Jerzy Marczak

Effect of Paraquat Intoxication and Ambroxol Treatment on Hydrogen Peroxide Production and Lipid Peroxidation in Selected Organs of Rat

Paraquat (Pq) is a herbicide which is very toxic to all animals and to man. It generates free radicals and leads to acute or chronic lung injury and usually to death. So far, the role of lipid peroxidation of cell membranes in the mechanism of its toxicity has not been proved satisfactorily and therefore in the present study we examined the concentration of hydrogen peroxide (H2O2) and various lipid peroxidation products (LPP)such as conjugated dienes (CD), lipid hydroperoxides (LH), malonyldialdehyde (MDA) and Schiff bases in selected organs of the rat given a single intraperitoneal dose 35 mg kg−1 Pq. We also evaluated the influence of a mucolytic and probably antioxidant drug, ambroxol, on Pq‐induced changes in the concentration of H2O2 and LPP. Paraquat increased the hepatic concentration of H2O2, CD, LH and MDA by approximately fourfold. Though the dose of Pq was nearly twice the LD50 dose, we did not notice any changes in the concentration of these substances in the critical organ, lung or heart and kidney. Ambroxol alleviated the increase of H2O2 in the liver but did not reduce the concentration of LPP. Moreover, the drug administered alone induced lipid peroxidation in the liver. Our results indicate that Pq does not induce H2O2 production and lipid peroxidation in the lung but it increases the concentration of H2O2 and LPP in the liver. Ambroxol inhibits the Pq‐induced increase in the concentration of H2O2 in the liver without protecting it against lipid peroxidation. Moreover, the drug alone may act as a pro‐oxidant.

Correlation between eicosanoids in bronchoalveolar lavage fluid and in exhaled breath condensate

Exhaled breath condensate (EBC) has been increasingly used as a new and non-invasive method to study airway inflammation. In this study we have compared the concentrations of lipid mediators in EBCwith concentrations in bronchoalveolar lavage fluid (BALF). We included 37 patients undergoing bronchoscopy (12 sarcoidosis, 12 COPD, 6 lung cancer, 5 chronic cough, 1 Wegener’s granulomatosis, 1 sclerodermia). Patients were not allowed to have exacerbation or any change in concomitant medication for at least 4 weeks prior to the study. In all patients, EBC was collected immediately prior to the bronchoscopy. The levels of cys-LTs, LTB4, 8-isoprostane were significantly higher in BALF compared to EBC (p < 0.0001, p < 0.001, p < 0.0001 for cys-LTs, LTB4, 8-isoprostane respectively). Moreover, there was a strong positive correlation between both leukotriene B4 and 8-isoprostane in BALF and EBC (r = 0.53 and r = 0.79, p < 0.01, respectively) in patients with sarcoidosis and COPD but there was no correlation between eicosanoids BALF and EBC in patients with chronic cough and lung cancer. This is the first study to compare EBC and BALF in different lung diseases which demonstrated significant correlations between the levels of eicosanoids in BALF and EBCin patientswith COPD and sarcoidosis. EBC may be useful inmeasuring inflammation in several inflammatory lung diseases.

Rhinosinusitis in COPD: symptoms, mucosal changes, nasal lavage cells and eicosanoids

The coexistence of upper airways disease with chronic obstructive pulmonary disease (COPD) is not well documented. The aim of this research was to assess sino-nasal inflammation in COPD by various tools, and look for the impact on quality of life, relation to smoking, disease severity and systemic inflammation. Current and ex-smokers with COPD (n = 42) and healthy never-smokers (n = 21) were included in this study. COPD severity was assessed by GOLD criteria and BODE index. Markers of systemic inflammation were measured. Nasal symptoms and general quality of life were assessed using the questionnaires; sino-nasal questionnaire (SNAQ-11) and St. Georges Respiratory Questionnaire (SGRQ). Nasal endoscopy and saccharine test were performed. Nasal lavages were collected for cytological examination and eicosanoids (cysteinyl leukotrienes, leukotriene B4, 8-isoprostane). Symptoms and endoscopic scores were higher in COPD (P ≤ 0.0001). Only SGRQ symptoms subscore correlated with SNAQ-11 (r = 0.34, P = 0.035). Mucociliary clearance was impaired only in current smokers (9.91 ± 0.49 versus 13.12 ± 0.68 minutes, P ≤ 0.001). 8-isoprostane was higher in COPD smokers compared to the controls (0.17 ± 0.04 versus 0.34 ± 0.09 pg/g protein, P < 0.05). Endoscopic score and mucociliary of impairment patients who currently smoked cigarettes correlated with concentrations of 8-isoprostane. None of the parameters correlated with disease severity and markers of systemic inflammation. We provide evidence of upper airways disease in COPD, which appears to be related more to patients who currently smoke than to disease severity.

Release of hydrogen peroxide by rat type II pneumocytes in the prolonged culture.

Type II pneumocytes (T II pneumocytes) produce hydrogen peroxide (H(2)O(2)), which may be potentially dangerous for the lung. These cells in culture differentiate to type I-like pneumocytes and it may reflect the differentiation which follows the injury of alveolar epithelium. This work was undertaken to estimate the H(2)O(2) release by T II pneumocytes, freshly isolated and cultured up to 8 days. The light and electron microscopy evaluation confirmed the differentiation of T II pneumocytes to type I-like cells. The release of H(2)O(2), estimated spectrofluorimetrically as homovanillic acid oxidation product obtained in the presence of horseradish peroxidase, was significantly higher at day 4 (0.63+/-0. 68nmol/mg protein/min, P</=0.02) and 6 (0.46+/-0.31, P</=0.001) compared to fresh cells (0.15+/-0.08). Phorbol esters increased H(2)O(2) release at day 2 (0.39+/-0.22 vs 0.16+/-0.08, P</=0.02) and the inhibition of protein kinase C resulted in the decrease at day 2 (0.14+/-0.06 vs 0.07+/-0.02, P</=0.025), day 6, (0.49+/-0.25 vs 0. 15+/-0.08, P</=0.005) and 8 (0.76+/-0.63 vs 0.23+/-0.29, P</=0.02). Inhibition of intracellular catalase resulted in a significant increase only at day 2 (0.23+/-0.1 vs 0.15+/-0.09, P</=0.05). Inhibition of mitochondrial respiratory chain decreased H(2)O(2) release at day 2 (0.13+/-0.11 vs 0.07+/-0.07, P</=0.002) and 4 (0. 75+/-0.88 vs 0.61+/-0.85, P</=0.002). These results indicate that alveolar epithelium may be a source of potentially dangerous ROS and that the cell differentiation is accompanied by the increase of H(2)O(2) production. Both mitochondrial respiratory chain and membrane-bound NADPH-oxidase may be responsible for the production of H(2)O(2) by T II pneumocytes.

Exhaled 8-isoprostane as a prognostic marker in sarcoidosis. A short term follow-up.

Background8-Isoprostane (8-IP) is a marker of lipid peroxidation. Elevated concentrations have been reported in BAL fluid and exhaled breath condensate (EBC) in sarcoidosis (S). To validate the prognostic value of this marker we tested whether: 1. high initial EBC 8-IP predispose to more severe disease; 2. low initial concentrations increase a chance of early remission; 3. remissions are connected with the decrease of EBC 8-IP.Methods40 patients (S) have been examined initially (V1) and after 8.5 ± 0.5 months (V2). EBC 8-IP concentrations were measured by ELISA. Chest X-ray, lung function test, serum ACE and Ca2+ concentrations, 24 hrs Ca2+loss, abdominal ultrasonography, symptoms evaluation were performed.ResultsWe confirmed higher concentrations of 8-IP in EBC of patients with sarcoidosis (p = 0.001). Relative risk (RR) of persistence of disease at V2 when initial 8-IP was above 20 pg/mL was 1.04, and the frequency distributions estimated by χ2 test were not significantly different. A chance (RR) of early complete remission when V1 8-IP was below DL, was 3.33 (p = 0.04 by χ2 test). A significant decrease of 8-IP at V2 was observed only in patients who received treatment (p = 0.03), but not in those with spontaneous remission.ConclusionsWe come to the conclusion, that low initial 8-IP may be a positive prognostic factor. A decrease of 8-IP in treated patients reflects a non-specific effect of treatment and is not related to mere regression of disease.

Exhaled breath 8-isoprostane as a marker of asthma severity.

Introduction Oxidative stress is a non-specific feature of airway inflammation in asthmatics. 8-Isoprostane (8-IP), a prostaglandin-F2α isomer, is a relatively new marker of oxidative stress and may be measured in exhaled breath condensate (EBC) of patients with asthma. This research study aimed to evaluate the usefulness of EBC 8-IP as a marker of severity and control of severe adult asthma. Material and methods Twenty-seven severe, never-smoking asthmatics were studied. According to positive or negative reversibility testing, this group was subdivided into reversible and irreversible asthma groups. All participants were observed for 8 weeks during which they completed daily diary observations including day and night symptoms, number of awakenings, peak expiratory flow (PEF) variability, daily rescue medication usage and oral steroids consumption. They attended the clinic 3 times and on these occasions spirometry assessments, EBC collection and asthma control tests (ACT) were done. Two control groups were included: 11 healthy never-smokers and 16 newly diagnosed and never-treated, non-smoking mild asthmatics. Results There were no statistically significant differences between severe asthma and healthy control or never-treated asthma groups in concentrations of EBC 8-IP (median and interquartile range: 4.67; 2.50-27.92 vs. 6.93; 2.5-12.98 vs. 3.80; 2.50-10.73, respectively). No correlations were found between EBC 8-IP and asthma control parameters, such as ACT results, night and day symptoms, consumption of rescue medication, percentage of days free of oral steroids, PEF diurnal variation, lung function test results, forced expiratory volume in the 1 s reversibility, and markers of systemic inflammation. Conclusions Our study results suggest that EBC 8-IP measurements are not useful for asthma monitoring.

Innate Immune Response to Viral Infections in Primary Bronchial Epithelial Cells is Modified by the Atopic Status of Asthmatic Patients

Purpose In order to gain an insight into determinants of reported variability in immune responses to respiratory viruses in human bronchial epithelial cells (HBECs) from asthmatics, the responses of HBEC to viral infections were evaluated in HBECs from phenotypically heterogeneous groups of asthmatics and in healthy controls. Methods HBECs were obtained during bronchoscopy from 10 patients with asthma (6 atopic and 4 non-atopic) and from healthy controls (n=9) and grown as undifferentiated cultures. HBECs were infected with parainfluenza virus (PIV)-3 (MOI 0.1) and rhinovirus (RV)-1B (MOI 0.1), or treated with medium alone. The cell supernatants were harvested at 8, 24, and 48 hours. IFN-α, CXCL10 (IP-10), and RANTES (CCL5) were analyzed by using Cytometric Bead Array (CBA), and interferon (IFN)-β and IFN-λ1 by ELISA. Gene expression of IFNs, chemokines, and IFN-regulatory factors (IRF-3 and IRF-7) was determined by using quantitative PCR. Results PIV3 and RV1B infections increased IFN-λ1 mRNA expression in HBECs from asthmatics and healthy controls to a similar extent, and virus-induced IFN-λ1 expression correlated positively with IRF-7 expression. Following PIV3 infection, IP-10 protein release and mRNA expression were significantly higher in asthmatics compared to healthy controls (median 36.03-fold). No differences in the release or expression of RANTES, IFN-λ1 protein and mRNA, or IFN-α and IFN-β mRNA between asthmatics and healthy controls were observed. However, when asthmatics were divided according to their atopic status, HBECs from atopic asthmatics (n=6) generated significantly more IFN-λ1 protein and demonstrated higher IFN-α, IFN-β, and IRF-7 mRNA expressions in response to PIV3 compared to non-atopic asthmatics (n=4) and healthy controls (n=9). In response to RV1B infection, IFN-β mRNA expression was lower (12.39-fold at 24 hours and 19.37-fold at 48 hours) in non-atopic asthmatics compared to atopic asthmatics. Conclusions The immune response of HBECs to virus infections may not be deficient in asthmatics, but seems to be modified by atopic status.

Switching from systemic steroids to ciclesonide restores the hypothalamic pituitary-adrenal axis.

Introduction Treatment of difficult asthma with oral corticosteroids (OCS) may suppress the hypothalamic-pituitary-adrenal axis. Aim In this study we have checked if the substitution of OCS with very high doses of ciclesonide may restore the adrenal function without losing the control of the disease. Material and methods In 5 patients with difficult, uncontrolled asthma despite treatment with OCS, inhaled and systemic glucocorticosteroids were replaced with very high doses of ciclesonide (1600–2400 µg/day). The symptoms of asthma and the lung function were assessed at baseline and on the 28th, 56th and 70th day of treatment, whereas the levels of cortisol and adrenocorticotropic hormone (ACTH) in the morning were measured at baseline and on the 28th and the 56th day of treatment. Results In all patients, the control of asthma symptoms, measured with Asthma Control Test questionnaire, improved from the mean score of 9.4 to 19.8 in 70 days. In 4 subjects force expiratory volume in 1 s improved gradually through the entire study reaching a mean improvement of 585 ml in 70 days. The ACTH levels were normalized in 3 patients after 28 days of observation and in all patients after 56 days. The cortisol level was normalized in 4 patients after 28 days and in another subject after 56 days of treatment with ciclesonide. Conclusions Switching from prednisone to very high doses of ciclesonide normalized the hypothalamic-pituitary adrenal axis function and also improved the disease control and the lung function in these 5 patients with difficult asthma.

Effectiveness of omalizumab in an asthmatic patient with severe airway and blood eosinophilia

Omalizumab is a monoclonal antibody raised against class E immunoglobulin (IgE), approved for the treatment of chronic severe (in the EU) or moderate-to-severe (in the USA) IgE-mediated asthma. Omalizumab is effective in reducing asthma exacerbations, hospitalizations and emergency visits due to exacerbations as well as in improving patients’ quality of life (QoL) [1–3].

[A 24-year-old male patient with pulmonary veno-occlusive disease - case report].

Pulmonary veno-occlusive disease (PVOD) is a rare cause of severe pulmonary hypertension characterised by poor prognosis. We report the case of a 24-year-old male patient with increasing dyspnea and exercise intolerance treated with calcium channel blockers and glucocorticosteroids, due to suspicion of pulmonary hypertension and interstitial lung disease, until lung biopsy was performed and a diagnosis of PVOD was established on the basis of the histological analysis of the lung biopsy sample. This case highlights that pulmonary veno-occlusive disease is a disorder that is difficult to diagnose and resistant to medical treatment, which is particularly poor prognostic factor. Due to poor response to medical therapy and high mortality in patients with PVOD, understanding the pathogenesis, differentiation with pulmonary arterial hypertension and the search for a new methods of treatment should be the key challenges for modern medicine.