Karmela Barišić

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.

Hsp70 silencing with siRNA in nanocarriers enhances cancer cell death induced by the inhibitor of Hsp90

Inducers of heat shock protein 70 (Hsp70) commonly promote cancer cell viability whereas inhibitors of Hsp90 reduce it. The anticancer agent celastrol, interferes with signal transduction pathways involving these heat shock proteins. The objective of this in vitro study was to silence inducible Hsp70 and to promote celastrol-induced tumor cell death. Hsp70 siRNA loaded chitosan-TPP carriers were prepared by ionic gelation and characterized by photon correlation spectroscopy and asymmetric flow field-flow fractionation combined with dynamic light scattering. Viability of human leukemia and glioblastoma cells and Hsp70 silencing was determined following treatment with chitosan-TPP-Hsp70 siRNA particles. The results showed that silencing of Hsp70 by chitosan-TPP-Hsp70 siRNA treatment significantly reduced cell viability, and enhanced antiproliferative effects of celastrol in leukemia and glioblastoma cells. In glioblastoma spheroids, higher concentrations of celastrol and Hsp70 siRNA in chitosan-TPP nanocarriers were necessary to induce cell death.

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.

Effect of non-genetic factors on paraoxonase 1 activity in patients undergoing hemodialysis

OBJECTIVES Hemodialyzed patients have lower paraoxonase 1 (PON1) activity. Higher mortality risk from cardiovascular disease observed in these patients could be due to the low antiathetrogenic activity of PON1. Understanding the mechanism that causes lower PON1 activity could provide the possibility for modulation of enzyme activity in purpose of preventing and/or decreasing development of atherosclerosis. DESIGN AND METHODS 87 healthy individuals and 71 hemodialyzed patients were enrolled in this study. RESULTS Hemodialyzed patients had reduced PON1 paraoxonase and arylesterase activity, concentrations of HDL, HDL(3) and HDL(2) and concentrations of free thiol groups. Distribution of HDL subfractions and distribution of PON1 phenotypes as well as concentrations of MDA were not different between two study groups. In the in vitro experiment high concentrations of urea, creatinine, uric acid and addition of patients sera ultrafiltrate did not significantly affect PON1 paraoxonase activity. CONCLUSION Decreased HDL concentration as well as lower PON1 concentration (shown indirectly by the enzyme arylesterase activity) might contribute, at least partly, to the reduced PON1 activity observed in hemodialyzed patients. Decreased concentration of free thiol groups in sera suggest that free thiol group (Cys284) in PON1 might also be oxidized, which can affect PON1 activity.

Levels changes of blood leukocytes and intracellular signalling pathways in COPD patients with respect to smoking attitude.

OBJECTIVES To examine whether the proportions of blood leukocyte subsets are altered in COPD, and to assess the disturbances in signalling pathways in the leukocytes of COPD patients, with respect to smoking history. DESIGN AND METHODS The study was performed at the Faculty of Pharmacy and Biochemistry and University Hospital for Lung Diseases, Zagreb, Croatia. Leukocyte counts were determined in 28 COPD male patients (11 smokers, 9 ex-smokers, 8 non-smokers) and 42 healthy male subjects (15 smokers, 13 ex-smokers, 14 non-smokers). We assessed activation of MAPKs (ERK, JNK, p38), and expression of Bcl-2 and Bax in the leukocytes by western blotting. RESULTS Neutrophil and monocyte percentages were significantly increased, and lymphocyte percentage significantly decreased in COPD patients compared with healthy subjects (p<0.05). However, no significant smoking effect regarding leukocyte subsets was observed when compared current or former smokers with non-smokers of each studied group. ERK was activated in non-smokers only, especially in healthy ones. In contrast, strong induction of JNK and p38 phosphorylation, decreased Bcl-2 levels and increased Bax levels were detected in COPD smokers and COPD ex-smokers, but also in healthy individuals who smoke compared with healthy non-smokers (p<0.05). CONCLUSION These results show that COPD and smoking affect intracellular signalling pathways. Understanding of the basic cellular and molecular mechanisms in COPD is essential for identification of molecules that may serve as targets for diagnosis and therapeutic interventions.

BpV (phen) induces apoptosis of RINm5F cells by modulation of MAPKs and MKP-1

We investigated the mechanism of toxicity of peroxovanadium complex bpV (phen) in RINm5F cells. Treatment with bpV (phen) provoked cell death, predominantly by apoptosis. This compound induced strong and sustained JNK and p38 MAPK activation. However, ERK phosphorylation was not affected. The level of expression of MAPK phosphatase MKP-1 was suppressed after bpV (phen) treatment. In addition, this compound did not stimulate proteolytic processing of procaspase-3, suggesting that caspase-3 is not activated during the course of bpV (phen)-induced apoptosis. A correlative inhibition of JNK activation by immunosuppressive drug FK 506 induced ERK activation and MKP-1 expression, and suppressed RINm5F cell death. A specific p38 inhibitor SB 203580 also stimulated ERK activation and cell survival. Furthermore, simultaneous pretreatment with both FK 506 and SB 203580 almost completely abolished cell death. Thus, our results suggest that stress kinases and MKP-1 have a role in bpV (phen)-induced apoptosis of RINm5F cells.

Changes of Glycoprotein Patterns in Sera of Humans under Stress

Stress exhibits adverse effects on many vital processes in which glycoproteins play a significant role(e.g. cell-cell/matrix interactions, immune response, neoplastic growth, implantation, prenatal development), yet only scarce attention has been directed towards studying stress induced changes in glycoprotein patterns. Using SDS-electrophoresis, blotting and digoxigenin-labelled lectins (Sambucus nigra agglutinin, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Maackia amurensis agglutinin and peanut (Arachis hypogaea) agglutinin),sera were analysed from 30 individuals chosen randomly from a severely stressed population of 309 male volunteers with no specific medical symptoms. Significant changes were found in glycoprotein pattern and content, compared with healthy controls of matching age and sex. Occasionally minor non-specific deviations from the reference values for several analytes (haemoglobin, glucose, bilirubin and alanine aminotransferase) were detected in the tested group, but glycoprotein GP4S (Mr = 45 000), detected by Datura stramonium agglutinin and Sambucus nigra agglutinin, appeared in 96.7% of samples of the stressed population. The same population also revealed an approximately 500-fold increase of GP37 in comparison with the control sera. These results suggest that stress, as a non-specific syndrome, induces specific biochemical changes, which could be of diagnostic relevance as risk makers before any more serious symptoms of stress-related consequences have developed.

Heat shock protein 70 and antibodies to heat shock protein 60 are associated with cerebrovascular atherosclerosis.

OBJECTIVES Heat shock proteins (Hsps) are produced by all cells, including vascular, to ensure stress protection. Damaged cells release Hsps in their local environment and systemic circulation. The aim of this study was to investigate the involvement and prognostic utility of serum Hsp60 and Hsp70, and the respective antibodies anti-Hsp60 and anti-Hsp70 in subjects with advanced atherosclerosis resulting in high degree of cerebrovascular stenosis. DESIGN AND METHODS Ultrasound Doppler examination of carotid arteries was used to discriminate between control and cerebrovascular atherosclerosis subjects. Twenty eight subjects without carotid obstruction were selected as controls. Fifty patients with obstruction of cerebrovascular blood flow were evaluated for the degree of stenosis of cerebral arteries by digital subtraction angiography. In parallel, serum concentrations of Hsp60, Hsp70, anti-Hsp60 and anti-Hsp70 were measured by ELISA kits. RESULTS Anti-Hsp60 was significantly higher (P=0.003) in the atherosclerosis group than in the control group (23.62ng/L vs. 15.28ng/L, respectively, expressed as median). Circulating Hsp70 was lower in the atherosclerosis than in the control group (P=0.048), with respective median values of 0.00μg/L vs. 0.22μg/L. Concentrations of Hsp60 and anti-Hsp70 did not differ significantly between the control and atherosclerosis group. CONCLUSIONS Higher circulating anti-Hsp60 is associated with advanced cerebrovascular atherosclerosis as a consequence of the autoimmunity part of the inflammation and bursting of atherosclerosis. Higher levels of Hsp70 observed in the control group could be protective in the development of atherosclerotic changes.

Association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-alpha (+489G/A) polymorphisms with COPD in Croatian population.

OBJECTIVE To test for possible association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-α (+489G/A) polymorphisms with chronic obstructive pulmonary disease (COPD) in Croatian population. METHODS Genotyping of DNA isolated from whole blood of 130 COPD patients (as defined by spirometry) and 95 healthy controls was performed. Fragment size analysis upon restriction enzyme digestion and/or sequencing was used for genotype/allele definition. Significance of findings was tested using χ(2) test. RESULTS hsp70-2 (+1267A/G) polymorphism was significantly associated with COPD. Results of genotyping analysis indicated that a genotype carrying G allele was preferentially associated with COPD; odds ratio (OR)=1.50, 95% confidence interval (CI)=1.00-2.24 and P=0.061. OR for the GG genotype was 3.47 with CI=1.26-9.56 and P=0.04. No association for hsp70-hom (+2437T/C), TNF-α (+489G/A) and HMOX-1 (number of GT repeats) polymorphisms were found. In addition, comparison of genotype frequencies among different stages of disease severity (GOLD II-IV) revealed no discrimination for any of the tested polymorphisms. CONCLUSION This study is supporting the association of hsp70-2 (+1267A/G) polymorphism and COPD. Higher frequency of G allele and GG genotype in Croatian COPD patients was observed. There was no evidence for the association of hsp70-hom (+2437T/C), TNF-α (+489G/A) SNPs and HMOX-1 (number of GT repeats) polymorphism with COPD. Allele and genotype frequencies for all of the tested polymorphisms show no association with disease severity (GOLD II-IV).