Lorraine Martin

Inflammatory related changes in bone mineral content in adults with cystic fibrosis

Background: Proinflammatory cytokines stimulate osteoclast activity and this could lead to increased bone resorption in patients with cystic fibrosis. The aim of this study was to determine whether markers of systemic inflammation are related to changes in bone mineral content (BMC) in adults with cystic fibrosis. Methods: Total body BMC was assessed by dual energy x ray absorptiometry in 100 patients (54 male) of mean (SD) age 25.6 (7.1) years and forced expiratory volume in 1 second (FEV1) 61.8 (24.1)% predicted on recruitment to the study and 1 year later. Blood was also taken at these time points to measure markers of systemic inflammation. Results: After 1 year BMC had reduced by 16.1 (62.1) g, p = 0.01; (0.6 (2.8)%). The change in BMC was related to mean levels of interleukin (IL)-6 (rs = −0.39, p<0.001) and C reactive protein (rs = −0.34, p = 0.002), intravenous antibiotic use (rs = −0.27, p = 0.006) and oral corticosteroid use (rs = −0.20, p = 0.045). Urinary markers of osteoclast activity were also related to IL-6 (rs  =  0.27, p = 0.02). Multiple linear regression revealed that IL-6 (coefficient –2.2 (95% CI –3.4 to –1.0) per pg/ml, p = 0.001), colonisation with Burkholderia cepacia (coefficient –46.8 (95% CI –75.5 to –18.1), p = 0.002), and annual change in BMI (coefficient 15.4 (95% CI 3.6 to 27.2) per kg/m2, p = 0.011) were independently significant predictors of annual change in BMC. Conclusions: These data suggest a pathophysiological mechanism by which chronic pulmonary infection results in bone loss in patients with cystic fibrosis.

Cathepsin S expression: An independent prognostic factor in glioblastoma tumours - A pilot Study

Cysteine proteinases have been implicated in astrocytoma invasion. We recently demonstrated that cathepsin S (CatS) expression is up‐regulated in astrocytomas and provided evidence for a potential role in astrocytoma invasion (Flannery et al., Am J Path 2003;163(1):175–82). We aimed to evaluate the significance of CatS in human astrocytoma progression and as a prognostic marker. Frozen tissue homogenates from 71 patients with astrocytomas and 3 normal brain specimens were subjected to ELISA analyses. Immunohistochemical analysis of CatS expression was performed on 126 paraffin‐embedded tumour samples. Fifty‐one astrocytoma cases were suitable for both frozen tissue and paraffin tissue analysis. ELISA revealed minimal expression of CatS in normal brain homogenates. CatS expression was increased in grade IV tumours whereas astrocytoma grades I–III exhibited lower values. Immunohistochemical analysis revealed a similar pattern of expression. Moreover, high‐CatS immunohistochemical scores in glioblastomas were associated with significantly shorter survival (10 vs. 5 months, p = 0.014). With forced inclusion of patient age, radiation dose and Karnofsky score in the Cox multivariate model, CatS score was found to be an independent predictor of survival. CatS expression in astrocytomas is associated with tumour progression and poor outcome in glioblastomas. CatS may serve as a useful prognostic indicator and potential target for anti‐invasive therapy.

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.

Detection of cathepsin S cysteine protease in human brain tumour microdialysates in vivo.

Microdialysis enables the chemistry of extracellular fluid in body tissues to be measured. Extracellular proteases such as the cysteine protease, cathepsin S (CatS), are thought to facilitate astrocytoma invasion. Microdialysates obtained from human brain tumours in vivo were subjected to cathepsin S activity and ELISA assays. Cathepsin S ELISA expression was detected in five out of 10 tumour microdialysates, while activity was detected in five out of 11 tumour microdialysates. Cathepsin S expression was also detected in microdialysate from the normal brain control although no activity was found in the same sample. While some refinements to the technique are necessary, the authors demonstrate the feasibility and safety of microdialysis in human astrocytomas in vivo. Characterisation of the extracellular environment of brain tumours in vivo using microdialysis may be a useful tool to identify the protease profile of brain tumours.

Binding and degradation of fibrinogen by Bacteroides fragilis and characterization of a 54 kDa fibrinogen-binding protein

Bacteroides fragilis is a bacterium that resides in the normal human gastro-intestinal tract; however, it is also the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses, and the most common cause of anaerobic bacteraemia. Abscess formation is important in bacterial containment, limiting dissemination of infection and bacteraemia. In this study, we investigated B. fragilis binding and degradation of human fibrinogen, the major structural component involved in fibrin abscess formation. We have shown that B. fragilis NCTC9343 binds human fibrinogen. A putative Bacteroides fragilis fibrinogen-binding protein, designated BF-FBP, identified in the genome sequence of NCTC9343, was cloned and expressed in Escherichia coli. The purified recombinant BF-FBP bound primarily to the human fibrinogen Bbeta-chain. In addition, we have identified fibrinogenolytic activity in B. fragilis exponential phase culture supernatants, associated with fibrinogenolytic metalloproteases in NCTC9343 and 638R, and cysteine protease activity in YCH46. All nine clinical isolates of B. fragilis examined degraded human fibrinogen; with eight isolates, initial Aalpha-chain degradation was observed, with varying Bbeta-chain and gamma-chain degradation. With one blood culture isolate, Bbeta-chain and gamma-chain degradation occurred first, followed by subsequent Aalpha-chain degradation. Our data raise the possibility that the fibrinogen-binding protein of B. fragilis, along with a variety of fibrinogenolytic proteases, may be an important virulence factor that facilitates dissemination of infection via reduction or inhibition of abscess formation.

Cystic fibrosis sputum stimulates CD18-independent neutrophil migration across endothelial cells.

Excessive neutrophil recruitment to the lung underlies inflammatory-mediated lung damage in cystic fibrosis (CF). Neutrophils can migrate to the lung using either a CD18-dependent or CD18-independent mechanism. To determine if one of these migratory pathways is preferentially utilized by neutrophils migrating to the CF airways, this study examined the CD18 dependency of neutrophil transendothelial migration stimulated by the soluble fraction of CF sputum (SOL). Results demonstrate the preferential use of the CD18-independent migratory mechanism by both control and CF neutrophils and suggest that selective blocking of the CD18-independent migration pathway may offer a means of decreasing neutrophil influx to the CF airways.

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.

Trypsin-like protease activity predicts disease severity and patient mortality in adults with cystic fibrosis

Introduction Serine trypsin-like (TL) proteases, which are excessively active in CF airways, promote activation of the epithelial sodium channel (ENaC) and airways dehydration; a key initiating factor for CF lung disease pathogenesis. Furthermore TL-proteases enhance mucin gene expression and mucus hypersecretion, yet whether there is any relationship between the activity of these enzymes and CF pulmonary disease is unknown. Objectives The primary objective of the current investigation was to determine whether TL-protease activity, measured in adult CF sputum sol, correlates with lung disease and patient outcome (survival). A secondary objective was to compare the strength of any relationships observed with that of neutrophil elastase (NE), an established protease biomarker. Methods In this cross sectional retrospective study we analysed CF sputum sol collected from 30 clinically stable adult CF patients. Protease activity was measured by monitoring the hydrolysis of peptide-based substrates. Biomarkers of inflammation (IL-8 and TNF-α) were measured by ELISA. Lung function was assessed by spirometry (FEV1). Mortality data was retrospectively obtained and time in months until death or transplantation used for subsequent survival analysis. Results TL-protease activity inversely correlated with lung function (FEV1) (r = −0.4, p = 0.031) however, no relationship with IL-8 and TNFα was observed. In contrast, NE was found to correlate with IL-8: r = 0.7, p < 0.001 and TNFα: r = 0.7, p < 0.001 but showed no relationship with lung function, indicating that these serine proteases play very distinct roles within the disease process. Kaplan-Meier analysis demonstrated significantly reduced survival for those individuals with above median TL-protease activity. Levels of NE activity showed no relationship with patient survival. Using a multivariate Cox regression analysis (adjusted for age and BMI) a significantly increased mortality hazard (HR 1.028, 95% CI: 1.007–1.049; p = 0.009) was also identified. These findings are supported by analysis of a validation cohort consisting of samples collected from a separate cohort of 33 adult CF patients. Conclusions TL-protease activity inversely correlates with lung function and patient survival. As such tryptic activity may warrant consideration when modelling CF survivorship and should be investigated further as a biomarker of CF lung disease and as a potential therapeutic target.

How specific are fluorogenic substrates designed to analyse active protease biomarkers of respiratory disease

Introduction Active proteases, such as neutrophil elastase (NE) and matrix metalloproteinases (MMPs), have been established as inflammatory biomarkers in lung diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Therefore, biological samples collected during clinical investigations are often analysed using fluorogenic substrates to determine protease activity and identify correlations with clinical and/or demographic parameters. Due to the nature of these diseases, samples often contain numerous active proteases from both human and bacterial origins which collectively have significant substrate crossover. This study investigates the ability of fluorogenic substrates to distinguish between proteases in complex clinical samples and provide an indication of the predictive capability of this assay type. Methods Expectorated sputum was randomly collected from patients with CF who were hospitalised for an acute exacerbation. Samples were processed within 30 minutes of collection, and the aqueous sol recovered, pooled, aliquoted and stored at −80°C until analysis. The capacity of sputum proteases to hydrolyse fluorogenic substrates with and without the presence of inhibitors specific for serine (multiple subclasses), metallo and cysteine proteases was examined. Fluorogenic substrates analysed include those for various inflammatory proteases including elastase-like (MeOSuc-AAPV-AMC), MMPs (MCA-PLGL-Dpa-AR-NH2), trypsin-like (Z-GGR-AMC) and chymotrypsin-like (Suc-AAPF-AMC). Substrate hydrolysis by a relevant recombinant enzyme (± inhibitors) was analysed as a control. Results Data analysis indicates that alternative enzymes actively hydrolyse substrates designed to be specific for one group of proteases. Inhibitors specific for metallo, trypsin-like, chymotrypsin-like and cysteine proteases all decreased elastase-like substrate turnover (∼10–50%). A similar trend was seen for chymotrypsin-like substrate using metallo, trypsin-like and elastase-like protease inhibitors (∼10–40%). Conclusion This investigation has suggested that there is significant non-specific hydrolysis and cross-reactivity when fluorogenic substrates are utilised to measure active proteases in complex biological samples. Thus, using such fluorogenic substrates may produce elevated readings, impacting on the accuracy of results when such assays are used for clinical or research purposes.