Search PubMed⌕ Search

Biomedical subjects

J Reiffers

Publications and source records attributed to J Reiffers.

At least 163 records · Page 9Linked to original sources
PubMed1995

Inhibition of chronic myelogenous leukemia cells harboring a BCR-ABL B3A2 junction by antisense oligonucleotides targeted at the B2A2 junction.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL hybrid gene. Two types of hybrid BCR-ABL mRNA have been found, B2A2 and B3A2. As the BCR-ABL rearrangement is specific to leukemic cells, selective inhibition of leukemic cell growth by BCR-ABL antisense oligonucleotides (ASO) has been reported in vitro for CML patients and cell lines. However, controversial results have been obtained from preclinical studies using anti-BCR-ABL ASO, as nonspecific inhibition of leukemic cell growth was evidenced in some cases. B3 exon secondary structure was deduced from its sequence and found to be a loop. According to this predictive structure of exon B3, a 56-mer antisense oligonucleotide targeting the polypurine bases from the B2A2 junction was devised which would inhibit proliferation (MTT assay) of B3A2 junction cell lines (K562 and a murine cell line Ba/F3 transfected with the B3A2 junctional sequence). This ASO had a hairpin-like secondary structure and was found to be much more resistant to the action of nucleases than control 18-mer standard oligonucleotides. Hybridization to its target mRNA occurs via formation of a triplex structure. A concentration of 5 microM of specific 56-mer B2A2 ASO was necessary to demonstrate 50% optical density (OD) reduction for K562 cell line and Ba/F3 transformed by B3A2 cDNA. Sense and non-sense 56-mer sequence or 18-mer linear ASO showed no effect for these concentrations. Western blot showed a partial inhibition of P210 protein; expression of P145abl remains unchanged. The 56-mer ASO also inhibited the proliferation of B2A2 junction cell line BV173 at the same concentration and showed no effect on the HL60 cell line used as control.

Base Sequence↗
PubMed1995

Chronic myelocytic leukemia patients achieving complete cytogenetic conversion under interferon alpha therapy: minimal residual disease follow-up.

We report minimal residual disease evaluation in 18 chronic myelocytic leukemia patients who achieved a durable complete cytogenetic conversion (CCC) under interferon alpha (IFN) therapy. Monitoring was performed every 3-6 months using bone marrow (BM) karyotypes and/or two-step reverse transcription polymerase chain reaction (RT-PCR) on peripheral blood samples. Median follow-up after first CCC was 47 months (range 15-69). All patients maintained complete hematological remission during follow-up. A median of five BM karyotypes were performed per patient (range: 3-11). The estimated chances of maintaining a major cytogenetic response (either CCC or less than 35% Ph positive metaphases were 93 +/- 13% (95% CI) at 36 months. One patient lost his cytogenetic response. A median of seven RT-PCR reactions were performed per patient (range: 1-11). A residual disease was detectable even in cases with long periods of CCC. However, in two patients, RT-PCRs were often negative; one, who had four successive negative RT-PCR was taken off IFN therapy and did not receive any other treatment; later in this case, RT-PCRs were again positive, but CCC was maintained for 39 months. Of the three who were taken off IFN and no longer treated, two maintained CCC (39+ and 33+ months); the third had a recurrence of 7% Ph-positive metaphases, and later returned to CCC. These results confirm that in most well-responding patients, the disease is not eradicated. However, it seems that the clonogenic potential of the residual leukemic clone is low. In patients taken off IFN therapy, IFN may have a particular remnant effect.

Adult↗
PubMed1995

Peripheral blood progenitor cell transplantation in France.

Autologous stem cell transplantation has increased in France over the last five years, mainly due to a marked increase in the number of autologous peripheral blood progenitor cell (PBPC) transplants, which represented about 66% of autologous transplants performed in 1994. This increase is related to the more rapid engraftment observed after PBPC transplantation than after bone marrow transplantation (BMT). The rate of haematopoietic recovery is significantly influenced by the dose of CFU-GM infused, by use of haematopoietic growth factors for mobilization and to a lesser extent after transplantation and by types of conditioning regimens. However, the outcome of patients does not seem to differ following PBPC as compared to bone marrow transplantation.

Blood Cell Count↗
PubMed1995

Expansion of blood CD34 positive cells: committed precursors expansion does not affect immature hematopoietic progenitors.

CD34 positive (CD34+) cells contain all hematopoietic progenitors from stem cells to committed precursors. Therefore the transplantation of purified bone marrow or blood CD34+ cells is sufficient for hematopoietic recovery after a myeloablative radiochemotherapy. Using different techniques, CD34+ progenitors can be induced to undergo terminal differentiation in a stroma-free liquid culture system in the presence of cytokines. In the present study, we have evaluated the functional potential of CD34+ blood progenitors after ex-vivo expansion cultures. CD34+ cells were isolated from 16 samples (PBSC n = 8 and Cord Blood (CB) n = 8) using either ISOLEX 50 (n=6), CEPRATE LC CD34 kit (n = 6) or MICROCELLECTOR T-25 Stem Cell kit (n = 4). CD34+ cells were cultured for seven days in the presence of 500 UI/ML of IL-1, 10 ng/ml of IL-3 and 10 ng/ml of SCF. We obtained an 8-fold expansion of nucleated cells. We observed a 59-fold expansion of GM-CSF responsive committed precursors, a 4.4-fold expansion of IL-1+IL-3+SCF+Epo responsive multilineage progenitors and a 2.2-fold expansion of the 5-FU resistant quiescent progenitors. We did not observe any significant difference in the amplification/expansion parameters between cultures initiated with CD34+ cells from PBSC or CB. Our data show that cytokine mediated ex-vivo expansion of blood CD34+ cells can produce a large number of committed precursors without affecting the compartment of the most immature progenitors. These results suggest that cytokine-mediated amplification technology could be of great interest in the autologous transplantation setting.

CD4 Antigens↗
PubMed1994

Radiobiological features of acute myeloblastic leukemia: comparison of self-renewal versus terminally differentiated populations.

PURPOSE: To evaluate the radiosensitivity of self-renewing progenitor cells in acute myeloblastic leukemia (AML), we have compared the radiosensitivity of the cells grown either in methylcellulose alone for 7 days, or first in suspension culture for 7 days before being plated in methylcellulose. Methylcellulose selects for terminal-dividing cells and suspension cultures have been developed because they allow self-renewal to occur: The exponential growth of the progenitors of AML cultured in suspension is due to self-renewal. METHODS AND MATERIALS: Cells were harvested from previously untreated leukemic human bone marrows. The myeloblastic lineage of the colonies was assessed by morphological, cytochemical, and immunophenotypic analysis, and by the use of growth factors that did not stimulate the growth of T-lymphocytes. The cell-cycle distribution of the blasts was analyzed by flow cytometry and was comparable for all samples. The irradiation was performed with gamma-photons at a dose-rate of 0.05 Gy/min, similar to the clinical conditions used in our institution for total body irradiation (TBI). RESULTS: The culture methods selected aggressive leukemias. There were large variations of the individual radiosensitivity whatever culture method was used. The progenitor cells capable of self-renewal were more radiosensitive than terminal dividing cells. In two cases, a shoulder was found in the initial part of the cell-survival curves of cells capable of self-renewal. In these two cases, the best fit for the data was the linear quadratic model (survival = e-alpha D-beta D2) with alpha/beta values of 1.49 Gy and 3.12 Gy, respectively. CONCLUSION: The very low values of alpha/beta suggest a reduced antileukemic effect in case of fractionated TBI, and may lead to more reliable screening methods to determine the most appropriate technique for radiation ablation of bone marrow prior to bone marrow transplantation (BMT).

Adolescent↗
PubMed1994

A controlled trial of recombinant human erythropoietin after bone marrow transplantation.

Recombinant human erythropoietin (rHuEPO) stimulates erythropoietic bone marrow cells and increases erythrocyte production. This prospective study was designed to evaluate the effects of rHuEPO on regeneration of erythropoiesis after allogeneic or autologous bone marrow transplantation (BMT). Seventeen centers participated in this randomized, double-blind, placebo-controlled multicenter trial. The randomization was performed centrally for each center and stratified according to allogeneic or autologous BMT and major ABO-blood group incompatibility. One hundred and six patients received rHuEPO after allogeneic BMT and 109 patients received placebo. After autologous BMT, 57 patients were treated with rHuEPO and 57 with placebo. Patients received either 150 IU/kg/day C127 mouse-cell-derived rHuEPO or placebo as continuous intravenous infusion. Therapy started after bone marrow infusion and lasted until independence from erythrocyte transfusions for 7 consecutive days with stable hemoglobin levels > or = 9 g/100 mL or until day 41. After allogeneic BMT, the reticulocyte counts were significantly higher with rHuEPO from day 21 to day 42 after BMT. The median time (95% confidence intervals) to erythrocyte transfusion independence was 19 days (range, 16.3 to 21.6) with rHuEPO and 27 days (range, 22.3 to > 42) with placebo (P < .003). The mean (+/- SD) numbers of erythrocyte transfusions until day 20 after BMT were 6.6 +/- 4.8 with rHuEPO and 6.0 +/- 3.8 with placebo. However, from day 21 to day 41, the rHuEPO-treated patients received 1.4 +/- 2.5 (median, 0) transfusions and the control group received 2.7 +/- 4.0 (median, 2) transfusions (P = .004). In the follow-up period from day 42 up to day 100, 2.4 +/- 5.6 transfusions were required with rHuEPO and 4.5 +/- 9.6 were required with placebo (P = .075). A multivariate analysis (ANOVA) showed that acute graft-versus-host disease (GVHD), major ABO-blood group incompatibility, age greater than 35 years, and hemorrhage significantly increased the number of transfusions. However, after day 20, rHuEPO significantly reduced the number of erythrocyte transfusions in these patient groups, as well as reducing incompatibility in the major ABO-blood group. For the whole study period, rHuEPO reduced the transfusion requirements in GVHD III and IV from 18.4 +/- 8.6 to 8.5 +/- 6.8 U (P = .05). After autologous BMT, there was no difference in the time to independence from erythrocyte transfusions and in the regeneration of reticulocytes. Marrow purging strongly increased the requirement for transfusions as well as the time to transfusion independence.

ABO Blood-Group System↗
PubMed1994

A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells.

A flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensation with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it was possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apoptotic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured polymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cell sorting of the presumed apoptotic population, followed by morphologic examination or DNA analysis of the sorted populations. The usefulness of such a method is discussed in terms of applications in the analysis of heterogeneous clinical samples, populations with low DNA degradation during apoptosis, and cell cycle position of the apoptotic cells.

Antineoplastic Agents↗
PubMed1994

Detection of cytarabine resistance in patients with acute myelogenous leukemia using flow cytometry.

Cytarabine (Ara-C) is currently used in the treatment of adult acute myeloid leukemia (AML). To predict the results of induction chemotherapy, it could be useful to detect leukemic cells that are resistant to Ara-C in patients with AML. Using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM), we have developed a cell resistance index to Ara-C (RI). The technique has been applied to 121 bone marrow (BM) samples from patients with de novo AML treated by a regimen containing Ara-C and daunorubicin (DNR). Ninety-seven patients achieved a complete remission (CR), and 24 patients did not and were considered drug-resistant (DR). The BM cells collected at diagnosis were cultured for 48 hours and underwent BrdUrd/DNA analysis. Among 25 patients with no or very low proliferative activity (<3% of cells in S-phase), the proportion of DR patients (nine of 25) was significantly higher than in a second group of 96 patients with detectable proliferative activity (15 of 96) (P < .025). Within this second group, there was a first group of nine patients with high RI values, which included only DR patients; a second group of 63 patients with low RI values, which included 62 CR patients; and a third group of 24 patients with intermediate RI values, which included 19 CR and five DR patients. In view of this series, our results show that it is possible to detect a majority of DR patients treated by Ara-C.

Acute Disease↗
PubMed1994

Autologous transplants for chronic myelogenous leukaemia: results from eight transplant groups.

Chronic myelogenous leukaemia (CML) can be cured by donor marrow transplant. Unfortunately, suitably HLA-matched related or unrelated donors are not available for the majority of patients. Transplant of stem cells derived from a patient's own marrow or peripheral blood (autologous transplant) avoids the need for an HLA-matched donor, is associated with a less complicated and shorter hospital course than donor transplantation, and has been successful in the treatment of other haematological malignancies. We report results of autologous transplants in 200 patients with CML at eight marrow transplant centres over seven years. This is the first multicentre analysis of autologous transplants for CML and reports on the largest number of patients studied to date. We show that autologous transplants provide a plateau in the survival curve not observed in conventional treatments. Autologous transplants are associated with a high engraftment rate, low mortality, and prompt return of both younger and older patients to normal activity levels. Our results suggest that autologous transplants provide an alternative to conventional treatment in the care of patients not eligible for donor transplant.

Adolescent↗
PubMed1994

The labelling of proliferating cells by Ki67 and MIB-1 antibodies depends on the binding of a nuclear protein to the DNA.

The antigen Ki-67 Ag, regarded as a marker for proliferating cells, was identified as a protein(s) (pKi-67) which can exist free or associated with DNA as evidenced by DNA digestion of cells before or after immunolabeling with Ki-67. The dual nature of this antigen was also supported by reconstitution of Ki-67 Ag from purified DNA and nuclear proteins extracted from the K562 cell line. The immunoreactivity of the resulting complexes was examined in solution using Ki-67 and MIB-1 antibodies. The interaction between Ki-67 or MIB-1 antibodies and pKi-67 was enhanced in the presence of undegraded ds DNA, indicating that ds DNA modulates the conformation of pKi-67 and that the altered conformation of pKi-67 is more reactive than the pure protein to both Ki-67 and MIB-1 antibodies.

Antibodies↗
PubMed1994

Peripheral blood progenitor cell transplantation for hematological malignancies.

It has been demonstrated that it is possible to mobilize hematopoietic progenitor cells from the bone marrow into the peripheral blood, from which they can be collected by apheresis and used for transplantation. Although the clinical results obtained using this approach have not been compared in a randomized trial to those obtained using bone marrow, it appears that this method has certain advantages. These, together with future developments, are discussed in this review.

Genetic Therapy↗
PubMed1994

Treatment of hematological malignancies relapsing after allogeneic bone marrow transplantation.

The rate of relapse after allogeneic bone marrow transplantation (BMT) varies between 15 and 60%. New therapeutic strategies are required urgently as no significant results have been obtained with standard chemotherapy. The best results of second allogeneic BMT have been obtained when the interval between the first and the second transplant was more than 6 to 20 months, depending on the study. Veno-occlusive disease was an important cause of non-leukemic death (13-65%). As the toxicity of second BMT is very high, other treatments have been considered: complete remissions were reported after sudden discontinuation of the immunosuppressive therapy. Interferon-alpha has been used for chronic myeloid leukemia patients and may achieve hematological and cytogenetic complete remission. More recently, donor leucocytes transfusions have been proposed and at least in some cases, have led to molecular complete remission (polymerase chain reaction with double amplification) in chronic myeloid leukemia patients. However, non predictable marrow aplasias and graft-versus-host reactions hamper the efficacy of this strategy. Finally, hemopoietic growth factors used to promote donor cell growth produce interesting results which deserve further studies.

Antineoplastic Combined Chemotherapy Protocols↗
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

Peripheral blood progenitor cell transplantation in 118 patients with hematological malignancies: analysis of factors affecting the rate of engraftment.

We retrospectively studied the factors affecting the rate of hematopoietic reconstitution (HR) in 118 patients with hematological malignancies who underwent peripheral blood progenitor cell (PBPC) transplantation at a single institution. The patients received a median number of 6.6 x 10(8) nucleated cells/kg corresponding to 9.5 x 10(4) (0.5-578) CFU-GM/kg and 6.8 x 10(6) (0.2-161) CD34-positive cells/kg. The median number of days to reach 500 polymorphonuclear cells/mm3 and 50,000 platelets/mm3 was 12.5 (6-93) and 14.5 (6-440) days, respectively. No patient died from infection during the aplastic phase. By multivariate analysis, we found that the dose of CFU-GM infused was the only factor that significantly affects the HR rate (p < 0.0001). Moreover, patients with acute myelogenous leukemia or those transplanted after busulfan or total-body irradiation conditioning regimens had a slower engraftment (p < 0.08). These results could lead to identifying patients who need growth factors posttransplantation and/or the reinfusion of "back-up" marrow together with PBPC.

Actuarial Analysis↗
PubMed1994

Allogeneic bone marrow transplantation in adult acute lymphoblastic leukemia in first complete remission: a comparative study. French Group of Therapy of Adult Acute Lymphoblastic Leukemia.

PURPOSE: Optimal postremission therapy remains controversial in adult patients with acute lymphoblastic leukemia (ALL). In a large multicentric trial (LALA87), we compared allogeneic bone marrow transplantation (BMT) with other postremission therapies (chemotherapy or autologous transplantation) using the result of the human leukocyte antigen (HLA) typing as a random allocation. PATIENTS AND METHODS: Patient eligibility requirements were as follows: (1) inclusion in LALA87 trial, (2) complete response to induction or salvage therapy, (3) age 15 to 40 years, and (4) at least one potential sibling donor. Patients with an HLA-identical sibling were assigned to the BMT group, while patients without a sibling donor constituted the control group. Allogeneic transplantation was scheduled for patients in the BMT group; in the control group, patients were randomly allocated to receive chemotherapy or autologous transplantation. RESULTS: Of 284 eligible points, 257 entered the study: 116 were allocated to the BMT group and 141 to the control group. The 5-year survival rates were not statistically significantly different between the two groups. When only patients with high-risk ALL were considered (those with [1] Philadelphia chromosome [Ph1] ALL, [2] null or undifferentiated ALL, or [3] c-ALL with either age greater than 35 years or WBC count > 30 x 10(9)/L or time to achieve complete remission > 4 weeks), overall survival (P = .03) and disease-free-survival (P = .01) were better for the BMT group compared with the control group (5-year overall survival rates, 44% v 20%; 5-year disease-free survival rates, 39% v 14%). CONCLUSION: Allogeneic transplantation does not improve survival in patients with standard-risk ALL and should be recommended only for patients with adverse prognostic factors.

Adolescent↗