Liquid chromatography/mass spectrometry analysis of exhaled leukotriene B(4 )in asthmatic children

BACKGROUND: The role of leukotriene (LT) B(4), a potent inflammatory mediator, in atopic asthmatic and atopic nonasthmatic children is largely unknown. The lack of a gold standard technique for measuring LTB(4 )in exhaled breath condensate (EBC) has hampered its quantitative assessment in this biological fluid. We sought to measure LTB(4 )in EBC in atopic asthmatic children and atopic nonasthmatic children. Exhaled nitric oxide (NO) was measured as an independent marker of airway inflammation. METHODS: Fifteen healthy children, 20 atopic nonasthmatic children, 25 steroid-naïve atopic asthmatic children, and 22 atopic asthmatic children receiving inhaled corticosteroids were studied. The study design was of cross-sectional type. Exhaled LTB(4 )concentrations were measured using liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS) with a triple quadrupole mass spectrometer. Exhaled NO was measured by chemiluminescence with a single breath on-line method. LTB(4 )values were expressed as the total amount (in pg) of eicosanoid expired in the 15-minute breath test. Kruskal-Wallis test was used to compare groups. RESULTS: Compared with healthy children [87.5 (82.5–102.5) pg, median and interquartile range], exhaled LTB(4 )was increased in steroid-naïve atopic asthmatic [255.1 (175.0–314.7) pg, p < 0.001], but not in atopic nonasthmatic children [96.5 (87.3–102.5) pg, p = 0.59)]. Asthmatic children who were receiving inhaled corticosteroids had lower concentrations of exhaled LTB(4 )than steroid-naïve asthmatics [125.0 (25.0–245.0) pg vs 255.1 (175.0–314.7) pg, p < 0.01, respectively]. Exhaled NO was higher in atopic nonasthmatic children [16.2 (13.5–22.4) ppb, p < 0.05] and, to a greater extent, in atopic steroid-naïve asthmatic children [37.0 (31.7–57.6) ppb, p < 0.001] than in healthy children [8.3 (6.1–9.9) ppb]. Compared with steroid-naïve asthmatic children, exhaled NO levels were reduced in asthmatic children who were receiving inhaled corticosteroids [15.9 (11.5–31.7) ppb, p < 0.01]. CONCLUSION: In contrast to exhaled NO concentrations, exhaled LTB(4 )values are selectively elevated in steroid-naïve atopic asthmatic children, but not in atopic nonasthmatic children. Although placebo control studies are warranted, inhaled corticosteroids seem to reduce exhaled LTB(4 )in asthmatic children. LC/MS/MS analysis of exhaled LTB(4 )might provide a non-invasive, sensitive, and quantitative method for airway inflammation assessment in asthmatic children

Collection of Aerosolized Human Cytokines Using Teflon® Filters

Background: Collection of exhaled breath samples for the analysis of inflammatory biomarkers is an important area of research aimed at improving our ability to diagnose, treat and understand the mechanisms of chronic pulmonary disease. Current collection methods based on condensation of water vapor from exhaled breath yield biomarker levels at or near the detection limits of immunoassays contributing to problems with reproducibility and validity of biomarker measurements. In this study, we compare the collection efficiency of two aerosol-to-liquid sampling devices to a filter-based collection method for recovery of dilute laboratory generated aerosols of human cytokines so as to identify potential alternatives to exhaled breath condensate collection. Methodology/Principal Findings: Two aerosol-to-liquid sampling devices, the SKC® Biosampler and Omni 3000™, as well as Teflon® filters were used to collect aerosols of human cytokines generated using a HEART nebulizer and single-pass aerosol chamber setup in order to compare the collection efficiencies of these sampling methods. Additionally, methods for the use of Teflon® filters to collect and measure cytokines recovered from aerosols were developed and evaluated through use of a high-sensitivity multiplex immunoassay. Our results show successful collection of cytokines from pg/m3 aerosol concentrations using Teflon® filters and measurement of cytokine levels in the sub-picogram/mL concentration range using a multiplex immunoassay with sampling times less than 30 minutes. Significant degradation of cytokines was observed due to storage of cytokines in concentrated filter extract solutions as compared to storage of dry filters. Conclusions: Use of filter collection methods resulted in significantly higher efficiency of collection than the two aerosol-to-liquid samplers evaluated in our study. The results of this study provide the foundation for a potential new technique to evaluate biomarkers of inflammation in exhaled breath samples

Continuous monitoring of the bronchial epithelial lining fluid by microdialysis

<p>Abstract</p> <p>Background</p> <p>Contents of the epithelial lining fluid (ELF) of the bronchi are of central interest in lung diseases, acute lung injury and pharmacology. The most commonly used technique broncheoalveolar lavage is invasive and may cause lung injury. Microdialysis (MD) is a method for continuous sampling of extracellular molecules in the immediate surroundings of the catheter. Urea is used as an endogenous marker of dilution in samples collected from the ELF. The aim of this study was to evaluate bronchial MD as a continuous monitor of the ELF.</p> <p>Methods</p> <p>Microdialysis catheters were introduced into the right main stem bronchus and into the right subclavian artery of five anesthetized and normoventilated pigs. The flowrate was 2 μl/min and the sampling interval was 60 minutes. Lactate and fluorescein-isothiocyanate-dextran 4 kDa (FD-4) infusions were performed to obtain two levels of steady-state concentrations in blood. Accuracy was defined as [bronchial-MD] divided by [arterial-MD] in percent. Data presented as mean ± 95 percent confidence interval.</p> <p>Results</p> <p>The accuracy of bronchial MD was calculated with and without correction by the arteriobronchial urea gradient. The arteriobronchial lactate gradient was 1.2 ± 0.1 and FD-4 gradient was 4.0 ± 1.2. Accuracy of bronchial MD with a continuous lactate infusion was mean 25.5% (range 5.7–59.6%) with a coefficient of variation (CV) of 62.6%. With correction by the arteriobronchial urea gradient accuracy was mean 79.0% (57.3–108.1%) with a CV of 17.0%.</p> <p>Conclusion</p> <p>Urea as a marker of catheter functioning enhances bronchial MD and makes it useful for monitoring substantial changes in the composition of the ELF.</p

Comparative analysis of selected exhaled breath biomarkers obtained with two different temperature-controlled devices

<p>Abstract</p> <p>Background</p> <p>The collection of exhaled breath condensate (EBC) is a suitable and non-invasive method for evaluation of airway inflammation. Several studies indicate that the composition of the condensate and the recovery of biomarkers are affected by physical characteristics of the condensing device and collecting circumstances. Additionally, there is an apparent influence of the condensing temperature, and often the level of detection of the assay is a limiting factor. The ECoScreen2 device is a new, partly single-use disposable system designed for studying different lung compartments.</p> <p>Methods</p> <p>EBC samples were collected from 16 healthy non-smokers by using the two commercially available devices ECoScreen2 and ECoScreen at a controlled temperature of -20°C. EBC volume, pH, NOx, LTB₄, PGE₂, 8-isoprostane and cys-LTs were determined.</p> <p>Results</p> <p>EBC collected with ECoScreen2 was less acidic compared to ECoScreen. ECoScreen2 was superior concerning condensate volume and detection of biomarkers, as more samples were above the detection limit (LTB₄and PGE₂) or showed higher concentrations (8-isoprostane). However, NOx was detected only in EBC sampled by ECoScreen.</p> <p>Conclusion</p> <p>ECoScreen2 in combination with mediator specific enzyme immunoassays may be suitable for measurement of different biomarkers. Using this equipment, patterns of markers can be assessed that are likely to reflect the complex pathophysiological processes in inflammatory respiratory disease.</p

Dissociating Markers of Senescence and Protective Ability in Memory T Cells

No unique transcription factor or biomarker has been identified to reliably distinguish effector from memory T cells. Instead a set of surface markers including IL-7Rα and KLRG1 is commonly used to predict the potential of CD8 effector T cells to differentiate into memory cells. Similarly, these surface markers together with the tumor necrosis factor family member CD27 are frequently used to predict a memory T cell's ability to mount a recall response. Expression of these markers changes every time a memory cell is stimulated and repeated stimulation can lead to T cell senescence and loss of memory T cell responsiveness. This is a concern for prime–boost vaccine strategies which repeatedly stimulate T cells with the aim of increasing memory T cell frequency. The molecular cues that cause senescence are still unknown, but cell division history is likely to play a major role. We sought to dissect the roles of inflammation and cell division history in developing T cell senescence and their impact on the expression pattern of commonly used markers of senescence. We developed a system that allows priming of CD8 T cells with minimal inflammation and without acquisition of maximal effector function, such as granzyme expression, but a cell division history similar to priming with systemic inflammation. Memory cells derived from minimal effector T cells are fully functional upon rechallenge, have full access to non-lymphoid tissue and appear to be less senescent by phenotype upon rechallenge. However, we report here that these currently used biomarkers to measure senescence do not predict proliferative potential or protective ability, but merely reflect initial priming conditions

Leukocyte Telomere Length in Major Depression: Correlations with Chronicity, Inflammation and Oxidative Stress - Preliminary Findings

Depression is associated with an unusually high rate of aging-related illnesses and early mortality. One aspect of “accelerated aging” in depression may be shortened leukocyte telomeres. When telomeres critically shorten, as often occurs with repeated mitoses or in response to oxidation and inflammation, cells may die. Indeed, leukocyte telomere shortening predicts early mortality and medical illnesses in non-depressed populations. We sought to determine if leukocyte telomeres are shortened in Major Depressive Disorder (MDD), whether this is a function of lifetime depression exposure and whether this is related to putative mediators, oxidation and inflammation.Leukocyte telomere length was compared between 18 unmedicated MDD subjects and 17 controls and was correlated with lifetime depression chronicity and peripheral markers of oxidation (F2-isoprostane/Vitamin C ratio) and inflammation (IL-6). Analyses were controlled for age and sex.The depressed group, as a whole, did not differ from the controls in telomere length. However, telomere length was significantly inversely correlated with lifetime depression exposure, even after controlling for age (p<0.05). Average telomere length in the depressed subjects who were above the median of lifetime depression exposure (≥9.2 years' cumulative duration) was 281 base pairs shorter than that in controls (p<0.05), corresponding to approximately seven years of “accelerated cell aging.” Telomere length was inversely correlated with oxidative stress in the depressed subjects (p<0.01) and in the controls (p<0.05) and with inflammation in the depressed subjects (p<0.05).These preliminary data indicate that accelerated aging at the level of leukocyte telomeres is proportional to lifetime exposure to MDD. This might be related to cumulative exposure to oxidative stress and inflammation in MDD. This suggest that telomere shortening does not antedate depression and is not an intrinsic feature. Rather, telomere shortening may progress in proportion to lifetime depression exposure

The effect of titanium dioxide nanoparticles on pulmonary surfactant function and ultrastructure

<p>Abstract</p> <p>Background</p> <p>Pulmonary surfactant reduces surface tension and is present at the air-liquid interface in the alveoli where inhaled nanoparticles preferentially deposit. We investigated the effect of titanium dioxide (TiO₂) nanosized particles (NSP) and microsized particles (MSP) on biophysical surfactant function after direct particle contact and after surface area cycling <it>in vitro</it>. In addition, TiO₂effects on surfactant ultrastructure were visualized.</p> <p>Methods</p> <p>A natural porcine surfactant preparation was incubated with increasing concentrations (50-500 μg/ml) of TiO₂NSP or MSP, respectively. Biophysical surfactant function was measured in a pulsating bubble surfactometer before and after surface area cycling. Furthermore, surfactant ultrastructure was evaluated with a transmission electron microscope.</p> <p>Results</p> <p>TiO₂NSP, but not MSP, induced a surfactant dysfunction. For TiO₂NSP, adsorption surface tension (γ_ads) increased in a dose-dependent manner from 28.2 ± 2.3 mN/m to 33.2 ± 2.3 mN/m (p < 0.01), and surface tension at minimum bubble size (γ_min) slightly increased from 4.8 ± 0.5 mN/m up to 8.4 ± 1.3 mN/m (p < 0.01) at high TiO₂NSP concentrations. Presence of NSP during surface area cycling caused large and significant increases in both γ_ads(63.6 ± 0.4 mN/m) and γ_min(21.1 ± 0.4 mN/m). Interestingly, TiO₂NSP induced aberrations in the surfactant ultrastructure. Lamellar body like structures were deformed and decreased in size. In addition, unilamellar vesicles were formed. Particle aggregates were found between single lamellae.</p> <p>Conclusion</p> <p>TiO₂nanosized particles can alter the structure and function of pulmonary surfactant. Particle size and surface area respectively play a critical role for the biophysical surfactant response in the lung.</p