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The aim of this study was to use a two-marker assay for the detection of breast can­cer cells circulating in patients' blood. We have applied a PCR-based methodology to follow up the possibility of the development of metastatic disease in stage I and II pa­tients who had undergone curative surgery. Since the number of circulating cancer cells in peripheral blood is very low, the technique for their detection needs to be not only highly sensitive, but also very specific. The reverse transcriptase-polymerase chain reaction (RT-PCR) technique may improve the sensitivity of breast cancer cell detection up to only a few cells per one million. The principle of the RT-PCR assay is to amplify a messenger RNA characteristic for breast epithelial cells in a blood sam­ple. Since we do not expect such cells to be circulating in peripheral blood of healthy subjects, detection of the characteristic mRNA should indicate the presence of circu­lating breast cancer cells. We analyzed the usefulness of three mRNA markers: cytokeratin 19 (CK19), mammaglobin (hMAM) and S subunit of human chorionic gonadotropin (S-hCG) for this test. Blood samples (112) were obtained from 55 patients, in stages I and II, with or without metastasis to regional lymph nodes (N0 or N1). We found that a two-marker assay increases the sensitivity of detection of breast cancer cells in com­parison with a single-marker one. Combination of two tumor-specific mRNA mark­ers, hMAM/CK19 or S-hCG/CK19, allowed the detection of circulating breast cancer cells in 65% of N1 patients and 38% of N0 patients. By comparison, the combination hMAM/-hCG allowed the detection of circulating breast cancer cells in the blood of 68% of N1 patients and 46% of N0 patients. Addition of the third marker did not significantly increase the detection sensitivity.
The experiment was performed on 20 adult Wistar rats aged 12 weeks, divided into two equal groups (control and experimental), each comprised of five males and five females. From the first day of the experiment, the experimental group rats were fed Murigran feed supplemented with ß-l,3/l,6-D-glucan at a dosage of 12-19 mg/rat/d, subject to body weight, while the control- group was administered the same feed without any additives. At the beginning of the experiment and then after 14 days, arterial blood samples were collected from the rats and diluted with heparin to measure and compare the phagocytic activity and oxidative metabolism of peripheral blood granulocytes and monocytes by flow cytometry. Statistically higher levels of the activity were observed in the group of rats administered glucan than in controls, expressed in terms of the percentage of phagocytic cells as well as average fluorescence intensity. ß-l,3/l,6-D-glucan also had a positive effect on the oxidative metabolism of both granulocytes and monocytes after stimulation with E. coli, and on the oxidative metabolism of granulocytes after stimulation with PMA.
As far as the detection of metal genotoxicity in fish is concerned, micronucleus (MN) test is considered an extremely suitable measure. In this study, frequencies of micronucleated erythrocytes were scored in peripheral blood of catfish, Heteropneustes fossilis (bloch) after acute in-vivo exposure of zinc at different concentrations (5, 10 and 30 ppm) in the laboratory condition. These three concentrations of zinc were tested at different durations such as 24h, 48h, 72h and 96h respectively. Highly significant (P < 0.001) increased values were obtained for MN frequencies in the peripheral erythrocytes of exposed fishes compared to control groups of fishes. These results confirm that dose- and time-dependent micronucleation in the peripheral erythrocytes of fish after short-term exposure to zinc could provide valuable information regarding zinc containing effluent quality and also help in genetic biomonitoring with this test model. In this context safe concentration of zinc vis-a-vis genotoxicity range could be evaluated for future studies.
The aim of the study was to evaluate the effect of Mannheimia haemolytica leukotoxin (Lkt) on cellular immune response in clinically-healthy calves given intravenously 25 µg per animal of M. haemolytica Al Lkt. The alternations of peripheral blood leukocytes were examined with a flow cytometry. The Lkt treated calves were compared with the non-treated controls before (0) and 1, 2, 3, 4, 5, 6, and 24 h after the treatment. The following parameters were assayed: white blood cell (WBC) count, percentage of polymorphonuclear leukocytes (PMNL), mid-size leukocytes, total percentage of lymphocytes and their subsets: CD2⁺ (T lymphocytes), CD4⁺ (T helper lymphocytes), and CD8⁺ (T suppressor/cytotoxic lymphocytes) with CD4⁺: CD8⁺ratio, and also WC4⁺ cells (B lymphocytes). The obtained results showed that in the treated calves, the WBC count, the percentages of PMNL, mid-size leukocytes, and some subpopulations of peripheral lymphocytes (CD2⁺, CD4⁺, CD8⁺) were significantly lower compared with the controls during the first of three hours of the experiment, and then the parameters increased and returned to the physiological level.
The study aimed to assess biophysical changes that take place in the peripheral blood mononuclear cell (PBMC) membranes when exposed in vitro to 10 μM quercetin or epigallocatechin gallate (EGCG) for 24 and 48 h. PBMCs isolated from hypercholesterolemia patients were compared to those from normocholesterolemia subjects. The membrane fluidity and transmembrane potential were evaluated and the results were correlated with biochemical parameters relevant to oxidative stress, assessed in the patients’ plasma. The baseline value of PBMC membrane anisotropy for the hypercholesterolemia patients was lower than that of the control group. These results correlated with the plasma levels of advanced glycation end products, which were significantly higher in the hypercholesterolemia group, and the total plasma antioxidant status, which was significantly higher in normocholesterolemia subjects. In the case of normocholesterolemia cells in vitro, polyphenols induced a decrease in membrane anisotropy (7.25–11.88% at 24 h, 1.82–2.26% at 48 h) and a hyperpolarizing effect (8.30–8.90% at 24 h and 4.58–13.00% at 48 h). The same effect was induced in hypercholesterolemia cells, but only after 48 h exposure to the polyphenols: the decrease in membrane anisotropy was 5.70% for quercetin and 2.33% for EGCG. After 48 h of in vitro incubation with the polyphenols, PBMCs isolated from hypercholesterolemia patients exhibited the effects that had been registered in cells from normocholesterolemia subjects after 24 h exposure. These results outlined the beneficial action of the studied polyphenols, quercetin and EGCG, as dietary supplements in normocholesterolemia and hypercholesterolemia patients.
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