Induced Sputum Differential Cell Counts Research Articles

Proinflammatory pattern in the lower airways of non-asthmatic obese adolescents

BackgroundObesity is known to be a pro-inflammatory condition affecting multiple organs. Obesity as a systemic pro-inflammatory state, might be associated with bronchial inflammation in non-smoking adolescents with a BMI ≥ 30 kg/m2 without evidence of concomitant chronic diseases. Materials and methodsWe studied non-asthmatic obese patients (n = 20; median age 15.8 years; BMI 35.0 kg/m2) compared to age matched healthy control subjects (n = 20; median age 17.5 years; BMI 21.5 kg/m2). Induced sputum differential cell counts and sputum mRNA levels were assessed for all study subjects. Serum levels of CRP, IL-6, and IL-8 were measured. Further, IL-5, IL-6, IL-8, IL-13, IL-17, TNF-α, IFN-γ, and IP-10 protein levels were analyzed in induced sputum was. ResultsSerum CRP levels, sputum inflammatory cell load and sputum eosinophils differed significantly between obese and non-obese subjects, for sputum neutrophils, a correlation was shown with BMI ≥ 30 kg/m2. Differences were also observed for sputum mRNA expression of IL6, IL8, IL13, IL17, IL23, and IFN-γ, as well as the transcription factors T-bet, GATA3, and FoxP3. ConclusionsIncreased bronchial inflammation, triggered by systemic or local inflammatory effects of obesity itself, may account for the higher rates of airway disease in obese adolescents.

HO-1 protects smokers exposed to artificial stone dust for pulmonary function tests deterioration.

Background: The Heme Oxygenase system, along with its catabolism products, is involved in a variety of crucial physiological functions, including cytoprotection, inflammation, anti-oxidative effects, apoptosis, angiogenesis, and vascular regulation. Objectives: To analyze the Heme Oxygenase -1 (HO-1) mediated effect of mild deterioration of pulmonary function testing (PFT) in exposed artificial stone smoking workers. Methods: One hundred stone workers divided into current smokers, ex-smokers and never smokers underwent Low Resolution Computed Tomography, PFT, induced sputum (IS) Particle Size Distribution (PSD) and Real Time PCR in IS samples. Results: Smoking status had no significant effect on PFT results but it altered the IS differential cell counts. There was significantly less decline in PFT over time for the smokers group. There was a significantly lower fraction of small particles (<2 μm) in the IS of the current smokers group compared to the never- and ex-smokers groups. HO-1 gene expression was higher among smokers compared to never- and ex-smokers groups. A low percentage of small particles (<5 μm) correlated negatively to the percentage of neutrophils and positively to the percentage of macrophages in the sputum of the smokers group. Conclusions: We found significantly lower risk for decreased PFT deterioration among smokers workers exposed to artificial stone dust with higher HO-1 gene expression suggesting a possible protective effect of smoking by the involvement of HO-1 mechanism. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 276-284).

P49 Can The Asthma Control Questionnaire (acq) And/or The Blood Eosinophil Count Accurately Detect Sputum Eosinophilia?

Induced sputum differential cell counts provide important information about airway inflammation and future risk in asthma, but are not universally available. We set out to ascertain the sensitivity and specificity of the Juniper Asthma Control Questionnaire (ACQ) and/or the peripheral blood eosinophil count to detect a sputum eosinophil count >3%. Methods We performed a retrospective, cross-sectional study of 165 subjects with asthma, aged 18–80, prescribed as-required bronchodilators, long-acting β-agonists, 0–4000 mcg inhaled beclometasone diproprionate equivalent or maintenance oral steroids (0–20 mg prednisolone) from our database. Current smokers were excluded. Spirometry, FeNO at 50 ml flow, sputum induction, Asthma Control Questionnaire (ACQ) and blood eosinophils (BEos) were recorded. Induced sputum eosinophils (SEos) defined eosinophilic (EA, SEos ≥3%) and non-eosinophilic (NEA, SEos 10 transformed and groups were compared with t-tests or Mann-Whitney-U using STATA and GraphPad. Receiver operating characteristic (ROC) curves determined cut-points of ACQ and BEos that identified SEos ≥3%, and these were tested retrospectively in a second population of adults with asthma (n = 48, 40% EA, mean FEV 1 82% predicted). Results The 31% with EA had a lower FEV 1 % predicted and FEV 1 /FVC ratio, a higher FeNO, BEos and ACQ compared to NEA (Table 1). There was a significant correlation between ACQ and sputum eosinophils (Pearson r 0.32, p The optimal cut-points of ACQ and BEos identifying a SEos of 3% were an ACQ >1.57 (sensitivity 55%, specificity 78%) and BEos ≥0.22 × 10 9 /L (sensitivity 84% and specificity 68%) respectively. Individuals with both ACQ of ≥1.57 AND a BEos of ≥0.22 the sensitivity was 45% and specificity of 100% for a SEos of 3%. Testing in the second population the ACQ≥1.57 had a sensitivity 47% and specificity 86% for SEos >3% while BEos ≥0.22 had sensitivity 68% and specificity 45%. The combination of ACQ ≥1.57 AND BEos ≥0.22 had a lower sensitivity of 37% but a high specificity of 93% and could be useful to exclude rather than identify the presence of sputum eosinophilia. Conclusion Combined testing using ACQ and BEos only identifies eosinophilia in 37% and can’t replace induced sputum, but may be useful to exclude sputum eosinophilia and prospective study is warranted.

Cellular Composition of Induced Sputum in Sarcoidosis

The aim of this study was to evaluate the cellular composition of induced sputum (IS) in sarcoidosis and its role in assessing the disease activity. The safety of the procedure was also determined. Sputum induction by inhalation of hypertonic saline was performed. Twenty-one samples from the healthy controls, 32 from patients with active disease, and 33 from subjects with inactive disease were analysed. The percentage of lymphocytes in IS was significantly higher in active sarcoidosis than in inactive disease and the control group (9.7% vs. 3.1% vs. 2.9%), and was the highest in the patients with parenchymal changes and active disease (13.3%). The percentage of macrophages was significantly lower in active sarcoidosis than in normal subjects (76.8% vs. 83.4%). It was also significantly lower in IS in active disease and stages II and III than in both subgroups with active and inactive stage I of sarcoidosis. There were no significant differences in the IS cell percentages between the whole sarcoidosis group and the controls. Sputum induction was well tolerated and stopped only four times, two of them because of dyspnoea or the decrease of PEF. The symptoms were well reversible after administering salbutamol. Sputum induction by inhalation of hypertonic saline is safe, but the evaluation of IS differential cell counts is not useful in sarcoidosis diagnosing. However, it could be used in assessing the activity of the disease, especially in patients with interstitial lung changes.

Investigation of the association between dietary intake, disease severity and airway inflammation in asthma

Dietary intake is an important modifiable risk factor for asthma and may be related to disease severity and inflammation, through the effects of intake of anti-oxidant-rich foods and pro-inflammatory nutrients. This study aimed to examine dietary intake in asthma in relation to asthma severity, lung function, inhaled corticosteroid use, leptin levels and inflammation. Food frequency questionnaires, spirometry and hypertonic saline challenge were completed by 137 stable asthmatics and 65 healthy controls. Plasma leptin was analysed by immunoassay. Induced sputum differential cell counts were determined. Subjects with severe persistent asthma consumed more fat and less fibre as compared with healthy controls (odds ratio 1.04 (95% confidence interval: 1.01-1.07), P = 0.014) (odds ratio 0.94 (95% confidence interval: 0.90-0.99), P = 0.018). Among asthmatics, higher fat and lower fibre intakes were associated with lower forced expiratory volume in 1 s and airway eosinophilia. Leptin levels were increased in both male and female asthmatics as compared with healthy controls. No association existed among asthmatics between corticosteroid use and dietary intake. It was found that asthmatics within the subgroup of severe persistent asthma have a different pattern of dietary intake as compared with healthy controls, which was associated with lower lung function and increased airway inflammation.

Expiratory flows and airway inflammation in elderly asthmatic patients

Asthma in the elderly is often underrecognized and suboptimally treated, resulting in an increased morbidity and mortality. The characteristics of asthma-related bronchitis and its optimal treatment remain to be determined in this population. We aimed to compare lung function and airway inflammation in elderly and younger asthmatic subjects. Data from two induced sputum databases were analyzed in three groups of asthmatic subjects (18-30 y, n = 136; 31-59 y, n = 385; 60-72 y, n = 172) and one group of healthy elderly subjects (60-89 y, n = 16). Expiratory flows and induced sputum cell counts were analyzed. Airway obstruction was more marked in elderly asthmatics compared with healthy elderly or younger asthmatic subjects (p < 0.01). An increase in sputum neutrophils and a decrease in macrophages and lymphocytes were observed in elderly asthmatics (p < 0.0001). Neutrophil percentages significantly increased with asthma severity in the young and the middle-aged groups, while they remained similar in elderly asthmatics regardless of asthma severity (p < 0.05). Neutrophil percentages weakly correlated with the dose of ICS in all asthmatics (r = 0.17, p < 0.0001). Age and dose of ICS were independent predictors of neutrophil percentage in asthmatic subjects in a regression model (R(2) = 0.12). Asthma in the elderly is associated with a more marked airway obstruction and sputum neutrophilia. Both age and the dose of corticosteroids need to be considered in the interpretation of the clinical relevance of sputum neutrophil count.

The allergen specificity of the late asthmatic reaction

Allergen inhalation challenge in asthma may induce both early (EAR) and late (LAR) asthmatic reactions. The EAR is IgE and mast cell dependent. The mechanism of the LAR is less well defined and we have hypothesized may be allergen dependent. The aim of this study was to investigate the allergen specificity of the LAR to allergen inhalation in asthma. In a randomized, double-blind, crossover design six asthmatic volunteers with dual sensitization to house dust mite (HDM) allergen and grass pollen (GP) allergen underwent inhalation allergen challenge with these separate allergens on two occasions separated by 14 days. Lung function changes were followed for 8-h postchallenge. Bronchial reactivity (histamine PC(20)) and airway inflammation, assessed by induced sputum differential cell count, were measured 24-h pre and postallergen challenge. The allergen inhalation challenges were matched to achieve the same magnitude of EAR. Despite comparable group mean EAR percent falls in FEV(1) (25.8% following GP and 28.0% following HDM (P = 0.917), the LAR was statistically greater on the HDM challenge day (13.0%vs 22.8% [P = 0.046]) and was associated with a significant airway eosinophil recruitment (mean (SD) of 5.4 (4.8)% to 22.1 (18.2)% (P = 0.028) that was not evident on the GP allergen challenge day. These findings identify the allergen specificity of the LAR and indicate that factors independent of IgE contribute to the LAR. Such findings have relevance both to the understanding of the allergen-induced airway responses in asthma and the need for homogeneity in inhaled-allergen challenge studies in asthma.

Protective Effect of Fish Oil Supplementation on Exercise-Induced Bronchoconstriction in Asthmatic Subjects

PURPOSE: Previous research has demonstrated that fish oil supplementation has a protective effect on exercise-induced bronchoconstriction (EIB) in elite athletes, which may be attributed to its antiinflammatory properties. Since EIB in asthma involves pro inflammatory mediator release, it is feasible that fish oil supplementation may reduce the severity of EIB in asthmatic subjects. The main aim of the study was to determine the efficacy offish oil supplementation on severity of EIB in subjects with asthma. METHODS: Sixteen asthmatic patients with documented EIB participated in a randomized, double-blind crossover study. Subjects entered the study on their normal diet, and then received either fish oil capsules containing 3.2 g eicosapentaenoic acid 2.0 g docohexaenoic acid (fish oil diet; n=8) or placebo capsules (placebo diet; n=8) taken daily for 3 weeks. At the beginning of the study (normal diet) and at the end of each treatment phase the following pre- and post-exercise measures were assessed: (1) pulmonary function; (2) induced sputum (IS) differential cell count percentage and pro inflammatory eicosanoid metabolite (LTC4-E4 and PGD2) and cytokine (IL-1β and TNF-α) concentrations; (3) eicosanoid metabolites LTB4 and LTB5 generation from activated polymorp ho nuclear leukocytes (PMNLs). RESULTS: On the normal and placebo diet subjects exhibited EIB. However, the fish oil diet improved pulmonary function to below the diagnostic EIB threshold, with a concurrent reduction in broncho dilator use. IS differential cell count percentage and concentration of LTC4-E4, PGD2, IL-1β and TNF-α were significantly reduced before and following exercise on the fish oil diet compared to the normal and placebo diet. There was a significant reduction in LTB4 and a significant increase in LTB5 generation from activated PMNLs on the fish oil diet compared to the normal and placebo diet. CONCLUSIONS: Our data suggest that fish oil supplementation may represent a potentially beneficial nonpharmacological intervention for asthmatic subjects with EIB.

Protective Effect of Fish Oil Supplementation on Exercise-Induced Bronchoconstriction in Asthma

Previous research has demonstrated that fish oil supplementation has a protective effect on exercise-induced bronchoconstriction (EIB) in elite athletes, which may be attributed to its antiinflammatory properties. Since EIB in asthma involves proinflammatory mediator release, it is feasible that fish oil supplementation may reduce the severity of EIB in asthmatic subjects. To determine the efficacy of fish oil supplementation on severity of EIB in subjects with asthma. Randomized, double-blind, crossover study. Lung function and exercise testing in a university research laboratory. Sixteen asthmatic patients with documented EIB entered the study on their normal diet and then received either fish oil capsules containing 3.2 g of eicosapentaenoic acid and 2.0 g of docohexaenoic acid (fish oil diet, n = 8) or placebo capsules (placebo diet, n = 8) daily for 3 weeks. At the beginning of the study (normal diet) and at the end of each treatment phase, the following pre-exercise and postexercise measures were assessed: (1) pulmonary function; (2) induced sputum differential cell count percentage and proinflammatory eicosanoid metabolite (leukotriene C4 [LTC4]-leukotriene E4 [LTE4] and prostaglandin D2 [PGD2]) and cytokine (interleukin [IL]-1beta and tumor necrosis factor [TNF]-alpha) concentrations; and (3) eicosanoid metabolites leukotriene B4 (LTB4) and leukotriene B5 (LTB(5)) generation from activated polymorphonuclear leukocytes (PMNLs). On the normal and placebo diet, subjects exhibited EIB. However, the fish oil diet improved pulmonary function to below the diagnostic EIB threshold, with a concurrent reduction in bronchodilator use. Induced sputum differential cell count percentage and concentrations of LTC4-LTE4, PGD2, IL-1beta, and TNF-alpha were significantly reduced before and following exercise on the fish oil diet compared to the normal and placebo diets. There was a significant reduction in LTB4 and a significant increase in LTB5 generation from activated PMNLs on the fish oil diet compared to the normal and placebo diets. Our data suggest that fish oil supplementation may represent a potentially beneficial nonpharmacologic intervention for asthmatic subjects with EIB.

The Influence of Age on Induced Sputum Differential Cell Counts in Normal Subjects

Study objectives: Sputum induction is increasingly used as a research technique and as a clinical tool. In order to evaluate abnormal results, normal ranges need to be fully developed. Although a number of studies have described normal ranges, none have investigated the effect of the age of the subject on these results. This study was undertaken to assess whether there are age-related differences in sputum cell differential cell counts in a population of normal, healthy volunteers. Study design and participants: Induced sputum samples were obtained from 66 healthy, nonsmoking subjects (24 men) with a mean age of 44 years (age range, 18 to 74 years). Differential cell counts were related to age. Results: Sputum neutrophil counts were found to correlate significantly with the age of the volunteers (r = 0.58; p < 0.001). Macrophage counts showed a proportionate, inverse correlation with increasing age (p < 0.01), but no correlation was seen for any other cell type. On subanalysis according to age range, the mean neutrophil differential increased from 26.9% (SD, 19.8%) [17 patients] in the group of patients who were 0 to 29 years of age to 68.5% (SD, 20.6%) [11 patients] in the group of patients who were > 60 years of age. Conclusion: In our healthy volunteer population, the induced sputum differential neutrophil count increased significantly with age. These findings highlight the need for age matching in controlled studies. Sputum induction is increasingly used as a research technique and as a clinical tool. In order to evaluate abnormal results, normal ranges need to be fully developed. Although a number of studies have described normal ranges, none have investigated the effect of the age of the subject on these results. This study was undertaken to assess whether there are age-related differences in sputum cell differential cell counts in a population of normal, healthy volunteers. Induced sputum samples were obtained from 66 healthy, nonsmoking subjects (24 men) with a mean age of 44 years (age range, 18 to 74 years). Differential cell counts were related to age. Sputum neutrophil counts were found to correlate significantly with the age of the volunteers (r = 0.58; p < 0.001). Macrophage counts showed a proportionate, inverse correlation with increasing age (p < 0.01), but no correlation was seen for any other cell type. On subanalysis according to age range, the mean neutrophil differential increased from 26.9% (SD, 19.8%) [17 patients] in the group of patients who were 0 to 29 years of age to 68.5% (SD, 20.6%) [11 patients] in the group of patients who were > 60 years of age. In our healthy volunteer population, the induced sputum differential neutrophil count increased significantly with age. These findings highlight the need for age matching in controlled studies.

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.

Effects of inhaled tumour necrosis factor alpha in subjects with mild asthma

Background: Inhaled tumour necrosis factor alpha (TNFα ) has previously been shown to induce airway neutrophilia and increased airway reactivity in normal subjects. It was hypothesised that a similar challenge...

The safety and success rate of sputum induction using a low output ultrasonic nebuliser

Induced sputum differential cell counts have been advocated as a method of non-invasively assessing airway inflammation in asthma and other airway diseases. Since sputum induction usually involves delivering hypertonic saline via a high output ultrasonic nebulizer there have been concerns about its safety in asthma. There are relatively little data on the effects of sputum induction in large numbers of patients. We have examined the success rate and effect of sputum induction on forced expiratory volume in 1 sec (FEV 1) in 100 inductions performed on 79 patients using a low output nebulizer. Thirty-seven patients had asthma, 29 had miscellaneous conditions (mainly chronic cough) and 13 were subjects without respiratory symptoms. Sputum was induced 10 min after 200 μg of inhaled salbutamol by sequential 5-min inhalations of 3, 4 and 5% saline delivered via a Fisoneb ultrasonic nebulizer and FEV 1 was measured after each inhalation. Sputum induction resulted in a sample suitable for analysis in 92% of asthmatics, 90% of those with miscellaneous conditions and 100% of normal subjects. The mean ( sem) maximum per cent fall in FEV 1 was 5·4% (0·1), 4·3% (1·0) and 2·6% (1·1) in subjects with asthma, miscellaneous conditions and in asymptomatic subjects respectively. Only 13 inductions resulted in a>10% fall in FEV 1, and only three of these resulted in a>20% fall. The maximum per cent fall in FEV 1 did not correlate with baseline FEV 1 % predicted ( r= −0·17), the log sputum eosinophil count ( r= −0·12), or the methacholine PC 20 ( r= −0·14). We conclude that sputum induction using a relatively low output ultrasonic nebulizer with premedication with salbutamol is successful and safe in the majority of patients with asthma and other airway conditions.