Exhaled nitric oxide : Variability and association with bronchial hyperresponsiveness and atopy

Airway inflammation is a key feature of bronchial asthma. In asthma management, according to international guidelines, the gold standard is anti-inflammatory treatment. Currently, only conventional procedures (i.e., symptoms, use of rescue medication, PEF-variability, and lung function tests) were used to both diagnose and evaluate the results of treatment with anti-inflammatory drugs. New methods for evaluation of degree of airway inflammation are required. Nitric oxide (NO) is a gas which is produced in the airways of healthy subjects and especially produced in asthmatic airways. Measurement of NO from the airways is possible, and NO can be measured from exhaled air. Fractional exhaled NO (FENO) is increased in asthma, and the highest concentrations are measured in asthmatic patients not treated with inhaled corticosteroids (ICS). Steroid-treated patients with asthma had levels of FENO similar to those of healthy controls. Atopic asthmatics had higher levels of FENO than did nonatopic asthmatics, indicating that level of atopy affected FENO level. Associations between FENO and bronchial hyperresponsiveness (BHR) occur in asthma. The present study demonstrated that measurement of FENO had good reproducibility, and the FENO variability was reasonable both short- and long-term in both healthy subjects and patients with respiratory symptoms or asthma. We demonstrated the upper normal limit for healthy subjects, which was 12 ppb calculated from two different healthy study populations. We showed that patients with respiratory symptoms who did not fulfil the diagnostic criteria of asthma had FENO values significantly higher than in healthy subjects, but significantly lower than in asthma patients. These findings suggest that BHR to histamine is a sensitive indicator of the effect of ICS and a valuable tool for adjustment of corticosteroid treatment in mild asthma. The findings further suggest that intermittent treatment periods of a few weeks’ duration are insufficient to provide long-term control of BHR in patients with mild persistent asthma. Moreover, during the treatment with ICS changes in BHR and changes in FENO were associated. FENO level was associated with BHR measured by a direct (histamine challenge) or indirect method (exercise challenge) in steroid-naïve symptomatic, non-smoking asthmatics. Although these associations could be found only in atopics, FENO level in nonatopic asthma was also increased. It can thus be concluded that assessment of airway inflammation by measuring FENO can be useful for clinical purposes. The methodology of FENO measurements is now validated. Especially in those patients with respiratory symptoms who did not fulfil the diagnostic criteria of asthma, FENO measurement can aid in treatment decisions. Serial measurement of FENO during treatment with ICS can be a complementary or an alternative method for evaluation in patients with asthma.

Temperature effect on hydrocarbon-enhanced nitric oxide conversion using a dielectric barrier discharge reactor

Experimental investigations into the effect of temperature on conversion of NO in the presence of hydrocarbons (ethylene, acetylene and n-hexane) are presented. An AC energized dielectric barrier discharge reactor was used as the plasma reactor. The experiments were carried out at different temperatures up to 200 degreesC. The discharge powers were measured at all the temperatures. The discharge power was found to increase with temperature. NO conversion in the presence of ethylene and n-hexane was better than that of acetylene at all temperatures. The addition of acetylene at room temperature showed no better conversion of NO compared to no additive case. While at higher temperatures, it could enhance the conversion of NO. A slight enhancement in NO and NOx removal was observed in the presence of water vapor. (C) 2003 Elsevier Science B.V. All rights reserved.

Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress Major histocompatibility complex II expression during Mycobacterium bovis Bacillus Calmette-Guerin infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guerin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-gamma-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Nitric oxide is the key mediator of death induced by fisetin in human acute monocytic leukemia cells

Nitric oxide ( NO) has been shown to be effective in cancer chemoprevention and therefore drugs that help generate NO would be preferable for combination chemotherapy or solo use. This study shows a new evidence of NO as a mediator of acute leukemia cell death induced by fisetin, a promising chemotherapeutic agent. Fisetin was able to kill THP-1 cells in vivo resulting in tumor shrinkage in the mouse xenograft model. Death induction in vitro was mediated by an increase in NO resulting in double strand DNA breaks and the activation of both the extrinsic and the intrinsic apoptotic pathways. Double strand DNA breaks could be reduced if NO inhibitor was present during fisetin treatment. Fisetin also inhibited the downstream components of the mTORC1 pathway through downregulation of levels of p70 S6 kinase and inducing hypo-phosphorylation of S6 Ri P kinase, eIF4B and eEF2K. NO inhibition restored phosphorylation of downstream effectors of mTORC1 and rescued cells from death. Fisetin induced Ca2+ entry through L-type Ca2+ channels and abrogation of Ca2+ influx reduced caspase activation and cell death. NO increase and increased Ca2+ were independent phenomenon. It was inferred that apoptotic death of acute monocytic leukemia cells was induced by fisetin through increased generation of NO and elevated Ca2+ entry activating the caspase dependent apoptotic pathways. Therefore, manipulation of NO production could be viewed as a potential strategy to increase efficacy of chemotherapy in acute monocytic leukemia.

I. The rate and mechanism of the vapor-phase reaction between water and parts-per-million concentrations of nitrogen dioxide. II. The rate and mechanism of the air oxidation of parts-per-million concentrations of nitric oxide in the presence of water vapor.

Part I

A study of the thermal reaction of water vapor and parts-per-million concentrations of nitrogen dioxide was carried out at ambient temperature and at atmospheric pressure. Nitric oxide and nitric acid vapor were the principal products. The initial rate of disappearance of nitrogen dioxide was first order with respect to water vapor and second order with respect to nitrogen dioxide. An initial third-order rate constant of 5.5 (± 0.29) x 10⁴ liter² mole^-2 sec^-1 was found at 25˚C. The rate of reaction decreased with increasing temperature. In the temperature range of 25˚C to 50˚C, an activation energy of -978 (± 20) calories was found.

The reaction did not go to completion. From measurements as the reaction approached equilibrium, the free energy of nitric acid vapor was calculated. This value was -18.58 (± 0.04) kilocalories at 25˚C.

The initial rate of reaction was unaffected by the presence of oxygen and was retarded by the presence of nitric oxide. There were no appreciable effects due to the surface of the reactor. Nitric oxide and nitrogen dioxide were monitored by gas chromatography during the reaction.

Part II

The air oxidation of nitric oxide, and the oxidation of nitric oxide in the presence of water vapor, were studied in a glass reactor at ambient temperatures and at atmospheric pressure. The concentration of nitric oxide was less than 100 parts-per-million. The concentration of nitrogen dioxide was monitored by gas chromatography during the reaction.

For the dry oxidation, the third-order rate constant was 1.46 (± 0.03) x 10⁴ liter² mole^-2 sec^-1 at 25˚C. The activation energy, obtained from measurements between 25˚C and 50˚C, was -1.197 (±0.02) kilocalories.

The presence of water vapor during the oxidation caused the formation of nitrous acid vapor when nitric oxide, nitrogen dioxide and water vapor combined. By measuring the difference between the concentrations of nitrogen dioxide during the wet and dry oxidations, the rate of formation of nitrous acid vapor was found. The third-order rate constant for the formation of nitrous acid vapor was equal to 1.5 (± 0.5) x 10⁵ liter² mole^-2 sec^-1 at 40˚C. The reaction rate did not change measurably when the temperature was increased to 50˚C. The formation of nitric acid vapor was prevented by keeping the concentration of nitrogen dioxide low.

Surface effects were appreciable for the wet tests. Below 35˚C, the rate of appearance of nitrogen dioxide increased with increasing surface. Above 40˚C, the effect of surface was small.

Thermodynamic and kinetic behavior of an argon plasma jet. Decomposition of nitric oxide between 1300 - 1750 degrees K in an argon plasma

In the first part of the study, an RF coupled, atmospheric pressure, laminar plasma jet of argon was investigated for thermodynamic equilibrium and some rate processes.

Improved values of transition probabilities for 17 lines of argon I were developed from known values for 7 lines. The effect of inhomogeneity of the source was pointed out.

The temperatures, T, and the electron densities, n_e , were determined spectroscopically from the population densities of the higher excited states assuming the Saha-Boltzmann relationship to be valid for these states. The axial velocities, v_z, were measured by tracing the paths of particles of boron nitride using a three-dimentional mapping technique. The above quantities varied in the following ranges: 10¹² ˂ n_e ˂ 10¹⁵ particles/cm³, 3500 ˂ T ˂ 11000 °K, and 200 ˂ v_z ˂ 1200 cm/sec.

The absence of excitation equilibrium for the lower excitation population including the ground state under certain conditions of T and n_e was established and the departure from equilibrium was examined quantitatively. The ground state was shown to be highly underpopulated for the decaying plasma.

Rates of recombination between electrons and ions were obtained by solving the steady-state equation of continuity for electrons. The observed rates were consistent with a dissociative-molecular ion mechanism with a steady-state assumption for the molecular ions.

In the second part of the study, decomposition of NO was studied in the plasma at lower temperatures. The mole fractions of NO denoted by x_NO were determined gas-chromatographically and varied between 0.0012 ˂ x_NO ˂ 0.0055. The temperatures were measured pyrometrically and varied between 1300 ˂ T ˂ 1750°K. The observed rates of decomposition were orders of magnitude greater than those obtained by the previous workers under purely thermal reaction conditions. The overall activation energy was about 9 kcal/g mol which was considerably lower than the value under thermal conditions. The effect of excess nitrogen was to reduce the rate of decomposition of NO and to increase the order of the reaction with respect to NO from 1.33 to 1.85. The observed rates were consistent with a chain mechanism in which atomic nitrogen and oxygen act as chain carriers. The increased rates of decomposition and the reduced activation energy in the presence of the plasma could be explained on the basis of the observed large amount of atomic nitrogen which was probably formed as the result of reactions between excited atoms and ions of argon and the molecular nitrogen.

Efeitos de diferentes dietas hiperlipídicas na via L-arginina-óxido nítrico e no stress oxidativo em eritrócitos de camundongos C57BL/6

Introdução: o óxido nítrico (NO) é um gás inorgânico com uma meia-vida curta e tem um papel crítico na manutenção da homeostase vascular e fluidez sanguínea. O NO é sintetizado a partir do aminoácido L-arginina por uma família de enzimas NO sintases (NOS). Estudos têm mostrado que eritrócitos expressam NOS endotelial (eNOS) funcional, que serve como uma fonte de NO intraluminal. Além disso, eritrócitos participam da defesa antioxidante removendo os radicais livres e prevenindo o dano oxidativo às membranas biológicas e a destruição do NO. Dietas hiperlípidicas estão associadas a um risco aumentado de doença cardiovacular e síndrome metabólica, mas os exatos mecanismos não estão completamente esclarecidos. O objetivo deste estudo foi investigar os efeitos de diferentes dietas hiperlípidicas na via L-arginina-NO e o estresse oxidativo em eritrócitos de camundongos. Metodologia: camundongos machos C57BL/6 de três meses de idade receberam diferentes dietas por 10 semanas: dieta normolipídica ou dieta hiperlipídica contendo banha de porco (HB), óleo de oliva (HO), óleo de girassol (HG) ou óleo de canola (HC). Foram analisados o transporte de L-arginina mediado pelos transportadores catiônicos y+ e y+L, a atividade da NOS, a expressão da eNOS e da NOS induzível (iNOS), a formação de substâncias reativas ao ácido tiobarbitúrico (TBARS) e a atividade das enzimas antioxidantes catalase (CAT) e superóxido dismutase (SOD). Resultados: o transporte total de L-arginina estava aumentado no grupo HO em comparação aos controles e aos outros grupos com dieta hiperlipídica. Quando o transporte foi fracionado, o sistema y+ estava mais ativado no grupo HO em relação aos controles e outros grupos que receberam dieta hiperlipídica. O transporte de L-arginina via sistema y+L estava maior nos grupos HO, HG e HC comparados aos grupos controle e HB. Adicionalmente, a atividade basal da NOS e a expressão de eNOS estavam aumentadas em eritrócitos independente do tipo de dieta hiperlípidica insaturada. Observou-se uma maior expressão da iNOS no grupo HO comparado ao controle. Em contraste, o grupo HB apresentou uma inibição da via L-arginina-NO. A análise da peroxidação lipídica, através da formação de TBARS, e da atividade da enzima antioxidante CAT não revelou diferenças entre os grupos, ao contrário do grupo HO, que induziu uma ativação de outra enzima antioxidante, a SOD. Conclusões: o presente estudo proporciona a primeira evidência de que os sistemas y+ e y+L regulam o transporte aumentado de L-arginina em eritrócitos de camundongos do grupo HO. Além disso, todas as dietas hiperlipídicas insaturadas induzem um aumento da atividade basal da NOS associada a uma expressão elevada da eNOS. É possível que diferentes mudanças na composição lipídica da membrana plasmática induzidas pelas dietas possam afetar transportadores e enzimas nos eritrócitos. Além disso, a inibição da via L-arginina-NO no grupo HB pode contribuir para o desenvolvimento da aterosclerose, enquanto dietas hiperlipídicas insaturadas podem ter um efeito protetor via aumento da geração de NO.

Alveolar nitric oxide and its role in pediatric asthma control assessment

Resumen Background: Nitric oxide can be measured at multiple flow rates to determine proximal (maximum airway nitric oxide flux; Jaw(NO)) and distal inflammation (alveolar nitric oxide concentration; CA(NO)). The main aim was to study the association among symptoms, lung function, proximal (maximum airway nitric oxide flux) and distal (alveolar nitric oxide concentration) airway inflammation in asthmatic children treated and not treated with inhaled glucocorticoids. Methods: A cross-sectional study with prospective data collection was carried out in a consecutive sample of girls and boys aged between 6 and 16 years with a medical diagnosis of asthma. Maximum airway nitric oxide flux and alveolar nitric oxide concentration were calculated according to the two-compartment model. In asthmatic patients, the asthma control questionnaire (CAN) was completed and forced spirometry was performed. In controls, differences between the sexes in alveolar nitric oxide concentration and maximum airway nitric oxide flux and their correlation with height were studied. The correlation among the fraction of exhaled NO at 50 ml/s (FENO50), CA(NO), Jaw(NO), forced expiratory volume in 1 second (FEV1) and the CAN questionnaire was measured and the degree of agreement regarding asthma control assessment was studied using Cohen's kappa. Results: We studied 162 children; 49 healthy (group 1), 23 asthmatic participants without treatment (group 2) and 80 asthmatic patients treated with inhaled corticosteroids (group 3). CA(NO) (ppb) was 2.2 (0.1-4.5), 3 (0.2-9.2) and 2.45 (0.1-24), respectively. Jaw(NO) (pl/s) was 516 (98.3-1470), 2356.67 (120-6110) and 1426 (156-11805), respectively. There was a strong association (r = 0.97) between FENO50 and Jaw(NO) and the degree of agreement was very good in group 2 and was good in group 3. There was no agreement or only slight agreement between the measures used to monitor asthma control (FEV1, CAN questionnaire, CA(NO) and Jaw(NO)). Conclusions: The results for CA(NO) and Jaw(NO) in controls were similar to those found in other reports. There was no agreement or only slight agreement among the three measure instruments analyzed to assess asthma control. In our sample, no additional information was provided by CA(NO) and Jaw(NO).

Avaliação do estrese oxidativo e da via L-arginina-óxido nítrico em plaquetas na gestação normal e na pré-eclâmpsia

A Pré-eclâmpsia (PE) é uma síndrome hipertensiva específica da gravidez, atualmente compreendida como uma doença sistêmica que cursa com inflamação, distúrbios da coagulação, desordens metabólicas, disfunção endotelial e desequilíbrio entre agentes vasoconstrictores e vasodilatadores. Contudo, a fisiopatologia da PE ainda não foi completamente elucidada. Este estudo investigou, em 51 gestantes, o estresse oxidativo, a via L-arginina-óxido nítrico e agregação plaquetária, na gestação normal (n=27) e na PE (n=24). Amostras de soro e plaquetas de gestantes normotensas e com PE, foram utilizadas para a medida de espécies reativas ao ácido tiobarbitúrico (TBARS), a carbonilação de proteínas, a atividade das enzimas superóxido dismutase (SOD), catalase (CAT) e glutationa peroxidase (GPx), assim como a produção de óxido nítrico (NO) pela formação de nitrito. Nas plaquetas foram avaliados, também, o transporte de L-arginina, a atividade da óxido nítrico sintase (NOS) e a agregação plaquetária. Em amostras de plasma foram realizadas medidas da proteína C reativa ultra-sensível (PCRus) e do aminoácido L-arginina. Estes resultados demonstram que não há diferença nas medidas da proteína C reativa e da L-arginina entre gestantes normotensas e com PE. A formação de nitritos e a atividade das enzimas antioxidantes SOD, CAT e GPx se encontram reduzidas no soro de gestantes com PE, enquanto a medida de TBARS e a carbonilação de proteínas não foi diferente das gestantes normotensas. Em plaquetas, o transporte de L-arginina pelo sistema y+L está diminuído na PE, ao passo que, a atividade da NOS, a formação de nitrito e a agregação plaquetária não modificaram em relação a gestação normal. A carbonilação de proteínas está aumentada em plaquetas na PE e a atividade da CAT está reduzida. Concluiu-se, que a menor formação de nitrito no soro sugere uma menor produção de NO em pacientes com PE. Este dado correlaciona com uma redução da defesa antioxidante observada pela menor atividade das enzimas SOD, CAT, GPx que deve contribuir para uma maior inativação do NO. Apesar dos níveis plasmáticos normais de L-arginina na PE, o sistema de transporte via y+L está reduzido em plaquetas, o que pode estar associado em parte ao estresse oxidativo que contribui para alterações físicas e estruturais da membrana, podendo afetar o influxo do aminoácido. Apesar do transporte de L-arginina reduzido e do aumento do estresse oxidativo em plaquetas na PE, não houve alteração da atividade da NOS, da produção de NO e da agregação plaquetária, indicando que mecanismos compensatórios possam estar contribuindo para a manutenção da produção de NO e de sua função modulatória sobre a agregação