Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

beta-Sitosterol stimulates ceramide metabolism in differentiated Caco2 cells

Previous studies from our laboratory on tumor cells suggest that phytosterols stimulate ceramide production, which was associated with cell growth inhibition and stimulation of apoptosis. The objective of the present study was to examine the effect of phytosterols on ceramide metabolism in small intestinal cells that represent the first cells in contact with dietary phytosterols. Caco(2) cells, an accepted model for human intestinal epithelial cells, were used in this study. Ceramide and ceramide-containing lipids were examined by labeling the ceramide pool with (3)H-serine. Cells were supplemented with 16 microM of sterols (cholesterol, beta-sitosterol or campesterol) for 16 days postconfluence and continued to differentiate. Of the two phytosterols, beta-sitosterol, but not campesterol, induced more than double the serine labeling when compared with cholesterol. This increase was uniform in sphingomyelin (SM), ceramide and sphingosine labeling. Sterols had no effect on SM concentration in the cells. In addition, sterol had no effect on the activity of SM synthase or sphingomyelinases. There was an inhibition of ceramidases with campesterol supplementation. These data suggest that the observed increases in SM and sphingosine labeling were due to an increase in ceramide turnover. The increase in ceramide turnover with beta-sitosterol supplementation was not associated with growth inhibition but was with increases in ceramide glycosylation products such as cerebrosides and gangliosides. It was concluded that beta-sitosterol has no effect on differential Caco(2), a model of normal small intestinal cells. The increase in the glycosylated ceramide products may offer a means to protect the cells from the harmful effect of ceramide by excreting them with lipoproteins.     

Source gene composition and gene conversion of the AluYh and AluYi lineages of retrotransposons

Background  

Alu elements are a family of SINE retrotransposons in primates. They are classified into subfamilies according to specific diagnostic mutations from the general Alu consensus. It is now believed that there may be several retrotranspositionally-competent source genes within an Alu subfamily. In this study, subfamilies falling on the AluYi and AluYh lineages, and the AluYg6 subfamily, are assessed for the presence of secondary source genes, and the influence of gene conversion on the AluYh and AluYi lineages is also described.     

Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters

It has been suggested that phytosterol and phytostanol esters possess similar cholesterol-lowering properties, however, whether mechanisms responsible are identical has not been addressed. To address this question, cholesterol plasma levels, absorption, biosynthesis, and turnover were measured in 15 hypercholesterolemic subjects consuming prepared diets each over 21 d using a cross-over design. Diets contained either i) margarine (M), ii) margarine with phytosterol esters (MSE) (1.84 g/d), or iii) margarine with phytostanol esters (MSA) (1.84 g/d). Cholesterol absorption was measured using the ratio of [(13)C]cholesterol(oral):D(7)-cholesterol(IV); biosynthesis using D incorporation from D(2)O and turnover by D(7)-cholesterol(IV) decay rates. Plasma total cholesterol level at d 21/22 was lower (P < 0. 05) for MSE (13.4%) but not MSA (10.2%) versus M (6.0%) diets. Plasma low density lipoprotein-cholesterol (LDL-C) mean reductions at d 21/22 were larger (P < 0.05) for MSE (12.9%) and MSA (7.9%) compared with M (3.9%). Plasma TG and high density lipoprotein-cholesterol (HDL-C) levels did not differ across diets. Cholesterol absorption was reduced (P < 0.05) 36.2 and 25.9% at d 21 for MSE and MSA versus M, while cholesterol biosynthesis was reciprocally increased (P < 0.05) 53.3 and 37.8% for MSE and MSA versus M, respectively. Cholesterol turnover was not influenced by diet.These data indicate that plant sterol and stanol esters differentially lower circulating total and LDL cholesterol levels by suppression of cholesterol absorption in hypercholesterolemic subjects.     

Gender effects of tall oil versus soybean phytosterols as cholesterol-lowering agents in hamsters

To examine the effect of gender on the mechanisms of action of phytosterols extracted from tall oil (TO) and soybean (SB) on cholesterol and phytosterol metabolism, male and female hamsters were fed cholesterol-enriched diets containing 0.5 or 1% (w/w) TO or SB phytosterols for 90 days. Plasma lipoprotein cholesterol profile and tissue phytosterol and cholesterol biosynthesis levels were determined. Mean plasma total-cholesterol level in females fed 1% (w/w) SB was reduced (p<0.05) by 44%, while in males it was lowered (p<0.05) by 25% compared with their respective controls. Moreover, mean plasma total-cholesterol level was reduced (p<0.05) in male hamsters by -31% and female hamsters by -32% when fed 1% (w/w) TO. Cholesterol biosynthesis was higher (p<0.05) by twofold in groups fed TO at 0.5 and 1% (w/w) concentrations, compared with SB. Hamsters fed TO at 0.5 and 1% (w/w) levels also had higher (p<0.05) hepatic and enterocytic campesterol contents than SB-fed animals. These findings demonstrate gender differences in cholesterol metabolism in TO- and SB-fed hamsters. The results suggest that TO, conversely to SB phytosterol, is a more effective cholesterol-lowering agent in male, but not as much in female, hamsters, over a feeding period of 90 days.     

Analysis of the features and source gene composition of the AluYg6 subfamily of human retrotransposons

Background  

Alu elements are a family of SINE retrotransposons in primates. They are classified into subfamilies according to specific diagnostic mutations from the general Alu consensus. It is now believed that there may be several retrotranspositionally-competent source genes within an Alu subfamily. To investigate the evolution of young Alu elements it is critical to have access to complete subfamilies, which, following the release of the final human genome assembly, can now be obtained using in silico methods.