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1.  Cluster Analysis and Clinical Asthma Phenotypes 
Rationale
Heterogeneity in asthma expression is multidimensional, including variability in clinical, physiologic, and pathologic parameters. Classification requires consideration of these disparate domains in a unified model.
Objectives
To explore the application of a multivariate mathematical technique, k-means cluster analysis, for identifying distinct phenotypic groups.
Methods
We performed k-means cluster analysis in three independent asthma populations. Clusters of a population managed in primary care (n = 184) with predominantly mild to moderate disease, were compared with a refractory asthma population managed in secondary care (n = 187). We then compared differences in asthma outcomes (exacerbation frequency and change in corticosteroid dose at 12 mo) between clusters in a third population of 68 subjects with predominantly refractory asthma, clustered at entry into a randomized trial comparing a strategy of minimizing eosinophilic inflammation (inflammation-guided strategy) with standard care.
Measurements and Main Results
Two clusters (early-onset atopic and obese, noneosinophilic) were common to both asthma populations. Two clusters characterized by marked discordance between symptom expression and eosinophilic airway inflammation (early-onset symptom predominant and late-onset inflammation predominant) were specific to refractory asthma. Inflammation-guided management was superior for both discordant subgroups leading to a reduction in exacerbation frequency in the inflammation-predominant cluster (3.53 [SD, 1.18] vs. 0.38 [SD, 0.13] exacerbation/patient/yr, P = 0.002) and a dose reduction of inhaled corticosteroid in the symptom-predominant cluster (mean difference, 1,829 μg beclomethasone equivalent/d [95% confidence interval, 307–3,349 μg]; P = 0.02).
Conclusions
Cluster analysis offers a novel multidimensional approach for identifying asthma phenotypes that exhibit differences in clinical response to treatment algorithms.
doi:10.1164/rccm.200711-1754OC
PMCID: PMC3992366  PMID: 18480428
taxonomy; corticosteroid response; multivariate classification
2.  Superoxide Dismutase Expression Attenuates Cigarette Smoke– or Elastase-generated Emphysema in Mice 
Rationale
Oxidants are believed to play a major role in the development of emphysema.
Objectives
This study aimed to determine if the expression of human copper–zinc superoxide dismutase (CuZnSOD) within the lungs of mice protects against the development of emphysema.
Methods
Transgenic CuZnSOD and littermate mice were exposed to cigarette smoke (6 h/d, 5 d/wk, for 1 yr) and compared with nonexposed mice. A second group was treated with intratracheal elastase to induce emphysema.
Measurements
Lung inflammation was measured by cell counts and myeloperoxidase levels. Oxidative damage was assessed by immunofluorescence for 3-nitrotyrosine and 8-hydroxydeoxyguanosine and lipid peroxidation levels. The development of emphysema was determined by measuring the mean linear intercept (Lm).
Main Results
Smoke exposure caused a fourfold increase in neutrophilic inflammation and doubled lung myeloperoxidase activity. This inflammatory response did not occur in the smoke-exposed CuZnSOD mice. Similarly, CuZnSOD expression prevented the 58% increase in lung lipid peroxidation products that occurred after smoke exposure. Most important, CuZnSOD prevented the onset of emphysema in both the smoke-induced model (Lm, 68 exposed control vs. 58 exposed transgenic; p < 0.04) and elastase-generated model (Lm, 80 exposed control vs. 63 exposed transgenic; p < 0.03). These results demonstrate for the first time that antioxidants can prevent smoke-induced inflammation and can counteract the proteolytic cascade that leads to emphysema formation in two separate animal models of the disease.
Conclusions
These findings indicate that strategies aimed at enhancing or supplementing lung antioxidants could be effective for the prevention and treatment of this disease.
doi:10.1164/rccm.200506-850OC
PMCID: PMC3982860  PMID: 16387805
emphysema; inflammation; oxidants
3.  Statin Use and Risk of Delirium in the Critically Ill 
Rationale
Delirium is common in intensive care unit (ICU) patients and is a predictor of worse outcomes and neuroinflammation is a possible mechanism. The antiinflammatory actions of statins may reduce delirium.
Objectives
To determine whether critically ill patients receiving statin therapy had a reduced risk of delirium than those not on statins.
Methods
A prospective cohort analysis of data from consecutive ICU patients admitted to a UK mixed medical and surgical critical care unit between August 2011 and February 2012; the Confusion Assessment Method for ICU was used to determine the days each patient was assessed as being free of delirium during ICU admission.
Measurements and Main Results
Delirium-free days, daily administration of statins, and serum C-reactive protein (CRP) were recorded. Four hundred and seventy consecutive critical care patients were followed, of whom 151 patients received statins. Using random-effects multivariable logistic regression, statin administration the previous evening was associated with the patient being assessed as free of delirium (odds ratio, 2.28; confidence interval, 1.01–5.13; P < 0.05) and with lower CRP (β = −0.52; P < 0.01) the following day. When the association between statin and being assessed as free of delirium was controlled for CRP, the effect size became nonsignificant (odds ratio, 1.56; confidence interval, 0.64–3.79; P = 0.32).
Conclusions
Ongoing statin therapy is associated with a lower daily risk of delirium in critically ill patients. An ongoing clinical trial, informed by this study, is investigating if statins are a potential therapy for delirium in the critically ill.
doi:10.1164/rccm.201306-1150OC
PMCID: PMC3974585  PMID: 24417431
delirium; statin; inflammation; C-reactive protein; critical care
4.  Therapeutic Effects of Human Mesenchymal Stem Cells in Ex Vivo Human Lungs Injured with Live Bacteria 
Rationale: Mesenchymal stem cells secrete paracrine factors that can regulate lung permeability and decrease inflammation, making it a potentially attractive therapy for acute lung injury. However, concerns exist whether mesenchymal stem cells’ immunomodulatory properties may have detrimental effects if targeted toward infectious causes of lung injury.
Objectives: Therefore, we tested the effect of mesenchymal stem cells on lung fluid balance, acute inflammation, and bacterial clearance.
Methods: We developed an Escherichia coli pneumonia model in our ex vivo perfused human lung to test the therapeutic effects of mesenchymal stem cells on bacterial-induced acute lung injury.
Measurements and Main Results: Clinical-grade human mesenchymal stem cells restored alveolar fluid clearance to a normal level, decreased inflammation, and were associated with increased bacterial killing and reduced bacteremia, in part through increased alveolar macrophage phagocytosis and secretion of antimicrobial factors. Keratinocyte growth factor, a soluble factor secreted by mesenchymal stem cells, duplicated most of the antimicrobial effects. In subsequent in vitro studies, we discovered that human monocytes expressed the keratinocyte growth factor receptor, and that keratinocyte growth factor decreased apoptosis of human monocytes through AKT phosphorylation, an effect that increased bacterial clearance. Inhibition of keratinocyte growth factor by a neutralizing antibody reduced the antimicrobial effects of mesenchymal stem cells in the ex vivo perfused human lung and monocytes grown in vitro injured with E. coli bacteria.
Conclusions: In E. coli–injured human lungs, mesenchymal stem cells restored alveolar fluid clearance, reduced inflammation, and exerted antimicrobial activity, in part through keratinocyte growth factor secretion.
doi:10.1164/rccm.201206-0990OC
PMCID: PMC3678109  PMID: 23292883
acute lung injury; bacterial pneumonia; cell-based therapy; keratinocyte growth factor
5.  Plasma Angiopoietin-2 Predicts the Onset of Acute Lung Injury in Critically Ill Patients 
Rationale: Current clinical prediction scores for acute lung injury (ALI) have limited positive predictive value. No studies have evaluated predictive plasma biomarkers in a broad population of critically ill patients or as an adjunct to clinical prediction scores.
Objectives: To determine whether plasma angiopoietin-2 (Ang-2), von Willebrand factor (vWF), interleukin-8 (IL-8), and/or receptor for advanced glycation end products (sRAGE) predict ALI in critically ill patients.
Methods: Plasma samples were drawn from critically ill patients (n = 230) identified in the emergency department. Patients who had ALI at baseline or in the subsequent 6 hours were excluded, and the remaining patients were followed for development of ALI.
Measurements and Main Results: Nineteen patients developed ALI at least 6 hours after the sample draw. Higher levels of Ang-2 and IL-8 were significantly associated with increased development of ALI (P = 0.0008, 0.004, respectively). The association between Ang-2 and subsequent development of ALI was robust to adjustment for sepsis and vasopressor use. Ang-2 and the Lung Injury Prediction Score each independently discriminated well between those who developed ALI and those who did not (area under the receiver operating characteristic curve, 0.74 for each), and using the two together improved the area under the curve to 0.84 (vs. 0.74, P = 0.05). In contrast, plasma levels of sRAGE and vWF were not predictive of ALI.
Conclusions: Plasma biomarkers such as Ang-2 can improve clinical prediction scores and identify patients at high risk for ALI. In addition, the early rise of Ang-2 emphasizes the importance of endothelial injury in the early pathogenesis of ALI.
doi:10.1164/rccm.201208-1460OC
PMCID: PMC3678110  PMID: 23328529
acute respiratory distress syndrome; acute lung injury; receptor for advanced glycation end products; angiopoietin-2; Lung Injury Prediction Score
6.  Childhood Obesity and Asthma Control in the GALA II and SAGE II Studies 
Rationale: Obesity is associated with increased asthma morbidity, lower drug responsiveness to inhaled corticosteroids, and worse asthma control. However, most prior investigations on obesity and asthma control have not focused on pediatric populations, considered environmental exposures, or included minority children.
Objectives: To examine the association between body mass index categories and asthma control among boys and girls; and whether these associations are modified by age and race/ethnicity.
Methods: Children and adolescents ages 8–19 years (n = 2,174) with asthma were recruited from the Genes-environments and Admixture in Latino Americans (GALA II) Study and the Study of African Americans, Asthma, Genes, and Environments (SAGE II). Ordinal logistic regression was used to estimate odds ratios (OR) and their confidence intervals (95% CI) for worse asthma control.
Measurements and Main Results: In adjusted analyses, boys who were obese had a 33% greater chance of having worse asthma control than their normal-weight counterparts (OR, 1.33; 95% CI, 1.04–1.71). However, for girls this association varied with race and ethnicity (P interaction = 0.008). When compared with their normal-weight counterparts, obese African American girls (OR, 0.65; 95% CI, 0.41–1.05) were more likely to have better controlled asthma, whereas Mexican American girls had a 1.91 (95% CI, 1.12–3.28) greater odds of worse asthma control.
Conclusions: Worse asthma control is uniformly associated with increased body mass index in boys. Among girls, the direction of this association varied with race/ethnicity.
doi:10.1164/rccm.201211-2116OC
PMCID: PMC3678111  PMID: 23392439
obesity; asthma control; race and ethnicity; age; sex
7.  Patients with Idiopathic Pulmonary Fibrosis with Antibodies to Heat Shock Protein 70 Have Poor Prognoses 
Rationale: Diverse autoantibodies are present in most patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that specific autoantibodies may associate with IPF manifestations.
Objectives: To identify clinically relevant, antigen-specific immune responses in patients with IPF.
Methods: Autoantibodies were detected by immunoblots and ELISA. Intrapulmonary immune processes were evaluated by immunohistochemistry. Anti–heat shock protein 70 (HSP70) IgG was isolated from plasma by immunoaffinity. Flow cytometry was used for leukocyte functional studies.
Measurements and Main Results: HSP70 was identified as a potential IPF autoantigen in discovery assays. Anti-HSP70 IgG autoantibodies were detected by immunoblots in 3% of 60 control subjects versus 25% of a cross-sectional IPF cohort (n = 122) (P = 0.0004), one-half the patients with IPF who died (P = 0.008), and 70% of those with acute exacerbations (P = 0.0005). Anti-HSP70 autoantibodies in patients with IPF were significantly associated with HLA allele biases, greater subsequent FVC reductions (P = 0.0004), and lesser 1-year survival (40 ± 10% vs. 80 ± 5%; hazard ratio = 4.2; 95% confidence interval, 2.0–8.6; P < 0.0001). HSP70 protein, antigen–antibody complexes, and complement were prevalent in IPF lungs. HSP70 protein was an autoantigen for IPF CD4 T cells, inducing lymphocyte proliferation (P = 0.004) and IL-4 production (P = 0.01). IPF anti-HSP70 autoantibodies activated monocytes (P = 0.009) and increased monocyte IL-8 production (P = 0.049). ELISA confirmed the association between anti-HSP70 autoreactivity and IPF outcome. Anti-HSP70 autoantibodies were also found in patients with other interstitial lung diseases but were not associated with their clinical progression.
Conclusions: Patients with IPF with anti-HSP70 autoantibodies have more near-term lung function deterioration and mortality. These findings suggest antigen-specific immunoassays could provide useful clinical information in individual patients with IPF and may have implications for understanding IPF progression.
doi:10.1164/rccm.201203-0506OC
PMCID: PMC3678112  PMID: 23262513
B cells; T cells; adaptive immunity; interstitial lung disease
8.  ADCYAP1R1 and Asthma in Puerto Rican Children 
Rationale: Epigenetic and/or genetic variation in the gene encoding the receptor for adenylate-cyclase activating polypeptide 1 (ADCYAP1R1) has been linked to post-traumatic stress disorder in adults and anxiety in children. Psychosocial stress has been linked to asthma morbidity in Puerto Rican children.
Objectives: To examine whether epigenetic or genetic variation in ADCYAP1R1 is associated with childhood asthma in Puerto Ricans.
Methods: We conducted a case-control study of 516 children ages 6–14 years living in San Juan, Puerto Rico. We assessed methylation at a CpG site in the promoter of ADCYAP1R1 (cg11218385) using a pyrosequencing assay in DNA from white blood cells. We tested whether cg11218385 methylation (range, 0.4–6.1%) is associated with asthma using logistic regression. We also examined whether exposure to violence (assessed by the Exposure to Violence [ETV] Scale in children 9 yr and older) is associated with cg11218385 methylation (using linear regression) or asthma (using logistic regression). Logistic regression was used to test for association between a single nucleotide polymorphism in ADCYAP1R1 (rs2267735) and asthma under an additive model. All multivariate models were adjusted for age, sex, household income, and principal components.
Measurements and Main Results: Each 1% increment in cg11218385 methylation was associated with increased odds of asthma (adjusted odds ratio, 1.3; 95% confidence interval, 1.0–1.6; P = 0.03). Among children 9 years and older, exposure to violence was associated with cg11218385 methylation. The C allele of single nucleotide polymorphism rs2267735 was significantly associated with increased odds of asthma (adjusted odds ratio, 1.3; 95% confidence interval, 1.02–1.67; P = 0.03).
Conclusions: Epigenetic and genetic variants in ADCYAP1R1 are associated with asthma in Puerto Rican children.
doi:10.1164/rccm.201210-1789OC
PMCID: PMC3733434  PMID: 23328528
methylation; ADCYAP1R1; childhood asthma; Puerto Ricans; violence
9.  Diminazene Attenuates Pulmonary Hypertension and Improves Angiogenic Progenitor Cell Functions in Experimental Models 
Rationale: Studies have demonstrated that angiotensin-converting enzyme 2 (ACE2) plays a protective role against lung diseases, including pulmonary hypertension (PH). Recently, an antitrypanosomal drug, diminazene aceturate (DIZE), was shown to exert an “off-target” effect of enhancing the enzymatic activity of ACE2 in vitro.
Objectives: To evaluate the pharmacological actions of DIZE in experimental models of PH.
Methods: PH was induced in male Sprague Dawley rats by monocrotaline, hypoxia, or bleomycin challenge. Subsets of animals were simultaneously treated with DIZE. In a separate set of experiments, DIZE was administered after 3 weeks of PH induction to determine whether the drug could reverse PH.
Measurements and Main Results: DIZE treatment significantly prevented the development of PH in all of the animal models studied. The protective effects were associated with an increase in the vasoprotective axis of the lung renin-angiotensin system, decreased inflammatory cytokines, improved pulmonary vasoreactivity, and enhanced cardiac function. These beneficial effects were abolished by C-16, an ACE2 inhibitor. Initiation of DIZE treatment after the induction of PH arrested disease progression. Endothelial dysfunction represents a hallmark of PH pathophysiology, and growing evidence suggests that bone marrow–derived angiogenic progenitor cells contribute to endothelial homeostasis. We observed that angiogenic progenitor cells derived from the bone marrow of monocrotaline-challenged rats were dysfunctional and were repaired by DIZE treatment. Likewise, angiogenic progenitor cells isolated from patients with PH exhibited diminished migratory capacity toward the key chemoattractant stromal-derived factor 1α, which was corrected by in vitro DIZE treatment.
Conclusions: Our results identify a therapeutic potential of DIZE in PH therapy.
doi:10.1164/rccm.201205-0880OC
PMCID: PMC3733435  PMID: 23370913
pulmonary hypertension; ACE2; angiogenic progenitor cells; diminazene
10.  β-Catenin in the Alveolar Epithelium Protects from Lung Fibrosis after Intratracheal Bleomycin 
Rationale: Alveolar epithelial cells (AECs) play central roles in the response to lung injury and the pathogenesis of pulmonary fibrosis.
Objectives: We aimed to determine the role of β-catenin in alveolar epithelium during bleomycin-induced lung fibrosis.
Methods: Genetically modified mice were developed to selectively delete β-catenin in AECs and were crossed to cell fate reporter mice that express β-galactosidase (βgal) in cells of AEC lineage. Mice were given intratracheal bleomycin (0.04 units) and assessed for AEC death, inflammation, lung injury, and fibrotic remodeling. Mouse lung epithelial cells (MLE12) with small interfering RNA knockdown of β-catenin underwent evaluation for wound closure, proliferation, and bleomycin-induced cytotoxicity.
Measurements and Main Results: Increased β-catenin expression was noted in lung parenchyma after bleomycin. Mice with selective deletion of β-catenin in AECs had greater AEC death at 1 week after bleomycin, followed by increased numbers of fibroblasts and enhanced lung fibrosis as determined by semiquantitative histological scoring and total collagen content. However, no differences in lung inflammation or protein levels in bronchoalveolar lavage were noted. In vitro, β-catenin–deficient AECs showed increased bleomycin-induced cytotoxicity as well as reduced proliferation and impaired wound closure. Consistent with these findings, mice with AEC β-catenin deficiency showed delayed recovery after bleomycin.
Conclusions: β-Catenin in the alveolar epithelium protects against bleomycin-induced fibrosis. Our studies suggest that AEC survival and wound healing are enhanced through β-catenin–dependent mechanisms. Activation of the developmentally important β-catenin pathway in AECs appears to contribute to epithelial repair after epithelial injury.
doi:10.1164/rccm.201205-0972OC
PMCID: PMC3733436  PMID: 23306543
apoptosis; idiopathic pulmonary fibrosis; wound healing
12.  Exercise-induced Pulmonary Hypertension 
Exercise stresses the pulmonary circulation through increases in cardiac output (Q.) and left atrial pressure. Invasive as well as noninvasive studies in healthy volunteers show that the slope of mean pulmonary artery pressure (mPAP)–flow relationships ranges from 0.5 to 3 mm Hg⋅min⋅L−1. The upper limit of normal mPAP at exercise thus approximates 30 mm Hg at a Q. of less than 10 L⋅min−1 or a total pulmonary vascular resistance at exercise of less than 3 Wood units. Left atrial pressure increases at exercise with an average upstream transmission to PAP in a close to one-for-one mm Hg fashion. Multipoint PAP–flow relationships are usually described by a linear approximation, but present with a slight curvilinearity, which is explained by resistive vessel distensibility. When mPAP is expressed as a function of oxygen uptake or workload, plateau patterns may be observed in patients with systolic heart failure who cannot further increase Q. at the highest levels of exercise. Exercise has to be dynamic to avoid the increase in systemic vascular resistance and abrupt changes in intrathoracic pressure that occur with resistive exercise and can lead to unpredictable effects on the pulmonary circulation. Postexercise measurements are unreliable because of the rapid return of pulmonary vascular pressures and flows to the baseline resting state. Recent studies suggest that exercise-induced increase in PAP to a mean higher than 30 mm Hg may be associated with dyspnea-fatigue symptomatology.
doi:10.1164/rccm.201211-2090CI
PMCID: PMC3733438  PMID: 23348976
pulmonary circulation; pulmonary vascular resistance; pulmonary arterial compliance; cardiac output; exercise capacity
14.  Interaction between Pseudomonas and CXC Chemokines Increases Risk of Bronchiolitis Obliterans Syndrome and Death in Lung Transplantation 
Rationale: Pseudomonas aeruginosa is the most commonly isolated gram-negative bacterium after lung transplantation and has been shown to up-regulate glutamic acid–leucine–arginine–positive (ELR+) CXC chemokines associated with bronchiolitis obliterans syndrome (BOS), but the effect of pseudomonas on BOS and death has not been well defined.
Objectives: To determine if the influence of pseudomonas isolation and ELR+ CXC chemokines on the subsequent development of BOS and the occurrence of death is time dependent.
Methods: A three-state model was developed to assess the likelihood of transitioning from lung transplant (state 1) to BOS (state 2), from transplant (state 1) to death (state 3), and from BOS (state 2) to death (state 3). This Cox semi-Markovian approach determines state survival rates and cause-specific hazards for movement from one state to another.
Measurements and Main Results: The likelihood of transition from transplant to BOS was increased by acute rejection, CXCL5, and the interaction between pseudomonas and CXCL1. The pseudomonas effect in this transition was due to infection rather than colonization. Movement from transplant to death was facilitated by pseudomonas infection and single lung transplant. Transition from BOS to death was affected by the length of time in state 1 and by the interactions between any pseudomonas isolation and CXCL5 and aspergillus, either independently or in combination.
Conclusions: Our model demonstrates that common post-transplantation events drive movement from one post-transplantation state to another and influence outcomes differently depending upon when after transplantation they occur. Pseudomonas and the ELR+ CXC chemokines may interact to negatively influence lung transplant outcomes.
doi:10.1164/rccm.201207-1228OC
PMCID: PMC3733405  PMID: 23328531
transplantation; lung; BOS; pseudomonas; chemokine
15.  Is Passive Diagnosis Enough? 
Rationale: Tuberculosis (TB) is characterized by a subclinical phase (symptoms absent or not considered abnormal); prediagnostic phase (symptoms noticed but diagnosis not pursued); and clinical phase (care actively sought). Diagnostic capacity during these phases is limited.
Objectives: To estimate the population-level impact of TB case-finding strategies in the presence of subclinical and prediagnostic disease.
Methods: We created a mathematical epidemic model of TB, calibrated to global incidence. We then introduced three prototypical diagnostic interventions: increased sensitivity of diagnosis in the clinical phase by 20% (“passive”); early diagnosis during the prediagnostic phase at a rate of 10% per year (“enhanced”); and population-based diagnosis of 5% of undiagnosed prevalent cases per year (“active”).
Measurements and Main Results: If the subclinical phase was ignored, as in most models, the passive strategy was projected to reduce TB incidence by 18% (90% uncertainty range [UR], 11–32%) by year 10, compared with 23% (90% UR, 14–35%) for the enhanced strategy and 18% (90% UR, 11–28%) for the active strategy. After incorporating a subclinical phase into the model, consistent with population-based prevalence surveys, the active strategy still reduced 10-year TB incidence by 16% (90% UR, 11–28%), but the passive and enhanced strategies’ impact was attenuated to 11% (90% UR, 8–25%) and 6% (90% UR, 4–13%), respectively. The degree of attenuation depended strongly on the transmission rate during the subclinical phase.
Conclusions: Subclinical disease may limit the impact of current diagnostic strategies for TB. Active detection of undiagnosed prevalent cases may achieve greater population-level TB control than increasing passive case detection.
doi:10.1164/rccm.201207-1217OC
PMCID: PMC3733406  PMID: 23262515
tuberculosis; diagnostic techniques and procedures; models; theoretical; epidemiology
16.  Clinical Risk Factors for Primary Graft Dysfunction after Lung Transplantation 
Rationale: Primary graft dysfunction (PGD) is the main cause of early morbidity and mortality after lung transplantation. Previous studies have yielded conflicting results for PGD risk factors.
Objectives: We sought to identify donor, recipient, and perioperative risk factors for PGD.
Methods: We performed a 10-center prospective cohort study enrolled between March 2002 and December 2010 (the Lung Transplant Outcomes Group). The primary outcome was International Society for Heart and Lung Transplantation grade 3 PGD at 48 or 72 hours post-transplant. The association of potential risk factors with PGD was analyzed using multivariable conditional logistic regression.
Measurements and Main Results: A total of 1,255 patients from 10 centers were enrolled; 211 subjects (16.8%) developed grade 3 PGD. In multivariable models, independent risk factors for PGD were any history of donor smoking (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.2–2.6; P = 0.002); FiO2 during allograft reperfusion (OR, 1.1 per 10% increase in FiO2; 95% CI, 1.0–1.2; P = 0.01); single lung transplant (OR, 2; 95% CI, 1.2–3.3; P = 0.008); use of cardiopulmonary bypass (OR, 3.4; 95% CI, 2.2–5.3; P < 0.001); overweight (OR, 1.8; 95% CI, 1.2–2.7; P = 0.01) and obese (OR, 2.3; 95% CI, 1.3–3.9; P = 0.004) recipient body mass index; preoperative sarcoidosis (OR, 2.5; 95% CI, 1.1–5.6; P = 0.03) or pulmonary arterial hypertension (OR, 3.5; 95% CI, 1.6–7.7; P = 0.002); and mean pulmonary artery pressure (OR, 1.3 per 10 mm Hg increase; 95% CI, 1.1–1.5; P < 0.001). PGD was significantly associated with 90-day (relative risk, 4.8; absolute risk increase, 18%; P < 0.001) and 1-year (relative risk, 3; absolute risk increase, 23%; P < 0.001) mortality.
Conclusions: We identified grade 3 PGD risk factors, several of which are potentially modifiable and should be prioritized for future research aimed at preventative strategies.
Clinical trial registered with www.clinicaltrials.gov (NCT 00552357).
doi:10.1164/rccm.201210-1865OC
PMCID: PMC3733407  PMID: 23306540
lung transplantation; clinical risk factors; primary graft dysfunction
17.  Mechanisms of Cardiac and Renal Dysfunction in Patients Dying of Sepsis 
Rationale: The mechanistic basis for cardiac and renal dysfunction in sepsis is unknown. In particular, the degree and type of cell death is undefined.
Objectives: To evaluate the degree of sepsis-induced cardiomyocyte and renal tubular cell injury and death.
Methods: Light and electron microscopy and immunohistochemical staining for markers of cellular injury and stress, including connexin-43 and kidney-injury-molecule-1 (Kim-1), were used in this study.
Measurements and Main Results: Rapid postmortem cardiac and renal harvest was performed in 44 septic patients. Control hearts were obtained from 12 transplant and 13 brain-dead patients. Control kidneys were obtained from 20 trauma patients and eight patients with cancer. Immunohistochemistry demonstrated low levels of apoptotic cardiomyocytes (<1–2 cells per thousand) in septic and control subjects and revealed redistribution of connexin-43 to lateral membranes in sepsis (P < 0.020). Electron microscopy showed hydropic mitochondria only in septic specimens, whereas mitochondrial membrane injury and autophagolysosomes were present equally in control and septic specimens. Control kidneys appeared relatively normal by light microscopy; 3 of 20 specimens showed focal injury in approximately 1% of renal cortical tubules. Conversely, focal acute tubular injury was present in 78% of septic kidneys, occurring in 10.3 ± 9.5% and 32.3 ± 17.8% of corticomedullary-junction tubules by conventional light microscopy and Kim-1 immunostains, respectively (P < 0.01). Electron microscopy revealed increased tubular injury in sepsis, including hydropic mitochondria and increased autophagosomes.
Conclusions: Cell death is rare in sepsis-induced cardiac dysfunction, but cardiomyocyte injury occurs. Renal tubular injury is common in sepsis but presents focally; most renal tubular cells appear normal. The degree of cell injury and death does not account for severity of sepsis-induced organ dysfunction.
doi:10.1164/rccm.201211-1983OC
PMCID: PMC3733408  PMID: 23348975
sepsis; apoptosis; necrosis; autophagy; kidney
18.  Intermittent Hypoxia and Stem Cell Implants Preserve Breathing Capacity in a Rodent Model of Amyotrophic Lateral Sclerosis 
Rationale: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1G93A rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure.
Objectives: To preserve or restore phrenic nerve activity in SOD1G93A rats at disease end stage.
Methods: SOD1G93A rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage.
Measurements and Main Results: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival.
Conclusions: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.
doi:10.1164/rccm.201206-1072OC
PMCID: PMC3733409  PMID: 23220913
ventilatory control; respiratory plasticity; spinal cord; growth factors; motor neuron
19.  Immunostimulatory Oligonucleotides Attenuate Airways Remodeling in Allergic Monkeys 
To determine whether inhaled immunostimulatory DNA sequence oligonucleotides containing CpG motifs mitigate the pathophysiologic manifestation of the asthmatic phenotype (airways hyperresponsiveness and airways remodeling), rhesus monkeys with experimentally induced allergic airways disease were treated seven times with inhaled immunostimulatory oligonucleotides (or sham) periodically for 33 weeks. Airways hyperresponsiveness was reduced twofold in immunostimulatory DNA sequence–treated compared with sham-treated monkeys. Airways from immunostimulatory oligonucleotide-treated monkeys had thinner reticular basement membranes, fewer mucous cells, fewer eosinophils, and fewer mast cells than sham-treated allergic monkeys. We conclude that inhaled immunostimulatory oligonucleotides can attenuate the magnitude of airway hyperreactivity and airways remodeling produced in nonhuman primates with experimentally induced allergic airways disease.
doi:10.1164/rccm.200404-533OC
PMCID: PMC3927836  PMID: 15306532
airway wall alterations; allergic asthma; immunostimulatory DNA sequence oligonucleotides; nonhuman primate
20.  Association of Large-Airway Lymphocytic Bronchitis with Bronchiolitis Obliterans Syndrome 
Rationale: Lung transplantation offers great promise for otherwise terminal lung diseases, but the development of bronchiolitis obliterans syndrome (BOS) continues to limit survival. Although acute rejection and lymphocytic bronchiolitis have been identified as risk factors for the development of BOS, it is unclear whether large-airway lymphocytic inflammation conveys the same risk.
Objectives: We evaluated lymphocytic bronchitis on endobronchial biopsies as a risk factor for BOS and mortality.
Methods: Endobronchial biopsies were collected and graded during surveillance after lung transplantation. We assessed samples with negative cultures collected in the first 90 days from 298 subjects and compared large-airway lymphocytic bronchitis assessed by a 0–2 “E-score” and with standard A and BR pathology scores for acute rejection and small-airway lymphocytic bronchiolitis, respectively.
Measurements and Main Results: We found surprisingly little association between large- and small-airway lymphocytic inflammation scores from a given bronchoscopy. Endobronchial lymphocytic bronchitis was more prevalent in subjects in BOS stage 0p and BOS stages 1–3 at the time of biopsy. Within 90 days after transplantation, increasing maximum E-score was associated with greater risk of BOS (adjusted hazard ratio, 1.76; 95% confidence interval, 1.11–2.78; P = 0.02) and in this analysis 90-day maximum E-scores were the only score type predictive of BOS (P < 0.01).
Conclusions: These results support a multicenter study to evaluate endoscopic biopsies for the identification of patients at increased risk for BOS. The association of endobronchial lymphocytic inflammation and BOS may have mechanistic implications.
doi:10.1164/rccm.201206-1025OC
PMCID: PMC3603592  PMID: 23239157
bronchiolitis obliterans; graft rejection; bronchoscopy; lung transplantation
23.  Superoxide Generated at Mitochondrial Complex III Triggers Acute Responses to Hypoxia in the Pulmonary Circulation 
Rationale: The role of reactive oxygen species (ROS) signaling in the O2 sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses.
Objectives: We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA).
Methods: A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC).
Measurements and Main Results: In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca2+]i. In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca2+]i of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice.
Conclusions: Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca2+]i that cause acute hypoxic pulmonary vasoconstriction.
doi:10.1164/rccm.201207-1294OC
PMCID: PMC3603595  PMID: 23328522
oxygen sensing; Rieske iron-sulfur protein; reactive oxygen species; roGFP; hypoxic pulmonary vasoconstriction
24.  Epigenetic Regulation of miR-17∼92 Contributes to the Pathogenesis of Pulmonary Fibrosis 
Rationale: Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung fibrosis with a high mortality rate. In organ repair and remodeling, epigenetic events are important. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and can target epigenetic molecules important in DNA methylation. The miR-17∼92 miRNA cluster is critical for lung development and lung epithelial cell homeostasis and is predicted to target fibrotic genes and DNA methyltransferase (DNMT)-1 expression.
Objectives: We investigated the miR-17∼92 cluster expression and its role in regulating DNA methylation events in IPF lung tissue.
Methods: Expression and DNA methylation patterns of miR-17∼92 were determined in human IPF lung tissue and fibroblasts and fibrotic mouse lung tissue. The relationship between the miR-17∼92 cluster and DNMT-1 expression was examined in vitro. Using a murine model of pulmonary fibrosis, we examined the therapeutic potential of the demethylating agent, 5′-aza-2′-deoxycytidine.
Measurements and Main Results: Compared with control samples, miR-17∼92 expression was reduced in lung biopsies and lung fibroblasts from patients with IPF, whereas DNMT-1 expression and methylation of the miR-17∼92 promoter was increased. Several miRNAs from the miR-17∼92 cluster targeted DNMT-1 expression resulting in a negative feedback loop. Similarly, miR-17∼92 expression was reduced in the lungs of bleomycin-treated mice. Treatment with 5′-aza-2′-deoxycytidine in a murine bleomycin-induced pulmonary fibrosis model reduced fibrotic gene and DNMT-1 expression, enhanced miR-17∼92 cluster expression, and attenuated pulmonary fibrosis.
Conclusions: This study provides insight into the pathobiology of IPF and identifies a novel epigenetic feedback loop between miR-17∼92 and DNMT-1 in lung fibrosis.
doi:10.1164/rccm.201205-0888OC
PMCID: PMC3603596  PMID: 23306545
microRNA; miR-17∼92; pulmonary fibrosis; DNA methylation; DNMT-1
25.  The Transmembrane Protein 16A Ca2+-activated Cl− Channel in Airway Smooth Muscle Contributes to Airway Hyperresponsiveness 
Rationale: Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca2+-activated Cl− [Cl(Ca)] channels are inferred to be involved in AHR, yet their molecular nature and the cell type they act within to mediate this response remain unknown.
Objectives: Transmembrane protein 16A (TMEM16A) and TMEM16B are Cl(Ca) channels, and activation of Cl(Ca) channels in airway smooth muscle (ASM) contributes to agonist-induced airway contraction. We hypothesized that Tmem16a and/or Tmem16b encode Cl(Ca) channels in ASM and mediate AHR.
Methods: We assessed the expression of the TMEM16 family, and the effects of niflumic acid and benzbromarone on AHR and airway contraction, in an ovalbumin-sensitized mouse model of chronic asthma. We also cloned TMEM16A from ASM and examined the Cl− currents it produced in HEK293 cells. We further studied the impacts of TMEM16A deletion on Ca2+ agonist–induced cell shortening, and on Cl(Ca) currents activated by Ca2+ sparks (localized, short-lived Ca2+ transients due to the opening of ryanodine receptors) in mouse ASM cells.
Measurements and Main Results: TMEM16A, but not TMEM16B, is expressed in ASM cells and its expression in these cells is up-regulated in ovalbumin-sensitized mice. Niflumic acid and benzbromarone prevent AHR and contraction evoked by methacholine in ovalbumin-sensitized mice. TMEM16A produces Cl(Ca) currents with kinetics similar to native Cl(Ca) currents. TMEM16A deletion renders Ca2+ sparks unable to activate Cl(Ca) currents, and weakens caffeine- and methacholine-induced cell shortening.
Conclusions: Tmem16a encodes Cl(Ca) channels in ASM and contributes to Ca2+ agonist–induced contraction. In addition, up-regulation of TMEM16A and its augmented activation contribute to AHR in an ovalbumin-sensitized mouse model of chronic asthma. TMEM16A may represent a potential therapeutic target for asthma.
doi:10.1164/rccm.201207-1303OC
PMCID: PMC3603598  PMID: 23239156
TMEM16A; airway smooth muscle; airway hyperresponsiveness

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