Marcus W. Butler

RNA-Seq quantification of the human small airway epithelium transcriptome

BackgroundThe small airway epithelium (SAE), the cell population that covers the human airway surface from the 6th generation of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq).ResultsThe data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype.ConclusionsThese observations provide insights into the unique biology of human SAE by providing quantit-ative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking.

Population genetic structure of the people of Qatar

People of the Qatar peninsula represent a relatively recent founding by a small number of families from three tribes of the Arabian Peninsula, Persia, and Oman, with indications of African admixture. To assess the roles of both this founding effect and the customary first-cousin marriages among the ancestral Islamic populations in Qatars population genetic structure, we obtained and genotyped with Affymetrix 500k SNP arrays DNA samples from 168 self-reported Qatari nationals sampled from Doha, Qatar. Principal components analysis was performed along with samples from the Human Genetic Diversity Project data set, revealing three clear clusters of genotypes whose proximity to other human population samples is consistent with Arabian origin, a more eastern or Persian origin, and individuals with African admixture. The extent of linkage disequilibrium (LD) is greater than that of African populations, and runs of homozygosity in some individuals reflect substantial consanguinity. However, the variance in runs of homozygosity is exceptionally high, and the degree of identity-by-descent sharing generally appears to be lower than expected for a population in which nearly half of marriages are between first cousins. Despite the fact that the SNPs of the Affymetrix 500k chip were ascertained with a bias toward SNPs common in Europeans, the data strongly support the notion that the Qatari population could provide a valuable resource for the mapping of genes associated with complex disorders and that tests of pairwise interactions are particularly empowered by populations with elevated LD like the Qatari.

Elafin prevents lipopolysaccharide-induced AP-1 and NF-kappaB activation via an effect on the ubiquitin-proteasome pathway.

The serine anti-protease elafin is expressed by monocytes, alveolar macrophages, neutrophils, and at mucosal surfaces and possesses antimicrobial activity. It is also known to reduce lipopolysaccharide-induced neutrophil influx into murine alveoli as well as to abrogate lipopolysaccharide-induced production of matrix metalloprotease 9, macrophage inhibitory protein 2, and tumor necrosis factor-α by as-yet unidentified mechanisms. In this report we have shown that elafin inhibits the lipopolysaccharide-induced production of monocyte chemoattractant protein-1 in monocytes by inhibiting AP-1 and NF-κB activation. Elafin prevented lipopolysaccharide-induced phosphorylation of AP-1, c-Jun, and JNK but had no effect on phosphorylation of p38. The lipopolysaccharide-induced degradation of IL-1R-associated kinase 1, IκBα, and IκBβ was inhibited by elafin but phosphorylation of IκBα was unaffected. Polyubiquitinated protein including polyubiquitinated IκBα was shown to accumulate in the presence of elafin. These results suggest that inhibition by elafin of lipopolysaccharide-induced AP-1 and NF-κB activation occurs via an effect on the ubiquitin-proteasome pathway.

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5—Lys-6 and Val-9—Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6—Gln-57 and Ser-10—Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.

Rapid needle-out patient-rollover time after percutaneous CT-guided transthoracic biopsy of lung nodules: effect on pneumothorax rate.

PURPOSE To assess the effect of a rapid needle-out patient-rollover time approach on the rate of pneumothorax after computed tomography (CT)-guided transthoracic needle biopsy of pulmonary nodules. MATERIALS AND METHODS The institutional review board approved the study, and all patients gave written informed consent. Between January 2008 and December 2009, percutaneous CT-guided lung biopsy was performed in 201 patients. Eighty-one biopsies were performed without (group 1) and 120 were performed with (group 2) a rapid needle-out patient-rollover time approach (defined as the time between removal of the biopsy needle and placing the patient biopsy-side down). Multivariate analysis was performed between groups for risk factors for pneumothorax, including patient demographic characteristics, lesion characteristics, and biopsy technique. RESULTS Mean rapid needle-out patient-rollover time (± standard deviation) was 9.5 seconds ± 4.8. Seventy-six percent of patients (75 of 98) achieved a needle-out patient-rollover time of 10 seconds or less. Unsuitability for the rapid needle-out patient-rollover time technique resulted in exclusion of 1.8% of patients. An increased number of pneumothoraces (25 [37%] vs 22 [23%]; P = .04) and an increased number of drainage catheter insertions were noted in group 1 compared with group 2 (10 [15%] versus four [4%], respectively; P = .029). At multiple regression analysis for group 1, lesion size and emphysema along the needle track were independent risk factors for pneumothorax (P = .032 and .021, respectively), and emphysema along the needle track was an independent predictor for insertion of a drainage catheter (P = .005). No independent predictor was identified for pneumothorax or insertion of a drainage catheter in group 2. CONCLUSION Rapid needle-out patient-rollover time during percutaneous CT-guided transthoracic lung biopsy reduces the rate of overall pneumothorax and pneumothorax necessitating a drainage catheter. Use of this technique attenuates the influence of traditional risk factors for pneumothorax.

Modulation of Cystatin A Expression in Human Airway Epithelium Related to Genotype, Smoking, COPD, and Lung Cancer

The cathepsin inhibitor Cystatin A (CSTA) has antiapoptotic properties linked with neoplastic changes in squamous cell epithelium, where it has been proposed as a diagnostic and prognostic marker of lung cancer. Notably, cystatin A is upregulated in dysplastic epithelium, prompting us to hypothesize that it might be modulated in chronic obstructive pulmonary disease (COPD), a small airway epithelial (SAE) disorder that is a risk factor for non-small cell lung cancer (NSCLC) in a subset of smokers. Here we report that genetic variation, smoking, and COPD can all elevate levels of CSTA expression in lung small airway epithelia, with still further upregulation in squamous cell carcinoma (SCC), an NSCLC subtype. We examined SAE gene expression in 178 individuals, including healthy nonsmokers (n = 60), healthy smokers (n = 82), and COPD smokers (n = 36), with corresponding large airway epithelium (LAE) data included in a subset of subjects (n = 52). Blood DNA was genotyped by SNP microarray. Twelve SNPs upstream of the CSTA gene were found to associate with its expression in SAE. Levels were higher in COPD smokers than in healthy smokers, who, in turn, had higher levels than nonsmokers. CSTA gene expression in LAE was also smoking-responsive. Using publicly available NSCLC expression data we also found that CSTA was upregulated in SCC versus LAE and downregulated in adenocarcinoma versus smoke-exposed SAE. All phenotypes were associated with different proportional expression of CSTA to cathepsins. Our findings establish that genetic variability, smoking, and COPD all influence CSTA expression, as does SCC, supporting the concept that CSTA may make pivotal contributions to NSCLC pathogenesis in both early and late stages of disease development.

Glutathione S-transferase Copy Number Variation Alters Lung Gene Expression

The glutathione S-transferase (GST) enzymes catalyse the conjugation of xenobiotics to glutathione. Based on reports that inherited copy number variations (CNVs) modulate some GST gene expression levels, and that the small airway epithelium (SAE) and alveolar macrophages (AMs) are involved early in the pathogenesis of smoking-induced lung disease, we asked: do germline CNVs modulate GST expression levels in SAE and AMs? Microarrays were used to survey GST gene expression and determine CNVs genotypes in SAE and AMs obtained by bronchoscopy from current smokers and nonsmokers. 26% of subjects were null for both GSTM1 alleles, with reduced GSTM1 mRNA levels seen in both SAE and AMs. 30% of subjects had homozygous deletions of GSTT1, with reduced mRNA levels in both tissues. Interestingly, GSTT2B exhibited homozygous deletion in the blood of 27% of subjects and was not expressed in SAE in the remainder of subjects, but was expressed in AMs of heterozygotes and wild-type subjects, proportionate to genotype. These data show a germline CNV-mediated linear relationship of genotype with expression level, suggesting minimal compensation of gene expression levels in heterozygotes, consistent with GST polymorphisms playing a role in the risk of smoking-associated, xenobiotic-induced lung disease.

Obstructive shock in a 47 year old female with a deep venous thrombosis due to intravascular leiomyomatosis: a case report

IntroductionIntra cardiac tumours and tumour thrombi can present in a manner resembling a massive pulmonary embolism. Intravascular leiomyomatosis with intracardiac extension is one such rare tumour. Survival from obstructive shock in this condition has not been previously reported.Case presentationA case is presented of a female who presented with recurrent syncope, cyanosis and then circulatory shock. An intravascular and intracardiac mass was suspected. Due to refractory shock, she ultimately underwent single stage median sternotomy and exploratory laparotomy, with excision of an intravascular leiomyoma.ConclusionIntravascular leiomyoma with intracardiac extension should be suspected in the differential diagnosis of a female with a history of uterine fibroids or hysterectomy and presenting with right heart obstructive symptoms.

Glucagon‐like peptide‐1 receptor expression on human eosinophils and its regulation of eosinophil activation

Glucagon‐like peptide‐1 (GLP‐1) and its receptor are part of the incretin family of hormones that regulate glucose metabolism. GLP‐1 also has immune modulatory roles.

Quinolone-associated tendonitis: a potential problem in COPD?

BackgroundQuinolones have traditionally had limited application in the area of community-acquired respiratory tract infections due to poor cover againstStreptococcus pneumoniae. This trend is changing with the broader spectrum of newer fluoroquinoiones. A rare serious side effect of fluoroquinolones is tendinopathy.AimsThis study describes two cases of levofloxacin-associated tendinopathy in patients with severe chronic obstructive pulmonary disease (COPD) and the implications and mechanisms involved are discussed.ConclusionsThe finding of two cases of levofloxacin-induced tendinopathy in our patients suggests that the problem may be more frequent than previously considered. Patients with COPD treated with fluoroquinolones may have other risk factors for tendinopathy such as advanced age, corticosteroid use and renal impairment and merit vigilance for signs of tendonitis.