Multidrug resistance of leukemia cells to cytostatics--the functional fluorescent dye efflux assay evaluated by flow cytometry

The functional fluorescent dye efflux assays are used in the study of the multidrug resistance of the malignant cells to the cytotoxic drugs which is caused by the overexpression of P-glycoprotein (P-gp) or another membrane transport system--a multidrug resistance related protein (MRP). P-glycoprotein and a multidrug resistance related protein are involved in the efflux of cytotoxic drugs out of the cell and are responsible for the resistance. Fluorescent dye efflux mediated by these proteins could be evaluated by the flow cytometry. This test seems to be an optimal approach to study the multidrug resistance. With help of the specific inhibitors such as verapamil and cyclosporine A, the functional capacity of these proteins and the possibility to overcome the multiresistant phenotype can be revealed. The cell line K562 with transfected P-glycoprotein gene serves as a model system for the studying of the transport function with the use of the fluorescent substrates and P-glycoprotein inhibitors.     

Detection of multiple myeloma cells using multicolor immunofluorescence and flow cytometry

The advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by the immunofluorescence using flow cytometry. We have examined the co-expression of CD19, CD38, CD45, CD54, CD56, and CD138 molecules in cells of peripheral blood and bone marrow aspirates in patients with multiple myeloma by 3-color flow cytometry. For the detection and characterization of multiple myeloma cells, combinations of following antibodies were used: anti-CD19 FITC, anti-CD38 FITC, anti-CD38 PE, anti-CD54 FITC, anti-CD56 PE-Cy5, anti-CD45 PE, anti-CD45 PE-Cy5 (Immunotech) and anti CD138 PE (Serotec). The samples were analyzed using EPICS XL (Coulter) flow cytometer, and the analysis was based on at least 10,000 events. Samples from 17 patients were analyzed. The percentage of multiple myeloma cells ranged between 0.3% and 54.2% in bone marrow aspirates and between 0.0 and 11.8% in periferal blood. The expression of CD138, CD38, CD54 and CD56 molecules was found in 100%, 100%, 85% and 68% of examined cases, respectively. In our opinion, multiple myeloma cells are best characterized by following combinations of antibodies: CD38 FITC/CD138 PE/CD45 PE-Cy5, CD54 FITC/CD138 PE/CD56 PE-Cy5 or CD54 FITC/CD38 PE/CD 56 PE-Cy5. The identification of a malignant clone is the first and the most important step in the characterization of the disease, determination of its prognosis and the detection of residual disease after treatment. Three-color flow cytometry represents a method which can meet these goals.     

The effect of glucagon on the glycosaminoglycan content of canine arteries

Summary Hypophysectomized young beagles were treated for 21 days with pharmacological doses of glucagon, administered intramuscularly in two daily injections of 0.5 mg each. Hyaluronic acid, heparan, dermatan and isomeric chondroitin sulphates were determined in aorta, carotid, iliac, renal, mesenteric and coronary arteries. In comparison with age-matched hypophysectomized controls, glucagon-treated dogs showed a reduction of the heparan sulphate concentration in their carotid arteries and thoracic aorta. These results indicate that glucagon has a very limited effect on arterial glycosaminoglycan content.     

Results of radical surgical correction of ventricular septal defect

This study evaluates results of radical correction of simple and complex ventricular septal defect in 85 children. 35 children were under 3 years of age. Two babies operated at the age of 6 and 8 months in a critical condition died after surgery. Total operation mortality was 2.3%. In 51 patients examined one year and more after surgery, the long-term results are in 90% of the cases excellent and in 10% favourable. In none of the patients did the authors find an unfavourable result of the operation. The authors regard radical surgical correction of ventricular septal defect as a method of choice regardless of the patient's age and weight.     

Importance of selected laboratory indicators in the differential diagnosis and monitoring of multiple myeloma

Multiple myeloma is one of the most common haematologic malignancies. Currently there are numerous studies looking for new prognostic markers in multiple myeloma. The most important of them are the markers related to proliferative activity of neoplastic cells or to size of tumor mass. The subject of this paper are the results obtained from investigation of some such laboratory markers in a group of patients with monoclonal gammopathies diagnosed at our department in the last 3 years. We analyzed blood and bone marrow samples from 51 patients with new diagnosed monoclonal gammopathies, 14 of them were patients with monoclonal gammopathy of undetermined significance and 37 patients had multiple myeloma. 17 patients with multiple myeloma were treated by high-dose chemotherapy regimen. We assessed significance of selected laboratory markers for differential diagnosis of monoclonal gammopathies and for monitoring of activity of multiple myeloma. Among the investigated parameters, we verified the significance of cell cycle analysis of bone marrow plasmatic population and of the determination of the number of circulating myeloma cells in differential diagnosis of monoclonal gammopathies. In our opinion, the determination of soluble CD138, beta 2-microglobulin and neopterin serum levels can be also recommended as helpful markers for a solution of this problem. Except of beta 2-microglobulin serum level we did not find statistically significant correlation with activity of multiple myeloma in any of the investigated parameters.     

Expression of mitochondrial uncoupling protein 3 and adenine nucleotide translocase 1 genes in developing rat heart: putative involvement in control of mitochondrial membrane potential

Postnatal maturation of the heart depends on the switch from glycolytic to oxidative metabolism and it is associated with decreasing tolerance to oxygen deprivation. Therefore, changes in composition and function of cardiac mitochondria during postnatal development require detailed characterization. Left-ventricular myocardium of prenatal, and 1-, 2-, 5-, 10-, 20-, 28-, 50-, 60-, and 90-d-old male Wistar rats was studied. The expression of uncoupling proteins (UCPs), adenine nucleotide translocase (ANT), and peroxisome proliferator-activated receptor alpha (PPARalpha) genes was characterized by northern blotting (UCP2), real-time quantitative RT-PCR (UCP2, UCP3, ANT1, ANT2, and PPARalpha), and by immunoblotting (UCP3). In isolated mitochondria, cytochromes a + a(3) were quantified by a spectrophotometry, and mitochondrial membrane potential (MMP) was measured using Rhodamine 123 (by spectrofluorimetry and flow cytometry). The specific content of cytochromes in mitochondria increased two-fold between birth and day 30, similarly, as the expression of ANT1 and PPARalpha genes. Postnatal activation in the expression of UCP2, UCP3, ANT1 and PPARalpha genes resulted in the expression maxima between days 20 and 30. The content/expression declined following day 20 (UCP2, UCP3, and PPARalpha) or 30 (cytochromes and ANT1), while expression of ANT2 declined continuously during the first month of life. In 1-d-old animals a single population of mitochondria with a relatively high MMP was observed; with increasing age, a second population of mitochondria with a significantly lower MMP appeared. The results support the view that mitochondrial energy conversion in heart changes during ontogeny and suggest the involvement of UCP3 and/or ANT1 in the control mechanism.