Ian K. Anderson

Expression of the T Helper 17-Associated Cytokines IL-17A and IL-17F in Asthma and COPD

Background: Asthma and COPD are characterized by airway dysfunction and inflammation. Neutrophilic airway inflammation is a common feature of COPD and is recognized in asthma, particularly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated in the development of neutrophilic airway inflammation, but their expression in asthma and COPD is uncertain. Methods: We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and 27 with COPD. Results: The median (interquartile range) IL-17A cells/mm2 submucosa was increased in mild to moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe asthma (P = .04). In COPD, IL-17A+ cells/mm2 submucosa were increased (0.5 [3.7]) compared with nonsmoking control subjects (0 [0]) but not compared with smoking control subjects (P = .046). IL-17F+ cells/mm2 submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) (P = .001) but was not increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutrophilic inflammation, but IL-17F was correlated with the submucosal eosinophil count (rs = 0.5, P = .005). The sputum IL-17 concentration in COPD was increased compared with asthma (2 [0-7] pg/mL vs 0 [0-2] pg/mL, P < .0001) and was correlated with post-bronchodilator FEV1% predicted (r = −0.5, P = .008) and FEV1/FVC (r = −0.4, P = .04). Conclusions: Our findings support a potential role for the Th17 cytokines IL-17A and IL-17F in asthma and COPD, but do not demonstrate a relationship with neutrophilic inflammation.

Elevated Sputum Interleukin-5 and Submucosal Eosinophilia in Obese Individuals with Severe Asthma

RATIONALE The relationship between airway inflammation and obesity in severe asthma is poorly understood. OBJECTIVES We sought to determine the relationship between sputum mediator profiles and the distribution of eosinophilic inflammation and obesity in people with severe asthma. METHODS Clinical parameters and eight mediators in sputum were assessed in 131 subjects with severe asthma from a single center categorized into lean, overweight, and obese groups defined by their body mass index. In an independent group of people with severe asthma (n = 45) and healthy control subjects (n = 19) eosinophilic inflammation was enumerated in bronchial submucosa, blood, and sputum and related to their body mass index. MEASUREMENTS AND MAIN RESULTS Sputum IL-5 geometric mean (95% confidence interval) (pg/ml) was elevated in the obese (1.8 [1.2-2.6]) compared with overweight (1.1 [0.8-1.3]; P = 0.025) and lean (0.9 [0.6-1.2]; P = 0.018) subjects with asthma and was correlated with body mass index (r = 0.29; P < 0.001). There was no relationship among body mass index, the sputum cell count, or other sputum mediators. In the bronchoscopy group the submucosal eosinophil number in the subjects with asthma was correlated with body mass index (Spearman rank correlation, rs = 0.38; P = 0.013) and the median (interquartile range) number of submucosal eosinophils was increased in obese (19.4 [11.8-31.2]) (cells per square millimeter) versus lean subjects (8.2 [5.4-14.6]) (P = 0.006). There was no significant association between sputum or peripheral blood eosinophil counts and body mass index. CONCLUSIONS Sputum IL-5 and submucosal eosinophils, but not sputum eosinophils, are elevated in obese people with severe asthma. Whether specific antieosinophilic therapy is beneficial, or improved diet and lifestyle in obese asthma has antiinflammatory effects beyond weight reduction, requires further study.

A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis

Background Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF. Methodology and Principal Findings miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts. Conclusion These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing.

A potent human anti-eotaxin1 antibody, CAT-213: isolation by phage display and in vitro and in vivo efficacy.

The CC chemokine, eotaxin1 (CCL11) is an important regulator of eosinophil function. A marked accumulation of eosinophils in tissues has been correlated with the up-regulation of eotaxin1 expression in several diseases. The potential therapeutic value of neutralizing the effects of eotaxin1 in inflammatory conditions (including asthma) is under investigation. A human single-chain fragment variable antibody that neutralizes human eotaxin1 (CAT-212) was produced using antibody phage display and converted to whole antibody IgG4 format (CAT-213). A novel approach to lead optimization in which the length of the variable heavy chain complementarity-determining region 3 was reduced by one amino acid resulted in an increase in potency of >1000-fold compared with the parent anti-eotaxin1 antibody. The optimized antibody binds eotaxin1 with high affinity (80.4 pM) and specificity. CAT-213 and CAT-212 do not bind or neutralize a range of other human proteins including human monocyte chemoattractant protein-1, a structurally similar chemokine. CAT-213 neutralizes the ability of eotaxin1 to cause an increase in intracellular calcium signaling (with an IC50 value of 2.86 nM), migration of CCR3-expressing L1.2 cells (with an IC50 value of 0.48 nM), and inhibition of the eotaxin1-evoked shape change of human eosinophils in vitro (with an IC50 of 0.71 nM). Local administration of CAT-213 to mice (1–100 μg kg–1) attenuates dermal eosinophilia induced by human eotaxin1, achieving >90% inhibition of eosinophil influx. CAT-213 may therefore be of therapeutic value in inhibiting diseases in which eotaxin1 and eosinophils play a major role, for example, severe asthma.

TGF -Induced Contraction Is Not Promoted by Fibronectin-Fibronectin Receptor Interaction, or SMA Expression

PURPOSE Transforming growth factor (TGF)-beta is a potent inducer of both transdifferentiation and contraction, which are regarded as critical processes that underpin tissue fibrosis. Consequently, transdifferentiation is believed to drive TGFbeta-mediated contraction. This study was conducted to determine the relationship between transdifferentiation of human lens epithelial cells and matrix contraction. METHODS Real-time PCR was used to investigate gene expression of transdifferentiation markers in the human lens cell line FHL 124 and native lens epithelia. Contraction was assessed with a patch-contraction assay, whereby all areas covered by cells were measured with imaging techniques after fixation and cell staining with Coomassie blue. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. To prevent fibronectin-fibronectin receptor interaction 100 microM RGDS peptide was used. Suppression of TGFbeta-induced alphaSMA expression was mediated by siRNA technology. RESULTS Real-time PCR analysis showed 10 ng/mL TGF-beta1 or -beta2 significantly increased expression of alphaSMA, fibronectin, and alpha5beta1 integrin (fibronectin receptor components) in FHL 124 cells and human lens epithelia. Cultures maintained in TGFbeta and RGDS showed a marked increase in the rate of contraction relative to TGF-beta alone. RGDS alone did not differ significantly from the control. Real-time PCR and Western blots showed reduced levels of message and alphaSMA protein when transfected with siRNA. alphaSMA knockdown did not prevent TGFbeta-induced contraction. CONCLUSIONS A targeted inhibition approach demonstrated that key elements associated with transdifferentiation are not critical for TGFbeta-induced matrix contraction.

Engineering design education – the integration of disciplines

The concept of the design process is not well understood by the general public. Indeed industry is now looking for graduates with the core skills of mathematics and science but enhanced by a firm grounding in the engineering design process. At Southampton a number of initiatives have been implemented in teaching practices and further activities are being constructed to increase the undergraduates awareness of the order and execution of the modern design process. The demands of manufacture on design and the abilities of the undergraduate to use high grade CAD/CAM computer packages to perform these tasks is the focus of the developments. The exact package that is being used is not important, more so the thinking processes required in using them to their best advantage. The paper will describe the concepts behind these initiatives and how the engineering education process must itself become an example of the integration of disciplines.