Breath condensate hydrogen peroxide correlates with both airway cytology and epithelial lining fluid ascorbic acid concentration in the horse

The relationship between hydrogen peroxide (H2O2) concentration in expired breath condensate (EBC) and cytology of the respiratory tract obtained from tracheal wash (TW) or bronchoalveolar lavage (BAL), and epithelial lining fluid (ELF) antioxidant status is unknown. To examine this we analysed the concentration of H2O2 in breath condensate from healthy horses and horses affected by recurrent airway obstruction (RAO), a condition considered to be an animal model of human asthma. The degree of airway inflammation was determined by assessing TW inflammation as mucus, cell density and neutrophil scores, and by BAL cytology. ELF antioxidant status was determined by measurement of ascorbic acid, dehydroascorbate, reduced and oxidised glutathione, uric acid and alpha-tocopherol concentrations. RAO-affected horses with marked airway inflammation had significantly higher concentrations of breath condensate H2O2 than control horses and RAO-affected horses in the absence of inflammation (2.0 +/- 0.5 micromol/l. 0.4 +/- 0.2 micromol/l and 0.9 +/- 0.2 micromol/l H2O2, respectively; p < 0.0001). The concentration of breath condensate H2O2 was related inversely to the concentration of ascorbic acid in ELF (r = -0.80; p < 0.0001) and correlated positively with TW inflammation score (r = 0.76, p < 0.0001) and BAL neutrophil count (r = 0.80, p < 0.0001). We conclude that the concentration of H2O2 in breath condensate influences the ELF ascorbic acid concentration and provides a non-invasive diagnostic indicator of the severity of neutrophilic airway inflammation.     

Elevated amount of Toll-like receptor 4 mRNA in bronchial epithelial cells is associated with airway inflammation in horses with recurrent airway obstruction

Recurrent airway obstruction (RAO) is characterized by neutrophilic airway inflammation and obstruction, and stabling of susceptible horses triggers acute disease exacerbations. Stable dust is rich in endotoxin, which is recognized by Toll-like receptor (TLR) 4. In human bronchial epithelium, TLR4 stimulation leads to elevation of interleukin (IL)-8 mRNA expression. The zinc finger protein A20 negatively regulates this pathway. We hypothesized that TLR4 and IL-8 mRNA and neutrophil numbers are elevated and that A20 mRNA is not increased in RAOs during stabling compared with controls and with RAOs on pasture. We measured the maximal change in pleural pressure (DeltaPpl(max)), determined inflammatory cell counts in bronchoalveolar lavage fluid (BAL), and quantified TLR4, IL-8, and A20 mRNA in bronchial epithelium by quantitative RT-PCR. We studied six horse pairs, each pair consisting of one RAO and one control horse. Each pair was studied when the RAO-affected horse had airway obstruction induced by stabling and after 7, 14, and 28 days on pasture. Stabling increased BAL neutrophils, DeltaPpl(max), and TLR4 (4.14-fold change) significantly in RAOs compared with controls and with RAOs on pasture. TLR4 correlated with IL-8 (R2 = 0.75). Whereas stabling increased IL-8 in all horses, A20 was unaffected. IL-8 was positively correlated with BAL neutrophils (R2 = 0.43) and negatively with A20 (R2 = 0.44) only in RAO-affected horses. Elevated TLR4 expression and lack of A20 upregulation in bronchial epithelial cells from RAO-affected horses may contribute to elevated IL-8 production, leading to exaggerated neutrophilic airway inflammation in response to inhalation of stable dust.     

Evaluation of H2O2 and pH in exhaled breath condensate samples: methodical and physiological aspects

Abstract

This veterinary study is aimed at further standardization of H₂O₂ and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the ‘ECoScreen’ in healthy calves (body weight 63–98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H₂O₂, concentrations of H₂O₂ in EBC, blood and ambient air were determined with the biosensor system ‘ECoCheck’. In EBC, the concentration of H₂O₂ was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H₂O₂ concentrations at 06:00 varied within the range 138–624 nmol l^?1 EBC or 0.10–0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H₂O₂ concentrations in EBC and blood, and EBC–H₂O₂ was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H₂O₂ measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H₂O₂. In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC–pH (r=0.89, R²=79.3%, p≤0.001). Physiological pH data in non-degassed EBC samples varied between 5.3 and 6.5, and were not significantly different between subjects, but were significantly higher in the evening compared with the morning. EBC–pH was not dependent on variables of spontaneous breathing pattern or ambient conditions, and no significant correlation was found between serum and EBC for pH.     

Persistent mucus accumulation: a consequence of delayed bronchial mucous cell apoptosis in RAO-affected horses?

This study examined the contribution of delayed apoptosis of bronchial mucous cells to mucus accumulation in equine recurrent airway obstruction (RAO). In pilot studies, Bcl-2, an apoptosis inhibitor, was detected in airway mucous cells of RAO-affected horses in remission and during acute disease, when most mucus was secreted. To study whether delayed apoptosis results in an increase in the number of mucous cells during disease recovery, six RAO-affected and six control horses were fed hay for 5 days to induce inflammation and then pellets for 7 days to partially resolve RAO before euthanasia. RAO-affected horses had more airway obstruction and luminal mucus than control horses under both management systems. At the time of euthanasia, RAO-affected horses had more inflammation and Bcl-2-positive bronchial mucous cells than control animals. In horses with >10 and <10 neutrophils per microliter of bronchoalveolar lavage fluid, >50% and <10% of mucous cells stained positive for Bcl-2, respectively. No differences in mucous cell number or amount of stored mucosubstance were observed between RAO-affected and control horses, but in RAO-affected animals, the amount of stored mucosubstance decreased as the number of neutrophils in bronchoalveolar lavage fluid increased. Because the number of mucous cells was similar in both groups of horses but only mucous cells of RAO-affected horses expressed Bcl-2 during recovery from acute disease, a conclusive role for Bcl-2 in prolonging bronchial mucous cell life could not be determined. Future studies are needed to compare horses that are kept in remission for prolonged periods when all mucous cells are fully developed.     

Fractionated breath condensate sampling: H2O2 concentrations of the alveolar fraction may be related to asthma control in children

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.     

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.     

Persistent mucin glycoprotein alterations in equine recurrent airway obstruction

Horses with the episodic asthmalike condition of recurrent airway obstruction (RAO) have bouts of inflammation and bronchoconstriction associated with indoor housing. To assess the potential differences in airway secretions between RAO-affected and control horses, methods to quantify mucus secretions were developed and applied to bronchoalveolar lavage fluid. The relative difference in the amount of mucin glycoproteins between control and RAO-affected horses was assessed with a carbohydrate side chain-specific monoclonal antibody (4E4) in an enzyme-linked immunosorbent assay and by carbohydrate-specific enzyme-linked lectin assays. Significantly increased levels of 4E4-immunoreactive glycoprotein and the mucin-associated carbohydrates fucose (alpha-1,2 linkage) and N-acetylglucosamine were detected in RAO-affected horses in acute disease. RAO-affected horses in remission maintained significantly elevated levels of alpha-1,2-fucose and N-acetylglucosamine, whereas the 4E4-immunoreactive glycoprotein levels displayed a trend toward an increase over control levels. These results indicated that persistent changes in the quantity and/or quality of mucus glycoproteins occurred in the RAO-affected horses.     

Exhaled breath condensate as matrix for toluene detection: A preliminary study

Abstract

The study was designed to investigate whether exhaled breath condensate, obtained by cooling exhaled air in spontaneous breathing, could be a suitable matrix for toluene quantitative analyses. Nine healthy subjects were exposed for a short period (20 min) to a known concentration of toluene. Exhaled breath condensate samples were collected before and at the end of the exposure, while the environmental concentration of toluene was continuously monitored. Toluene was analysed by head-space gas-chromatography mass spectrometry, and assay repeatability was also estimated in vitro. Baseline and post-exposure measurement of hippuric acid, the urinary toluene metabolite, was performed to assess current toluene exposure. Before the exposure toluene concentrations in the exhaled breath condensate were lower than the detectable limit in all subjects, while after the exposure toluene was detectable with a median value 0.35 µg l^?1 (range 0.15–0.55 µg l^?1) in all the exhaled breath condensate samples. As compared with the standard calibration in distilled water, the curves obtained by exhaled breath condensate were linear and comparable with the range examined in vivo for toluene. A significant correlation was found between the environmental toluene levels and toluene in the exhaled breath condensate at the end of exposure. Furthermore, a significant relationship between increased exhaled breath condensate toluene levels and urinary hippuric acid after the exposure was found. In conclusion, exhaled breath condensate is a promising matrix for toluene assessment, although its application in humans requires further investigations.     

Increase of pro-oxidants with no evidence of lipid peroxidation in exhaled breath condensate after a 10-km race in non-athletes

It is a well-established fact that exercise increases pro-oxidants and favors oxidative stress; however, this phenomenon has been poorly studied in human lungs. Pro-oxidative generation (H₂O₂, NO₂ ^?), lipid peroxidation markers (MDA), and inflammation (pH) in exhaled breath condensate (EBC) have been determined through data from 10 active subjects who ran 10 km; samples were obtained immediately before, at 20, and at 80 min post-exertion. In EBC, the concentration of H₂O₂ at 80 min post-exertion was increased. NO₂ ^? concentration showed a tendency to increase at 80 min post-exertion, with no variations in MDA and pH. No variations of NO₂ ^? were found in plasma, while there was an increase of NO₂ ^? at 80 min post-exertion in the relation between EBC and plasma. NO₂ ^? in EBC did not correlate to plasmatic NO₂ ^?, while it did correlate directly with H₂O₂ in EBC, suggesting a localized origin for the exercise-related NO₂ ^? increase in EBC. MDA in plasma did not increase nor correlate with MDA in EBC. In conclusion, high-intensity exercise increases lung-originated pro-oxidants in non-athlete subjects with no evidence of early lipid peroxidation and changes in the pH value in EBC.     

Optimising hydrogen peroxide measurement in exhaled breath condensate

Abstract

Background: Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing airway pathology. A number of substances, including hydrogen peroxide (H₂O₂), have been measured in EBC, without adequate published details of validation and optimisation.

Objectives: To explore factors that affect accurate quantitation of H₂O₂ in EBC.

Materials and Methods: H₂O₂ was measured in EBC samples using fluorometry with 4-hydroxyphenylacetic acid. A number of factors that might alter quantitation were studied including pH and buffering conditions, reagent storage, and assay temperature.

Results: Standard curve slope was significantly altered by pH, leading to a potential difference in H₂O₂ quantification of up to 42%. These differences were resolved by increasing the buffering capacity of the reaction mix. H₂O₂ added to EBC remained stable for 1 h when stored on ice. The assay was unaffected by freezing assay reagents. The limit of detection for H₂O₂ ranged from 3.4 nM to 8.8 nM depending on the buffer used.

Conclusions: The reagents required for this assay can be stored for several months allowing valuable consistency in longitudinal studies. The quantitation of H₂O₂ in EBC is pH-dependent but increasing assay buffering reduces this effect. Sensitive reproducible quantitation of H₂O₂ in EBC requires rigorous optimisation.     

The in vivo and in vitro influence of methyl jasmonate on oxidative processes in Arabidopsis thaliana leaves

In Arabidopsis thaliana leaves a strong increase of H₂O₂ content was induced by application of methyl jasmonate (JAMe) through the root system, but the induction only slightly depended on JAMe concentration. The activity of superoxide dismutase and ascorbic acid peroxidase increased at lower JAMe concentrations and decreased at higher ones. Catalase activity decreased proportionally to JAMe concentration (in comparison with control plants). The sum of ascorbic acid and dehydroascorbate content at 10⁻⁶ M JAMe was similar to the control, but at higher concentrations it increased, especially due to a higher ascorbate accumulation. Methyl jasmonate applied directly to the extract of leaves (in vitro experiment) also induced a strong increase in H₂O₂ level, even at a low concentration (10⁻⁸ M). Since lower JAMe concentrations induced weak superoxide dismutase and did not change catalase and peroxidase activity, it is suggested that in this case a high level of hydrogen peroxide was not the result of the activity of the mentioned enzymes. JAMe-induction of H₂O₂ increase at the highest JAMe concentration resulted from SOD activity. Our in vivo and in vitro experiments suggest that jasmonate can influence oxidative stress not only through gene expression but also by its direct effect on enzyme activity.     

Light emission from the Fe2+–EDTA–ascorbic acid–H2O2 system strongly enhanced by plant phenolic acids

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra‐weak photon emission (UPE). We investigated the UPE from the Fe²⁺–EDTA (ethylenediaminetetraacetic acid)–AA (ascorbic acid)–H₂O₂ (hydrogen peroxide) system with a multitube luminometer (Peltier‐cooled photon counter, spectral range 380–630 nm). The UPE, of 92.6 μmol/L Fe²⁺, 185.2 μmol/L EDTA, 472 μmol/L AA, 2.6 mmol/L H₂O₂, reached 1217 ± 118 relative light units during 2 min measurement and was about two times higher (P < 0.001) than the UPE of incomplete systems (Fe²⁺–AA–H₂O₂, Fe²⁺–EDTA–H₂O₂, AA–H₂O₂) and medium alone. Substitution of Fe²⁺ with Cr²⁺, Co²⁺, Mn²⁺ or Cu²⁺ as well as of EDTA with EGTA (ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid) or citrate powerfully inhibited UPE. Experiments with scavengers of reactive oxygen species (dimethyl sulfoxide, mannitol, sodium azide, superoxide dismutase) revealed the dependence of UPE only on hydroxyl radicals. Dimethyl sulfoxide at the concentration of 0.74 mmol/L inhibited UPE by 79 ± 4%. Plant phenolics (ferulic, chlorogenic and caffec acids) at the concentration of 870 μmol/L strongly enhanced UPE by 5‐, 13.9‐ and 46.8‐times (P < 0.001), respectively. It is suggested that augmentation of UPE from Fe²⁺–EDTA–AA–H₂O₂ system can be applied for detection of these phytochemicals.     

Hydrogen peroxide and catalase are inversely related in adult patients undergoing cardiopulmonary bypass: implications for antioxidant protection

Abstract

Adult patients undergoing cardiopulmonary bypass (CPB) surgery are subjected to increased oxidative stress and show a spectrum of lung injury. Increased levels of hydrogen peroxide (H₂O₂)are often seen during episodes of oxidative stress, such as the use of high FiO₂s, and this molecule plays a key role in the formation of highly damaging oxidants such as the hydroxyl radical. Oxidative damage to plasma proteins was assessed by measuring free thiol groups, and antioxidant protection against H₂O₂ by measuring catalase activity. CPB patients (n =39) receiving either 100% or 50% oxygen at the end of bypass were studied by measuring levels of H₂O₂ in breath condensate and levels of catalase in their plasma, and comparing these to pre-bypass levels. Post-bypass, all CPB patients exhaled significantly lower levels of H₂O₂ (P < 0.0001) at a time when they had significantly increased activity (0.809 ± 0.11 versus 1.688 ±18 U/mg protein) of catalase in their plasma. There were no significant differences in these parameters between the 100% and 50% oxygen groups. At a time when oxidative stress is greatest, there appears to be a corresponding plasma increase in the antioxidant catalase. Whether this change is fortuitous or a response to oxidative stress is at present under consideration.     

Association of Lung Inflammatory Cells with Small Airways Function and Exhaled Breath Markers in Smokers – Is There a Specific Role for Mast Cells?

Background

Smoking is associated with a mixed inflammatory infiltrate in the airways. We evaluated whether airway inflammation in smokers is related to lung function parameters and inflammatory markers in exhaled breath.

Methods

Thirty-seven smokers undergoing lung resection for primary lung cancer were assessed pre-operatively by lung function testing including single-breath-nitrogen washout test (sb-N2-test), measurement of fractional exhaled nitric oxide (FeNO) and pH/8-isoprostane in exhaled breath condensate (EBC). Lung tissue sections containing cancer-free large (LA) and small airways (SA) were stained for inflammatory cells. Mucosal (MC_T) respectively connective tissue mast cells (MC_TC) and interleukin-17A (IL-17A) expression by mast cells was analysed using a double-staining protocol.

Results

The median number of neutrophils, macrophages and mast cells infiltrating the lamina propria and adventitia of SA was higher than in LA. Both MCTC and MC_T were higher in the lamina propria of SA compared to LA (MC_TC: 49 vs. 27.4 cells/mm²; MC_T: 162.5 vs. 35.4 cells/mm²; P<0.005 for both instances). IL-17A expression was predominantly detected in MC_TC of LA. Significant correlations were found for the slope of phase III % pred. of the sb-N2-test (r_s= -0.39), for the FEV₁% pred. (r_s= 0.37) and for FEV₁/FVC ratio (r_s=0.38) with MC_T in SA (P<0.05 for all instances). 8-isoprostane concentration correlated with the mast cells in the SA (r_s=0.44), there was no correlation for pH or FeNO with cellular distribution in SA.

Conclusions

Neutrophils, macrophages and mast cells are more prominent in the SA indicating that these cells are involved in the development of small airway dysfunction in smokers. Among these cell types, the best correlation was found for mast cells with lung function parameters and inflammatory markers in exhaled breath. Furthermore, the observed predominant expression of IL-17A in mast cells warrants further investigation to elucidate their role in smoking-induced lung injury, despite the lack of correlation with lung function and exhaled breath parameters.     

Immediate response of Chara braunii exposed to zinc and hydrogen peroxide

The immediate effect of zinc (Zn) and hydrogen peroxide (H₂O₂) in Chara braunii was analyzed in short-time exposure experiments. The exposure concentrations were 12.3, 18.4, and 24.5 μmol L^?1 H₂O₂, 12, 60, and 120 mg L^?1 Zn, and 12.3 μmol L^?1 H₂O₂ + 12 mg L^?1 Zn, 12.3 μmol L^?1 H₂O₂ + 60 mg L^?1 Zn, and 18.4 μmol L^?1 H₂O₂ + 12 mg L^?1 Zn. The stress response of C. braunii was analyzed by measuring photosynthetic photosystem II activity, chlorophyll a and b and carotenoid contents, the H₂O₂ concentration, and antioxidant enzyme activities of ascorbic peroxidase, catalase, and guaiacol peroxidase. The short-term addition of Zn reduced pigment contents in C. braunii. Chlorophyll a and b and carotenoid contents in H₂O₂-exposed C. braunii were as high as in control plants. Photosynthesis was reduced in H₂O₂-treated C. braunii and the short-term addition of Zn did not affect the electron transport rate. H₂O₂ concentration and antioxidant enzyme activities in C. braunii were not significantly different between control and exposed plants. Trends of enzymatic adaptation were described: the H₂O₂-induced stress response was characterized by increased antioxidant enzyme activities, whereas Zn inactivated catalase in C. braunii.     

Free radicals in exhaled breath condensate in cystic fibrosis and healthy subjects

Many markers of airway inflammation and oxidative stress can be measured non-invasively in exhaled breath condensate (EBC). However, no attempt has been made to directly detect free radicals using electron paramagnetic resonance (EPR) spectroscopy. Condensate was collected in 14 children with cystic fibrosis (CF) and seven healthy subjects. Free radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide. EPR spectra were recorded using a Bruker EMX^® spectrometer. Secondly, to study the source of oxygen centered radical formation, catalase or hydrogen peroxide was added to the condensate. Radicals were detected in 18 out of 21 condensate samples. Analysis of spectra indicated that both oxygen and carbon centered radicals were trapped. Within-subject reproducibility was good in all but one subject. Quantitatively, there was a trend towards higher maximal peak heights of both oxygen and carbon centered radicals in the children with CF. Catalase completely suppressed the signals in condensate. Addition of hydrogen peroxide resulted in increased radical signal intensity. Detection of free radicals in EBC of children with CF and healthy subjects is feasible using EPR spectroscopy.     

Increased hypoxia-inducible factor 1alpha expression in lung cells of horses with recurrent airway obstruction

ABSTRACT: BACKGROUND: Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition caused by exposure of susceptible horses to organic dusts in hay. The immunological processes responsible for the development and the persistence of airway inflammation are still largely unknown. Hypoxia-inducible factor (Hif) is mainly known as a major regulator of energy homeostasis and cellular adaptation to hypoxia. More recently however, Hif also emerged as an essential regulator of innate immune responses. Here, we aimed at investigating the potential involvement of Hif1-alpha in myeloid cells in horse with recurrent airway obstruction. RESULTS: In vitro, we observed that Hif is expressed in equine myeloid cells after hay dust stimulation and regulates genes such as tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A). We further showed in vivo that airway challenge with hay dust upregulated Hif1-alpha mRNA expression in myeloid cells from the bronchoalveolar lavage fluid (BALF) of healthy and RAO-affected horses, with a more pronounced effect in cells from RAO-affected horses. Finally, Hif1-alpha mRNA expression in BALF cells from challenged horses correlated positively with lung dysfunction. CONCLUSION: Taken together, our results suggest an important role for Hif1-alpha in myeloid cells during hay dust-induced inflammation in horses with RAO. We therefore propose that future research aiming at functional inactivation of Hif1 in lung myeloid cells could open new therapeutic perspectives for RAO.     

Effect of oxygen on ascorbic acid uptake and concentration in embryonic chick brain

The effects of oxygen on ascorbic acid concentration and transport were studied in chick embryo (Gallus gallus domesticus). During normoxic incubations, plasma ascorbic acid concentration peaked on fetal day 12 and then fell, before increasing again on day 20 when pulmonary respiration began. In contrast, cerebral ascorbic acid concentration rose after day 6, was maintained at a relatively high level during days 8–18, and then fell significantly by day 20. Exposure of day 16 embryos for 48 h to 42% ambient O₂ concentration decreased ascorbic acid concentration by four-fifths in plasma and by one-half in brain, compared to values in normoxic (21% O₂) or hypoxic (15% O₂) controls. Hyperoxic preincubation of embryos also inhibited ascorbic acid transport, as evidenced by decreased initial rates of saturable and Na⁺-dependent [¹⁴C]ascorbic acid uptake into isolated brain cells. It may be concluded that changes in ascorbic acid concentration occur in response to oxidative stress, consistent with a role for the vitamin in the detoxification of oxygen radicals in fetal tissues. However, changing O₂ levels have less effect on ascorbic acid concentration in brain than in plasma, indicating regulation of the vitamin by brain cells. Furthermore, the effect of hyperoxia on cerebral vitamin C may result, in part, from inhibition of cellular ascorbic acid transport.     

Effect of Selenium and Vitamin C on Clinical Outcomes, Trace Element Status, and Antioxidant Enzyme Activity in Horses with Acute and Chronic Lower Airway Disease. A Randomized Clinical Trial

Excess production of reactive oxygen species is involved in the pathogenesis of airway disorders in horses. Trace element antioxidants have a beneficial role in oxidant/antioxidant balance. The aim of the present study was to evaluate the effect of a combination of sodium selenite and ascorbic acid on clinical outcome, antioxidant enzymes, and trace elements status in horses with lower airway disease. For this purpose, 40 draft horses with lower airway disease were randomly selected (acute, n?=?20; chronic, n?=?20). Both acute and chronic cases were randomly allocated into two subgroups (ten each). Groups 1 and 2 were the horses with acute disease, while groups 3 and 4 were chronically ill. For all groups, each horse was administered antibiotic, non-steroidal anti-inflammatory, and mucolytic drug. In addition, groups 2 and 4 were injected with 15 mg/kg sodium selenite and 30 mg/kg ascorbic acid every 24 h for successive 4 weeks. Venous blood samples were obtained from diseased horses on three occasions; at first examination, and at 2 and 4 weeks post-treatment. Clinically, antioxidant supplementation improved the clinical signs with significant decrease (p?<?0.05) of the clinical index score in both acute and chronic cases. In supplemented groups compared with non-supplemented, there was a significant increase (p?<?0.05) in the levels of copper, zinc, selenium, and iron as well as in the activity of glutathione-S-transferase and catalase. Meanwhile, there was a significant decrease (p?<?0.05) in the levels of manganese, malondialdehyde, hydrogen peroxide, and low-density lipoprotein and in the activity of glutathione reductase. The results of the present study indicate that administration of sodium selenite and ascorbic acid may have beneficial effect on clinical outcome and antioxidant balance in horses with acute and chronic lower airway disease.