Radioiodine therapy accelerates apoptosis in peripheral blood lymphocytes of patients with differentiated thyroid cancer.

Both apoptosis and micronuclei formation reflect cytogenetic damage in cells and could contribute to cell homeostasis. The aim of this study was to evaluate apoptosis in peripheral blood lymphocytes (PBLs) of patients with differentiated thyroid cancer (DTC) before and after 131-iodine (131-I)-therapy and its correlation with micronuclei (MN) frequency. The study population included 18 DTC patients and 18 healthy donors. Apoptotic cells were detected using the Annexin V-FITC/7-AAD kit and MN frequency by cytokinesis-block MN assay. The difference between early apoptosis in PBLs of DTC patients before therapy and controls (9.88 ± 4.99% vs. 6.64 ± 2.07%, p = 0.003) was significant, as well as between early apoptosis in PBLs of DTC patients before and after 131-I-therapy (9.88 ± 4.99% vs. 13.53 ± 6.57%, p = 0.008). The MN frequency and early apoptosis in PBLs of DTC patients was positively correlated before (r = 0.540, p= 0.021) and after 131-I-therapy (r = 0.585, p= 0.014). Thyroid cancer patients had a significantly increased early apoptosis in PBLs, which further increased after 131-I-therapy in association with MN frequency.

Free light chains ratio as a marker to estimate prognosis and survival in patients with multiple myeloma and primary amyloidosis.

PURPOSE: The aim of this study was to evaluate the significance of free light chains ratio (FLC ratio) as a prognostic factor for remission, progression and survival in patients with multiple myeloma (MM) and primary amyloidosis. METHODS: The concentrations of immunoglobulins and FLC ratio were measured using immunonephelometry. A total of 101 patients from 3 different disease groups were investigated during a 7-year period: 1) MM (n=95); 2) nonsecretory multiple myeloma (NSMM) (n=3); and 3) primary amyloidosis (n=3). Reference range for FLC ratio was 0.26-1.65. RESULTS: According to the International Staging System (ISS) for MM, abnormal serum FLC ratio was < 0.03 or > 32. Patients with MM and highly or intermediately abnormal FLC ratio and a combination of adverse risk factors (56.9%) had median survival of 26 months (range 16-38), as opposed to patients with normal or slightly changed values of FLC ratio without adverse risk factors (43.1%) with median survival of 45 months (range 27-69). Also, all of the patients with NSMM had slightly changed values of FLC ratio corresponding to low risk of disease progression. In patients with primary amyloidosis, 33.3% had slightly changed values of FLC ratio corresponding to low risk of disease progression, as opposed to 66.7% with abnormal FLC ratio, corresponding to high risk. CONCLUSION: Abnormal FLC ratio in the examined groups could be an independent risk factor of disease progression and worse prognosis.

Antioxidant enzymes activities and plasma levels of oxidative stress markers in B-chronic lymphocytic leukemia patients.

PURPOSE: Overproduction of reactive oxygen species (ROS) intermediates above the functional capability of cellular antioxidants may result in instability of important macromolecules and represents the molecular basis of many diseases including inflammation processes, cardiovascular alterations, cancer etc. The purpose of this study was to determine plasma level of superoxide anion, hydrogen-peroxide and malondialdehyde (MDA) as markers of oxidative stress and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) as antioxidant enzymes in B-chronic lymphocytic leukemia (B-CLL) patients. METHODS: The study included 29 untreated B-CLL patients in stage A, and 21 in stages B and C, classified according to the Binet system; 31 healthy volunteers formed the control group. After centrifugation of heparinized peripheral blood, plasma levels of all investigated parameters were determined using spectrophotometric methods. RESULTS: Plasma CAT activity was increased in B-CLL patients compared with control subjects; also, progression of disease was related with significantly higher plasma activity of CAT. Also, B-CLL patients showed significantly higher plasma concentration of MDA compared with controls. No statistically significant differences of superoxide anion and hydrogen peroxide as well as plasma activity of SOD and GPx between the tested groups were noted. CONCLUSION: Increase of CAT activity in B-CLL patients indicates that there is stimulation of the antioxidant enzyme system, while the increase of MDA concentration shows increased lipid peroxidation level. According to these results it could be concluded that an imbalance exists between oxidants and antioxidants in the plasma of B-CLL patients.

In vitro induction of apoptotic cell death in chronic lymphocytic leukemia by two natural products: preliminary study.

PURPOSE: B cell chronic lymphocytic leukemia (B-CLL) is an neoplastic disorder characterized by alterations in the pathways of programmed cell death (apoptosis). Deregulation of apoptosis pathways also contributes to chemoresistance of B-CLL cells. Therefore, it is not surprising that induction and acceleration of apoptosis represent key point in novel B-CLL therapeutic protocols. The present study was designed to investigate the effects of two natural products, Immunarc forte and Korbazol on the in vitro survival of leukemic cells. METHODS: peripheral blood mononuclear cells (PBMC) from 20 B-CLL patients and 20 healthy donors were used for cytotoxicity studies. Cytotoxic activity of the tested products were assessed by the MTT colorimetric assay and the type of cell death was determined by flow cytometry. RESULTS: we found that Korbazol was selectively cytotoxic against B-CLL cells, but the cytotoxic activity of Immunarc forte was much weaker. Of note, synergy was shown between these two drugs, and this effect was also selective, without affecting the normal mononuclear cells. According to Annexin-V binding, Korbazol and Immunarc forte induced apoptotic type of cell death in B-CLL cells. Moreover, treatment with Korbazol, but not with Immunarc forte, decreased spontaneous apoptosis in cultured normal polymorphonuclear cells. CONCLUSION: our findings imply that Korbazol is as potential therapeutic agent that induces apoptosis of B-CLL cells. The resistance of normal mononuclear cells and anti-apoptotic effects on normal polymorphonuclear cells, as well as its ability to synergize with Immunarc forte, warrants further investigation and supports their therapeutic application in the treatment of B-CLL.

Endoplasmic reticulum stress associated with caspases-4 and -2 mediates korbazol-induced B-chronic lymphocytic leukemia cell apoptosis.

PURPOSE: B-cell chronic lymphocytic leukemia (B-CLL) is an incurable disease that rapidly develops drug resistance. Therefore there is a need for identifying new agents that will improve the therapeutic outcome. Korbazol is a natural product known to exert cytotoxic effect on the in vitro survival of leukemic cells. The aim of this study was to investigate the mechanism of korbazol-induced apoptosis in B-CLL leukemic cells. METHODS: peripheral blood mononuclear cells from 10 B-CLL patients were used for assessing the effect of caspase inhibitors and chelator of intracellular Ca(2)+. RESULTS: cell death rate induced by the tested compound was decreased with the caspase-3 inhibitor Ac-DEVD-CHO, and the inhibitors of caspase-2 (Z-VDVAD-FMK) and -4 (ZYVAD- FMK), but not with the caspase-9 inhibitor z-LEHD-FMK and caspase-8 inhibitor z-IETD-FMK. No significant release of cytochrome C (cyt C) from mitochondria to the cytosol of B-CLL cells treated with korbazol was observed. Moreover, chelating of intracellular Ca(2)+ with BAPTA-AM almost completely abolished the cytotoxic effect of korbazol. CONCLUSION: engagement of caspases-2 and -4 and mobilization of intracellular Ca(2)+ indicate involvement of endoplasmic reticulum (ER) stress in apoptosis induced by korbazol.

Oxidative stress accelerates spontaneous apoptosis of B-chronic lymphocytic leukemia lymphocytes.

PURPOSE: B-chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of small immature B lymphocytes which do not undergo apoptosis due to an underlying defect. One potential mechanism of defective apoptosis could be irregular oxidative stress. The goal of our investigation was to determine in vitro production of oxidative stress markers by lymphocytes of B-CLL patients. PATIENTS AND METHODS: 30 untreated stage A B-CLL patients, as well as 20 stage B and C patients and 30 healthy volunteers as a control group were examined. Nitric oxide (NO), superoxide anion, hydrogen peroxide and malondialdehyde (MDA) were measured by spectrophotometry in supernatants of lymphocytes cultures of all 3 investigational groups. The method applied for detecting apoptosis was fluorescence microscopic analysis using acridine orange/ethidium bromide (AO/EB) double staining. RESULTS: In vitro lymphocyte production of superoxide anion, hydrogen peroxide and MDA was increased in B-CLL patients, while there were no statistical significantly differences of NO production among the tested groups. Compared with the spontaneous apoptosis observed in control subjects lymphocytes, B-CLL lymphocytes showed increased percentages of apoptotic cells after incubation for 24 h. Disease progression was not followed with significant differences in spontaneous apoptosis of B-CLL lymphocytes. CONCLUSION: This intensive oxidative stress markers production in cultures of B-CLL lymphocytes could be one of the potential mechanisms in the pathogenesis of abnormal apoptosis.

High-performance liquid chromatographic assay of fleroxacin in human serum using fluorescence detection.

A HPLC method has been developed for the direct assay of fleroxacin in serum, without previous extraction. Serum samples, after the addition of sodium dodecylsulfate (0.5%), were injected directly into an LC Hisep column. The mobile phase consisted of acetonitrile, water and triethylamine in a per cent volume ratio 18:80:2. The pH of the mobile phase was adjusted to 6.50 with the addition of phosphoric acid. The drug was detected fluorometrically at lambda (ex )=280 nm and lambda (em )=450 nm . The linear concentration range of fleroxacin was between 0.01 and 2.0mg/l with a detection limit of 1ng/ml.

Systemic response of peripheral blood leukocytes and their phagocytic activity during acute myocardial infarction.

OBJECTIVES: To determine changes in leukocyte counts and phagocytic activity of peripheral blood mononuclear (MN) and polymorphonuclear (PMN) cells as potential cellular markers of systemic immunological events in acute myocardial infarction (AMI). PATIENTS AND METHODS: Thirty patients with a first AMI and 30 healthy volunteers were examined. Immunological analyses were performed at admission and repeated at one and seven days after the acute event. MN and PMN cells were obtained from heparinized whole blood after centrifugation and separation on a density gradient, and incubated with a fixed number of heat-inactivated and labelled yeast particles. Total leukocyte counts, leukocyte populations and some parameters of phagocytic activity were determined: percentage phagocytosis, phagocytic index, absolute phagocytic index, count of phagocytes in a fixed volume of peripheral blood (CP) and phagocytic capacity. RESULTS: Patients with AMI had increased total leukocyte counts accompanied by increased PMN counts, while there were no significant differences in total MN count and MN populations. Except for the phagocytic index, all phagocytic parameters of MN and PMN cells were increased in patients with AMI at admission and on the first day of disease. On the seventh day after AMI only the CP of MN cells had increased significantly in patients with AMI, while percentage phagocytosis, CP and capacity of phagocytosis of PMN cells increased during the acute phase of AMI. CONCLUSIONS: These data suggest that AMI was followed with a strongly systemic inflammatory response to myocardial damage. Furthermore, activated MN and PMN cells may be a significant source of free radicals that may be involved in lipid peroxidation and produce tissue damage in the early postinfarction period.

Study of solution equilibria between aluminum(III) ion and ofloxacin.

The complex formation equilibria between aluminium(III) ion and ofloxacin in 0.1 mol/l(-1) ionic medium at 298 K were studied by glass electrode pH-metric and UV spectrophotometric measurements. Within ofloxacin to aluminium mole ratio ranging from 2:1 to 25:1 and in pH interval from 2.5 to 10.5, the obtained experimental results were explained by the formation of the following complexes: AI(Hoflo) (log beta1,1,1 = 15.93 +/- 0.03), Al(oflo)2 (log beta1,2,0= 14.84 +/- 0.07), Al(oflo) (log beta1,1,0 = 10.20 +/- 0.04) as well as several other mixed and pure hydrolytic complexes. The structure and mechanism of the formation of the complexes and their possible implications on aluminum toxicity were discussed.

Solution equilibria between ethylenediamine-N,N'-di-3-propionate and some transition metal(II) ions.

Solution equilibria between the ligand ethylenediamine-N,N'-di-3-propionate (eddp(2-)) and copper(II), nickel(II) or cobalt(II) ions were studied by glass electrode pH-metric and spectrophotometric measurements in 0.1 M NaCl ionic medium at 298.0+/-0.2 K. In the concentration limits 1.0

Protein interactions with bivalent tin. 1. Hydrolysis and complexation of tin(II) ion with glycine.

The complexation between tin(II) ion and glycine was studied in 0.15 mol/dm3 NaCl medium at 310 K using potentiometric glass electrode titrations. In the pH range 1.1-4.5 and concentration range of the tin(II) between 0.2 and 5.0 mmol/dm3, with variable glycine-to-tin molar ratio up to 10:1, the experimental data were explained by the formation of the following complexes and their overall stability constants: log(beta +/- sigma): Sn(HGly)+, (12.78 +/- 0.08); Sn(Gly)+, (10.02 +/- 0.07); Sn(OH)Gly, (7.34 +/- 0.03), as well as the pure hydrolytic complex Sn4(OH)2+(6), whose stability constant was determined in separate experiments and found to be -4.30 +/- 0.08, under the same experimental conditions as for complexation study. The precipitate formed in tin(II)-glycine system at pH ca. 5.0 was characterized by chemical and TG analysis, I. R. spectra, X-ray powder diffraction, and electron scanning microscopy measurements. It has been shown that the precipitate has the composition Sn(OH)Gly and crystallizes in a tetragonal system with unit cell dimensions a = b = 1.584 nm, c = 0.597 nm. The mechanism of the complex formation in solution is discussed.

Acid-base equilibria in substituted 4-quinolone carboxylic acid solutions.

The protonation constants of some substituted 4-quinolone carboxylic acid anions (ofloxacin, pefloxacin and ciprofloxacin) were determined in lithium chloride solution of various concentrations at 293, 303 and 313 K using the pH-metric and spectrophotometric methods. The values of thermodynamic parameters (deltaG, deltaH and deltaS) for the first protonation step of these anions in 0.1M lithium chloride at 293 K, are reported. The formation of intramolecular hydrogen bonds is discussed.

Potentiometric determination of the protonation constants of 1,3-propanediamine-N,N'-diacetate-N,N'-di-3-propionate.

The protonation constants of 1,3-propanediamine-N,N'-diacetate-N,N'-di-3-propionate (1,3-pddadp) were determined in sodium nitrate solution of various concentrations and in 0.1M potassium nitrate medium. The values of the thermodynamic functions (DeltaG, DeltaH and DeltaS) for the successive protonation equilibria, in 0.1M (Na)NO(3) medium, at 25 degrees , are also reported.