Authors: Mark W. Frampton; Ian A. Greaves

Authors: Dan L. Nicolae; Carole Ober

Authors: Claudia E. Kuehni; Ben D. Spycher; Michael Silverman

Author: Eliot R. Spindel

Author: Alexander Logan

Authors: Angela J. Rogers; Benjamin A. Raby; Jessica A. Lasky-Su; Amy Murphy; Ross Lazarus; Barbara J. Klanderman; Jody S. Sylvia; John P. Ziniti; Christoph Lange; Juan C. Celedón; Edwin K. Silverman; Scott T. Weiss

Rationale: Association studies have implicated many genes in asthma pathogenesis, with replicated associations between single-nucleotide polymorphisms (SNPs) and asthma reported for more than 30 genes. Genome-wide genotyping enables simultaneous evaluation of most of this variation, and facilitates more comprehensive analysis of other common genetic variation around these candidate genes for association with asthma.

Objectives: To use available genome-wide genotypic data to assess the reproducibility of previously reported associations with asthma and to evaluate the contribution of additional common genetic variation surrounding these loci to asthma susceptibility.

Methods: Illumina Human Hap 550Kv3 BeadChip (Illumina, San Diego, CA) SNP arrays were genotyped in 422 nuclear families participating in the Childhood Asthma Management Program. Genes with at least one SNP demonstrating prior association with asthma in two or more populations were tested for evidence of association with asthma, using family-based association testing.

Measurements and Main Results: We identified 39 candidate genes from the literature, using prespecified criteria. Of the 160 SNPs previously genotyped in these 39 genes, 10 SNPs in 6 genes were significantly associated with asthma (including the first independent replication for asthma-associated integrin β3 [ITGB3]). Evaluation of 619 additional common variants included in the Illumina 550K array revealed additional evidence of asthma association for 15 genes, although none were significant after adjustment for multiple comparisons.

Conclusions: We replicated asthma associations for a minority of candidate genes. Pooling genome-wide association study results from multiple studies will increase the power to appreciate marginal effects of genes and further clarify which candidates are true “asthma genes.”

Authors: Simon Francis Thomsen; Sophie van der Sluis; Lone G. Stensballe; Danielle Posthuma; Axel Skytthe; Kirsten O. Kyvik; David L. Duffy; Vibeke Backer; Hans Bisgaard

Rationale: Severe respiratory syncytial virus (RSV) infection is associated with asthma but the nature of this association is imperfectly understood.

Objectives: To examine the nature of the association between severe RSV infection and asthma in a population-based sample of twins.

Methods: Data on hospitalization due to RSV infection was gathered for all twins born in Denmark between 1994 and 2000 (8,280 pairs) and linked to information on asthma obtained from hospital discharge registries and parent-completed questionnaires. Genetic variance components models and direction of causation models were fitted to the observed data.

Measurements and Main Results: RSV hospitalization and asthma were positively associated (r = 0.43), and genetic determinants for the two disorders overlapped completely. Modeling the direction of causation between RSV hospitalization and asthma showed that a model in which asthma “causes” RSV hospitalization fitted the data significantly better (P = 0.39 for deterioration in model fit) than a model in which RSV hospitalization “causes” asthma (P < 0.001 for deterioration in model fit), even when sex, birth weight, and maternal smoking during pregnancy were accounted for.

Conclusions: RSV infection that is severe enough to warrant hospitalization does not cause asthma but is an indicator of the genetic predisposition to asthma.

Authors: Stephan Urs Sixt; Michael Adamzik; Daniel Spyrka; Boris Saul; Jan Hakenbeck; Jeremias Wohlschlaeger; Ulrich Costabel; Alexander Kloß; Jan Giesebrecht; Burkhardt Dahlmann; Jürgen Peters

Rationale: Repair mechanisms resulting in alveolar protein degradation in acute respiratory distress syndrome (ARDS) are largely unknown.

Objectives: To test whether the 20S proteasome is present and functional in the alveolar space in patients with ARDS.

Methods: Proteasome antigenic concentration in bronchoalveolar lavage (BAL) supernatants was measured by ELISA in patients with ARDS (n = 64), acute lung injury (ALI) (n = 8), sarcoidosis (n = 13), and in healthy subjects (n = 8). Cleavage of specific fluorogenic substrates (±epoxomicin), I¹²⁵ albumin degradation rate, and gel filtration were used to quantify and characterize proteasomal activity. The presence of proteasomes was confirmed independently by electron microscopic techniques.

Measurements and Main Results: Proteasome concentrations in patients with ARDS were markedly increased (1,069 ± 1,194 ng/ml) in comparison to healthy subjects (60.8 ± 49.8; P < 0.001), ALI (154 ± 43; P = 0.006), and sarcoidosis (97.6 ± 42.2; P = 0.037). All fluorogenic substrates were hydrolyzed (Suc-LLVY-AMC, 3.6 ± 8.8 pkat/mg; BZ-VGR-AMC, 1.8 ± 3.1; Suc-LLE-AMC, 1 ± 1.7) by BAL supernatants of patients with ARDS, with inhibition by epoxomicin (P = 0.0001), and the majority of proteolytic activity was detected in BAL supernatant. Maximum hydrolyzing activity occurred at 660 kD and 20S proteasome was seen microscopically after purification and being released by pneumocytes type II. Proteasomal activity and albumin degradation rate in patients with ARDS were approximately 17-fold lower than in healthy subjects. Proteasomal activity in normal BAL was inhibited by BAL aliquots from patients with ARDS but not by denatured BAL, and returned to normal by purification.

Conclusions: For the first time, we identified extracellular, biologically active 20S proteasome in the alveolar space of patients with ARDS in concentrations much higher than in normal subjects or in those with ALI.

Authors: Murali Shyamsundar; Scott T. W. McKeown; Cecilia M. O'Kane; Thelma R. Craig; Vanessa Brown; David R. Thickett; Michael A. Matthay; Clifford C. Taggart; Janne T. Backman; J. Stuart Elborn; Daniel F. McAuley

Rationale: Simvastatin inhibits inflammatory responses in vitro and in murine models of lung inflammation in vivo. As simvastatin modulates a number of the underlying processes described in acute lung injury (ALI), it may be a potential therapeutic option.

Objectives: To investigate in vivo if simvastatin modulates mechanisms important in the development of ALI in a model of acute lung inflammation induced by inhalation of lipopolysaccharide (LPS) in healthy human volunteers.

Methods: Thirty healthy subjects were enrolled in a double-blind, placebo-controlled study. Subjects were randomized to receive 40 mg or 80 mg of simvastatin or placebo (n = 10/group) for 4 days before inhalation of 50 μg LPS. Measurements were performed in bronchoalveolar lavage fluid (BALF) obtained at 6 hours and plasma obtained at 24 hours after LPS challenge. Nuclear translocation of nuclear factor-κB (NF-κB) was measured in monocyte-derived macrophages.

Measurements and Main Results: Pretreatment with simvastatin reduced LPS-induced BALF neutrophilia, myeloperoxidase, tumor necrosis factor-α, matrix metalloproteinases 7, 8, and 9, and C-reactive protein (CRP) as well as plasma CRP (all P < 0.05 vs. placebo). There was no significant difference between simvastatin 40 mg and 80 mg. BALF from subjects post-LPS inhalation induced a threefold up-regulation in nuclear NF-κB in monocyte-derived macrophages (P < 0.001); pretreatment with simvastatin reduced this by 35% (P < 0.001).

Conclusions: Simvastatin has antiinflammatory effects in the pulmonary and systemic compartment in humans exposed to inhaled LPS.

Clinical trial registered with www.controlled-trials.com (ISRCTN21056528).

Authors: Michelle L. Bell; Keita Ebisu; Roger D. Peng; Jonathan M. Samet; Francesca Dominici

Rationale: There are unexplained geographical and seasonal differences in the short-term effects of fine particulate matter (PM2.5) on human health. The hypothesis has been advanced to include the possibility that such differences might be due to variations in the PM2.5 chemical composition, but evidence supporting this hypothesis is lacking.

Objectives: To examine whether variation in the relative risks (RR) of hospitalization associated with ambient exposure to PM2.5 total mass reflects differences in PM2.5 chemical composition.

Methods: We linked two national datasets by county and by season: (1) long-term average concentrations of PM2.5 chemical components for 2000–2005 and (2) RRs of cardiovascular and respiratory hospitalizations for persons 65 years or older associated with a 10-μg/m³ increase in PM2.5 total mass on the same day for 106 U.S. counties for 1999 through 2005.

Measurements and Main Results: We found a positive and statistically significant association between county-specific estimates of the short-term effects of PM2.5 on cardiovascular and respiratory hospitalizations and county-specific levels of vanadium, elemental carbon, or nickel PM2.5 content.

Conclusions: Communities with higher PM2.5 content of nickel, vanadium, and elemental carbon and/or their related sources were found to have higher risk of hospitalizations associated with short-term exposure to PM2.5.

Authors: Ioannis Kotsianidis; Evangelia Nakou; Irene Bouchliou; Argyrios Tzouvelekis; Emmanouil Spanoudakis; Paschalis Steiropoulos; Ioannis Sotiriou; Vassilis Aidinis; Dimitrios Margaritis; Costas Tsatalas; Demosthenes Bouros

Rationale: The implication of T cells in the pathogenesis of idiopathic pulmonary fibrosis (IPF) is controversial. CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) are pivotal in maintaining immune homeostasis, but their role in IPF pathophysiology has not yet been studied.

Objectives: To explore Treg dynamics and function in IPF.

Methods: Treg levels and dynamics were analyzed by flow cytometry in the peripheral blood (PB) and bronchoalveolar lavage (BAL) of 21 patients with IPF, 35 patients with lung diseases other than IPF (patients without IPF), 20 patients with collagen vascular diseases with pulmonary parenchymal involvement (CVD-IP), and 28 healthy volunteers. The suppression of autologous CD4⁺CD25⁻ cell-proliferative responses and cytokine release by magnetic bead–isolated Tregs was evaluated by proliferation assays and cytometric bead array. Correlations of Treg function and levels with lung function parameters were also performed.

Measurements and Main Results: In patients with IPF, both BAL and PB Tregs were reduced compared with those of healthy volunteers and patients without IPF, although not always significantly. Treg levels were not affected by the administration of low-dose prednisone in four nonresponding patients. The suppressor potential of BAL and PB Tregs was compromised in patients with IPF and patients with CVD-IP, compared with healthy volunteers and patients without IPF. Similarly, the Treg-induced suppression of helper T-cell type 1 and 2 cytokine secretion was impaired in the BAL of patients with IPF and patients with CVD-IP. Moreover, the defective function of BAL Tregs correlated highly with parameters of disease severity.

Conclusions: This study provides the first evidence of global Treg impairment in IPF that strongly correlates with disease severity, suggesting a role for Tregs in the fibrotic process.

Authors: Soon-Kyung Hwang; Hwang-Tae Lim; Arash Minai-Tehrani; Eun-Sun Lee; Jongmin Park; Seung Bum Park; George R. Beck; Myung-Haing Cho

Rationale: Difficulties in achieving long-term survival of patients with lung cancer treated with conventional therapies suggest that novel approaches are required. Recent advances in aerosol-mediated gene delivery have provided the possibility of an alternative for the safe and effective treatment of lung cancer.

Objectives: To investigate the repeated effect of carboxyl-terminal modulator protein (CTMP) on multistage lung tumorigenesis. In this study, we addressed this question by studying the effects of lentivirus-based CTMP in the lungs of 9- and 13-week-old K-ras^LA1 mice, a model of lung cancer.

Methods: An aerosol of lentivirus-based CTMP was delivered into 9- and 13-week-old K-ras^LA1 mice, a model of lung cancer, through a nose-only inhalation system twice a week for 4 weeks. The effects of CTMP on lung cancer progression and Akt-related signals were evaluated.

Measurements and Main Results: Long-term repeated delivery of CTMP effectively reduced tumor progression in the lungs at different stages of development. Lentiviral-CTMP inhibited protein synthesis and cell cycle and altered Akt signaling pathway in the lungs of 9-week-old K-ras^LA1 mice, and increased apoptosis was observed in the lungs of 13-week-old K-ras^LA1 mice.

Conclusions: Long-term repeated viral delivery of CTMP may provide a useful tool for designing lung tumor treatment.

Authors: Laura Paleari; Eva Negri; Alessia Catassi; Michele Cilli; Denis Servent; Rolando D'Angelillo; Alfredo Cesario; Patrizia Russo; Massimo Fini

Rationale: Studies strongly suggest that the nicotinic acetylcholine receptors for nicotine (nAChRs) play a significant role in lung cancer predisposition and natural history. The nAChR α7 subunit has been found to be pivotal in the control of nicotine-induced lung cancer development and in growth signal transduction induced by nicotine binding to nAChRs.

Objectives: To investigate the anticancer effects of α7-nAChR antagonists.

Methods: (1) To check the correlation between α7-nAChR presence and α-cobratoxin (α-CbT) sensitivity, binding experiments were performed in various normal human cells, lung cancer cell lines, and primary tumoral cells; (2) to demonstrate that α-CbT might be an efficient adjuvant therapy for non–small cell lung cancer (NSCLC) we expanded our previous observations to a panel of NSCLCs of various subtypes orthotopically grafted on nonobese diabetic/severe combined immunodeficient mice; (3) to gain insight into the mechanism of α-CbT–induced tumor reduction, the cells obtained after enzymatic digestion of tumors were analyzed for procaspase-9, Bax, Bad, and Bcl-XL protein; and (4) Snail/E-cadherin expression was evaluated to acquire information about the chemoresistance of cancer cells to α-CbT.

Measurements and Main Results: We report herein the results of an experimental strategy aimed at investigating the antitumor effects of a powerful α7-nAChR antagonist, α-CbT, in an in vivo setting set to mimic the clinical setting of lung cancer; in addition, a possible explanation for α-CbT selectivity toward cancer cells is presented.

Conclusions: We report the prolonged survival of α-CbT–treated animals in our mouse model of NSCLC, which is most likely the result of multiple mechanisms, including various antiproliferative and antiangiogenic effects.

Authors: Christophe Guilluy; Saadia Eddahibi; Christian Agard; Christophe Guignabert; Mohamed Izikki; Ly Tu; Laurent Savale; Marc Humbert; Elie Fadel; Serge Adnot; Gervaise Loirand; Pierre Pacaud

Rationale: The complex and multifactorial pathogenesis of pulmonary hypertension (PH) involves constriction, remodeling, and in situ thrombosis of pulmonary vessels. Both serotonin (5-HT) and Rho kinase signaling may contribute to these alterations.

Objectives: To investigate possible links between the 5-HT transporter (5-HTT) and RhoA/Rho kinase pathways, as well as their involvement in the progression of human and experimental PH.

Methods: Biochemical and functional analyses of lungs, platelets, and pulmonary artery smooth muscle cells (PA-SMCs) from patients with idiopathic PH (iPH) and 5-HTT overexpressing mice.

Measurements and Main Results: Lungs, platelets, and PA-SMCs from patients with iPH were characterized by marked elevation in RhoA and Rho kinase activities and a strong increase in 5-HT binding to RhoA indicating RhoA serotonylation. The 5-HTT inhibitor fluoxetine and the type 2 transglutaminase inhibitor monodansylcadaverin prevented 5-HT–induced RhoA serotonylation and RhoA/Rho kinase activation, as well as 5-HT–induced proliferation of PA-SMCs from iPH patients that was also inhibited by the Rho kinase inhibitor fasudil. Increased Rho kinase activity, RhoA activation, and RhoA serotonylation were also observed in lungs from SM22–5-HTT⁺mice, which overexpress 5-HTT in smooth muscle and spontaneously develop PH. Treatment of SM22–5-HTT⁺ mice with either fasudil or fluoxetine limited PH progression and RhoA/Rho kinase activation.

Conclusions: RhoA and Rho kinase activities are increased in iPH, in association with enhanced RhoA serotonylation. Direct involvement of the 5-HTT/RhoA/Rho kinase signaling pathway in 5-HT–mediated PA-SMC proliferation and platelet activation during PH progression identify RhoA/Rho kinase signaling as a promising target for new treatments against PH.

Authors: George T. O'Connor; Brian Caffo; Anne B. Newman; Stuart F. Quan; David M. Rapoport; Susan Redline; Helaine E. Resnick; Jonathan Samet; Eyal Shahar

Rationale: Cross-sectional epidemiologic studies show an association between sleep-disordered breathing and hypertension, but only one cohort study has examined sleep-disordered breathing as a risk factor for incident hypertension.

Objectives: To examine whether sleep-disordered breathing increases the risk of incident hypertension among persons 40 years of age and older.

Methods: In a prospective cohort study, we analyzed data from 2,470 participants who at baseline did not have hypertension, defined as blood pressure of at least 140/90 mm Hg or taking antihypertensive medication. The apnea-hypopnea index (AHI), the number of apneas plus hypopneas per hour of sleep, was measured by overnight in-home polysomnography. We estimated odds ratios for developing hypertension during 5 years of follow-up according to baseline AHI.

Measurements and Main Results: The odds ratios for incident hypertension increased with increasing baseline AHI; however, this relationship was attenuated and not statistically significant after adjustment for baseline body-mass index. Although not statistically significant, the observed association between a baseline AHI greater than 30 and future hypertension (odds ratio, 1.51; 95% confidence interval, 0.93–2.47) does not exclude the possibility of a modest association.

Conclusions: Among middle-aged and older persons without hypertension, much of the relationship between AHI and risk of incident hypertension was accounted for by obesity. After adjustment for body mass index, the AHI was not a significant predictor of future hypertension, although a modest influence of an AHI greater than 30 on hypertension could not be excluded.

Author: Jerome M. Reich

Authors: Richard D. Kim; David E. Greenberg; Kenneth N. Olivier; Steven M. Holland

Author: Richard M. Effros

Authors: Ron Do; Karen Bartlett; Helen Dimich-Ward; Winnie Chu; Susan Kennedy

Authors: Nicoletta Carlo-Stella; Laura Belloli; Roberta Barbera; Camilla Gambaro; Giacomo Rando; Alberto Malesci; Bianca Marasini

Authors: Edoardo Savarino; Massimo Ghio; Giorgio Sammito; Francesco Indiveri; Vincenzo Savarino