In Vitro modelling of RSV infection and cytopathogenesis in well-differentiated human primary airway epithelial cells (WD-PAECs).

The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis.

EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from “mucogenic” strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.

Reduced epithelial suppressor of cytokine signalling 1 in severe eosinophilic asthma

Severe asthma represents a major unmet clinical need. Eosinophilic inflammation persists in the airways of many patients with uncontrolled asthma, despite high-dose inhaled corticosteroid therapy. Suppressors of cytokine signalling (SOCS) are a family of molecules involved in the regulation of cytokine signalling via inhibition of the Janus kinase-signal transducers and activators of transcription pathway. We examined SOCS expression in the airways of asthma patients and investigated whether this is associated with persistent eosinophilia.

Healthy controls, mild/moderate asthmatics and severe asthmatics were studied. Whole genome expression profiling, quantitative PCR and immunohistochemical analysis were used to examine expression of SOCS1, SOCS2 and SOCS3 in bronchial biopsies. Bronchial epithelial cells were utilised to examine the role of SOCS1 in regulating interleukin (IL)-13 signalling in vitro.

SOCS1 gene expression was significantly lower in the airways of severe asthmatics compared with mild/moderate asthmatics, and was inversely associated with airway eosinophilia and other measures of T-helper type 2 (Th2) inflammation. Immunohistochemistry demonstrated SOCS1 was predominantly localised to the bronchial epithelium. SOCS1 overexpression inhibited IL-13-mediated chemokine ligand (CCL) 26 (eotaxin-3) mRNA expression in bronchial epithelial cells.

Severe asthma patients with persistent airway eosinophilia and Th2 inflammation have reduced airway epithelial SOCS1 expression. SOCS1 inhibits epithelial IL-13 signalling, supporting its key role in regulating Th2-driven eosinophilia in severe asthma.

A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause dis- ease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consist- ing of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar com- pound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aerugi- nosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.

Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis.

INTRODUCTION: The dichotomization of non-small cell carcinoma (NSCLC) subtype into squamous (SQCC) and adenocarcinoma (ADC) has become important in recent years and is increasingly required with regard to management. The aim of this study was to determine the utility of a panel of commercially available antibodies in refining the diagnosis on small biopsies and also to determine whether cytologic material is suitable for somatic EGFR genotyping in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer. METHODS: Thirty-two consecutive cases of NSCLC were first tested using a panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, 34betaE12, and a D-PAS stain for mucin, to determine their value in refining diagnosis of NSCLC. After this test phase, two further pathologists independently reviewed the cases using a refined panel that excluded 34betaE12 because of its low specificity for SQCC, and refinement of diagnosis and concordance were assessed. Ten cases of ADC, including eight derived from cytologic samples, were sent for EGFR mutation analysis. RESULTS: There was refinement of diagnosis in 65% of cases of NSCLC to either SQCC or ADC in the test phase. This included 10 of 13 cases where cell pellets had been prepared from transbronchial needle aspirates. Validation by two further pathologists with varying expertise in lung pathology confirmed increased refinement and concordance of diagnosis. All samples were adequate for analysis, and they all showed a wild-type EGFR genotype. CONCLUSION: A panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced with refining a diagnosis of NSCLC to SQCC or ADC. These small samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.