Kelly Moffitt

Neutrophil Elastase Activity is Associated with Exacerbations and Lung Function Decline in Bronchiectasis

Rationale: Sputum neutrophil elastase and serum desmosine, which is a linked marker of endogenous elastin degradation, are possible biomarkers of disease severity and progression in bronchiectasis. This study aimed to determine the association of elastase activity and desmosine with exacerbations and lung function decline in bronchiectasis. Methods: This was a single‐center prospective cohort study using the TAYBRIDGE (Tayside Bronchiectasis Registry Integrating Datasets, Genomics, and Enrolment into Clinical Trials) registry in Dundee, UK. A total of 433 patients with high‐resolution computed tomography‐confirmed bronchiectasis provided blood samples for desmosine measurement, and 381 provided sputum for baseline elastase activity measurements using an activity‐based immunosassay and fluorometric substrate assay. Candidate biomarkers were tested for their relationship with cross‐sectional markers of disease severity, and with future exacerbations, mortality and lung function decline over 3 years. Measurement and Main Results: Elastase activity in sputum was associated with the bronchiectasis severity index (r = 0.49; P < 0.0001) and was also correlated with the Medical Research Council dyspnea score (r = 0.34; P < 0.0001), FEV1% predicted (r = −0.33; P < 0.0001), and the radiological extent of bronchiectasis (r = 0.29; P < 0.0001). During a 3‐year follow‐up, elevated sputum elastase activity was associated with a higher frequency of exacerbations (P < 0.0001) but was not independently associated with mortality. Sputum elastase activity was independently associated with FEV1 decline (&bgr; coefficient, −0.139; P = 0.001). Elastase showed good discrimination for severe exacerbations with an area under the curve of 0.75 (95% confidence interval [CI], 0.72‐0.79) and all‐cause mortality (area under the curve, 0.70; 95% CI, 0.67‐0.73). Sputum elastase activity increased at exacerbations (P = 0.001) and was responsive to treatment with antibiotics. Desmosine was correlated with sputum elastase (r = 0.42; P < 0.0001) and was associated with risk of severe exacerbations (hazard ratio 2.7; 95% CI, 1.42‐5.29; P = 0.003) but not lung function decline. Conclusions: Sputum neutrophil elastase activity is a biomarker of disease severity and future risk in adults with bronchiectasis.

Inflammatory markers in cystic fibrosis patients with transmissible Pseudomonas aeruginosa

Chronic Pseudomonas aeruginosa infection in cystic fibrosis (CF) leads to a damaging host inflammatory response. There are an increasing number of reports of P. aeruginosa cross-infection at CF centres. The clinical significance of acquisition of a transmissible strain for patients who already harbour P. aeruginosa is unclear. In this study, levels of inflammatory markers in clinically stable adult CF patients who harbour transmissible and sporadic strains of P. aeruginosa have been compared. Patients with CF and chronic P. aeruginosa infection were grouped into those who harbour a transmissible P. aeruginosa and those who harbour their own sporadic strains. Total white cell and differential counts, sputum neutrophil elastase (NE), interleukin (IL)‐8, tumour necrosis factor (TNF)‐α, plasma IL‐6 and NE/α1‐antitrypsin complexes, serum C‐reactive protein, and urine TNF receptor 1 were all measured in clinically stable patients 4–6 weeks following completion of intravenous antibiotic therapy. The two groups (both n=20) were well matched for per cent predicted forced expiratory volume in one second, per cent predicted forced vital capacity and body mass index. There were no significant differences in levels of white cell counts or inflammatory markers between the two groups. At times of clinical stability, cystic fibrosis patients infected with transmissible Pseudomonas aeruginosa do not have a heightened inflammatory response above that of those harbouring sporadic strains.

Association of airway cathepsin B and S with inflammation in cystic fibrosis.

Irreversible tissue damage within the cystic fibrosis (CF) lung is mediated by proteolytic enzymes during an inflammatory response. Serine proteinases, in particular neutrophil elastase (NE), have been implicated however, members of the cysteine proteinase family may also be involved. The aim of this study was to determine cathepsin B and S levels in cystic fibrosis (CF) sputum and to assess any relationship to recognized markers of inflammation such as sputum NE, interleukin‐8 (IL‐8), tumor necrosis factor alpha (TNF‐α), urine TNF receptor 1 (TNFr1), plasma IL‐6, and serum C‐reactive protein (CRP). Proteinase activities were measured in the sputum of 36 clinically stable CF patients using spectrophotometric and fluorogenic assays. Immunoblots were also used to confirm enzyme activity data. All other parameters were measured by ELISA. Patients had a mean age of 27.2 (8.2) years, FEV. of 1.6 (0.79) L and BMI of 20.7 (2.8). Both cathepsin B and S activities were detected in all samples, with mean concentrations of 18.0 (13.5) µg/ml and 1.6 (0.88) µg/ml, respectively and were found to correlate not only with each other but with NE, TNF‐α and IL‐8 (in all cases . < 0.05). Airway cathepsin B further correlated with circulatory IL‐6 and CRP however, no relationship for either cathepsin was observed with urine TNFr1. This data indicates that cathepsin B and S may have important roles in the pathophysiology of CF lung disease and could have potential as markers of inflammation in future studies. Pediatr. Pulmonol. 2010; 45:860–868.

The emerging role of serine proteases in apoptosis

Unregulated apoptosis can be due to a disruption in the balance and control of both intra- and inter-cellular proteolytic activities leading to various disease states. Many proteases involved in apoptotic processes are yet to be identified; however, several are already well characterized. Caspases traditionally held the predominant role as prime mediators of execution. However, latterly, evidence has accumulated that non-caspases, including calpains, cathepsins, granzymes and the proteasome have roles in mediating and promoting cell death. Increasingly, research is implicating serine proteases within apoptotic processing, particularly in the generation of nuclear events such as condensation, fragmentation and DNA degradation observed in late-stage apoptosis. Serine proteases therefore are emerging as providing additional or alternative therapeutic targets.

From sentencing to execution--the processes of apoptosis.

Objectives Cell proliferation and apoptosis play a major role in maintaining homeostasis and as such any disruption within these processes can lead to disease states. Apoptosis occurs in three non‐distinct phases – induction, effector and degradation – and can be executed through both the extrinsic and intrinsic pathways in addition to recognised sub‐pathways such as the p53 and lysosomal pathways. This review article highlights these pathways, incorporating an overview of the molecular regulators of apoptosis.

Proteases implicated in apoptosis: old and new.

Objectives The role of proteases in the regulation of apoptosis is becoming increasingly apparent. Whilst many of these proteases are already characterised, some have yet to be identified. Traditionally caspases held the traditional role as the prime mediators of apoptosis; however, attention is now turning towards the contribution made by serine proteases.

Inflammatory and immunological biomarkers are not related to survival in adults with Cystic Fibrosis

BACKGROUND Chronic Pseudomonas aeruginosa pulmonary infection is associated with a decline in lung function and reduced survival in people with Cystic Fibrosis (CF). Damaging inflammatory and immunological mediators released in the lungs can be used as markers of chronic infection, inflammation and lung tissue damage. METHODS Clinical samples were collected from CF patients and healthy controls. Serum IgG and IgA anti-Pseudomonas antibodies, sputum IL-8 and TNFα, plasma IL-6 and urine TNFr1 were measured by ELISA. Sputum neutrophil elastase (NE), cathepsin S and cathepsin B were measured by spectrophotometric and fluorogenic assays. The relationship between IgG and IgA, inflammatory mediators and long-term survival was determined. RESULTS IgG and IL-6 positively correlated with mortality. However, multivariate analysis demonstrated that after adjusting for FEV(1), IgG was not independently related to mortality. A relationship was observed between IgG and IL-6, TNFα, TNFr1 and between IgA and IL8, cathepsin S and cathepsin B. CONCLUSIONS These data indicate that biomarkers of inflammation are not independent predictors of survival in people with CF.

Chymotrypsin-like serine proteinases are involved in the maintenance of cell viability

An increasing number of studies have implicated serine proteinases in the development of apoptosis. In this study, we assessed the ability of a set of highly specific irreversible inhibitors (activity probes), incorporating an α-amino alkane diphenyl phosphonate moiety, to modulate cell death. In an initial assessment of the cellular toxicity of these activity probes, we discovered that one example, N-α-tetramethylrhodamine phenylalanine diphenylphosphonate {TMR-Phe(P)(OPh)(2)} caused a concentration-dependent decrease in the viability of HeLa and U251 mg cells. This reduced cell viability was associated with a time-dependent increase in caspase-3 activity, PARP cleavage and phosphatidylserine translocation, establishing apoptosis as the mechanism of cell death. SDS-PAGE analysis of cell lysates prepared from the HeLa cells treated with TMR-Phe(P)(OPh)(2), revealed the presence of a fluorescent band of molecular weight 58 kDa. Given that we have previously reported on the use of this type of activity probe to reveal active proteolytic species, we believe that we have identified a chymotrypsin-like serine proteinase activity integral to the maintenance of cell viability.

181* Development of a novel tool for the rapid detection of neutrophil elastase as a marker of inflammation within the clinic

Neutrophil elastase (NE) is a biomarker of infection and inflammation which has been shown to correlate with the severity of several respiratory diseases, including cystic fibrosis (CF). Utilising our unique Protease-Tag molecules, we have developed a novel methodology for the capture and detection of active proteases in complex samples such as CF sol. The aim of this work was to provide initial clinical validation of the NE-Tag assay in CF. Sputum (n = 45) was recovered from CF patients hospitalised for acute exacerbation. Sol was recovered and analysed for NE activity using the NE-Tag ELISA and two fluorogenic substrate-based assays [1. Suc-AAPV-AMC (Sigma) and 2. InnozymeTM Immunocapture assay (Calbiochem)]. NE activity between assays and with a range of clinical parameters was correlated. A highly significant correlation was shown between assays. NE activity (NETag) further correlated appropriately with clinical parameters: inversely with FEV1 (p = 0.036) and positively with CRP (p = 0.035), neutrophils and total white cell counts (p = 0.000). The InnozymeTM assay showed similar correlations with the clinical parameters (with the exception of CRP). No correlations with any of the clinical parameters were observed when NE was measured using the standard fluorogenic substrate. NE as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. The NE-Tag assay has advantages over the InnoZymeTM assay in terms of time, ease of use and no dependency on a kinetic readout; it has also the capability of being transferred to a lateral flow device for routine monitoring.

The development of a novel immunoassay for the quantification of active tryptase

Introduction: Tryptase, a serine protease found in the secretory granules of mast cells, has been associated with several diseases including allergic asthma and Chronic Obstructive Pulmonary Disease (COPD). Elevated levels of tryptase occur in biological fluids, including blood, following mast cell degranulation, in response to allergic stimulation. In COPD, tryptase levels in sputum correlate with the severity of disease. Aims and Objectives ProAxsis has developed a range of ProteaseTags ® which selectively bind to the active forms of proteases, only. When combined with an appropriate antibody, they provide activity-based immunoassays (ABIs) for the quantification of active proteases. Here we report the development of a novel ProteaseTag ® ABI for the quantification of active mast cell tryptase. Methods: A ProteaseTag ® , designed to irreversibly inhibit mast cell tryptase, was synthesised using methodologies developed in-house. The desired irreversible inhibition of tryptase was established kinetically using a fluorogenic activity assay and confirmed with electroblotting techniques. The ProteaseTag ® was subsequently incorporated into an ABI, which was evaluated for the quantification of active tryptase. Results: Our novel tryptase ProteaseTag ® ABI proved successful for the quantification of active tryptase, with detection limits ranging from 7.8125 ng/ml to 500 ng/ml. Conclusions We have developed a novel ABI for the measurement of active tryptase, which could allow rapid quantification of the protease in patient samples. The use of our ABI could facilitate the adoption of active tryptase as a validated biomarker in several respiratory diseases, including asthma and COPD.