Antiinflammatory Effect of N-Acetylcysteine Combined with Exogenous Surfactant in Meconium-Induced Lung Injury.

Neonatal meconium aspiration syndrome (MAS) can be treated by exogenous surfactant (S). However, aspirated meconium initiates local inflammation and oxidation which may inactivate surfactant and reduce its action. This experimental study estimated whether combined use of surfactant and the antioxidant N-acetylcysteine (NAC) can enhance effectiveness of therapy. Meconium-instilled rabbits were non-treated (M), treated with monotherapies (M + S, M + NAC), combined therapy (M + S + NAC), or received saline instead of meconium (controls, C). Surfactant therapy consisted of two lung lavages (BAL) with diluted Curosurf (5 mg phospholipids/ml, 10 ml/kg) followed by undiluted Curosurf (100 mg phospholipids/kg). N-acetylcysteine (Acc Injekt, 10 mg/kg) was given intravenously in M + S + NAC group 10 min after surfactant therapy. Animals were oxygen-ventilated for additional 5 h. Then, differential white cell count in the blood (WBC) was determined. Left lung was saline-lavaged and differential cell count in BAL was determined. In right lung tissue, wet/dry weight ratio, oxidation markers (TBARS, 3NT) and interleukines (IL-2, IL-6, IL-13, and TNFα) using ELISA and RT-PCR were estimated. Combined S + NAC therapy significantly decreased W/D ratio, TBARS, 3NT, and IL, whereas the effect of monotherapies (either S or NAC) was less obvious. In conclusion, addition of NAC to surfactant treatment may enhance the therapeutic outcome in MAS.

Effects of S-Nitroso-N-Acetyl-Penicillamine (SNAP) on Inflammation, Lung Tissue Apoptosis and iNOS Activity in a Rabbit Model of Acute Lung Injury.

Acute lung injury is characterized by lung edema, surfactant dysfunction, and inflammation. The main goal of our study was to evaluate effects of S-nitroso-N-acetyl-penicillamine (SNAP) on migration of cells into the lung and their activation, inducible NO synthase (iNOS) activity, and apoptosis in experimental acute lung injury (ALI) in rabbits. ALI was induced by repetitive lung lavage with saline. The animals were divided into the following groups: (1) ALI without therapy, (2) lung injury treated with SNAP (ALI + SNAP), and (3) healthy animals (Control). After 5 h of ventilation, total and differential counts of cells in the bronchoalveolar lavage fluid (BALF) were assessed. Concentrations of interleukins (IL)-1ß, IL-6, and IL-8, endogenous secretory receptor for advanced glycation endproducts (esRAGE), sphingosine-1-phosphate receptor (S1PR)3, caspase-3, and mRNA expression of inducible NO synthase (iNOS) in lung tissue and nitrite/nitrate in plasma were analyzed. In the right lung, apoptotic cells were evaluated by TUNEL assay. In the animals with ALI, higher counts of cells, mainly neutrophils, in BALF and increased production of pro-inflammatory substances were observed compared with controls. SNAP therapy reduced a leak of cells into the lung and decreased concentrations of pro-inflammatory and apoptotic markers, reduced mRNA expression of iNOS, and decreased apoptotic index in the lung.

Bronchodilator and Anti-Inflammatory Action of Theophylline in a Model of Ovalbumin-Induced Allergic Inflammation.

Phosphodiesterases (PDEs) represent a super-family of 11 enzymes hydrolyzing cyclic nucleotides into inactive 5' monophosphates. Inhibition of PDEs leads to a variety of cellular effects, including airway smooth muscle relaxation, inhibition of cellular inflammation, and immune responses. In this study we focused on theophylline, a known non-selective inhibitor of PDEs. Theophylline has been used for decades in the treatment of chronic inflammatory airway diseases. It has a narrow therapeutic window and belongs to the drugs whose plasma concentration should be monitored. Therefore, the main goal of this study was to evaluate the plasma theophylline concentration and to determine its relevance to pharmacological effects after single and longer term (7 days) administration of theophylline at different doses (5, 10, 20, and 50 mg/kg) in guinea pigs. Airway hyperresponsiveness was assessed by repeated exposure to ovalbumin. Theophylline reduced specific airway resistance in response to histamine nebulization, measured in a double chamber body plethysmograph. A decrease in tracheal smooth muscle contractility after cumulative doses of histamine and acetylcholine was confirmed in vitro. A greater efficacy of theophylline after seven days long treatment indicates the predominance of its anti-inflammatory activity, which may be involved in the bronchodilating action.

High-Frequency Jet Ventilation against Small-Volume Conventional Mechanical Ventilation in the Rabbit Models of Neonatal Acute Lung Injury.

Patients with acute lung injury are ventilated by conventional mechanical ventilation (CMV) rather than high-frequency jet ventilation (HFJV). This study estimated the potential usefulness of HFJV in acute lung injury. The issue was addressed by comparing the effects on lung function of CMV and HFJV in two rabbit models of neonatal acute lung injury: repetitive saline lung lavage (LAV) and meconium aspiration syndrome (MAS) induced by intratracheal meconium instillation. The animals were then ventilated with either HFJV or CMV for 4 h. Ventilatory pressures, blood gases, and indexes of gas exchange were assessed. Lung edema formation was expressed as wet-dry lung weight ratio. Both LAV and MAS significantly decreased lung compliance, increased airway resistance, and caused severe hypoxemia, hypercarbia, and acidosis. Although CMV was superior to HFJV at 1 h of ventilation, there were no clinically relevant differences in lung function or edema formation between CMV and LAV in both models of respiratory insufficiency at 4 h of ventilation. We conclude that, HFJV may be used for ventilation in acute non-homogenous lung injury.

Effects of Selective Inhibition of PDE4 by YM976 on Airway Reactivity and Cough in Ovalbumin-Sensitized Guinea Pigs.

Phosphodiesterases (PDEs) are enzymes involved in the degradation of cAMP and cGMP. Selective PDE4 inhibitors (e.g., roflumilast) are effective in therapy of chronic obstructive pulmonary disease associated with neutrophil inflammation. The aim of this study was to evaluate the effects of a selective PDE4 inhibitor, YM976, on citric acid-induced cough, in vivo and in vitro airway smooth muscle reactivity to histamine, and on inflammatory mediators in ovalbumin-sensitized guinea pigs, with experimentally induced eosinophil inflammation. The YM976 was administered intraperitoneally at a dose of 1.0 mg/kg once daily for 7 days. Sensitization with ovalbumin led to a significant increase in the number of coughs, and in vivo and in vitro airway reactivity. Also, increased plasma levels of IL-4, IL-5, and PAF were observed, with a significant increase in the differential count of eosinophils in both blood and bronchoalveolar lavage fluid. The YM976 suppressed the number of coughs, the airway reactivity in tracheal tissue strips, and the IL-4 level. The findings indicate that PDE4 inhibition by YM976 exerts antitussive and anti-inflammatory effects in guinea pigs with ovalbumin-induced eosinophilic inflammation.

Influence of roflumilast on airway reactivity and apoptosis in ovalbumin-sensitized Guinea pigs.

Chronic inflammatory diseases, associated with airway obstruction and cough, are usually treated with bronchodilating and anti-inflammatory drugs. Inhibition of phosphodiesterases (PDE) leads to both of these effects and influences apoptosis of immune cells. In chronic obstructive pulmonary disease, roflumilast, a selective PDE4 inhibitor, has been recently approved for pharmacotherapy. The aim of this study was to evaluate the effect of long-term administration of roflumilast in experimental allergic inflammation in guinea pigs. Male adult guinea pigs were used in the study. There were four experimental groups sensitized with ovalbumin for 14 days and thereafter treated per os, by inhalation, and intraperitoneally for 7 days with roflumilast or vehicle. A control group was left without sensitization. Roflumilast reduced specific airway resistance after nebulization of histamine, as measured in a double-chamber whole-body plethysmograph. This effect was confirmed in in vitro organ bath, with significant decreases in tracheal and lung smooth muscle contractility after cumulative doses of histamine. Suppression of hematological and immunological markers of inflammation and enhanced apoptosis in animals treated with roflumilast points to the possibility of a beneficial effect of roflumilast in allergic inflammation.

N-acetylcysteine alleviates the meconium-induced acute lung injury.

Meconium aspiration in newborns causes lung inflammation and injury, which may lead to meconium aspiration syndrome (MAS). In this study, the effect of the antioxidant N-acetylcysteine on respiratory and inflammatory parameters were studied in a model of MAS. Oxygen-ventilated rabbits were intratracheally given 4 mL/kg of meconium (25 mg/mL) or saline. Thirty minutes later, meconium-instilled animals were administered N-acetylcysteine (10 mg/kg; i.v.), or were left without treatment. The animals were oxygen-ventilated for additional 5 h. Ventilatory pressures, oxygenation, right-to-left pulmonary shunts, and leukocyte count were measured. At the end of experiment, trachea and lung were excised. The left lung was saline-lavaged and a total and differential count of cells in bronchoalveolar lavage fluid (BAL) was determined. Right lung tissue strips were used for detection of lung edema (expressed as wet/dry weight ratio) and peroxidation (expressed by thiobarbituric acid-reactive substances, TBARS). In lung and tracheal strips, airway reactivity to acetylcholine was measured. In addition, TBARS and total antioxidant status were determined in the plasma. Meconium instillation induced polymorphonuclear-derived inflammation and oxidative stress. N-acetylcysteine improved oxygenation, reduced lung edema, decreased polymorphonuclears in BAL fluid, and diminished peroxidation and meconium-induced airway hyperreactivity compared with untreated animals. In conclusion, N-acetylcysteine effectively improved lung functions in an animal model of MAS.

The influence of L-NAME on iNOS expression and markers of oxidative stress in allergen-induced airway hyperreactivity.

Nitric oxide (NO) effects in airways are influenced by the activity of NO-synthase isoforms and NO metabolism. Inducible NO-synthase (iNOS), which produces large amounts of NO, is active during the inflammatory process. NO quickly reacts, producing reactive oxygen species (ROS). In this study we attempted to detect the expression of iNOS and markers of ROS in the airway hyperreactivity (AHR) condition. The study was performed in guinea pigs, divided into four groups. Two groups were treated with the non-selective inhibitor of NO-synthase L-NAME. The other two groups were used as controls. Exhaled NO was monitored in vivo, AHR was assessed both in vivo and in vitro, and the expression of iNOS in lung homogenate, and oxidative stress markers were measured in the venous blood. L-NAME significantly affected the AHR only in in vitro condition, blocked the expression of iNOS in control but not in sensitized animals, and decreased the level of exhaled NO. The results concerning the oxidative stress markers are equivocal. The study confirmed that NO is involved in the regulation of AHR; the effects being mediated via iNOS and ROS activity.

Effects on Lung Function of Small-Volume Conventional Ventilation and High-Frequency Oscillatory Ventilation in a Model of Meconium Aspiration Syndrome.

For treatment of severe neonatal meconium aspiration syndrome (MAS), lung-protective mechanical ventilation is essential. This study compared short-term effects of small-volume conventional mechanical ventilation and high-frequency oscillatory ventilation on lung function in experimentally-induced MAS. In conventionally-ventilated rabbits, MAS was induced by intratracheal instillation of meconium suspension (4 ml/kg, 25 mg/ml). Then, animals were ventilated conventionally with small-volume (f-50/min; VT-6 ml/kg) or with high frequency ventilation (f-10/s) for 4 h, with the evaluation of blood gases, ventilatory pressures, and pulmonary shunts. After sacrifice, left lung was saline-lavaged and cells in bronchoalveolar lavage fluid (BALF) were determined. Right lung was used for the estimation of lung edema formation (wet/dry weight ratio). Thiobarbituric acid-reactive substances (TBARS), oxidative damage markers, were detected in lung tissue and plasma. Meconium instillation worsened gas exchange, and induced inflammation and lung edema. Within 4 h of ventilation, high frequency ventilation improved arterial pH and CO2 elimination compared with conventional ventilation. However, no other significant differences in oxygenation, ventilatory pressures, shunts, BALF cell counts, TBARS concentrations, or edema formation were observed between the two kinds of ventilation. We conclude that high frequency ventilation has only a slight advantage over small-volume conventional ventilation in the model of meconium aspiration syndrome in that it improves CO2 elimination.

Alterations in vagal-immune pathway in long-lasting mental stress.

We studied a potential impact of chronic psychosocial load on the allostatic biomarkers--cardiac vagal activity, inflammation, and oxidative stress in healthy undergraduate students. Continuous resting ECG signals were monitored in a group of 16 female healthy students (age: 23.2±0.2 years, BMI: 20.9±0.5 kg/m2) at two time periods: right after holiday (rest period) and a day before final exams (stress period). Vagal activity was quantified by spectral analysis of heart rate variability at high frequency band (HF-HRV). The immune response was assessed from the level of tumor necrosis factor-alpha (TNF-α) in plasma. In addition, mean RR intervals were evaluated. We found that HF-HRV was significantly reduced and the TNF-α was increased in the stress period compared with the rest period. No significant changes were found in the RR interval. In conclusion, allostatic load induced by stress and the accompanying greater immune response decreased cardiovagal regulation in healthy young subjects. These findings may help understand the pathway by which stress can influence health and disease.

Cardiovascular effects of N-acetylcysteine in meconium-induced acute lung injury.

Anti-inflammatory drugs are increasingly used for treatment of neonatal meconium aspiration syndrome (MAS), but their adverse effects are poorly known. Therefore, the aim of this study was to evaluate the effects of the antioxidant N-acetylcysteine on cardiovascular parameters in an animal model of MAS. Oxygen-ventilated rabbits were intratracheally instilled 4 mL/kg of meconium suspension (25 mg/mL) or saline. Thirty minutes later, meconium-instilled animals were given N-acetylcysteine (10 mg/kg, i.v.) or the same volume of saline. Changes in cardiovascular parameters (blood pressure, heart rate, and heart rate variability) were recorded over a 5-min course of solution administration, over 5 min after its end, and then hourly for 5 h. Oxidation markers (thiobarbituric acid-reactive substances (TBARS) and total antioxidant status) and aldosterone, as a non-specific marker of cardiovascular injury, were determined in plasma. Meconium instillation did not evoke any significant cardiovascular changes, but induced oxidative stress and elevated plasma aldosterone. N-acetylcysteine significantly reduced the mentioned markers of injury. However, its administration was associated with short-term increases in blood pressure and in several parameters of heart rate variability. Considering these effects of N-acetylcysteine, its intravenous administration in newborns with MAS should be carefully monitored.

Inflammatory Activity in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder in early childhood characterized by impairment in communication and behavior. Recent research is focused on the immune dysregulation as a potential pathomechanism leading to ASD. Thus, we addressed the hypothesis that inflammatory activity might be enhanced in children suffering from ASD. We examined 15 children with ASD (13 boys/2 girls, mean age of 9.3 ± 0.7 years) and 20 age/gender-matched healthy subjects as a control group. All children were medication free and in good health. Hematological parameters in venous blood and plasma levels of pro-inflammatory cytokines - tumor necrosis factor alpha (TNF-α), interleukin 1ß (IL-1ß), and interleukin 8 (IL-8) - were assessed in each subject using human ultra-sensitive ELISA kits. In addition, TBARS as a marker of oxidative stress was evaluated. We found that the level of IL-8 was significantly increased in the ASD children, whereas the other markers remained unappreciably changed compared to controls (p = 0.003). In conclusion, the study demonstrates a discrete immune dysfunction in ASD of pro-inflammatory character.

Pulmonary surfactant and prostaglandin E2 in airway smooth muscle relaxation of human and male guinea pigs.

Pulmonary surfactant serves as a barrier to respiratory epithelium but can also regulate airway smooth muscle (ASM) tone. Surfactant (SF) relaxes contracted ASM, similar to ß2-agonists, anticholinergics, nitric oxide, and prostanoids. The exact mechanism of surfactant relaxation and whether surfactant relaxes hyperresponsive ASM remains unknown. Based on previous research, relaxation requires an intact epithelium and prostanoid synthesis. We sought to examine the mechanisms by which surfactant causes ASM relaxation. Organ bath measurements of isometric tension of ASM of guinea pigs in response to exogenous surfactant revealed that surfactant reduces tension of healthy and hyperresponsive tracheal tissue. The relaxant effect of surfactant was reduced if prostanoid synthesis was inhibited and/or if prostaglandin E2-related EP2 receptors were antagonized. Atomic force microscopy revealed that human ASM cells stiffen during contraction and soften during relaxation. Surfactant softened ASM cells, similarly to the known bronchodilator prostaglandin E2 (PGE2) and the cell softening was abolished when EP4 receptors for PGE2 were antagonized. Elevated levels of PGE2 were found in cultures of normal human bronchial epithelial cells exposed to pulmonary surfactant. We conclude that prostaglandin E2 and its EP2 and EP4 receptors are likely involved in the relaxant effect of pulmonary surfactant in airways.

Anti-inflammatory treatment in dysfunction of pulmonary surfactant in meconium-induced acute lung injury.

Inflammation, oxidation, lung edema, and other factors participate in surfactant dysfunction in meconium aspiration syndrome (MAS). Therefore, we hypothesized that anti-inflammatory treatment may reverse surfactant dysfunction in the MAS model. Oxygen-ventilated rabbits were given meconium intratracheally (25 mg/ml, 4 ml/kg; Mec) or saline (Sal). Thirty minutes later, meconium-instilled animals were treated by glucocorticoids budesonide (0.25 mg/kg, i.t.) and dexamethasone (0.5 mg/kg, i.v.), or phosphodiesterase inhibitors aminophylline (2 mg/kg, i.v.) and olprinone (0.2 mg/kg, i.v.), or the antioxidant N-acetylcysteine (10 mg/kg, i.v.). Healthy, non-ventilated animals served as controls (Con). At the end of experiments, left lung was lavaged and a differential leukocyte count in sediment was estimated. The supernatant of lavage fluid was adjusted to a concentration of 0.5 mg phospholipids/ml. Surfactant quality was evaluated by capillary surfactometer and expressed by initial pressure and the time of capillary patency. The right lung was used to determine lung edema by wet/dry (W/D) weight ratio. Total antioxidant status (TAS) in blood plasma was evaluated. W/D ratio increased and capillary patency time shortened significantly, whereas the initial pressure increased and TAS decreased insignificantly in Sal vs. Con groups. Meconium instillation potentiated edema formation and neutrophil influx into the lungs, reduced capillary patency and TAS, and decreased the surfactant quality compared with both Sal and Con groups (p > 0.05). Each of the anti-inflammatory agents reduced lung edema and neutrophil influx into the lung and partly reversed surfactant dysfunction in the MAS model, with a superior effect observed after glucocorticoids and the antioxidant N-acetylcysteine.

Cardiovascular side effects of aminophylline in meconium-induced acute lung injury.

As inflammation plays an important role in the pathogenesis of neonatal meconium aspiration syndrome (MAS), anti-inflammatory agents including inhibitors of phosphodiesterases (PDE) are increasingly used in the treatment. To evaluate side effects of PDE inhibitors, this study analyzed changes in blood pressure, heart rate (HR) and heart rate variability (HRV) during and after intravenous aminophylline in the animal model of MAS. Oxygen-ventilated rabbits were given meconium intratracheally (25 mg/ml, 4 ml/kg) or saline. Thirty minutes later, the animals were treated by intravenous aminophylline (Syntophyllin, 2 mg/kg) or saline (sham-treated controls). A second dose of the treatment was given 2 h later. During (5 min) and immediately after (5 min) the treatment, and during 5 h after the treatment, mean blood pressure in the femoral artery (MAP), HR and HRV were evaluated. In meconium-instilled animals, increases in MABP, HR, and HRV were observed already 5 min after aminophylline administration, while in saline-instilled animals aminophylline increased HR and caused inconsistant changes in HRV parameters compared to sham-treated animals. Within 5 h after the treatment administration, MAP, HR, and HRV parameters gradually returned to the initial values. Concluding, intravenous aminophylline may lead to acute cardiovascular changes. Thus, if aminophylline is used for treatment of MAS, its possible cardiovascular effects should be considered, particularly in patients with cardiovascular instability.

Sex differences in plasma metabolites in a guinea pig model of allergic asthma.

Sex seems to be a contributing factor in the pathogenesis of bronchial asthma. This study aimed to find sex-related differences in metabolome measured by hydrogen-1 nuclear magnetic resonance ((1)H NMR) spectroscopy in healthy and ovalbumin (OVA)-sensitized guinea pigs. Adult male and female animals were divided into controls and OVA-sensitized groups. OVA-sensitization was performed by OVA systemic and inhalational administration within 14 days; on day 15, animals were killed by anesthetic overdose followed by exsanguination. Blood was taken and differential white blood cell count was measured. Left lung was saline-lavaged and differential cell count in the bronchoalveolar lavage fluid (BALF) was measured. After blood centrifugation, plasma was processed for (1)H NMR analysis. Metabolomic data was evaluated by principal component analysis (PCA). Eosinophil counts elevated in the BALF confirming eosinophil-mediated inflammation in OVA-sensitized animals of both sexes. Sex differences for lactate, glucose, and citrate were found in controls, where these parameters were lower in males than in females. In OVA-sensitized males higher glucose and lower pyruvate were found compared to controls. OVA-sensitized females showed lower lactate, glucose, alanine, 3-hydroxy-butyrate, creatine, pyruvate, and succinate concentrations compared to controls. In OVA-sensitized animals, lactate concentration was lower in males. Data from females (healthy and OVA-sensitized) were generally more heterogeneous. Significant sex differences in plasma concentrations of metabolites were found in both healthy and OVA-sensitized animals suggesting that sex may influence the metabolism and may thereby contribute to different clinical picture of asthma in males and females.

Efficacy of surfactant therapy of ARDS induced by hydrochloric acid aspiration followed by ventilator-induced lung injury - an animal study.

The development of acute respiratory distress syndrome (ARDS) is known to be independently attributable to aspiration-induced lung injury. Mechanical ventilation as a high pressure/volume support to maintain sufficient oxygenation of a patient could initiate ventilator-induced lung injury (VILI) and thus contribute to lung damage. Although these phenomena are rare in the clinic, they could serve as the severe experimental model of alveolar-capillary membrane deterioration. Lung collapse, diffuse inflammation, alveolar epithelial and endothelial damage, leakage of fluid into the alveoli, and subsequent inactivation of pulmonary surfactant, leading to respiratory failure. Therefore, exogenous surfactant could be considered as a therapy to restore lung function in experimental ARDS. This study aimed to investigate the effect of modified porcine surfactant in animal model of severe ARDS (P/F ratio

Herbal compounds in the treatment of pulmonary silicosis.

Herbal compounds including those already well-established in traditional Chinese medicine have been increasingly tested in the treatment of various diseases. Recent studies have shown that herbal compounds can be of benefit also for pulmonary silicosis as they can diminish changes associated with silica-induced inflammation, fibrosis, and oxidative stress. Due to a lack of effective therapeutic strategies, development of novel approaches which may be introduced particularly in the early stage of the disease, is urgently needed. This review summarizes positive effects of several alternative plant-based drugs in the models of experimental silicosis with a potential for subsequent clinical investigation and use in future.

Acute lung injury - from pathophysiology to treatment.

Acute lung injury is characterized by acute respiratory insufficiency with tachypnea, cyanosis refractory to oxygen, decreased lung compliance, and diffuse alveolar infiltrates on chest X-ray. The 1994 American-European Consensus Conference defined "acute respiratory distress syndrome, ARDS" by acute onset after a known trigger, severe hypoxemia defined by PaO2/FiO2

Exhaled and Nasal Nitric Oxide - Impact for Allergic Rhinitis.

FeNO measurement is a validated non-invasive technique, which is used for diagnosis and monitoring of asthma. It would be desirable to find a reliable method to monitor allergic rhinitis (AR) via measurement of FeNO, and/or nasal nitric oxide (nNO). The aim of our study was the assessment of the efficacy of FeNO and nNO as markers in AR treatment. FeNO and nNO were measured with the portable NO analyser (NIOX MINO®) in healthy participants and in patients with AR. The patients were examined during the pollen season and out of it. The effect of local corticosteroids and antihistamine therapy was observed in patients with AR during pollen season after three weeks of therapy. There are significant differences between FeNO and nNO in patients with AR compared to healthy controls at all set points of measurements. While FeNO responded well to the treatment with both antihistamines and combined therapy, nNO decreased only after combined therapy with antihistamines and nasal corticosteroids. nNO monitoring alone is not a suitable method to monitor inflammation of the upper airways in AR and its suppression by anti-allergic treatment and should be correlated with other markers as FeNO or symptom scores.