Effect of Physical and Chemical Methods of Homogenization on Inflammatory Mediators in Sputum of Asthma Patients

Grebski, Elzbieta; Peterson, Christer; Tc, Medici

doi:10.1378/chest.119.5.1521

Cited by 29 publications

(28 citation statements)

References 15 publications

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“…Compared to other mediators, MPO concentration is greatly affected by the method used for its detection or for sputum sample processing [24][25][26], and sputum MPO concentrations have been reported to be extremely variable in different studies, with a large standard deviation [27]. In the samples in this study, an acceptable interassay coefficient of variation and a fairly good recovery of MPO were found, which, however, was lower in sputum induced by HS than in sputum induced by IS inhalation.…”

Section: Discussionmentioning

confidence: 54%

Granulocyte markers in hypertonic and isotonic saline-induced sputum of asthmatic subjects

Cianchetti

Bacci²,

Ruocco³

et al. 2004

European Respiratory Journal

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The aim of this study was to assess whether hyperosmolarity affects granulocyte mediator levels in induced sputum of asthmatic subjects.A total of 32 mild-to-moderate asthmatics, who inhaled either hypertonic (HS; 4.5% NaCl) or isotonic (IS; 0.9% NaCl) solutions for 15 min, were studied. Selected sputum was used for analysis. Eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO) and free neutrophil elastase (NE) were measured in sputum supernatant.Sample weight, total and differential cell counts, as well as viability and squamous cell percentage were no different after the two tests. No significant differences in ECP, EPX, MPO or NE levels were observed between HS-and IS-induced sputum. Repeatability of the two tests was good for macrophages, neutrophils, eosinophils, ECP, EPX and NE, but not for lymphocytes and MPO.In conclusion, hyperosmolarity does not affect sputum cell counts and the levels of most granulocyte degranulation markers examined in this study, confirming that both hypertonic and isotonic solutions can be reliably used to induce sputum in asthmatics. Hypertonic saline (HS) inhalation is currently used to collect sputum from the airways of asthmatic subjects who do not produce sputum spontaneously. However, in high-risk subjects, inhalation with isotonic saline (IS) is recommended, at least at the beginning of sputum induction [1]. Thus, it is important that sputum samples collected after either induction method have a similar composition. Several studies have compared the cell composition of sputum obtained after the two different methods of induction and showed good concordance for inflammatory cell percentages [2-4]; however, few studies have compared the concentrations of soluble mediators in the sputum supernatant [5].A large number of in vivo and in vitro studies have shown that hypertonic solutions modify mediator release from inflammatory cells by acting as a strong stimulus for cell activation [6-8], or, conversely, by inhibiting leukocyte degranulation [5,9]. In sputum induction, the hypertonic solution might increase osmolarity of the airway lining fluid, thus influencing mediator release from granulocytes during inhalation or sputum processing, or both.Therefore, the aim of this study was to compare preformed soluble mediator levels derived from eosinophils and neutrophils, such as eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO) and neutrophil elastase (NE), in induced sputum obtained by means of either HS or IS inhalation in mild-to-moderate asthmatic subjects. In addition, the total and differential cell counts were also evaluated in the same sputum samples. Subjects and methods SubjectsA total of 44 mild-to-moderate asthmatic subjects, who were recruited from the authors9 asthma clinic, were examined. Asthma was diagnosed at the time of the first examination, according to internationally accepted criteria [10], after assessing reversible airway obstruction and/or nonspecific bronchial hyperresponsiveness ...

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Section: Discussionmentioning

confidence: 54%

Granulocyte markers in hypertonic and isotonic saline-induced sputum of asthmatic subjects

Cianchetti

Bacci²,

Ruocco³

et al. 2004

European Respiratory Journal

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“…To assess the impact of sputum on the antibacterial activity of NB-401, we repeated the standard planktonic testing for the 12 biofilm-forming strains in the presence of CF sputum. We used a mixture of sputum from 15 CF patients to avoid interpatient variation in macromolecule and high-molecular-weight DNA compositions and in ionic conditions and applied mechanical shearing only to minimize changes to the native microenvironment (22). The activity of NB-401 against VOL.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Activities of a Novel Nanoemulsion against Burkholderia and Other Multidrug-Resistant Cystic Fibrosis-Associated Bacterial Species

LiPuma¹,

Rathinavelu²,

Foster³

et al. 2009

Antimicrob Agents Chemother

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Respiratory tract infection, most often involving opportunistic bacterial species with broad-spectrum antibiotic resistance, is the primary cause of death in persons with cystic fibrosis (CF). Species within the Burkholderia cepacia complex are especially problematic in this patient population. We investigated a novel surfactant-stabilized oil-in-water nanoemulsion (NB-401) for activity against 150 bacterial isolates recovered primarily from CF respiratory tract specimens. These specimens included 75 Burkholderia isolates and 75 isolates belonging to other CF-relevant species including Pseudomonas, Achromobacter, Pandoraea, Ralstonia, Stenotrophomonas, and Acinetobacter. Nearly one-third of the isolates were multidrug resistant, and 20 (13%) were panresistant based on standard antibiotic testing. All isolates belonging to the same species were genotyped to ensure that each isolate was a distinct strain. The MIC 90 of NB-401 was 125 g/ml. We found no decrease in activity against multidrug-resistant or panresistant strains. MBC testing showed no evidence of tolerance to NB-401. We investigated the activity of NB-401 against a subset of strains grown as a biofilm and against planktonic strains in the presence of CF sputum. Although the activity of NB-401 was decreased under both conditions, the nanoemulsion remained bactericidal for all strains tested. These results support NB-401's potential role as a novel antimicrobial agent for the treatment of infection due to CF-related opportunistic pathogens.

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“…In experiments using ultrasonication to disperse sputum followed by addition of DTT (final concentration 5 mM) to aliquots of sputum, DTT has been found to have no effect on ECP or eosinophil protein X (EPX) level but reduces recovery of eosinophil peroxidase and MPO [105]. In spiking studies, DTT, when added for sputum processing, does not interfere with measurement of ECP, EPX, IL-8, tryptase or immunoglobulin A [2,9].…”

Section: Factors Specific To the Soluble Mediatormentioning

confidence: 99%

“…Physical methods of dispersal using ultracentrifugation [8], glass homogenisation [33] or ultrasonication [105] may be used. Ultracentrifugation, with separation into a sol and gel phase, may exclude mediators that remain bound to the gel phase [109], and mediators may "stick" to glass beads.…”

Section: Factors Specific To the Soluble Mediatormentioning

confidence: 99%

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Analysis of fluid phase mediators

Keatings

Leigh²,

Peterson³

et al. 2002

European Respiratory Journal

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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...

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Effect of Physical and Chemical Methods of Homogenization on Inflammatory Mediators in Sputum of Asthma Patients

Cited by 29 publications

References 15 publications

Granulocyte markers in hypertonic and isotonic saline-induced sputum of asthmatic subjects

Granulocyte markers in hypertonic and isotonic saline-induced sputum of asthmatic subjects

In Vitro Activities of a Novel Nanoemulsion against Burkholderia and Other Multidrug-Resistant Cystic Fibrosis-Associated Bacterial Species

Analysis of fluid phase mediators

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