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.

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.

Relationship of serum levels of tumor markers with tissue expression of gene products in ovarian carcinoma.

PURPOSE: The aim of this prospective study was to determine serum levels and tissue expression of CA125, CA 15-3, p53, HER-2 and nm23 tumor markers, which are used in the detection and follow up of patients with ovarian carcinoma. PATIENTS AND METHODS: 19 patients with malignant and benign ovarian tumors were included in this study. Serum levels of CA125, CA 15-3 and p53 tumor markers were detected in preoperative and postoperative blood samples using ELISA technique. Tissue expression of p53, HER-2 and nm23 were examined using immunohistochemistry. RESULTS: All serum tumor markers were elevated in patients with ovarian carcinoma. Serum level of CA 15-3 was increased in patients with ovarian carcinoma (median 48.33 U/ml, normal range 0-36), while it was normal in patients with benign ovarian tumors (median 20.67 U/ml; p >0.05). CA125 serum values were strikingly increased in ovarian carcinoma (median 264.16 IU/ml, normal range 0-35) and benign ovarian tumors (median 119.59 IU/ml; p <0.05). Serum levels of p53 in patients with ovarian carcinoma were increased (median 0.69 U/ml, normal range 0-0.50) compared to patients with benign tumors (0.32 U/ml; p <0.05). Histological HER-2 overexpression was detected in 7 cases, including 4 with strong (score 3+ and 2+) and 3 with weak or no HER-2 expression (score 1+ and 0) in ovarian carcinoma tissue; in benign tumors HER-2 overexpression was detected in 1 case (p >0.05). Strong overexpression of p53 was detected in 3 cases with malignant and none with benign tumors (p >0.05); and strong overexpression of nm23 was detected in 5 cases with malignant and 2 with benign tumors (p >0.05). CONCLUSION: Serum levels of CA125, CA 15-3 and p53 are strikingly increased, as well as the expression of HER-2 and p53 in carcinomatous tissue. Detection and analysis of multiple tumor-specific markers in serum and tissue can give useful clinical information for the management of ovarian carcinoma and can also improve the sensitivity and specificity of these markers.

Preliminary study of mononuclear phagocytosis during breast cancer therapy.

PURPOSE: To evaluate the eventual changes in the number and phagocytic functions of blood monocytes in breast cancer patients during surgical treatment and chemotherapy. MATERIALS AND METHODS: The absolute and relative number of peripheral blood leukocytes and monocyte phagocytic functions were determined at the time of diagnosis (I), following surgery (II), during (III) and after chemotherapy (IV) in 30 patients diagnosed with breast cancer. The control group consisted of 30 age-matched healthy women. RESULTS: The mean number of monocytes was significantly lower in cancer patients at diagnosis, while they increased following surgery reaching the control values. There were no postchemotherapy changes in the number of monocytes. Monocyte phagocytic activity was decreased at the time of diagnosis. Following surgery, the capacity of phagocytosis (CP) recovered to normal values, but the index of phagocytosis (IP) remained decreased. During and after chemotherapy, as well as one year after surgery, the IP still remained decreased. CONCLUSION: Our results showed that some properties of monocytes' phagocytic activity in cancer patients were decreased at diagnosis, returning back to normal range after surgical therapy. However, time is needed to confirm whether the alteration of IP may provide additional information when monitoring breast cancer patients.

Serum HER2 and CA 15-3 in breast cancer patients.

PURPOSE: The HER2 oncogene encodes a transmembrane tyrosine kinase receptor. This molecule could be a new marker for prognosis and response to therapy in patients with advanced breast cancer. However, the extracellular domain of c-erbB-2 (HER2) transmembrane receptor undergoes proteolytic cleavage from the fulllength protein by metalloproteases, and is shed into the blood as a circulating antigen. To determine the clinical utility of this oncoprotein, the soluble form of HER2 was assayed in the serum of breast cancer patients. PATIENTS AND METHODS: Serum levels of breast carcinoma antigens CA 15-3 and HER2 were determined in 60 patients, 40 with localized (group A) and 20 with metastatic (group B) breast carcinoma. CA 15-3 measurements served as "gold standard" to which HER2 diagnostic and/or prognostic value was compared. Sera from 10 healthy women served as controls. RESULTS: Serum levels of the tested tumor markers HER2 and CA 15-3 were significantly higher in cancer patients compared to controls. CA 15-3 correlated with bulky initial tumor, whereas HER2 showed no differences between healthy individuals and group A patients. The serum levels of the tested markers in group B patients were significantly higher (p <0.001) than the serum levels of patients in group A. Striking increase in serum levels of HER2 was found in 52.7% and CA 15-3 in 52.9% of patients with metastatic cancer. A combination of markers was more sensitive than using one marker alone. In this regard, 90% of the patients with metastasis had at least one of the markers increased. CONCLUSION: The results of this study suggest that the HER2 oncoprotein may be potentially useful in detecting recurrence of breast cancer. However, to improve sensitivity and specificity in the diagnosis and monitoring of breast cancer, the use of multiple tumor markers should be employed.

High-performance liquid chromatographic method for the assay of dexamethasone and xylometazoline in nasal drops containing methyl p-hydroxybenzoate.

A rapid and sensitive high-performance liquid chromatographic method has been developed for the determination of dexamethasone sodium phosphate (DSP), xylometazoline hydrochloride (XMC) and methyl p-hydroxybenzoate (MHB). An assay of the compounds has been performed on a HPLC system GBC 1210, at controlled room temperature, on a Nucleosil C8 column (250x3 mm, 5 microm). The mobile phase was acetonitrile-water (35:65, v/v), at a flow-rate of 1 ml min(-1). The parameters for validation such as linearity (r>0.9996), precision (RSD: 0.51-(1.93%), limit of detection and quantification (2.032 x 10(-4) and 4.063 x 10(-4) mg ml(-1) for DSP, 9.7 x 10(-5) and 1.953 x 10(-4) mg ml(-1) for XMC, 1.953 x 10(-4) and 3.096 x 10(-4) mg ml(-1) for MHB) have also been reported. The method was applied to the determination of DSP, XMC and MHB in nasal drops. The statistical parameters were found to be satisfactory, with recovery values ranging from 98.69 to 101.60% (RSD: 0.32-1.03%). The method is simple and accurate and therefore suitable for the simultaneous determination of these compounds in dosage form.

Simultaneous assay of ephedrine hydrochloride, theophylline, papaverine hydrochloride and hydroxyzine hydrochloride in tablets using RP-LC.

The analytical problem was to control the quality of imported antiasthmatic tablets containing ephedrine hydrochloride, theophylline, papaverine hydrochloride and hydroxyzine hydrochloride. The aim of the analytical method for the assay was to separate, identify and quantify all compounds, at the same time. A gradient capable RP-LC system was used, using a commercially packed Nucleosil C18 column connected to a dual channel variable, programmable wavelength detector. The analysis was performed in the gradient program of increasing concentration of acetonitrile in water. The influence of pH of the mobile phase was established. The proposed method is reliable, reproducible, easy to perform and satisfies the aim.

High-performance liquid chromatographic assay of 8-hydroxyquinoline sulfate and its stability in immunobiological preparations.

Because of the ability of 8-hydroxyquinoline sulfate (8-HQS) to irreversibly bind metals from rubber stoppers, the stability of 8-HQS in tuberculin solutions was investigated. For the determination of 8-HQS, a simple and sensitive reversed-phase HPLC method with detection at 240 nm was developed and validated. Rapid decreases in concentrations of 8-HQS were found in samples stored in original vials which were exposed to different temperatures and vial positions.

[The effect of complete thymic extract on motility and progressive motility of spermatozoa in asthenozoospermia]. / Dejstvo celokupnog ekstrakta timusa na motilitet i progresivni motilitet spermatozoida kod astenozoospermije.

The thymic gland is involved in immunologic and endocrinologic heomostasis of the body. Neonatal thymectomy of experimental animals leads to gonadal dysgenesis. Both eostrogen and androgen receptors have been identified in thymic epitelial cells, and sex steroids can be modulated by secretion of thymosin. High levels of thymosin alpha-1 in seminal plasma are correlated with sperm count, viability and motility in fertile males as compared to infertile ones. In this study we examined the effects of complete thymic extract on motility and progressive motility of spermatozoa obtained from asthenozoospermic men. In the first part of experiment, we studied the influence of complete thymic extract, as a bioactive medium component, on movement of spermatozoa samples with initial motility lower than 30%, in vitro. The study was carried out on two groups of sperm samples, 40 samples each, obtained by masturbation after a 72-hour-abstinence. After completed liquefaction at room temperature (30-40 min.), we determined the usual parameters of sperm quality. Total motility (M%) and progressive motility (PM%) of spermatozoa were analyzed in 10 microscopic view fields. Each group of sperm samples was prepared by swim-up and swim-down method. Then, we estimated the total motility and progressive motility of "washed" spermatozoa in every sample. In addition, every sample was divided in two equal parts. One part was immediately left in thermostat (control). To the second part we added 0.2 mg of complete thymic extract before incubation (experimental group). Total and progressive motilities were analyzed after 1, 4, 12 and 24 hours of incubation. The t-test was used for statistical analysis of results. The second part of the study was carried out on a group of 10 asthenozoospermic men (volunteers). We examined the influence of complete thymic extract, as intra-muscular injection, in a doze of 150 mg daily, during seven days, on motility and progressive motility of spermatozoa. Sperm samples were obtained by masturbation after four days of abstinence. We compared the parameters before and after the therapy. They were statistically processed by a method of difference. The results showed that the complete thymic extract increased the total and progressive motilities of spermatozoa separated by swim-down method (p < 0.001), in vitro (Table 1-4). The parenteral application of complete thymic extract, statistically significantly increased motility and progressive motility of spermatozoa in asthenozoospermic men (p < 0.05) (Table 5). These findings could be useful in assisted reproductive technology.

[Microsurgical treatment of tubal factors in sterility]. / Mikrohirursko lijecenje tubarnog faktora steriliteta.

In the period from 1982-1988, 164 patients were operated due to the tubal factor sterility. Indications for operations were based on HSG and laparoscopy. Microsurgical process was applied. 57 (34.75%) of 164 operated patients conceived. There were 41 (25.90%) intrauterine and 16 (9.14%) ectopic pregnancies. The most frequently pregnancies happened in the patients who had been treated by fimbroplasty and isolated adhesiolysis. The patients, who had been treated by salpingostomy terminalis and tubal implantation had lower pregnancy percentages.

Endometrial adenocarcinoma associated with drug-induced hyperprolactinemia.

A 54-year-old woman with hyperprolactinemia and amenorrhea that occurred with phenothiazine and tricyclic antidepressant use developed a stage IBG2 endometrial adenocarcinoma. This is believed to be the first case reported of endometrial carcinoma associated with drug-induced hyperprolactinemia.

Results of our 15-year study into the biological effects of microwave exposure.

The results obtained during 15 years of clinical and experimental examinations of biological microwave exposure effects are briefly surveyed. Some important results are reported. Based on their experience, the authors present their attitudes concerning harmful microwave effects on living matter. They consider that microwave effects, either direct or indirect, are the results of hyperthermia. Exposure of the living body to irradiation intensities not causing thermal effects do not induce important pathological alterations in the irradiated organisms. Also, it has been pointed out that the term "injury" is more suitable than the term "microwave sickness" when harmful effects of microwaves to the living organism are concerned. According to the authors, the term "microwave sickness" is not acceptable as a synonym for professional diseases of persons working with sources of microwave energy, since it refers to the complex of insufficiently defined symptoms of uncertain etiology.