Biomedical subjects

P Dumain

Publications and source records attributed to P Dumain.

28 records · Page 2Linked to original sources

PubMed1994

Cytokine-mediated expansion of 5-FU resistant peripheral blood stem cells and bone marrow: self-renewal and commitment capacity.

To further characterize the primitive stem cell subpopulation present in chemotherapy mobilized peripheral blood stem cells (PBSC), we evaluated the functional characteristics of 5-FU-resistant PBSC and normal bone marrow (BM) cells after 7 days incubation with IL-1 + IL-3+SCF. The resulting 5-FU-resistant cells were evaluated for (1) the production of GM-CSF-responsive clonogenic elements (CE), (2) the production of IL-3+GM-CSF-responsive CE, and (3) their self-renewal capacity (production of IL-1+IL-3+SCF-responsive CE). We also evaluated the percentage of CD34+ cells, the percentage of cells in S phase of the cell cycle, and the number of nucleated cells before and after cytokine-mediated expansion. We demonstrated an overall loss in nucleated cells after cytokine-mediated expansion in all cell fractions. We demonstrated a significantly greater increase in the percentage of CD34+ cells in the 5-FU-resistant PBSC fraction as compared to 5-FU-resistant BM cells (p = 0.012). We also showed that 5-FU-resistant PBSC have a greater capacity for self-renewal, amplification of IL-3+GM-CSF-responsive progenitors, and the production of committed GM-CSF-responsive progenitors as compared with BM cells, but this did not reach statistical significant. These results suggest that PBSC contain a truly primitive stem cell with an enhanced self-renewal and differentiative capacity that is recruited by 5-FU resistance and IL-1+IL-3+CSF-mediated expansion.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗

PubMed1994

Daunorubicin (DNR) accumulation in fresh leukemic cells: correlation with clinical and biological features.

The DNR accumulation (DNR Acc) and the verapamil (VRP) index (percent increase of VRP on DNR accumulation) was studied by using flow cytometry. Fresh leukemic mononuclear bone marrow blasts from 80 unselected ANLL patients' samples were incubated with DNR in the presence or absence of VRP. The DNR accumulation was determined by flow cytometry. The median DNR Acc was 28 (range: 4-101) and the median VRP index was 4% (range 0-53). VRP significantly enhanced DNR Acc in 42 of the ANLL samples (52.5%). DNR Acc or VRP index were not influenced by age, sex, or WBC counts. Only the FAB subclassification and the blast immunophenotyping were found to influence the parameters studied here. The lowest DNR Acc was found in M0 and M6 blast cells (15 range 0-46 and 10.5 range 8-13 respectively). M4 and M5 ANLL samples accumulated significantly more DNR than M0 and M6 blast cells. The VRP index was significantly higher in M0 compared with M1 and M2 samples, as well as in M4 compared with M1 samples. A slightly positive correlation was found between the percentage of CD34-positive cells in the CD34-positive samples and DNR Acc. In this study, DNR Acc and the VRP index were not significantly correlated with the response to chemotherapy or survival. In conclusion, this study shows that ANLL leukemic cells differ in anthracyclin accumulation and response to VRP in vitro.

Acute Disease↗

PubMed1993

Flow cytometric estimation of poly(A)+ RNA by fluorescent in situ hybridization.

A method for the detection of poly(A)+ RNA in cell suspensions by in situ hybridization and flow cytometry is described. The hybridizing properties of oligothymidylate o(dT) was well preserved after coupling to fluorescein. This fluorescent oligonucleotide was used as a probe to determine the poly(A)+ RNA content of fixed HL60 leukemic cells by flow cytometry. Labeling was considerably reduced by treating the cells with RNAse, and by competitive hybridization with either poly(U) or free poly(A). Labeling was also decreased in a time-dependent fashion by incubating the cells with actinomycin D prior to fixation. This method represents an improvement on the methods measuring total RNA and could be of value in investigations on the effect of drugs on RNA metabolism.

Dactinomycin↗

PubMed1993

Treatment of relapsed acute myeloid leukemia using GM-CSF before intensive chemotherapy.

Ten patients with acute myeloblastic leukemia (AML) in first relapse were treated with high-dose cytosine-arabinoside (ara-C, 3 g/m2/12 hours x 4) and amsacrine (150 mg/m2/day x 5). In order to prime the cells, the patients were given rh-GM-CSF (3 micrograms/kg/d) for four days, the first infusion starting 48 hours before chemotherapy. Two patients died during the aplastic phase, seven patients achieved a second complete remission (CR2) and one patient remained leukemic. The median duration of aplasia was 17 days (14-21). These results were comparable to those obtained in our previous series of 27 patients treated for AML in first relapse with the same chemotherapy but without GM-CSF (66% CR2). After 48 hours of GM-CSF infusion, (before chemotherapy was started), seven patients had an increase in the white blood cell count with an increase in the absolute number of blast cells in five of these cases. Marrow blast cells percentages increased in 3 of the 8 patients analysed. Six of seven patients tested showed an increase in the percentage of cells in S-phase (studied by flow cytometry using the bromodeoxyuridine (BrdU/DNA) labelling technique and BrdU incorporation). GM-CSF used to prime leukemic cells may be safely administered but its clinical usefulness needs to be further evaluated.

Adolescent↗

PubMed1992

Quantitation of resistance of leukemic cells to cytosine arabinoside from BrdUrd/DNA bivariate histograms.

The cytotoxicity of ara-C derives from an inhibition of DNA synthesis after incorporation of ara-CTP into DNA. The rate of DNA synthesis can be determined from the amount of bromodeoxy-uridine (BrdUrd) incorporated into cells after a short exposure to BrdUrd. We developed a computer program to quantify the inhibition of the rate of DNA synthesis by analysis of the distribution of BrdUrd/DNA. Inhibition was evaluated in ara-C-sensitive and resistant cells after incubation with different doses of ara-C. An index of resistance to ara-C (RI) was expressed as the ratio of the amount of BrdUrd incorporated into S phase cells incubated with ara-C to that incorporated in the absence of ara-C. In the ara-C-sensitive and resistant HL60 cells, a linear relationship between RI and log ara-C concentration was observed. Small numbers of slightly resistant cells in mixtures of ara-C-sensitive and resistant cells could be determined using this method, making it suitable for clinical use to test the resistance of leukemic cells to ara-C.

Bromodeoxyuridine↗

PubMed1992

Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry.

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have been shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU-sensitive and DAU-resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance.

Benzimidazoles↗

PubMed1991

Modalities of synthesis of Ki67 antigen during the stimulation of lymphocytes.

The antibody Ki67 is currently used to evaluate the proliferative fraction of solid tumors and some hematological malignancies. We have used phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes as a model to study the entry of quiescent cells into cell cycle and to follow their progress to the next cycle. Flow cytometric analysis of lymphocyte samples stained with the antibody Ki67 and a DNA marker has allowed us to follow the expression of Ki67 antigen (Ki67 Ag) as a function of the position of the cells in the cell cycle. The use of drugs blocking the stimulated lymphocytes in different phases of the cell cycle permitted us to demonstrate that Ki67 Ag expression started from the beginning of the first S phase. The level of Ki67 Ag increased during S phase until mitosis, when its expression was maximal. After division, the cells in G1 phase showed a decrease in Ki67 Ag expression (possibly corresponding to degradation) until they reentered S phase, when the level of Ki67 Ag increased again. The results confirm that the expression of Ki67 Ag is related to the proliferative state of the cells and suggest that it may be used to determine the proliferative cell fraction in hematopoietic tissues.

Antigens, Surface↗

PubMed1990

Flow cytometric study of the activation of polymorphonuclear cells.

The activation of human polymorphonuclear cells (PMN) by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or the protein kinase C activator, phorbol myristate acetate (PMA), was studied using flow cytometry. Two probes were used to evaluate PMN activation: 1) a monoclonal antibody (MoF11) directed against an antigen (Ag) expressed on the membrane of monocytes and of activated PMN; 2) rhodamine phalloidin was used at the cytoplasmic level to measure the F-actin content. The expression of MoF11 antigen was found to be 3 to 5 times greater on the membrane of PMN activated by either FMLP or PMN as compared with membrane expression of the same Ag on resting PMN. This increase was found to be dose dependent for the two activators. Kinetic studies showed that a maximum response was observed in 1 to 2 min at 37 degrees C when FMLP was used, whilst a similar response required 10 min when PMA was used. The same discrepancy with activators was observed when actin polymerization was measured by labelling with rhodamine phalloidin. However, pretreatment of PMN with cytochalasin B inhibited actin polymerization whilst MoF11 antigen expression was increased, suggesting that the MoF11 antigen could be stored in granules of resting PMN. The study of actin polymerization and of MoF11 antigen expression, separately or in combination, could be a useful tool for the detection of activated PMN in biological samples.

Actins↗

PubMedDate not supplied

Fluorescence-based selection of retrovirally transduced cells in congenital erythropoietic porphyria: direct selection based on the expression of the therapeutic gene.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is an inherited disease caused by a deficiency of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor with death occurring in early adult life and available treatments are only symptomatic and unsatisfactory. In vitro gene transfer experiments have documented the feasibility of gene therapy via haematopoietic stem cells to treat this disease. To facilitate future ex vivo gene therapy in humans, the design of efficient selection procedures to increase the frequency of genetically corrected cells prior to autologous transplantation is a critical step. METHODS: An alternative selection procedure based upon expression of a transferred gene was performed on a lymphoblastoid (LB) cell line from a patient with congenital erythropoietic porphyria to obtain high frequencies of genetically modified cells. The presence of exogeneous delta-aminolevulinic acid (ALA), a haem precursor, induces an increase in porphyrin accumulation in LB deficient cells. Porphyrins exhibit a specific fluorescent emission and can be detected by cytofluorimetry under ultraviolet excitation. RESULTS: In genetically modified cells, the restored metabolic flow from ALA to haem led to a lesser accumulation of porphyrins in the cells, which were easily separated from the deficient cells by flow cytometry cell sorting. CONCLUSION: This selection process represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Adult↗

PubMedDate not supplied

Role of blast cell immunophenotyping for the diagnosis and prognosis of acute myeloid leukemia.

Bone marrow blast cell antigen expression from 86 patients with de novo acute myeloid leukemias (AML) was studied and correlated with FAB classification and clinical outcome. Among a panel of 14 monoclonal antibodies routinely used for the diagnosis of acute leukemias we studied the expression of six antibodies (CD13, CD15, VIM2, CD33, CD14, CD34) of the granulomonocytic lineage and found that some of them were useful for diagnosis and/or prognosis. For FAB subclassification of AML, the CD13 or VIM2 antigen expression was of no benefit. Monocytic leukemias (M4 + M5PD + M5WD) more frequently expressed CD34 antigen (28/31) than granulocytic (M1 + M2 + M3) subtypes (33/53) (P < 0.01). Finally, the most striking differences were found with CD14 antigen expression: CD14 antigen was more frequently expressed in M4 + M5 leukemias (21/31) than in M1 + M2 + M3 subtypes (12/33) (P < 0.01). The mean percentage of CD14 positive blast cells was accordingly higher in monocytic leukemias than in granulocytic leukemias and the difference was highly significant (P < 0.0001). The CD15 antigen was more frequently expressed in differentiated leukemias (M2 + M3 + M4 + M5WD) (35/44) than in poorly differentiated forms (M1 + M5PD) (17/37) (P < 0.001). The statistical difference was higher when the mean percentage of CD15 positive blast cells were compared (P < 0.0003). Moreover these latter percentages were different in M1 and M2 subtypes (P < 0.003). The blast cell expression of CD13, CD14, CD15 or CD33 was not predictive of the length of CR or survival. Moreover, our results support previously published findings suggesting a longer overall survival duration for patients whose leukemic cells do not express the CD34 antigen (P < 0.01). We also confirm that patients with the more differentiated subtypes of AML (CD13-, CD34+) tend to survive longer than patients with the less differentiated subtypes of AML (CD13-, CD34+) (P < 0.001).

Adolescent↗