Ivana Čepelak

Azithromycin modulates neutrophil function and circulating inflammatory mediators in healthy human subjects

Effects on human neutrophils and circulating inflammatory mediators were studied in 12 volunteers who received azithromycin (500 mg/day, p.o.) for 3 days. Blood was taken 1 h before treatment, 2.5, 24 h and 28 days after the last dose. An initial neutrophil degranulating effect of azithromycin was reflected in rapid decreases in azurophilic granule enzyme activities in cells and corresponding increases in serum. The oxidative response to a particulate stimulus was also acutely enhanced. These actions were associated with high plasma and neutrophil drug concentrations. A continuous fall in chemokine and interleukin-6 serum concentrations, within the non-pathological range, accompanied a delayed down-regulation of the oxidative burst and an increase in apoptosis of neutrophils up to 28 days after the last azithromycin dose. Neutrophils isolated from blood at this time point still contained detectable drug concentrations. Acute neutrophil stimulation could facilitate antibacterial effects of azithromycin, while delayed, potentially anti-inflammatory activity may curtail deleterious inflammation.

High efficiency entrapment of superoxide dismutase into mucoadhesive chitosan-coated liposomes.

Superoxide dismutase (SOD), antioxidative enzyme and potential anti-inflammatory agent, was encapsulated into mucoadhesive chitosan-coated liposomes in order to increase its releasing time and to facilitate its cellular penetration. Positively, neutrally and negatively charged liposomes were prepared using soybean lecithin, stearylamine, phosphatidyl glycerol and cholesterol. The effects of liposomal lipid composition and protein to lipid ratio on the encapsulation parameters were studied in three preparation methods: dehydration-rehydration, hydration and proliposome methods. The highest efficiency of SOD entrapment, 39-65%, was achieved by the proliposome method. Vesicles prepared by the hydration method entrapped 1-13% and vesicles prepared by dehydration-rehydration entrapped 2-3% of SOD. Stability tests for SOD-loaded liposomes prepared by the proliposome method showed no significant loss of the enzyme activity within 1 month at 4 degrees C or within 2 days at 37 degrees C. Positively, neutrally and negatively charged liposomes, prepared by the proliposome method, were successfully coated with two types of low and medium molecular weight chitosans. Both types of chitosan coating increased the mucoadhesive characteristics of all three types of vesicles. Using the proliposome method and subsequent chitosan coating, highly efficient SOD-loaded vesicles for drug targeting on mucosal tissues could be produced.

Platelet count, mean platelet volume and smoking status in stable chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD), an increasing global health problem, may be complicated by acute atherothrombotic events. Although systemic inflammation plays the leading role in atherothrombotic processes, platelet activation and increased coagulation together with oxidative stress can significantly exacerbate atherosclerosis in COPD patients. In this study we determined platelet count, mean platelet volume (MPV) and classical markers of systemic inflammation – serum C-reactive protein (CRP), white blood cell (WBC) count and the relative proportion of segmented neutrophils in COPD patients, and compared them to those from the healthy controls. The most important and novel finding of this study was that patients with COPD had a significantly increased platelet count, along with a reduced MPV when compared to healthy controls (286 vs. 260 × 109/l; 9.6 vs. 8.7 fL, respectively). Cigarette smoking had no influence on these results. The presence of systemic inflammation was clearly proved by the increase in classical inflammatory markers (CRP, WBC and segmented neutrophil count).

Arginase, a new marker of mammary carcinoma

Activities of arginase, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were determined in sera obtained in a group of healthy women, women with verified carcinoma of the breast, benign mastopathy, a group of patients with carcinoma of various organs and a group of patients with acute viral hepatitis. Preoperative values of serum arginase activity in patients with breast carcinoma were up to 4-fold those found in healthy women. Sensitivity of the test was 86%. After the surgery, the activity decreased abruptly during the first week and normalised within 15-30 days. In benign diseases of the breast, the activity of arginase was normal. Serum arginase activity is raised in both benign and malignant liver diseases, however, the quotients alanine aminotransferase/arginase, aspartate aminotransferase/arginase and alkaline phosphatase/arginase differ significantly. Thus, use of alanine aminotransferase/arginase quotient implies a high degree of confidence in differentiating between increased arginase activity in mammary carcinoma (alanine aminotransferase/arginase = 0.572 +/- 0.278) and high arginase activity in hepatitis (alanine aminotransferase/arginase = 12.226 +/- 1.822).

Iron and Ferritin Concentrations in Exhaled Breath Condensate of Children with Asthma

Maintenance of iron homeostasis is of utmost importance for the respiratory system physiology and pathophysiology. Local iron deficiency or accumulation may result in particular respiratory function impairment. The aim of the present study was to find out whether iron and ferritin could be determined in exhaled breath condensate (EBC) of healthy children and children with asthma. Oxidative stress was verified by determination of EBC superoxide dismutase (SOD) activity, and the airway inflammatory process by determination of exhaled nitric oxide (FENO). EBC was collected from 39 children (22 healthy children as a control group and 17 asthmatics) using an EcoScreen condenser. Iron, ferritin, and SOD were determined on optimization and validation for low concentrations. In comparison with a control group, asthma patients had a statistically significantly lower iron concentration (p = 0.0001) and higher SOD catalytic activity (p = 0.0160), with no significant difference in ferritin levels (p = 0.5252), although percentile values indicated elevated ferritin concentration in about half of asthma patients. FENO values were significantly higher in the asthma group (p = 0.0047). This preliminary study demonstrated the possibility of determining iron and ferritin concentrations and SOD activity in EBC, and a significant difference in EBC iron and SOD between asthma patients and healthy children.

Exhaled breath condensate: a new method for lung disease diagnosis

Abstract Analysis of exhaled breath composition in lung disease patients can indirectly point to biochemical changes that occur in the fluid lining airway surfaces. The parameters of redox and acid-base changes, and of inflammatory changes relevant in the pathogenesis of most pulmonary diseases are currently most widely determined in exhaled breath condensate. The collection of exhaled breath condensate is a safe, non-invasive, easy and simple diagnostic procedure that is suitable for longitudinal studies and applicable in patients of all age groups, irrespective of the disease severity. In spite of many scientific studies involving lung disease patients, methodology for exhaled breath condensate collection and analysis has not yet been realized for daily utilization. Additional studies of the exact origin of condensate constituents and standardization of the overall analytical process, including collection, storage, analysis and result interpretation, are needed. Irrespective of these limitations, further investigation of this sample type is fully justified by the fact that classical specimens used in the management of pulmonary disease are either obtained by invasive procedures (e.g., induced sputum, biopsy, bronchoalveolar lavage) or cannot provide appropriate information (e.g., urine, serum). Analysis of exhaled breath condensate in the future might contribute significantly to our understanding of the physiological and pathophysiological processes in lungs, to early detection, diagnosis and follow up of disease progression, and to evaluation of therapeutic response. Clin Chem Lab Med 2007;45:945–52.

Expression of Hsp70 in kidney cells exposed to ochratoxin A

Abstract. Ochratoxin A (OTA) is a possible etiological agent of endemic nephropathy, a chronic renal disease with high prevalence in limited geographic areas. Ochratoxicosis has many characteristics of different pathological states in which heat shock proteins (Hsps) are usually induced. The most inducible heat shock proteins belong to the Hsp70 family. We determined the level of expression of Hsp70 by the Western blot analysis in kidneys of rats treated with low doses of OTA and in LLC-PK1 and MDCK cells exposed to OTA. Estimation of cell viability and release of lactate dehydrogenase (LDH) confirmed the toxic effects of OTA on cultured cells. OTA affects the relative distribution of two Hsp70 isoforms (68-kDa and 74-kDa isoforms), but does not change total amount of Hsp70 in rat kidney. No changes in the Hsp70 level were detected in LLC-PK1 and MDCK cells treated with OTA, although the cells were seriously injured, as was seen from the reduced cell viability and increased release of LDH. Both cell lines were capable of having Hsp70 induced following a heat shock. However, exposure of the cells to OTA before the heat shock challenge prevented Hsp70 induction. Results of the study show that OTA does not induce Hsp70 in rat kidney or in cultured kidney cells. The absence of Hsp70 protective effects in the cells and tissues might be a possible explanation for the cumulative destructive effects of OTA and a silent onset of endemic nephropathy in humans and of OTA-induced experimental nephrotoxicity in animals.

Cut-off values for total serum immunoglobulin E between non-atopic and atopic children in north-west Croatia

Abstract Background: The aim of this study was to determine cut-off values for total serum immunoglobulin E (IgE) between non-atopic and atopic children with respiratory symptoms. Children of 0–16years of age were evaluated for respiratory symptoms of >4-week duration. Methods: Children were divided into two groups: non-atopic children (n=3355) who were non-IgE-sensitized with undetectable allergen-specific IgE (<0.35kIUA/L), and atopic children (n=4620) who were sensitized to ≥1 allergens (specific IgE ≥0.35 kIUA/L). Upper and lower centiles were determined and cut-off values calculated using receiver operating characteristic (ROC) analysis. Results: Serum total IgE increased with age in both groups, although at a variable level and rate, and reached a plateau at 9 and 10years in non-atopic and atopic children, respectively. Atopic children had on average 14-fold higher serum total IgE compared to non-atopic children. In both groups, the median was lower than the corresponding mean and the distribution skewness was always positive (group I, 0.87; group II, 0.91). In almost all age groups, the 95th percentile for non-atopic children corresponded to the calculated cut-off values, whereas the 10th percentile for atopic children corresponded to the respective cut-off values only until the age of 8years, after which greater differences between the cut-off values and the 10th percentile were recorded. Cut-off values for total serum IgE in children up to 16years were determined with diagnostic sensitivity, specificity and area under the ROC curve of 96%, 91% and 0.950, respectively. Conclusions: The 95th percentile for total IgE in non-atopic children and the 10th percentile in atopic children could be taken as cut-off values in children up to 8years of age, after which significant percentile discrepancies between non-atopic and atopic children were recorded. Since atopic subjects show a more irregular centile distribution, cut-off values are best determined by ROC analysis.

Exhaled breath condensate – from an analytical point of view

Over the past three decades, the goal of many researchers is analysis of exhaled breath condensate (EBC) as noninvasively obtained sample. A total quality in laboratory diagnostic processes in EBC analysis was investigated: pre-analytical (formation, collection, storage of EBC), analytical (sensitivity of applied methods, standardization) and post-analytical (interpretation of results) phases. EBC analysis is still used as a research tool. Limitations referred to pre-analytical, analytical, and post-analytical phases of EBC analysis are numerous, e.g. low concentrations of EBC constituents, single-analyte methods lack in sensitivity, and multi-analyte has not been fully explored, and reference values are not established. When all, pre-analytical, analytical and post-analytical requirements are met, EBC biomarkers as well as biomarker patterns can be selected and EBC analysis can hopefully be used in clinical practice, in both, the diagnosis and in the longitudinal follow-up of patients, resulting in better outcome of disease.

Glutathione cycle in stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidant/antioxidant imbalance. Glutathione is the most abundant cellular low‐molecular weight thiol and the glutathione redox cycle is the fundamental component of the cellular antioxidant defence system. Concentration of total glutathione and catalytic activities of glutathione peroxidase and glutathione reductase were determined in peripheral blood of patients (n = 109) and healthy subjects (n = 51). Concentration of total glutathione in patients was not changed in comparison to healthy controls. However, we found statistically significant difference between patients with moderate and severe disease stages. Glutathione reductase activity was increased, while glutathione proxidase activity was decreased in the patients with COPD, when compared to healthy controls. We found no significant difference in glutathione peroxidase and glutathione reductase activities between stages. Patients who smoked had lower concentration of total glutathione compared with former smokers and never‐smoking patients. Lung function parameters were inversely associated with glutathione level. Evidence is presented for differential modulation of glutathione peroxidase and glutathione reductase activities in peripheral blood of patients with stable COPD. We suppose that in addition to glutathione biosynthesis, glutathione reductase‐dependent regulation of the glutathione redox state is vital for protection against oxidative stress. Copyright