main Results: Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7–16 wk) and the canalicular (gestational age: 17–26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV1 (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV1/FVC (combined P = 0.003).
Conclusions: Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.
doi:10.1164/rccm.200907-1009OC
PMCID: PMC2822972  PMID: 19926868
asthma; lung development; lung function; genetic variation; gene expression
13.  Serum Amyloid A Regulates Granulomatous Inflammation in Sarcoidosis through Toll-like Receptor-2 
Rationale: The critical innate immune mechanisms that regulate granulomatous inflammation in sarcoidosis are unknown. Because the granuloma-inducing component of sarcoidosis tissues has physicochemical properties similar to those of amyloid fibrils, we hypothesized that host proteins capable of forming poorly soluble aggregates or amyloid regulate inflammation in sarcoidosis.
Objectives: To determine the role of the amyloid precursor protein, serum amyloid A, as an innate regulator of granulomatous inflammation in sarcoidosis.
Methods: Serum amyloid A expression was determined by immunohistochemistry in sarcoidosis and control tissues and by ELISA. The effect of serum amyloid A on nuclear factor (NF)-κB induction, cytokine expression, and Toll-like receptor-2 stimulation was determined with transformed human cell lines and bronchoalveolar lavage cells from patients with sarcoidosis. The effects of serum amyloid A on regulating helper T cell type 1 (Th1) granulomatous inflammation were determined in experimental models of sarcoidosis, using Mycobacterium tuberculosis catalase–peroxidase.
Measurements and Main Results: We found that the intensity of expression and distribution of serum amyloid A within sarcoidosis granulomas was unlike that in many other granulomatous diseases. Serum amyloid A localized to macrophages and giant cells within sarcoidosis granulomas but correlated with CD3+ lymphocytes, linking expression to local Th1 responses. Serum amyloid A activated NF-κB in Toll-like receptor-2–expressing human cell lines; regulated experimental Th1-mediated granulomatous inflammation through IFN-γ, tumor necrosis factor, IL-10, and Toll-like receptor-2; and stimulated production of tumor necrosis factor, IL-10, and IL-18 in lung cells from patients with sarcoidosis, effects inhibited by blocking Toll-like receptor-2.
Conclusions: Serum amyloid A is a constituent and innate regulator of granulomatous inflammation in sarcoidosis through Toll-like receptor-2, providing a mechanism for chronic disease and new therapeutic targets.
doi:10.1164/rccm.200905-0696OC
PMCID: PMC2822973  PMID: 19910611
sarcoidosis; serum amyloid A; innate immunity; granuloma; cytokines
14.  Pulmonary Hypertension and Computed Tomography Measurement of Small Pulmonary Vessels in Severe Emphysema 
Rationale: Vascular alteration of small pulmonary vessels is one of the characteristic features of pulmonary hypertension in chronic obstructive pulmonary disease. The in vivo relationship between pulmonary hypertension and morphological alteration of the small pulmonary vessels has not been assessed in patients with severe emphysema.
Objectives: We evaluated the correlation of total cross-sectional area of small pulmonary vessels (CSA) assessed on computed tomography (CT) scans with the degree of pulmonary hypertension estimated by right heart catheterization.
Methods: In 79 patients with severe emphysema enrolled in the National Emphysema Treatment Trial (NETT), we measured CSA less than 5 mm2 (CSA<5) and 5 to 10 mm2 (CSA5−10), and calculated the percentage of total CSA for the lung area (%CSA<5 and %CSA5–10, respectively). The correlations of %CSA<5 and %CSA5–10 with pulmonary arterial mean pressure (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\overline{Ppa}\end{equation*}\end{document}) obtained by right heart catheterization were evaluated. Multiple linear regression analysis using \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\overline{Ppa}\end{equation*}\end{document} as the dependent outcome was also performed.
Measurements and Main Results: The %CSA<5 had a significant negative correlation with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\overline{Ppa}\end{equation*}\end{document} (r = −0.512, P < 0.0001), whereas the correlation between %CSA5–10 and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\overline{Ppa}\end{equation*}\end{document} did not reach statistical significance (r = −0.196, P = 0.083). Multiple linear regression analysis showed that %CSA<5 and diffusing capacity of carbon monoxide (DlCO) % predicted were independent predictors of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\overline{Ppa}\end{equation*}\end{document} (r2 = 0.541): %CSA <5 (P < 0.0001), and DlCO % predicted (P = 0.022).
Conclusions: The %CSA<5 measured on CT images is significantly correlated to \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\overline{Ppa}\end{equation*}\end{document} in severe emphysema and can estimate the degree of pulmonary hypertension.
doi:10.1164/rccm.200908-1189OC
PMCID: PMC2817812  PMID: 19875683
chronic obstructive pulmonary disease; emphysema; pulmonary hypertension; CT
15.  Milk Fat Globule Epidermal Growth Factor 8 Attenuates Acute Lung Injury in Mice after Intestinal Ischemia and Reperfusion 
Rationale: Milk fat globule epidermal growth factor 8 (MFG-E8) is a potent opsonin for the clearance of apoptotic cells and is produced by mononuclear cells of immune competent organs including the spleen and lungs. It attenuates chronic and acute inflammation such as autoimmune glomerulonephritis and bacterial sepsis by enhancing apoptotic cell clearance. Ischemia-reperfusion (I/R) injury of the gut results in severe inflammation, apoptosis, and remote organ damage, including acute lung injury (ALI).
Objectives: To determine whether MFG-E8 attenuates intestinal and pulmonary inflammation after gut I/R.
Methods: Wild-type (WT) and MFG-E8−/− mice underwent superior mesenteric artery occlusion for 90 minutes, followed by reperfusion for 4 hours. A group of WT mice was treated with 0.4 μg/20 g recombinant murine MFG-E8 (rmMFG-E8) at the beginning of reperfusion. Four hours after reperfusion, MFG-E8, cytokines, myeloperoxidase activity, apoptosis, and histopathology were assessed. A 24-hour survival study was conducted in rmMFG-E8– and vehicle-treated WT mice.
Measurements and Main Results: Mesenteric I/R caused severe widespread injury and inflammation of the small intestines and remote organs, including the lungs. MFG-E8 levels decreased in the spleen and lungs by 50 to 60%, suggesting impaired apoptotic cell clearance. Treatment with rmMFG-E8 significantly suppressed inflammation (TNF-α, IL-6, IL-1β, and myeloperoxidase) and injury of the lungs, liver, and kidneys. MFG-E8–deficient mice suffered from greatly increased inflammation and potentiated ALI, whereas treatment with rmMFG-E8 significantly improved the survival in WT mice.
Conclusions: MFG-E8 attenuates inflammation and ALI after gut I/R and may represent a novel therapeutic agent.
doi:10.1164/rccm.200804-625OC
PMCID: PMC2817813  PMID: 19892861
inflammation; apoptosis; organ failure
16.  Targeted Injury of Type II Alveolar Epithelial Cells Induces Pulmonary Fibrosis 
Rationale: Ineffective repair of a damaged alveolar epithelium has been postulated to cause pulmonary fibrosis. In support of this theory, epithelial cell abnormalities, including hyperplasia, apoptosis, and persistent denudation of the alveolar basement membrane, are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar epithelial cells have been identified in familial forms of pulmonary fibrosis. Although these findings are compelling, there are no studies that demonstrate a direct role for the alveolar epithelium or, more specifically, type II cells in the scarring process.
Objectives: To determine if a targeted injury to type II cells would result in pulmonary fibrosis.
Methods: A transgenic mouse was generated to express the human diphtheria toxin receptor on type II alveolar epithelial cells. Diphtheria toxin was administered to these animals to specifically target the type II epithelium for injury. Lung fibrosis was assessed by histology and hydroxyproline measurement.
Measurements and Main Results: Transgenic mice treated with diphtheria toxin developed an approximately twofold increase in their lung hydroxyproline content on Days 21 and 28 after diphtheria toxin treatment. The fibrosis developed in conjunction with type II cell injury. Histological evaluation revealed diffuse collagen deposition with patchy areas of more confluent scarring and associated alveolar contraction.
Conclusions: The development of lung fibrosis in the setting of type II cell injury in our model provides evidence for a causal link between the epithelial defects seen in idiopathic pulmonary fibrosis and the corresponding areas of scarring.
doi:10.1164/rccm.200810-1615OC
PMCID: PMC2817814  PMID: 19850947
diphtheria toxin; lung; collagen; scarring
17.  Effectiveness of Radiation Therapy for Elderly Patients with Unresected Stage I and II Non–Small Cell Lung Cancer 
Rationale: Radiotherapy (RT) is considered the standard treatment for patients with stage I or II non–small lung cancer who are not surgical candidates because of comorbities or preferences against surgery.
Objectives: To compare the outcomes of patients treated with RT alone with those who were untreated to assess the effect of RT on survival.
Methods: Using the Surveillance, Epidemiology and End Results (SEER) registry linked to Medicare files, we identified 6,065 unresected patients with histologically confirmed stage I and stage II non–small cell lung cancer, diagnosed between 1992 and 2002. We used propensity score methods and instrumental variable analysis to control for the possible effects of known as well as unmeasured confounders.
Measurements and Main Results: Overall, 59% of patients received RT. The overall and lung cancer–specific survival of unresected patients treated with RT was significantly better compared with the untreated cases (P < 0.0001 for both comparisons). RT was associated with a 6-month improvement in median overall survival. Propensity score analyses showed that RT was associated with improved overall (hazard ratio, 0.74; 95% confidence interval, 0.70–0.78) and lung cancer–specific survival (hazard ratio, 0.73; 95% confidence interval, 0.69–0.78). Instrumental variable analysis also indicated improved outcomes among patients treated with RT.
Conclusions: RT improves survival of elderly patients with unresected stage I or II lung cancer. These results should be confirmed in prospective trials.
doi:10.1164/rccm.200907-1064OC
PMCID: PMC2817815  PMID: 19892859
18.  Increased Propensity for Central Apnea in Patients with Obstructive Sleep Apnea 
Rationale: There is increasing evidence of increased ventilatory instability in patients with obstructive sleep apnea (OSA), but previous investigations have not studied whether the hypocapnic apneic threshold is altered in this group.
Objectives: To compare the apneic threshold, CO2 reserve, and controller gain between subjects with and without OSA matched for age, sex, and body mass index.
Methods: Hypocapnia was induced via nasal mechanical ventilation for 3 minutes. Cessation of mechanical ventilation resulted in hypocapnic central hypopnea or apnea depending upon the magnitude of the hypocapnia. The apnea threshold (PetCO2–AT) was defined as the measured PetCO2 at which the apnea closest to the last hypopnea occurred. The CO2 reserve was defined as the change in PetCO2 between eupneic PetCO2 and PetCO2–AT. Controller gain was defined as the ratio of change in Ve between control and hypopnea or apnea to the ΔPetCO2.
Measurements and Main Results: Eleven pairs of subjects were studied. There was no difference in the PetCO2–AT between the two groups. However, the CO2 reserve was smaller in the subjects with OSA (2.2 ± 0.6 mm Hg) compared with the control subjects (4.5 ± 1.4 mm Hg; P < 0.001). The controller gain was increased in the subjects with OSA (3.7 ± 1.3 L/min/mm Hg) compared with the control subjects (1.6 ± 0.5 L/min/mm Hg; P < 0.001). Controller gain decreased and CO2 reserve increased in seven subjects restudied after using continuous positive airway pressure for 1 month.
Conclusions: Ventilatory instability is increased in subjects with OSA and is reversible with the use of continuous positive airway pressure.
doi:10.1164/rccm.200810-1658OC
PMCID: PMC2809244  PMID: 19762565
control of breathing; obstructive sleep apnea; controller gain; apneic threshold; complex sleep apnea; central sleep apnea
19.  Transcriptomic Analysis of Human Lung Development 
Rationale: Current understanding of the molecular regulation of lung development is limited and derives mostly from animal studies.
Objectives: To define global patterns of gene expression during human lung development.
Methods: Genome-wide expression profiling was used to measure the developing lung transcriptome in RNA samples derived from 38 normal human lung tissues at 53 to 154 days post conception. Principal component analysis was used to characterize global expression variation and to identify genes and bioontologic attributes contributing to these variations. Individual gene expression patterns were verified by quantitative reverse transcriptase–polymerase chain reaction analysis.
Measurements and Main Results: Gene expression analysis identified attributes not previously associated with lung development, such as chemokine-immunologic processes. Lung characteristics attributes (e.g., surfactant function) were observed at an earlier-than-anticipated age. We defined a 3,223 gene developing lung characteristic subtranscriptome capable of describing a majority of the process. In gene expression space, the samples formed a time-contiguous trajectory with transition points correlating with histological stages and suggesting the existence of novel molecular substages. Induction of surfactant gene expression characterized a pseudoglandular “molecular phase” transition. Individual gene expression patterns were independently validated. We predicted the age of independent human lung transcriptome profiles with a median absolute error of 5 days, supporting the validity of the data and modeling approach.
Conclusions: This study extends our knowledge of key gene expression patterns and bioontologic attributes underlying early human lung developmental processes. The data also suggest the existence of molecular phases of lung development.
doi:10.1164/rccm.200907-1063OC
PMCID: PMC2797628  PMID: 19815808
microarrays; surfactant; principal component analysis
20.  Evaluation of Quantitative IFN-γ Response for Risk Stratification of Active Tuberculosis Suspects 
Rationale: The contribution of interferon-γ release assays (IGRAs) to appropriate risk stratification of active tuberculosis suspects has not been studied.
Objectives: To determine whether the addition of quantitative IGRA results to a prediction model incorporating clinical criteria improves risk stratification of smear-negative–tuberculosis suspects.
Methods: Clinical data from tuberculosis suspects evaluated by the San Francisco Department of Public Health Tuberculosis Control Clinic from March 2005 to February 2008 were reviewed. We excluded tuberculosis suspects who were acid fast–bacilli smear–positive, HIV-infected, or under 10 years of age. We developed a clinical prediction model for culture-positive disease and examined the benefit of adding quantitative interferon (IFN)-γ results measured by QuantiFERON-TB Gold (Cellestis, Carnegie, Australia).
Measurements and Main Results: Of 660 patients meeting eligibility criteria, 65 (10%) had culture-proven tuberculosis. The odds of active tuberculosis increased by 7% (95% confidence interval [CI], 3–11%) for each doubling of IFN-γ level. The addition of quantitative IFN-γ results to objective clinical data significantly improved model performance (c-statistic 0.71 vs. 0.78; P < 0.001) and correctly reclassified 32% of tuberculosis suspects (95% CI,11–52%; P < 0.001) into higher-risk or lower-risk categories. However, quantitative IFN-γ results did not significantly improve appropriate risk reclassification beyond that provided by clinician assessment of risk (4%; 95% CI, −7 to +22%; P = 0.14).
Conclusions: Higher quantitative IFN-γ results were associated with active tuberculosis, and added clinical value to a prediction model incorporating conventional risk factors. Although this benefit may be attenuated within highly experienced centers, the predictive accuracy of quantitative IFN-γ levels should be evaluated in other settings.
doi:10.1164/rccm.200906-0981OC
PMCID: PMC2797629  PMID: 19797760
interferon-gamma release assay; Quantiferon; risk prediction; risk reclassification
21.  Lung Dendritic Cell Expression of Maturation Molecules Increases with Worsening Chronic Obstructive Pulmonary Disease 
Rationale: Dendritic cells (DCs) have not been well studied in chronic obstructive pulmonary disease (COPD), yet their integral role in activating and differentiating T cells makes them potential participants in COPD pathogenesis.
Objectives: To determine the expression of maturation molecules by individual DC subsets in relationship to COPD stage and to expression of the acute activation marker CD69 by lung CD4+ T cells.
Methods: We nonenzymatically released lung leukocytes from human surgical specimens (n = 42) and used flow cytometry to identify three DC subsets (mDC1, mDC2, and pDC) and to measure their expression of three costimulatory molecules (CD40, CD80 and CD86) and of CD83, the definitive marker of DC maturation. Spearman nonparametric correlation analysis was used to identify significant correlations between expression of DC maturation molecules and COPD severity.
Measurements and Main Results: Expression of CD40 by mDC1 and mDC2 and of CD86 by mDC2 was high regardless of GOLD stage, but CD80 and CD83 on these two DC subsets increased with disease progression. pDC also showed significant increases in expression of CD40 and CD80. Expression of all but one of the DC molecules that increased with COPD severity also correlated with CD69 expression on lung CD4+ T cells from the same patients, with the exception of CD83 on mDC2.
Conclusions: This cross-sectional study implies that COPD progression is associated with significant increases in costimulatory molecule expression by multiple lung DC subsets. Interactions with lung DCs may contribute to the immunophenotype of CD4+ T cells in advanced COPD.
Clinical trial registered with www.clinicaltrials.gov (NCT00281229).
doi:10.1164/rccm.200904-0552OC
PMCID: PMC2796731  PMID: 19729666
human; flow cytometry; B70 costimulatory molecules; CD69 antigen; CD4+; T lymphocytes
22.  Heightened Endoplasmic Reticulum Stress in the Lungs of Patients with Chronic Obstructive Pulmonary Disease 
Rationale: Nuclear factor erythroid 2–related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD.
Objectives: To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD.
Methods: We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD.
Measurements and Main Results: Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death.
Conclusions: Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.
doi:10.1164/rccm.200903-0324OC
PMCID: PMC2796732  PMID: 19797762
Nrf2; proteasome system; endoplasmic reticulum stress; unfolded protein response; chronic obstructive pulmonary disease lungs
23.  Maternal Exposure to Particulate Matter Increases Postnatal Ozone-induced Airway Hyperreactivity in Juvenile Mice 
Rationale: Epidemiologic studies implicate air pollutant exposure during pregnancy as a risk factor for wheezing in offspring. Ozone exposure is linked to exacerbations of wheezing in children.
Objectives: To determine if maternal pulmonary exposure to traffic-related particles during pregnancy augments ozone–induced airway hyperresponsiveness in offspring.
Methods: C57BL6 time-mated mice were given NIST SRM#1648 (particulate matter [PM]) 0.48 mg, saline vehicle, or no treatment by tracheal insufflation twice weekly for 3 weeks. PM exposure augmented maternal lung inflammation and placental TNF-α, Keratinocyte-derived cytokine (KC), and IL-6 (measured at gestation Day 18). After parturition, dams and litters were exposed to air or ozone 1 ppm 3 h/d, every other day, thrice weekly for 4 weeks. Respiratory system resistance in pups was measured at baseline and after administration of nebulized methacholine.
Measurements and Main Results: Ozone increased airway hyperresponsiveness, but the increase was greatest in pups born to PM-treated dams. Whole-lung TNF-α, IL-1β, KC, IL-6, and MCP-1 were increased in ozone–treated pups, with the greatest increase in pups born to dams given PM. Airway epithelial mucous metaplasia estimated by periodic acid-Schiff Alcian blue staining was increased in ozone–exposed pups born to PM-treated dams. Alveolar development, determined by morphometry, and airway smooth muscle bulk, estimated using α-actin histochemistry, were unaffected by prenatal or postnatal treatment.
Conclusions: Maternal pulmonary exposure to PM during pregnancy augments placental cytokine expression and postnatal ozone–induced pulmonary inflammatory cytokine responses and ozone–induced airway hyperresponsiveness without altering airway structure.
doi:10.1164/rccm.200901-0116OC
PMCID: PMC2796733  PMID: 19762564
24.  Toll-like Receptor 9 Activation Is a Key Mechanism for the Maintenance of Chronic Lung Inflammation 
Rationale: Accumulating evidence supports the hypothesis that the continuous host response to a persistent challenge can polarize the cytokine environment toward a Th2 cytokine phenotype, but the mechanisms responsible for this skewing are not clear.
Objectives: We investigated the role of Toll-like receptor 9 (TLR9) in a Th2-driven pulmonary granulomatous response initiated via the embolization of Schistosoma mansoni eggs to the lungs of mice.
Methods: Mice were intravenously injected with S. mansoni eggs. Histological and flow cytometric analysis, cytokine measurement, adoptive transfer of bone marrow (BM)-derived dendritic cells (DCs), and in vitro T-cell treatments with antigen-presenting cells were examined.
Measurements and Main Results: In comparison to wild-type mice, TLR9−/− mice showed increased pulmonary granuloma size, augmented collagen deposition, increased Th2 cytokine phenotype, and impaired accumulation of DCs. BM-derived DCs, but not macrophages, recovered from animals with developed Th2-type lung granulomas promoted the production of type 2 cytokines from CD4+ T cells. BM-derived DCs from TLR9−/− mice induced impaired Th1 cytokine and enhanced Th2 cytokine production by T cells, compared with DCs from WT mice. Macrophages from TLR9−/− mice expressed a significantly higher alternatively activated (M2) phenotype characterized by increased “found in inflammatory zone-1” (FIZZ1) and arginase-1 expression. The adoptive transfer of BM-derived DCs from syngeneic WT mice into TLR9−/− mice restored the granuloma phenotype seen in WT mice.
Conclusions: These studies suggest that TLR9 plays an important mechanistic role in the maintenance of the pulmonary granulomatous response.
doi:10.1164/rccm.200906-0892OC
PMCID: PMC2796734  PMID: 19797157
granuloma; pulmonary fibrosis; innate immunity; dendritic cell; macrophage
25.  Two-Dimensional Differential In-Gel Electrophoresis Proteomic Approaches Reveal Urine Candidate Biomarkers in Pediatric Obstructive Sleep Apnea 
Rationale: Sleep studies are laborious, expensive, inaccessible, and inconvenient for diagnosing obstructive sleep apnea (OSA) in children.
Objectives: To examine whether the urinary proteome uncovers specific clusters that are differentially expressed in the urine of children with OSA.
Methods: Two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry proteomics followed by validation with western blot of ELISA.
Measurements and Main Results: Morning urine proteins from 60 children with polysomnographically confirmed OSA and from matched children with primary snoring (n = 30) and control subjects (n = 30) were assessed. A total of 16 proteins that are differentially expressed in OSA were identified, and 7 were confirmed by either immunoblots or ELISA. Among the latter, receiver–operator curve analyses of urinary concentrations of uromodulin, urocortin-3, orosomucoid-1, and kallikrein assigned favorable predictive properties to these proteins. Furthermore, combinatorial approaches indicated that the presence of values beyond the calculated cutoff concentrations for three or more of the proteins yielded a sensitivity of 95% and a specificity of 100%.
Conclusions: Proteomic approaches reveal that pediatric OSA is associated with specific and consistent alterations in urinary concentrations of specific protein clusters. Future studies aiming to validate this approach as a screening method of habitually snoring children appears warranted.
doi:10.1164/rccm.200905-0765OC
PMCID: PMC2796735  PMID: 19797158
sleep apnea; biomarkers; children; urine

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