Altered lung biology of healthy never smokers following acute inhalation of E-cigarettes.

BACKGROUND: Little is known about health risks associated with electronic cigarette (EC) use although EC are rising in popularity and have been advocated as a means to quit smoking cigarettes. METHODS: Ten never-smokers, without exposure history to tobacco products or EC, were assessed at baseline with questionnaire, chest X-ray, lung function, plasma levels of endothelial microparticles (EMP), and bronchoscopy to obtain small airway epithelium (SAE) and alveolar macrophages (AM). One week later, subjects inhaled 10 puffs of "Blu" brand EC, waited 30 min, then another 10 puff; n = 7 were randomized to EC with nicotine and n = 3 to EC without nicotine to assess biological responses in healthy, naive individuals. RESULTS: Two hr. post-EC exposure, subjects were again assessed as at baseline. No significant changes in clinical parameters were observed. Biological changes were observed compared to baseline, including altered transcriptomes of SAE and AM for all subjects and elevated plasma EMP levels following inhalation of EC with nicotine. CONCLUSIONS: This study provides in vivo human data demonstrating that acute inhalation of EC aerosols dysregulates normal human lung homeostasis in a limited cohort of healthy naïve individuals. These observations have implications to new EC users, nonsmokers exposed to secondhand EC aerosols and cigarette smokers using EC to quit smoking. TRIAL REGISTRATION: ClinicalTrials.gov NCT01776398 (registered 10/12/12), NCT02188511 (registered 7/2/14).

Persistence of circulating endothelial microparticles in COPD despite smoking cessation.

INTRODUCTION: Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation. METHODS: Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b-CD31+), pulmonary capillary EMP (CD42b-CD31+ACE+) and apoptotic EMP (CD42b-CD62E+/CD42b-CD31+) levels were quantified by flow cytometry. RESULTS: Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit. CONCLUSIONS: Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation. TRIAL REGISTRATION NUMBER: NCT00974064; NCT01776398.

Pulmonary Abnormalities in Young, Light-Use Waterpipe (Hookah) Smokers.

RATIONALE: Waterpipes, also called hookahs, are currently used by millions of people worldwide. Despite the increasing use of waterpipe smoking, there is limited data on the health effects of waterpipe smoking and there are no federal regulations regarding its use. OBJECTIVES: To assess the effects of waterpipe smoking on the human lung using clinical and biological parameters in young, light-use waterpipe smokers. METHODS: We assessed young, light-use, waterpipe-only smokers in comparison with lifelong nonsmokers using clinical parameters of cough and sputum scores, lung function, and chest high-resolution computed tomography as well as biological parameters of lung epithelial lining fluid metabolome, small airway epithelial (SAE) cell differential and transcriptome, alveolar macrophage transcriptome, and plasma apoptotic endothelial cell microparticles. MEASUREMENTS AND MAIN RESULTS: Compared with nonsmokers, waterpipe smokers had more cough and sputum as well as a lower lung diffusing capacity, abnormal epithelial lining fluid metabolome profile, increased proportions of SAE secretory and intermediate cells, reduced proportions of SAE ciliated and basal cells, markedly abnormal SAE and alveolar macrophage transcriptomes, and elevated levels of apoptotic endothelial cell microparticles. CONCLUSIONS: Young, light-use, waterpipe-only smokers have a variety of abnormalities in multiple lung-related biological and clinical parameters, suggesting that even limited waterpipe use has broad consequences on human lung biology and health. We suggest that large epidemiological studies should be initiated to investigate the harmful effects of waterpipe smoking.

Persistence of smoking-induced dysregulation of miRNA expression in the small airway epithelium despite smoking cessation.

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10th to 12th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p<0.01, fold-change >1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/ß-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.

Intraflagellar transport gene expression associated with short cilia in smoking and COPD.

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.

Prevalence of the apolipoprotein E Arg145Cys dyslipidemia at-risk polymorphism in African-derived populations.

Apolipoprotein E, a protein component of blood lipid particles, plays an important role in lipid transport. Different mutations in the apolipoprotein E gene have been associated with various clinical phenotypes. In an initiated study of Qataris, we observed that 17% of the African-derived genetic subgroup were heterozygotes for a rare Arg145Cys (R145C) variant that functions as a dominant trait with incomplete penetrance associated with type III hyperlipoproteinemia. On the basis of this observation, we hypothesized that the R145C polymorphism might be common in African-derived populations. The prevalence of the R145C variant was assessed worldwide in the "1000 Genomes Project" and in 1,012 whites and 1,226 African-Americans in New York, New York. The 1000 Genomes Project data demonstrated that the R145C polymorphism is rare in non-African-derived populations but present in 5% to 12% of Sub-Saharan African-derived populations. The R145C polymorphism was also rare in New York whites (1 of 1,012, 0.1%); however, strikingly, 53 of the 1,226 New York African-Americans (4.3%) were R145C heterozygotes. The lipid profiles of the Qatari and New York R145C heterozygotes were compared with those of controls. The Qatari R145C subjects had higher triglyceride levels than the Qatari controls (p <0.007) and the New York African-American R145C subjects had an average of 52% greater fasting triglyceride levels than the New York African-American controls (p <0.002). From these observations, likely millions of people worldwide derived from Sub-Saharan Africans are apolipoprotein E R145C. In conclusion, although larger epidemiologic studies are necessary to determine the long-term consequences of this polymorphism, the available evidence suggests it is a common cause of a mild triglyceride dyslipidemia.

Long-term follow-up assessment of a phase 1 trial of angiogenic gene therapy using direct intramyocardial administration of an adenoviral vector expressing the VEGF121 cDNA for the treatment of diffuse coronary artery disease.

On the basis of studies in experimental animals demonstrating that AdVEGF121, an E1(-)E3(-) serotype 5 adenovirus coding the 121 isoform of vascular endothelial growth factor (VEGF), could mediate the generation of new blood vessels and reverse coronary ischemia, a clinical study of direct myocardial administration of AdVEGF121 was initiated in patients with late-stage, diffuse coronary artery disease. This study provides long-term (median, 11.8 years) follow-up on these patients. From 1997 to 1999, AdVEGF121 was administered by direct myocardial injection to an area of reversible ischemia in 31 patients with severe coronary disease, either as an adjunct to conventional coronary artery bypass grafting (group A) or as minimally invasive sole (MIS) therapy, using a minithoracotomy (group B). There was no control group; the study participants served as the control subjects. The 5- and 10-year survival was 10 of 15 (67%) and 6 of 15 (40%) for the group A patients, and 11 of 16 (69%) and 5 of 16 (31%) for group B sole therapy patients, respectively. In comparison, maximal medical therapy in comparable groups in the literature have a 3- to 5-year survival rate of 52 to 59%. For the survivors, the angina score for group A was 3.4±0.5 at time 0 and 1.9±1.0 at last follow-up, and for group B it was 3.4±0.6 and 2.0±1.1, respectively. The incidences of malignancy and retinopathy were no greater than that expected for the age-matched general population. We conclude that adenovirus-mediated VEGF direct myocardial administration to patients with severe coronary artery disease is safe, and future larger trials are warranted to assess efficacy.

Threshold of biologic responses of the small airway epithelium to low levels of tobacco smoke.

RATIONALE: Epidemiologic data demonstrate that individuals exposed to low levels of tobacco smoke have decrements in lung function and higher risk for lung disease compared with unexposed individuals. Although this risk is small, low-level tobacco smoke exposure is so widespread, it is a significant public health concern. OBJECTIVES: To identify biologic correlates of this risk we hypothesized that, compared with unexposed individuals, individuals exposed to low levels of tobacco smoke have biologic changes in the small airway epithelium, the site of the first abnormalities associated with smoking. METHODS: Small airway epithelium was obtained by bronchoscopy from 121 individuals; microarrays were used to assess genome-wide gene expression; urine nicotine and cotinine were used to categorize subjects as "nonsmokers," "active smokers," and "low exposure." Gene expression data were used to determine the threshold and induction half maximal level (ID50) of urine nicotine and cotinine at which the small airway epithelium showed abnormal responses. MEASUREMENTS AND MAIN RESULTS: There was no threshold of urine nicotine without a small airway epithelial response, and only slightly above detectable urine cotinine threshold with a small airway epithelium response. The ID50 for nicotine was 25 ng/ml and for cotinine it was 104 ng/ml. CONCLUSIONS: The small airway epithelium detects and responds to low levels of tobacco smoke with transcriptome modifications. This provides biologic correlates of epidemiologic studies linking low-level tobacco smoke exposure to lung health risk, identifies the genes most sensitive to tobacco smoke, and defines thresholds at which the lung epithelium responds to low levels of tobacco smoke.

Confronting the issues of therapeutic misconception, enrollment decisions, and personal motives in genetic medicine-based clinical research studies for fatal disorders.

Genetic medicine-based therapies have unlocked the potential for ameliorating diseases previously considered inevitably fatal. Inherent in the clinical trials of genetic medicines are ethical issues of therapeutic misconception, enrollment decisions as they relate to the risks and benefits of research, and the complex relationships among funding sources, investigators, and the families of affected individuals. The purpose of this paper is to help define these complex issues relevant to the use of genetic medicines and to describe the strategy we have used to confront these issues in a phase I trial of adeno-associated virus-mediated gene transfer to the central nervous system of children with late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal lysosomal storage disease associated with progressive neurodegeneration and death by mid-childhood. Our approach to these challenges should provide a useful paradigm for investigators initiating other genetic medicine- based studies to treat inevitably fatal diseases.

Safety of local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions.

To help define the safety profile of the use of adenovirus (Ad) gene transfer vectors in humans, this report summarizes our experience since April 1993 of the local administration of E1(-)/E3(-) Ad vectors to humans using low (<10(9) particle units) or intermediate (10(9)-10(11) particle units) doses. Included in the study are 90 individuals and 12 controls, with diverse comorbid conditions, including cystic fibrosis, colon cancer metastatic to liver, severe coronary artery disease, and peripheral vascular disease, as well as normals. These individuals received 140 different administrations of vector, with up to seven administrations to a single individual. The vectors used include three different transgenes (human cystic fibrosis transmembrane conductance regulator cDNA, E. coli cytosine deaminase gene, and the human vascular endothelial growth factor 121 cDNA) administered by six different routes (nasal epithelium, bronchial epithelium, percutaneous to solid tumor, intradermal, epicardial injection of the myocardium, and skeletal muscle). The total population was followed for 130.4 patient-years. The study assesses adverse events, common laboratory tests, and long-term follow-up, including incidence of death or development of malignancy. The total group incidence of major adverse events linked to an Ad vector was 0.7%. There were no deaths attributable to the Ad vectors per se, and the incidence of malignancy was within that expected for the population. Overall, the observations are consistent with the concept that local administration of low and intermediate doses of Ad vectors appears to be well tolerated.

Analysis of risk factors for local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions.

In this study we analyze the adverse events and abnormal laboratory parameters following local administration of low (<10(9) particle units) and intermediate (10(9)-10(11) particle units) single and repetitive doses (140 total) of E1(-)E3(-) adenovirus (Ad) gene transfer vectors administered to the respiratory epithelium, solid tumors, skin, myocardium, and skeletal muscle in eight gene transfer trials since April 1993. In the accompanying paper by Harvey et al., (Hum. Gene Ther. 2002; 13:15-63), we conclude that for the total group, no deaths were attributable to the Ad vectors per se, and the incidence of major adverse events likely caused by an Ad vector was 0.7%. The present study analyzes the trials as a group to evaluate risk factors for the adverse events, abnormal values among laboratory parameters, and known deaths. Ten putative risk factors were assessed, including "patient-related" (age, sex, comorbid index and pretherapy anti-Ad antibodies), "vector-related" (dose, route, transgene, and number of vector administrations), and "trial-related" (trial in which the individual was enrolled, and whether surgery was part of the trial). While assessment of each factor individually suggested several possible associations with adverse events, abnormal laboratory parameters, or deaths, multivariate analysis identified only age, comorbid index, and surgery (comorbid index for death; age and surgery for non-death adverse events) as variables significantly associated with increased risk for a major (severity scale 3-4 of 4) adverse event for individuals enrolled in these gene transfer trials. Importantly, multivariate analysis suggested that vector-related parameters, including dose, route, transgene, or number of vector administrations at the doses and routes evaluated in these studies, do not appear to be significant risk factors for a major adverse event. With the caveat that these are phase I, uncontrolled trials, we conclude that (1) there is no definitive risk factor that will clearly predict a major adverse outcome resulting from local administration of low and intermediate doses of Ad gene transfer vectors; and (2) major adverse events in these gene transfer trials are associated primarily with the study population and/or trial procedures, not the Ad vectors themselves. This assessment is consistent with the concept that local administration of low and intermediate doses of Ad gene transfer vectors appears to be well tolerated.