Increased detection of interleukin-;5 in sputum by addition of protease inhibitors

Kelly, Margaret M.; Leigh, Richard; Carruthers, Stacey; Horsewood, Peter; Gleich, Gerald J.; Hargreave, Frederick E.; Cox, Gerard

doi:10.1183/09031936.01.00098501

Cited by 31 publications

(25 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have investigated whether and how DTE might interfere with the detection of eotaxin in sputum when using a commercial immunoassay. As a further step in validating the method, and after a report that proteases present in the airway lining fluid might have an effect on the stability and therefore detection of mediators such as IL-5, 13 we have addressed the question of whether there would be any benefit from adding protease inhibitors (PIs) to sputum processed with PBS. Finally, we have examined the effect of high-speed centrifugation during processing with respect to effects on cell contamination and eotaxin levels.…”

mentioning

confidence: 99%

Measurement of eotaxin (CCL11) in induced sputum supernatants: Validation and detection in asthma

Hadjicharalambous

Dent

May

et al. 2004

Journal of Allergy and Clinical Immunology

View full text Add to dashboard Cite

mentioning

confidence: 99%

Measurement of eotaxin (CCL11) in induced sputum supernatants: Validation and detection in asthma

Hadjicharalambous

Dent

May

et al. 2004

Journal of Allergy and Clinical Immunology

View full text Add to dashboard Cite

“…(The addition of CTAB is not recommended if other mediators are to be measured.) Processing sputum after addition of protease inhibitors has been shown to improve measurement of IL-5 (see appendix) [114].…”

Section: Factors Specific To the Soluble Mediatormentioning

confidence: 99%

“…STOCKLEY and BAYLEY [21] found that the addition of elastase to sputum resulted in decreased levels of secretory leucocyte protease inhibitor and MPO by immunoassay. The addition of protease inhibitors to sputum before processing has resulted in increased rates of detection and levels of IL-5, suggesting that proteases are interfering substances in some cases [114].…”

Section: What To Do If Validity Is Poormentioning

confidence: 99%

Analysis of fluid phase mediators

Keatings

Leigh²,

Peterson³

et al. 2002

European Respiratory Journal

Self Cite

View full text Add to dashboard Cite

Sputum cellular indices are valid, reliable and responsive to change [1][2][3][4][5][6][7]. Increasingly, numerous inflammatory mediators are being measured in the fluid phase of sputum; these include cytokines, chemokines, granulocyte proteins, markers of vascular leakage, eicosanoids, proteases and others. Many of these mediators are included in table 1, which gives details of methods used by investigators to process sputum and measure mediator levels. The table also provides the median/mean levels measured in studied subject groups to give an indication of the expected levels of these mediators in sputum. However, the reproducibility, precision and validity of many of these measurements in sputum have not been investigated and, therefore, their utility as a research and clinical tool remains uncertain and requires confirmation.The following issues are important in the analysis of mediators: 1) choice of methods for measuring fluidphase mediators; 2) points to consider when planning an immunoassay; and 3) evaluation of the measurement of a soluble mediator in sputum, i.e. validation of the measurement method. Methods for measuring fluid-phase mediatorsThe three main types of method used for the measurement of sputum fluid-phase mediators are bioassays, enzyme assays and immunoassays. BioassaysBioassays rely on the retention of biological activity and the ability to exert a measurable effect, such as proliferation of cells, bone marrow colony formation or chemotaxis [3,11,58]. Sputum processing with mucolytics such as dithiothreitol (DTT) or dithioerythritol, which are strong reducing agents, may decrease the biological activity of cytokines, many of which rely on disulphide bonds to provide a stable structure for bioactivity. Bioassays also have the disadvantage of being inconvenient, time-consuming and lacking in specificity [59]. In addition, the presence of commonly occurring endogenous cytokine inhibitors, although allowing an estimate of net activity, may result in significant underestimation of total cytokine levels [58]. Enzyme assaysMany of the mediators of the inflammatory response are enzymes, released from cellular sites of synthesis into an environment replete with enzyme inhibitors. Again, an estimate of net activity is important when evaluating their potential contribution to tissue responses. The proteases neutrophil elastase [20,22,60,61], cathepsin G [20, 61] and cathepsin B [22] have been assayed in sputum using specific chromogenic substrates, from which proteases release a coloured product that can be quantified spectrophotometrically. Net protease activity is determined using purified enzyme standards. Active forms of matrix metalloproteinases in sputum are identified by means of substrate gel zymography, and net activity can be quantified using radiolabelled substrates [62]. Other proteases, such as chymase and tryptase, are less robust. Their activity rapidly diminishes on freezing/thawing sputum samples, and immunoassays are the best means of detecting them (see below).The activity of e...

show abstract

“…Several research groups have investigated modified sputum processing techniques to optimize biomarker recovery [66][67][68]. However, these processing techniques are not fully validated and most of them prevented recovery of other mediators from the samples.…”

Section: Biomarkers In Fluid Phasementioning

confidence: 99%