Functional characterization of human CD34+ cells that express low or high levels of the membrane antigen CD111 (nectin 1).

Nectins are recently described adhesion molecules that are widely expressed on many tissues, including the hematopoietic tissue. Nectin 1 (CD111) is expressed on a higher proportion of mobilized peripheral blood (mPB) than cord blood (CB) CD34+ cells, and of CD34+/CD38+ cells when compared with CD34+/CD38- cells. We studied functional properties of human CB and mPB CD34+ cells that express low or high levels of CD111. CD34+/CD111(dim) cells contain a higher proportion of cells in G0/G1 phase than CD34+/CD111(bright) cells. CD34+/CD111(bright) cells contain more erythroid progenitors: CFU-E, than their counterparts, which on the opposite contain more HPP-CFC. Limiting dilution analyses demonstrate a higher frequency of immature progenitors: cobblestone-area colony-forming cells, CD34+/CD111(dim) than in CD34+/CD111(bright) cells. In vitro differentiation assays demonstrate a higher frequency of B-, T- and dendritic-cell precursors, but less NK-cell precursors in CD34+/CD111(dim) cells. Evaluation of engraftment in NOD-SCID mice shows that SCID repopulating cells are more frequent among mPB CD34+/CD111(dim) cells. Liquid culture of CD34+/CD111(dim) cells with erythropoietin shows that CD111 expression increases simultaneously with CD36, following CD71 and before glycophorin A expression. In conclusion, immature human hematopoietic progenitors express low levels of CD111 on their surface. During erythroid differentiation CD34+ cells acquire higher levels of the CD111 antigen.

A novel mechanism for thalassaemia intermedia.

Thalassaemia intermedia is a moderate form of thalassaemia resulting from various genetic defects. We report an undescribed mechanism leading to this condition: a somatic deletion of the beta-globin gene in the haemopoietic lineage of a heterozygous beta-thalassaemic patient. We did molecular studies and haemoglobin analysis of the patient and his parents. We found that the deletion gives rise to a mosaic of cells with either one or no functional beta-globin gene and it extends to a region of frequent loss of heterozygosity called LOH11A, which is located close to the beta-globin locus. Thus, loss of heterozygosity can be a cause of non-malignant genetic disease.

Forced expression of the Ikaros 6 isoform in human placental blood CD34(+) cells impairs their ability to differentiate toward the B-lymphoid lineage.

Studies in mice suggest that the Ikaros (Ik) gene encodes several isoforms and is a critical regulator of hematolymphoid differentiation. Little is known on the role of Ikaros in human stem cell differentiation. Herein, the biological consequences of the forced expression of Ikaros 6 (Ik6) in human placental blood CD34(+) progenitors are evaluated. Ik6 is one of the isoforms produced from the Ikaros premessenger RNA by alternative splicing and is thought to behave as a dominant negative isoform of the gene product because it lacks the DNA binding domain present in transcriptionally active isoforms. The results demonstrate that human cord blood CD34(+) cells that express high levels of Ik6 as a result of retrovirally mediated gene transfer have a reduced capacity to produce lymphoid B cells in 2 independent assays: (1) in vitro reinitiation of human hematopoiesis during coculture with the MS-5 murine stromal cell line and (2) xenotransplantation in nonobese diabetic-severe combined immunodeficient mice. These results suggest that Ikaros plays an important role in stem cell commitment in humans and that the balance between the different isoforms is a key element of this regulatory system; they support the hypothesis that posttranscriptional events can participate in the control of human hematopoietic differentiation.

Early progenitor cells from human mobilized peripheral blood express low levels of the flt3 receptor, but exhibit various biological responses to flt3-L.

The biological effects of flt3-L, and the expression of its tyrosine kinase receptor (flt3, CD135) were investigated on the immature subsets of human circulating peripheral blood progenitors obtained from cancer patients or normal volunteer donors, after mobilization with rhG-CSF or chemotherapy. flt3 was expressed at low levels, and its expression increased concomitantly with expression of CD38 within the CD34+ cell population. Despite this low-level expression, flt3-L exerted synergistic effects with a combination of c-kit ligand, IL-3, IL-6, GM-CSF and G-CSF, mainly to induce proliferation of CD34+/CD38- cells. In addition, flt3-L increased the detection of HPP-CFC, both immediately after cell selection, and after 7 and 14 d of cultures. We conclude that flt3-L is active on circulating early mobilized haemopoietic progenitors, despite the low- level expression of its receptor.

A neutralizing anti-TGF-beta1 antibody promotes proliferation of CD34+Thy-1+ peripheral blood progenitors and increases the number of transduced progenitors.

The subset of blood cells that expresses both CD34 and Thy1 (CD90) cell surface molecules is enriched in hematopoietic stem cell activity and can be obtained from the peripheral blood of cancer patients after mobilization by chemotherapy and granulocyte colony-stimulating factor (G-CSF). Because transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of hematopoietic progenitor proliferation and differentiation, in this study we analyzed the impact of neutralizing TGF-beta1 activity during culture and retroviral transduction of CD34+Thy1+ cells. When purified CD34+Thy1+ cells were cultured in the presence of a neutralizing antibody against TGF-beta1, the percentage of cycling cells, proliferation, and absolute number of clonogenic progenitors were increased in comparison to the cultures performed without the addition of antibody. Antibody-mediated neutralization of TGF-beta1 during retroviral transduction performed by coculture of CD34+Thy1+ cells with a MFG-S-nlsLacZ retroviral vector-producing cell line did not affect the percentage of transduced progenitors as assessed by direct X-Gal staining of colonies in clonogenic assays. However, due to the better expansion of CD34+Thy1+ cells in the presence of anti-TGF-beta1, the absolute number of transduced progenitors recovered at the end of the culture was increased.

Efficiency of retroviral transduction into hematopoietic cells by cocultivation procedure does not correlate with viral titer.

Relative transduction efficiency with retroviral vector-producing clones was assayed by cocultivating TF-1, a human CD34+ hematopoietic cell line and YR-2, a sheep B-lymphoid cell line, with LacZ containing vector-producing cells, and then by scoring the percentage of X-Gal+ cells. At the same time, viral titer was estimated by titration assay with murine fibroblasts. Results clearly demonstrated a lack of correlation between viral titer and efficiency of transduction into hematopoietic cells, which depends neither on the type of packaging cell line, PG-13 and GP-envAM12 in this study, nor on the type of LacZ containing retroviral vector. These results strongly favor consideration of interactions between producers and target cells of the study for the screening of producing cell lines.

Hematological reconstitution and gene therapy: retroviral transfer of the bacterial beta-galactosidase activity into human hematopoietic CD34+ cell populations and into T lymphocytes derived from the peripheral blood.

We report the possibility to transfer marker genes coding for beta-galactosidase activity using retroviral vectors into human peripheral blood CD34+ cells, peripheral blood T-lymphocytes and into the growth factor-dependent human hematopoietic cell line TF-1. Using the MFG-nisLacZ and the FLac vector and various packaging cell lines, we demonstrated retroviral transfer and high expression of a bacterial beta-galactosidase activity induced by the nisLacZ gene or the Sh-ble/LacZ gene. Kinetics of expression of the transgenes were analyzed both in primary cells and cell lines. Absence of cytotoxicity related to the expression of the bacterial beta-galactosidase was assessed in both cell types. These results open interesting prospectives for the use of the beta-galactosidase activity to mark and follow the fate of genetically modified cells isolated from patients prior to reimplantation.

Highly efficient retroviral gene transfer into human primary T lymphocytes derived from peripheral blood.

T lymphocytes are a promising cell vehicle for gene therapy purposes. By cocultivating retroviral vector producing cells and target cells, highly efficient gene transfer was achieved with activated human T lymphocytes derived from peripheral blood with vectors carrying different forms of the bacterial beta-galactosidase gene including the regular LacZ gene, the Sh-ble::LacZ gene and the nlsLacZ gene. Infection kinetics of T cells activated by a combination of monoclonal antibodies directed against CD2 and CD28 indicated that the highest efficiencies of transduction were obtained when the cocultivation began 4 days after stimulation. In fact, with the FLac vector, a new retroviral vector which expresses the Sh-ble::LacZ gene, we observed up to 78% transduction efficiency assessed by X-gal staining performed 2 days after the end of the cocultivation. Expression of the transduced genes was observed throughout the period of culture. Neither the cocultivation step nor the expression of the transduced Sh-ble::LacZ gene altered cell culture proliferation or the expression of selected cell surface antigens. In addition, we showed that CD4+ and CD8+ T cells were equally transduced.

Retroviral transfer of the nlsLacZ gene into human CD34+ cell populations and into TF-1 cells: future prospects in gene therapy.

Few data are available concerning behavior of reimplanted human hematopoietic cells after autologous stem cell transplantation. This paper reports the possibility to transfer gene markers coding for beta-galactosidase (beta-Gal) activity by retroviral vectors into a human leukemic growth factor-dependent cell line, TF-1, and into human hematopoietic progenitors isolated from peripheral blood or bone marrow. Using various combinations of retroviral vectors and packaging cell lines, we demonstrated high expression of a bacterial beta-Gal activity induced by the LacZ gene, the nlsLacZ gene, or the Sh-ble/LacZ gene, in human hematopoietic cells. The expression of the nlsLacZ construct was stable until the end of the culture in infected CD34+ cell-enriched cell populations, and a slow decrease of transgene expression was observed in a transduced TF-1 cell population during a 1-year long-term culture. Data obtained with the nlsLacZ gene demonstrate that both retroviral transfer and corresponding gene expression were not found to modify the pattern of cell proliferation and differentiation. These results open interesting prospectives for the use of the nlsLacZ gene to mark and follow the fate of progenitor cells isolated from patients with cancers prior to reimplantation.

Methotrexate test-dose protocol in the presence of 7-hydroxy-methotrexate.

Methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) plasma concentration-time curves (AUC) have been analyzed in 24 patients after different routes of MTX administration. After an i.v. bolus (50 mg/m2), the AUC for 7-OH-MTX is correlated with that for MTX and inversely correlated with the MTX plasma clearance. When MTX is administered with plasma steady level standardization, using the test-dose protocol, at a level of 10(-5) M over 36 hr (10(-5), 36 hr), 7-OH-MTX-AUC is still correlated with the i.v. bolus pharmacokinetic parameters. The dose prediction using the classical test-dose protocol provides a less efficient MTX dose adjustment at 5 X 10(-4) M over 8 hr (5.10(-4), 8 hr) and the hydroxylation process is no more correlated with the i.v. parameters. On the opposite, upon 6 successive infusions with 10(-5), 36 hr or 5.10(-4), 8 hr protocols, the plasma concentrations of 7-OH-MTX are not significantly modified. This suggests that the hydroxylation process is not inducible.

Kinetics of methotrexate and its metabolites in red blood cells.

It has been suggested that the intra-erythrocyte levels of methotrexate (MTX) and its polyglutamized derivatives could offer a method for evaluating intracellular MTX accumulation and its metabolism in children with acute lymphoblastic leukemia. We were interested in measuring the intra-erythrocyte levels of MTX in patients with solid tumors receiving high-dose MTX treatment. After a first incorporation stage occurring during infusion, MTX concentrations subsequently increased 9-12 days after the treatment as polyglutamized derivatives. Thirty days after the infusion, MTX and its polyglutamates were still measurable in erythrocytes. The percentage of polyglutamization varied on an individual basis, but two groups of patients could be separated according to their ability to form polyglutamates. We also noted the presence of 7-hydroxy-methotrexate (7-OH-MTX) appearing 48 hours after the beginning of the infusion which was still present in 17/20 samples 30 days after the treatment.

Methotrexate-vindesine association in head and neck cancer: modification of methotrexate's hydroxylation in presence of vindesine.

High-dose methotrexate (HD-MTX) infusions associated with vindesine (VDS) have been used in the treatment of head and neck cancer. In a previous study, VDS was shown to increase the apparent plasma clearance of MTX. We have studied the MTX hydroxylation process in the presence of VDS. Different routes of administration have been tested (IV pushes and 24-h or 36-h infusions). A radioimmunoassay has been developed to measure 7-OH-MTX. The defined protocols enabled us to show that VDS influences not only the pharmacokinetic behavior of methotrexate but also its hydroxylation, which is decreased in presence of VDS.

Methotrexate-vindesine association in leukemia: pharmacokinetic study.

Methotrexate (MTX) and vindesine (VDS) have both been found effective in the treatment of acute leukemia. With regard to their pharmacological effects at the cellular level they can reportedly interact. Administration of MTX at high dose levels has been suggested as both a curative and preventive treatment of blastic meningitis. The purpose of this work was to determine whether an injection of VDS leads to any change in MTX clearance and uptake in the cerebrospinal fluid (CSF). The plasma pharmacokinetic and CSF influx of MTX and VDS were assayed after high dose systemic MTX with an intravenous bolus of VDS administered in the treatment of acute lymphoblastic leukemia. The MTX concentration was determined by enzymatic assay and VDS by radio immunoassay. No interrelation between these drugs was found. MTX levels in the CSF were sufficient for therapeutic effectiveness but were not affected by intravenous VDS. Detectable amounts of VDS were observed in the CSF but were not altered by MTX.

Dosage predictions in high-dose methotrexate infusions. Part 1: Evaluation of the classic test-dose protocol.

A preliminary methotrexate (MTX) kinetic evaluation following administration of an IV bolus (test-dose) allowed individualization of high-dose infusions (HD-MTX). This approach combined with therapeutic drug monitoring was found to have good performance over a large scale of predicted steady-state levels (Css) (10(-5) to 10(-4) M over 24 to 36 h) corresponding to 17 to 650 mg/h deliveries (root mean squared error : rmse (precision) = 1.54 X 10(-5) M (21.4%) and mean error : me (bias) = 0.043 X 10(-5) M (NS)). However a significative (p less than 0.05) but rather low over-estimation of dosage (me = 7.38 X 10(-5) M (14.8%)) associated to a decrease in the prediction precision (rmse = 13.3 X 10(-5) M (26.6%)) occurred in 5 X 10(-4) M predicted Css over 8 h (970 to 1970 mg/h deliveries). However in a number of cases (6 out of 29) important deviations from predicted Css occurred, implying the need to stop the infusion before 8 h. These results indicated that MTX pharmacokinetics was linear from low test-dose bolus injections to high-dose infusions. This allowed dosage predictions based upon preliminary estimation of MTX clearance and associated to therapeutic drug monitoring during and following infusion.

Dosage predictions in high-dose methotrexate infusions. Part 2: Bayesian estimation of methotrexate clearance.

Population pharmacokinetics of methotrexate (MTX) was evaluated from intravenous test-dose (TD) data (n = 20 corresponding to 174 measured samples). Bayesian prediction of MTX clearance from TD experiments combining population data with measured levels (at times 0.5 and 6 h) was found to be feasible in routine situations with good performance (root mean squared error : rmse (precision) = 1.14 1.h-1 (11.2%) and mean error : me (bias) = 0.06 1.h-1 (NS) relatively to weighted least-square estimates, n = 50). The precision of Bayesian prediction was comparable to that of the model independent which is used in routine practice and involves 9 measured levels over 30 h, (rmse = 1.35 1.h-1 (10.9%), n = 50). However, the routine method presented a significative bias (me = -0.81 1.h-1, n = 50).

Methotrexate-vindesine association in the treatment of head and neck cancer influence of vindesine on methotrexate's pharmacokinetic behavior.

Determination of methotrexate (MTX) kinetics after an IV bolus (50 mg/m2) allows prediction of the steady-state plasma level of this drug during a constant infusion. This prediction allows high-dose MTX (HD-MTX) therapy without major toxicity. Patients with head and neck carcinoma received HD-MTX and vindesine (VDS) infusions concomitantly. The therapeutic survey of these patients showed that the predicted plasma level of MTX was not achieved in the presence of VDS. Moreover, the computed dose of MTX had to be increased by a larger amount if the MTX plasma clearance after the identification IV push was low (less than 9 l/h). In the presence of VDS, the creatinine clearance is lower than when MTX is infused alone, and MTX renal elimination is identical (MTX or MTX + VDS infusions). Thus it seems that the decrease of the MTX plasma level during MTX-VDS infusion could be due to an increase of cellular incorporation.

High-dose methotrexate: a clinical and pharmacokinetic evaluation. Treatment of advanced squamous cell carcinoma of the head and neck using a prospective mathematical model and pharmacokinetic surveillance.

Some of 66 patients with head and neck tumors were treated with high-dose methotrexate monochemotherapy. The use of a prospective mathematical model with pharmacokinetic surveillance proved to be reliable, practical, and useful. By this means chemotherapy could be individualized, with a resultant marked reduction in the frequency and severity of toxicity. The onset of clinical toxic manifestations was significantly correlated with a poor therapeutic response and poor prognosis. The patients were classified in to three groups according to poor, intermediate, and good pharmacokinetic parameters calculated after an intravenous identification dose of methotrexate. These group allocations had a very high prognostic value with regard to toxicity, and especially to the quality of therapeutic response to high-dose methotrexate. They are suggested as useful guidelines in the prescription of high-dose methotrexate chemotherapy.