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PubMed1999

Markers of nitric oxide metabolism in sputum and exhaled air are not increased in chronic obstructive pulmonary disease.

BACKGROUND: Nitric oxide (NO) is involved in inflammation and host defence of the lung. It has been found in increased concentrations in the airways in asthmatic subjects but its levels in patients with chronic obstructive pulmonary disease (COPD) have not been investigated. A study was undertaken to determine whether markers of NO metabolism (NO in exhaled air, iNOS expression in sputum cells, and nitrite + nitrate (NO2-/NO3-) in sputum supernatant) are increased in subjects with COPD, and whether they correlate with inflammatory indices in induced sputum. The associations of these markers with smoking were also assessed. METHODS: Sixteen subjects with COPD (median age 66 years, median forced expiratory volume in one second (FEV1) 63% predicted, eight current smokers) and 16 healthy subjects (median age 63 years, median FEV1 113% predicted, eight current smokers) participated in the study. NO was measured during tidal breathing and sputum was induced by inhalation of hypertonic saline. RESULTS: No differences were observed between subjects with COPD and healthy controls in exhaled NO excretion rate (median 5.15 and 6.25 nmol/min), sputum macrophage iNOS expression (14% and 12%), and sputum supernatant NO2-/NO3- (46 and 73 microM). NO in exhaled air correlated with the percentage of sputum eosinophils in patients with COPD (rho = 0.65, p = 0.009) but not in healthy individuals. Exhaled NO and supernatant NO2-/NO3- levels were lower in healthy smokers than in healthy non/ex-smokers. CONCLUSIONS: Our findings indicate that NO metabolism is not increased in patients with stable COPD. The close association between exhaled NO levels and sputum eosinophils suggests a role for NO in airway inflammation in COPD. Studies performed during exacerbations may clarify this role.

Aged↗
PubMed2005

Antioxidant properties of cystic fibrosis sputum.

Oxidative stress is a likely contributor to the pathogenesis of cystic fibrosis (CF) lung disease. However, hydrogen peroxide (H(2)O(2)), a physiological oxidant, is not elevated in CF exhalates. H(2)O(2) may be neutralized by antioxidants in CF airway secretions. The H(2)O(2)-detoxifying capacity of CF airway secretions, obtained via sputum induction, was studied in an in vitro H(2)O(2) cytotoxicity model. 16HBE14o- cells were exposed to H(2)O(2) in culture medium containing either 0 or 10% fetal bovine serum (FBS) or 10% CF sputum supernatant (extracted without use of dithiothreitol). The efficiency of H(2)O(2) neutralization was estimated by measuring intracellular oxidant levels (dihydrorhodamine 123) after 2 h and cell viability (propidium iodide) after 24 h of H(2)O(2) exposure. Furthermore, the presence of reduced thiols (DTNB assay) and reduced glutathione (recycling assay) in CF sputum samples was evaluated. CF sputum extracts completely prevented intracellular oxidant accumulation seen in cells incubated with H(2)O(2) in both control media (i.e., 0 or 10% FBS). Furthermore, CF sputum abolished cell death in 16HBE14o- cells exposed to up to 1 mM H(2)O(2). In contrast, there was 100% cytotoxicity in cells exposed to 600 microM H(2)O(2) in both control media. The H(2)O(2)-detoxifying potential of CF sputum was sustained after catalase and heme peroxidases were inactivated by sodium azide, which does not affect glutathione peroxidase. In addition, reduced protein thiols were found in abundance in CF sputum. In conclusion, CF sputum is capable to neutralize H(2)O(2) and abundant reduced thiols and/or glutathione peroxidase are fully sufficient to detoxify H(2)O(2).

Adult↗
PubMed2003

Thioredoxin liquefies and decreases the viscoelasticity of cystic fibrosis sputum.

The persistent and viscous nature of airway secretions in cystic fibrosis (CF) disease leads to airway obstruction, opportunistic infection, and deterioration of lung function. Thioredoxin (Trx) is a protein disulfide reductase that catalyzes numerous thiol-dependent cellular reductive processes. To determine whether Trx can alter the rheological properties of mucus, sputum obtained from CF patients was treated with TRX and its reducing system (0.1 microM thioredoxin reductase + 2 mM NADPH), and liquid phase-gel phase ratio (percent liquid phase) was assessed by compaction assay. Exposure to low Trx concentrations (1 microM) caused significant increases in the percentage of liquid phase of sputum. Maximal increases in percent liquid phase occurred with 30 microM Trx. Additional measurements revealed that sputum liquefaction by the Trx reducing system is dependent on NADPH concentration. The relative potency of the Trx reducing system also was compared with other disulfide-reducing agents. In contrast with Trx, glutathione and N-acetylcysteine were ineffective in liquefying sputum when used at concentrations <1 mM. Sputum viscoelasticity, measured by magnetic microrheometry, also was diminished significantly following 20-min treatment with 3, 10, or 30 microM Trx. Similarly, this reduction in viscoelasticty also was dependent on NADPH concentration. Further investigation has indicated that Trx treatment increases the solubility of high-molecular-weight glycoproteins and causes redistribution of extracellular DNA into the liquid phase of sputum. Recognizing that mucins are the major gel-forming glycoproteins in mucus, we suggest that Trx alters sputum rheology by enzymatic reduction of glycoprotein polymers present in sputum.

Adolescent↗
PubMedDate not supplied

Eotaxin level in induced sputum is increased in patients with bronchial asthma and in smokers.

BACKGROUND: Airway eosinophilia is one of the hallmarks of asthma. Eotaxin may play an important role in eosinophil recruitment. OBJECTIVES: To examine the relationship between eotaxin levels in the sputum and eosinophilic inflammation. METHODS: The sputum was obtained from 11 non-smokers, 14 smokers and 13 asthmatic patients using a sputum induction method. Eotaxin and interleukin (IL)-5 levels in the sputum were determined by ELISA and immunocytochemical analysis. RESULTS: Asthmatic patients had eosinophilia and smokers showed neutrophilia in their sputum. The eotaxin level in the sputum was significantly higher in smokers (median 412.5, range 91.1-872.2 pg/ml) and asthmatic patients (351.0, 185.0-928.0 pg/ml) compared with non-smokers (123.2, 0-369.0 pg/ml; both p < 0.05). IL-5 was detected in the sputum of 1 non-smoker, none of the smokers and 4 asthmatic patients. The percentage of eotaxin-positive cells was higher in smokers and asthmatic patients than in non-smokers, but the percentage of IL-5-positive cells was significantly higher only in asthmatic patients (p < 0.05). CONCLUSIONS: These findings suggest that the elevated eotaxin level in the sputum does not always accompany the increase in eosinophils, and cooperation with another cytokine such as IL-5 may be required for the recruitment of eosinophils.

Adult↗
PubMed1995

Eosinophil viability-enhancing activity in sputum from patients with bronchial asthma, Contributions of interleukin-5 and granulocyte/macrophage colony-stimulating factor.

To investigate the mechanisms of eosinophil activation in the airways of patients with asthma, we attempted to detect eosinophil-activating cytokines in sputum extracts obtained from asthmatic patients during acute attacks or in remission by eosinophil survival assay. Purified guinea pig eosinophils were cultured in the presence or absence of sputum extracts, and the eosinophil viability was measured on Day 4. Eosinophil viability in the presence of sputum extracts derived from patients during moderate or severe attacks was significantly higher than that for sputum obtained from patients in remission or during mild attacks or from those with other respiratory diseases, including bronchiectasis and diffuse panbronchiolitis (p < 0.05). The total symptom score during the week prior to sputum collection correlated with the eosinophil viability (rs = 0.79, p < 0.01). Eosinophil viability-enhancing activity (EVEA) in the sputum of asthmatic patients with moderate or severe attacks was neutralized by anti-IL-5 antibody and by anti-GM-CSF antibody by 19.9 +/- 13.7% and 76.9 +/- 8.2% (mean +/- SEM, n = 7), respectively. EVEA was completely neutralized by a combination of anti-IL-5 and anti-GM-CSF antibodies. There was a significant correlation between the concentration of eosinophil cationic protein (ECP) in sputum extracts and the eosinophil viability (rs = 0.54, p < 0.05). These findings suggest that IL-5 and GM-CSF are present in the sputum during asthma attacks and that these cytokines are at least partially responsible for eosinophil activation in asthma.

Animals↗
PubMed1996

Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements.

Methods to examine sputum for indices of airway inflammation are evolving. We have examined the repeatability and the validity of an improved method to measure sputum cells and fluid-phase eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin (EDN), albumin, fibrinogen, tryptase, and interleukin-5 (IL-5). Sputum was induced with hypertonic saline twice within 6 d in 10 healthy subjects, 19 stable asthmatics, and 10 smokers with nonobstructive bronchitis. The method included the processing of freshly expectorated sputum separated from saliva, treatment with a fixed proportion of dithiothreitol 0.1% followed by Dulbecco's phosphate-buffered saline, making cytospins, and collecting the supernatant. The reproducibility of measurements, calculated by the intraclass correlation coefficient, was high for all indices measured with the exception of total cell counts and proportion of lymphocytes. Asthmatics, in comparison with healthy subjects and smokers with bronchitis, had a higher proportion of sputum eosinophils (median percent 5.2 versus 0.5 and 0.3), metachromatic cells (0.3 versus 0.07 and 0.08), ECP (1,040 micrograms/L versus 288 and 352), MBP (1,176 micrograms/L versus 304 and 160), and EDN (1,512 micrograms/L versus 448 and 272). Asthmatics differed from healthy subjects, but not from smokers with bronchitis, in the proportion of neutrophils (46.9% versus 24.1%), albumin (704 versus 288 micrograms/mL), and fibrinogen (2,080 versus 440 ng/mL). Smokers with bronchitis showed a trend for a higher neutrophil count and levels of albumin and fibrinogen than healthy subjects. The proportion of sputum eosinophils correlated positively with ECP, MBP, EDN, albumin and fibrinogen levels, and metachromatic cell counts correlated with tryptase. In asthmatics, IL-5 correlated with eosinophil counts. There was a significant negative correlation between sputum indices and expiratory flows and methacholine PC20. Thus, the methods of measuring cell and fluid phase markers in induced sputum used in this study are reproducible and valid. They can therefore be used to reliably measure these indices of airway inflammation.

Adult↗
PubMed1997

Cell infiltration, ICAM-1 expression, and eosinophil chemotactic activity in asthmatic sputum.

We have applied the technique of sputum induction by hypertonic saline in asthmatics and nonatopic control subjects to study an array of indices of airway inflammation believed to be relevant to asthma pathogenesis. Compatible with a central role for eosinophils and mast cells in asthma, sputum of asthmatic subjects contained increased numbers of eosinophils and levels of eosinophil cationic protein (ECP) and mast cell tryptase. Eosinophil numbers, and ECP and histamine levels correlated with the degree of methacholine airways responsiveness, and ECP, tryptase, and histamine correlated with raised concentrations of albumin. Using the micro-Boyden chamber technique eosinophil chemotactic activity was identified only in the sputum from asthmatics. The correlation between the raised levels of total IgA, IL-8/IgA complexes, and tryptase and the degree of sputum eosinophilia and ECP levels, suggests possible mechanisms for eosinophil chemotaxis and activation in asthma. Row cytometric analysis of sputum lymphocytes showed an increase in CD4+ T cells and T cells expressing intercellular adhesion molecule-1 (ICAM-1) in asthma which, together with the finding of raised levels of soluble ICAM-1 in the sputum, indicates upregulation of this adhesion molecule. Finally, the proportion of CD16+ natural killer (NK) cells was reduced in the sputum of asthmatics. These observations highlight the importance of the airway inflammation in causing asthma and further confirm the usefulness of sputum induction as a tool in asthma research.

Adult↗
PubMed1997

Rheology of cystic fibrosis sputum after in vitro treatment with hypertonic saline alone and in combination with recombinant human deoxyribonuclease I.

Treatment with recombinant human deoxyribonuclease I (rhDNase) is currently used as therapy for cystic fibrosis (CF) lung disease. Hypertonic saline (HS) acts as an expectorant promoting mucus secretion and augmenting the volume of sputum. We evaluated the individual and combined effects of HS and rhDNase in vitro on the viscoelasticity of CF sputum. Sputum samples were collected from nine CF patients to use for in vitro testing. Aliquots of CF sputum (0.20 to 0.40 g) were subjected to the following protocols: (1) negative control sample without any treatment; (2) positive control sample, adding 10% volume of normal saline (0.9% NaCl); (3) application of hypertonic saline (HS-3% NaCl); (4) combining approximately 100 nM concentration of rhDNase with protocols 2 and 3. The samples in protocols 2 through 4 were incubated for 30 min at 37 degrees C. For each protocol, CF sputum was analyzed at baseline and at 30 min for spinnability by filancemeter and viscoelasticity by magnetic microrheometry. Spinnability decreased for the sputum samples that were treated with rhDNase, in combination with either HS or normal saline. Treatment with HS alone and combined treatment with rhDNase and HS decreased log G* (the principal viscoelasticity index) to the same degree. Saline alone and rhDNase in normal saline both increased the predicted cough clearability of the sputum; however, the combined treatment with rhDNase and hypertonic saline had the best overall effect on cough clearability. The change in predicted mucociliary clearability, although greatest after HS, was not significant. These in vitro results suggest that combined treatment with rhDNase and HS should be evaluated further as a potential mucotropic approach to augment the clearance of purulent sputum in CF lung disease.

Adolescent↗
PubMed1999

Induced sputum eicosanoid concentrations in asthma.

Further definition of the role of leukotrienes (LT) and prostaglandins (PG) in asthma would be helped by a noninvasive method for assessing airway production. The supernatant from sputum induced with hypertonic saline and dispersed using dithiotrietol has been successfully used to measure other molecular markers of airway inflammation and might be a useful method. We have measured induced sputum supernatant LTC(4)/D(4)/E(4) concentrations using enzyme immunoassay and PGE(2), PGD(2), TXB(2), and PGF(2alpha) using gas chromatography-negative ion chemical ionization-mass spectroscopy in 10 normal subjects and in 26 subjects with asthma of variable severity. Sputum cysteinyl-leukotrienes concentrations were significantly greater in subjects with asthma (median, 9.5 ng/ml) than in normal control subjects (6.4 ng/ml; p < 0.02) and greater in subjects with persistent asthma requiring inhaled corticosteroids (median, 11.4 ng/ml) or studied within 48 h of an acute severe exacerbation of asthma (13 ng/ml) than in subjects with episodic asthma treated with inhaled beta(2)-agonists only (7.2 ng/ml). There were no significant differences in the concentrations of other eicosanoids between groups, although there was a negative correlation between the percentage sputum eosinophil count and sputum PGE(2) concentration (r = -0.48; p < 0.01) in subjects with asthma. We conclude that induced sputum contains high concentrations of eicosanoids and that sputum LTC(4)/D(4)/E(4) concentrations are significantly greater in subjects with asthma than in normal subjects. The inverse relationship between eosinophilic airway inflammation and sputum PGE(2) concentration would be consistent, with the latter having an anti-inflammatory role.

Adult↗
PubMed2001

Induced sputum derives from the central airways: confirmation using a radiolabeled aerosol bolus delivery technique.

Indirect evidence suggests that induced sputum derives from the surfaces of the bronchial airways. To confirm this experimentally, we employed a radiolabeled aerosol bolus delivery technique that preferentially deposits aerosol in the central airways in humans. We hypothesized that there would be significantly more radioactivity recovered in an induced sputum sample, and greater airways clearance of radiolabeled particles, immediately after a central versus peripheral airways deposition. Ten healthy volunteers underwent radiolabeled aerosol deposition ((99m)Tc sulfur colloid particles) to the central and peripheral airways on separate occasions followed immediately by induced sputum or no sputum (control), while seated in front of a gamma camera. Radioactivity was measured in the selected sputum sample, processed cell pellet, and supernatant fraction. Significantly more radioactivity was present in all portions of the sputum sample after central versus peripheral airways deposition (i.e., selected sample: 15,607 counts +/- 2,985 versus 943 counts +/- 298, p = 0.001). Clearance from the whole lung was significantly greater 40 min after central versus peripheral airways deposition (48 +/- 3% versus 5 +/- 1%, p = 0.0001). Compared with control, induced sputum greatly enhanced clearance after central deposition (48 +/- 3% versus 11 +/- 6%, p = 0.0001), but not after peripheral deposition (5 +/- 1% versus 3 +/- 0.8%). These results provide direct evidence that induced sputum derives from the central airways with little or no contribution from the peripheral airways.

Administration, Inhalation↗
PubMed2001

Validity and safety of sputum induction by inhaled uridine 5'-triphosphate.

UNLABELLED: Inhalation of hypertonic saline during sputum induction causes bronchoconstriction. We studied the validity and safety of sputum induction by uridine 5'-triphosphate (UTP). Sputum was induced by a 5-min inhalation of hypertonic saline (3%) on Day 1 and UTP (5 mg/ml in 0.9% saline) on Days 8 and 15 in 16 healthy subjects and 16 patients with mild-to-moderate asthma. Inhaled UTP produced twofold greater amounts of sputum than did hypertonic saline. There were significant differences in oxygen desaturation and bronchoconstriction during the procedure between the two methods: the maximal fall in Sa(O(2)), the AUC of the Sa(O(2))-time response, and the fall in PEF were less in the subjects who received UTP than in those who received hypertonic saline. Sputum total cell and differential cell counts, with a high proportion of eosinophils in asthmatics, were similar between specimens obtained by hypertonic saline and UTP. When we compared two consecutive measurements on the UTP-induced sputum samples, the reproducibility calculated by the intraclass correlation coefficient was high for the proportion of eosinophils, neutrophils, and macrophages. Therefore, inhalation of UTP aerosols may provide an effective, relatively noninvasive, valid, and reproducible method of sputum induction for the assessment of airway inflammation in asthma. KEYWORDS: uridine triphosphate; induced sputum; airway inflammation; bronchoconstriction; asthma

Administration, Inhalation↗
PubMed1977

Detection of penumococcal antigens in the sputum in pneumococcal pneumonia.

Forty-seven patients with bacterial pneumonia were grouped by use of clinical criteria according to the relative certainty of a diagnosis of pneumococcal pneumonia. Sputums were tested for pneumococcal antigens by counterimmunoelectrophoresis with polyvalent pneumococcal antiserum. Antigens were detected in the sputum of 29 of 39 patients with evidence of pneumococcal pneumonia, and there was good correlation between the detection of antigens and the degree of certainty of the clinical diagnosis. Antigens persisted briefly in the sputum during therapy with antimicrobial drugs and could be detected during the first 48 hours of therapy in most cases of pneumococcal pneumonia. Pneumococci were isolated from the sputum in only 18 of 39 cases of pneumococcal pneumonia, and sputum cultures did not correlate as well as counterimmunoelectrophoresis with clinical diagnoses. In studies of 27 patients with chronic bronchitis without pneumonia, pneumococci were isolated from sputum in 10 cases, whereas counterimmunoelectrophoresis was positive in 5 cases. Counterimmunoelectrophoresis provides a simple and rapid method for detecting pneumococcal antigens in sputum, and it appears to be more reliable than sputum cultures in establishing a presumptive diagnosis in pneumococcal pneumonia.

Adult↗
PubMed1986

Sputum examination for the diagnosis of Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome.

The diagnostic utility of sputum examination in patients with Pneumocystis carinii pneumonia secondary to the acquired immunodeficiency syndrome (AIDS) has so far not been determined. Sputum was induced in 43 patients with AIDS or suspected AIDS just prior to fiberoptic bronchoscopy, scheduled because of an unexplained pulmonary infiltrate on a chest radiograph. Pneumocystis carinii pneumonia was diagnosed by sputum examination and/or by a bronchoscopic procedure in 20 patients. Of these, sputum samples were positive for Pneumocystis organisms in 11 (55%) of 20 patients tested, bronchial washings were positive in 11 (79%) of 14 patients tested, brush biopsies were positive in 9 (53%) of 17 patients tested, and transbronchial lung biopsies were positive in 18 (90%) of 20 patients tested. The presence of P. carinii cysts in sputum did not correlate with the presence of alveolar macrophages in sputum nor with the volume of sputum. Sputum examination for P. carinii organisms, employed as a first diagnostic step in patients with AIDS with pulmonary infiltrates, may frequently obviate the need for bronchoscopy.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Local and systemic inflammation in patients with chronic obstructive pulmonary disease: soluble tumor necrosis factor receptors are increased in sputum.

Chronic obstructive pulmonary disease (COPD) is characterized by significant chronic inflammation in the pulmonary compartment as well as in the circulation. This study aimed to elucidate the relationship between local and systemic inflammation in smoking-induced COPD by assessing levels of soluble (s) tumor necrosis factor (TNF) receptors, TNF-alpha, and interleukin-8 (IL-8) in induced sputum and in plasma. Sputum induction was performed in 18 subjects with COPD (FEV(1) 56% predicted) and 17 healthy smokers (FEV(1) 99% predicted). Patients with COPD showed significantly higher percentages of neutrophils and levels of sTNF-R55 and IL-8 in sputum as compared with control subjects, whereas sputum sTNF-R75 levels tended to be higher in COPD. Sputum TNF-alpha levels were similar in both groups. When comparing sTNF receptors in sputum and plasma, no direct correlations were found despite elevation of circulating sTNF-R75 levels in patients with COPD. In addition, sputum sTNF receptors were inversely related to the FEV(1) in patients with COPD, whereas circulating sTNF receptors were not, suggesting different regulation of inflammation in the pulmonary and systemic compartment. When subjects were divided according to their current smoking status, levels of sTNF-R55, sTNF-R75, and IL-8 in sputum were significantly elevated in ex-smoking versus currently smoking patients with COPD, suggesting ongoing inflammation in airways and circulation of patients with COPD after smoking cessation.

Aged↗
PubMed2003

Cellular profiles of induced sputum in children with stable cystic fibrosis: comparison with BAL.

Neutrophil-dominated endobronchial inflammation is a major characteristic of cystic fibrosis (CF) and there is increasing demand for easy-to-perform noninvasive monitoring for prediction and intervention. Fourteen stable paediatric CF patients (8-17 yrs; mean forced expiratory volume in one second 86.7% of the predicted value) were investigated once by fractional bronchoalveolar lavage (BAL) and by sputum induction on three occasions, 2-6 weeks apart. Sputum was induced by consecutive 10-min inhalations of 3, 4 and 5% saline. CF sputum cellular profiles were compared with BAL fluid cell counts and samples from age-matched healthy children, and between different time points to assess reproducibility. Adequate sputum was recovered on >95% of occasions. In all sputum fractions, CF patients showed higher neutrophil counts than healthy children. Neutrophil percentages were highest in the first BAL fraction (median 92%), followed by sputum, in which the percentages decreased in consecutive fractions (72, 66 and 64%), whereas counts were lowest in the pooled BAL fraction (53%). Increasing percentages of macrophages mirrored the decreases in neutrophil percentage. Results of sputum induction at different time points in the CF patients showed good reproducibility and nonoverlap with counts from healthy children. In conclusion, the results of sputum induction in children with mild stable cystic fibrosis adequately describe airway inflammation by providing cellular profiles with lower relative neutrophil counts than in the first ("bronchial") bronchoalveolar lavage fraction and higher relative neutrophil counts than in subsequent pooled ("more peripheral") bronchoalveolar lavage fractions.

Adolescent↗
PubMed2003

Reduced eosinophil apoptosis in induced sputum correlates with asthma severity.

This study was carried out to investigate the relationship between induced sputum eosinophil apoptosis and clinical severity score, airway obstruction and symptom scores in patients with chronic stable asthma. Altogether, 41 chronic stable asthmatic subjects of varying severity defined by Aas score and 17 control subjects underwent spirometry, symptom questionnaire and successful sputum induction. Sputum was processed and cytospins prepared for light microscopy to determine normal and apoptotic eosinophils. Mild asthmatic subjects had a significantly lower percentage sputum eosinophils and a significantly higher eosinophil apoptotic ratio (AR) than moderate or chronic severe asthmatics. Severe asthmatic subjects had a significantly greater age, duration of asthma and sputum eosinophil count x mL(-1) than mild asthmatic subjects. Asthmatic subjects' symptom scores, severity scores and age inversely correlated with AR and the percentage of sputum eosinophils. Baseline forced expiratory volume in one second inversely correlated with percentage sputum eosinophils and positively correlated with AR. The study demonstrates a relationship between reduced sputum eosinophil apoptosis and increased clinical severity of chronic stable asthma, providing additional evidence that eosinophil apoptosis may be important in the resolution of eosinophilic airway inflammation in asthma.

Adult↗
PubMed2004

Effect of saliva contamination on induced sputum cell counts, IL-8 and eosinophil cationic protein levels.

Excessive salivary contamination of induced sputum samples prevents the satisfactory examination of lower airway inflammation. The effects of salivary contamination on different sputum fluid phase measures and the levels of salivary contamination preventing analysis are not defined. The present study sought to examine this by investigating the effect of increasing salivary contamination on induced sputum samples. Sputum and saliva samples from subjects with asthma and healthy controls were collected, and treated with dithiothreitol (DTT). Saliva was then added to aliquots of dispersed sputum in increasing proportions (0% to 100%). The effect of increasing saliva contamination was assessed on sputum total cell count, viability, differential cell count and fluid phase levels of interleukin (IL)-8, eosinophil cationic protein (ECP) and total protein. The addition of saliva to induced sputum reduced total cell counts and absolute cell counts but did not change the differential cell count. Levels of fluid phase ECP and IL-8 were significantly reduced with increased salivary contamination. There was a progressive reduction in ECP and IL-8, which reached significance at 70% and 80% saliva contamination, respectively. IL-8 levels corrected for total protein showed no change with increasing saliva concentrations. Induced sputum differential cell counts expressed as the proportion of nonsquamous cells are robust measures that are not influenced by salivary contamination. Studies reporting total and absolute cell counts and fluid phase mediator levels require control for squamous contamination.

Adult↗
PubMed1997

Do sputum eosinophils and ECP relate to the severity of asthma?

There is much evidence that eosinophils play an important role in bronchial epithelial damage in asthma by releasing cationic proteins. However, the extent to which eosinophil inflammation relates to indices of asthma severity in chronic stable asthma is still a matter of debate. We studied 46 clinically stable patients with mild to severe chronic asthma (forced expiratory volume in one second (FEV1) 50-126% of predicted value). The clinical severity of asthma was graded from 1 to 4 according to the Aas scoring system. Twelve normal subjects were also studied as controls. Induction of sputum was performed by hypertonic saline to determine differential cell count, and eosinophil cationic protein (ECP) by the so-called "plug technique". The concentration of ECP was measured by a fluoroimmunoassay. Bronchial hyperresponsiveness was recorded by inhaling progressive concentrations of histamine, and the concentration that caused a 20% decrease in FEV1 (PC20) was calculated. Sputum eosinophils (range 0-61%), sputum ECP (range 24-10,800 microg x L[-1]) and serum ECP (range 4-61 microg x L[-1]) were significantly greater in asthmatics than in normal subjects, and distinguished the most severe group with the highest Aas score from the others. Sputum eosinophils and sputum ECP were strongly related to each other. The relationships between sputum or serum ECP and PC20 (range 0.016-7.5 mg x mL[-1]), and between sputum ECP and FEV1 were found to be weak. In conclusion, sputum outcomes of eosinophil activation and serum eosinophilic cationic protein appear to be useful indicators of disease. They do not accurately reflect current clinical or functional indices of asthma severity in chronic stable patients, and might therefore provide complementary data disease monitoring.

Adolescent↗