Flt-3 and its ligand are expressed in neural crest-derived tumors and promote survival and proliferation of their cell lines.

Flt-3 ligand (FL) is a cytokine that promotes the survival, proliferation, and differentiation of hematopoietic progenitors in synergy with other growth factors, such as stem cell factor. Previously we have demonstrated that stem cell factor and its receptor c-kit are expressed in neural crest-derived tumor cells and that a c-kit block induces their apoptosis. Here we have evaluated the expression of flt-3 and its ligand in 12 neuroectodermal tumor cell lines from neuroblastoma (NB), neuroepithelioma (NE), Ewing sarcoma (ES), and peripheral neuroectodermal tumor (PNET) and in 38 biopsies: 19 from NB and 19 from ES and PNET. RT-PCR demonstrated the expression of flt-3 and FL in all lines. Coexpression was observed in 42% of NB and in 74% of ES and PNET biopsies. Flow cytometry confirmed the presence of membrane and cytoplasmic flt-3 and membrane FL in all lines, whereas soluble FL protein was not measurable in their supernatants. Microphysiometric demonstration of acidification of the medium provided evidence of the specific response of cell lines to FL stimulation. Specific flt-3 phosphorylation after FL treatment was also demonstrated by Western blotting analysis. In cells growing in RPMI plus 1% fetal calf serum, FL revealed a significant proliferating activity, more evident in NB and NE lines (mean increase of viable cells, 73 +/- 26% after 1 day). Treatment with flt-3 antisense oligonucleotides significantly inhibited cell growth. FL also displayed an antiapoptotic activity: after a 12-hour culture in the presence of 0.1% fetal calf serum, FL caused a 50% reduction of apoptotic cells. These results provide further evidence that neuroectodermal and hematopoietic cells share common regulatory pathways, and could be of interest in the clinical management of neuroectodermal tumors.

Retinoic acid modulates stem cell factor secretion by human neuroblastoma cell lines.

The hemopoietin stem cell factor (SCF) and its receptor c-kit are expressed in some tumoral cells, including neuroblastoma (NB) cells. We have investigated the effect of retinoic acid (RA), one of the most active differentiating agents on human NB cells, on the SCF production by human neuroblastoma cell lines. SCF concentration was determined by immunoenzymatic assay in the supernatants of seven neuroblastoma cell lines. All cell lines except one showed detectable amounts of SCF in the supernatant in basal culture conditions. A progressive increase pattern of the SCF concentration over time, was common to all SCF secreting cell lines, both unstimulated and RA-stimulated. Moreover, after 48 and 72 hours-exposure to RA, SCF concentrations were higher than in the untreated controls (p < 0.01). Membrane SCF mRNA isoform was also detected by reverse-transcription polymerase chain reaction. These effects demonstrated that RA, besides inducing neuronal differentiation, enhanced SCF production in neuroblastoma cell lines.

Diamond-Blackfan anaemia in the Italian population.

Diamond-Blackfan anaemia (DBA) is a congenital disease characterized by defective erythroid progenitor maturation: 30% of patients have congenital malformations. The link between these malformations and defective erythropoiesis is unclear: a defect in a molecule acting both on embryo development and haemopoiesis has been proposed. Inheritance is autosomal dominant in most familial cases, but recessive families have also been reported. Many cases are sporadic. A DBA locus has been mapped on chromosome 19q13.2 (Gustavsson et al, 1997), but several families unlinked to this locus have also been reported (Gustavsson et al, 1998). This paper presents clinical, epidemiological and molecular data for DBA in the Italian population. Segregation analysis of 19q markers in patients with DBA showed exclusion of this locus in 5/12 families with inherited DBA. There was evidently locus heterogeneity for DBA in this population. A new microdeletion was identified in one patient. Other families, in which DBA segregates concordantly with the 19q critical region, suggest incomplete penetrance and expressivity of the DBA gene.

Cell adhesion molecules in cord blood hematopoietic progenitors.

We investigated the expression of different cell adhesion molecules on cord blood (CB) and bone marrow (BM) CD34+/CD38+ and CD34+/CD38- cells. CD11a and CD62L were more expressed in CB than in BM CD34+/CD38- subset, suggesting a possible advantage in homing and engraftment. A short exposure to various cytokines increased CD62L expression only in the more differentiated CB and BM CD34+/CD38+ cells.

Use of recombinant granulocyte colony-stimulating factor in Fanconi's anemia.

BACKGROUND AND OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) has been shown to improve the neutropenic status of patients with bone marrow failure. The side effects in prolonged treatment still need to be determined. DESIGN AND METHODS: We have studied the efficacy and the long-term side effects of G-CSF in four patients with Fanconi's anemia and severe neutropenia. RESULTS: Three patients responded with an increase in their absolute neutrophil count; neither improvement in platelet count and hemoglobin concentration nor effect on transfusion requirements was seen. CFU-GM and BFU-E were undetectable before, during and after treatment. Responders showed an important reduction in number and severity of infections, with a marked improvement of clinical status. The fourth patient developed acute myeloid leukemia after 4 weeks of G-CSF treatment. During maintenance, one patient was treated with G-CSF for 18 months, until she received bone marrow transplantation, without presenting side effects. In the second responding patient G-CSF treatment was stopped because of appearance of immature cells in peripheral blood and myeloid blasts in bone marrow. The third responding patient presented immature peripheral myeloid cells during the third year of G-CSF treatment: disappearance of immature cells was observed after G-CSF reduction. In two cases FISH analysis revealed monosomy 7 after G-CSF treatment. INTERPRETATION AND CONCLUSIONS: G-CSF use results in an improvement of clinical status, but long term administration may cause adverse experiences and requires a close hematological monitoring.

Cell adhesion molecule expression in cord blood CD34+ cells.

Self-renewal, proliferation, differentiation, homing, and mobilization of hematopoietic progenitor cells (HPCs) are regulated by a complex mechanism that involves the bone marrow (BM) microenvironment. Cell adhesion molecules (CAMs) expressed on HPCs and on endothelial and stromal cells play a pivotal role in this process. In this study, we have used three-color cytofluorometric analysis to compare CAM expression in the subsets of cord blood (CB) and BM HPCs and examined the effect of a short exposure to various cytokines on L-selectin expression. The study was carried out on unseparated samples to avoid any possible bias from positive CD34 selection. CAMs were highly expressed in both CB and BM CD34+CD38+ cells. In this population, L-selectin, H-CAM, and LFA-1 were significantly more expressed in BM than in CB. With regard to the more immature progenitors, the subsets of CD34+/CD38-/L-selectin+ and CD34+/CD38-/LFA1+ cells were significantly larger in CB than in BM. Since the expression of such CAMs has been related to the repopulating capacity of HPCs, our results suggest a possible advantage in homing and engraftment of more undifferentiated CB as opposed to BM HPCs. A 4/24-h exposure to various cytokines significantly increased the percentage of CB CD34+/CD38+/L-selectin+ cells, while HPCs were differentiated since the percentage of CD34+/CD38-/L-selectin+ cells was reduced. These data show that a short exposure to cytokines increases L-selectin expression in the more differentiated CB HPCs. This could improve their homing in a transplant setting.

Cytofluorimetric analysis of CD34 cells.

The cytofluorimetric enumeration of CD34-positive cells is a useful method for measuring haematopoietic stem cells. The large variation of results between different laboratories, in multicenter quality control studies, could be due to multiple technical problems. CD34 analysis must be performed in whole blood and the NH4Cl or the NH4Cl-derived reagents give the best results in the lysis procedure. The monoclonal antibody for CD34 detection must be a class III, if possible PE-conjugated. Any differences were found in the absolute CD34 count using different analysis protocols: Milan, ISHAGE, Dutch, or a commercial kit, when the sample is fresh and in good viable condition. The multiparametric approach, using three or four colours simultaneously, could be required in scoring apheresis products from patients with CD34-positive blast cells (ie ALL of B cell origin) and in deciding the optimum time to start the leukapheresis, choosing, with an equal CD34 number, the moment at which the highest number of more immature progenitor cells (CD38-negative or dim, CD90-positive) is present.

Stem cell factor suppresses apoptosis in neuroblastoma cell lines.

Stem cell factor (SCF) is a glycoprotein growth factor produced by marrow stromal cells that acts after binding to its specific surface receptor, which is the protein encoded by the protooncogene c-kit. SCF synergizes with specific lineage factors in promoting the proliferation of primitive hematopoietic progenitors, and has been administered to expand the pool of these progenitors in cancer patients treated with high-dose chemotherapy. SCF and its c-kit receptor are expressed by some tumor cells, including myeloid leukemia, breast carcinoma, small cell lung carcinoma, melanoma, gynecological tumors, and testicular germ cell tumors. Previous studies of SCF in neuroblastoma have produced conflicting conclusions. To explore the role of SCF in neuroblastoma, we studied five neuroblastoma lines (IMR-5, SK-N-SH, SK-N-BE, AF8, and SJ-N-KP) and the neuroepithelioma line CHP-100. All lines expressed mRNA for c-kit and c-kit protein at low intensity as measured by flow cytometry, and secreted SCF in medium culture as shown by ELISA. Exogenous SCF did not modify 3H thymidine uptake in the neuroblastoma and neuroepithelioma cell lines. After 6 days' culture in the presence of anti-c-kit, the number of viable neuroblastoma cells was significantly lower than the control, and terminal deoxynucleotidyl transferase assay showed a substantial increase of apoptotic cells: The percentage of positive cells was 1-3% in the control lines, whereas in the presence of anti c-kit it varied from 29% of SK-N-BE to 92% of CHP-100. After 9 days' culture in the presence of anti-c-kit, no viable cells were detectable. These data indicate that SCF is produced by some neuroblastoma cell lines via an autocrine loop to protect them from apoptosis.

Diamond-Blackfan anemia: expansion of erythroid progenitors in vitro by IL-9, but exclusion of a significant pathogenetic role for the IL-9 gene and the hematopoietic gene cluster on chromosome 5q.

Diamond-Blackfan anemia (DBA) is a congenital pure red blood cell aplasia that often requires lifelong transfusional therapy. Autosomal dominant and recessive inheritance have both been reported, suggesting genetic heterogeneity, but most cases occur sporadically. The origin of impaired erythropoiesis is unknown. Several erythroid growth factors have been thought to have a role in the pathogenesis of DBA. However, there is neither molecular nor clinical evidence for the involvement of erythropoietin (EPO), its receptor, stem cell factor (SCF), or interleukin (IL)-3, even if the addition of SCF to IL-3 and EPO does significantly increase the growth of erythroid progenitors in in vitro cultures in most patients. In this work we evaluated the possible role of another early-acting erythroid growth factor, IL-9. We found that the addition of IL-9 to SCF, IL-3, and EPO further increases burst-forming unit-erythroid growth in in vitro cultures of those DBA patients who responded to SCF. To investigate the role of the IL-9 gene, we evaluated its segregation in 22 families with members who have DBA by using a polymorphic microsatellite located within its intron 4. Lod score analysis ruled out any statistically significant involvement of the IL-9 gene in the pathogenesis of DBA. Moreover, linkage analysis with 11 highly polymorphic markers spanning 5q31.1-q33.2 excluded this region, which is included in the major cluster of genes active in hematopoiesis of the human genome.

Effect of different exposures to desferrioxamine on neuroblastoma cell lines.

Desferrioxamine (DFO) has shown anti-proliferative and cytotoxic effects on several tumor cells. DFO is used at present in the treatment of neuroblastoma in combination with chemotherapy (D-CECaT regimen: cyclophosphamide, etoposide, carboplatin, and thiotepa). We compared the effect of continuous or intermittent exposures to DFO on 3H-thymidine uptake, viability, and cell cycle of human neuroblastoma (NB) cell lines. Our results show that continuous exposures to DFO cause dose- and time-dependent cytotoxicity of NB cells, while intermittent exposures result in significant NB cell toxicity only when using high DFO concentrations. By 3H-thymidine uptake, a significant inhibition of proliferation was observed only in continuous exposures. In addition, a consistent arrest in G1 phase was detected only in cultures treated continuously with high DFO concentrations. Our data indicate that 3H-thymidine uptake, viability, and cell cycle changes are proportional to the extent of exposure and concentration of DFO, suggesting that in vivo DFO continuous infusion may improve anti-neuroblastoma activity.

HLA-haploidentical umbilical cord blood stem cell transplantation in a child with advanced leukemia: clinical outcome and analysis of hematopoietic recovery.

Growing attention has been focused on cord blood as a source of transplantable hematopoietic stem cells. However, clinical experience is rather limited. In this study we describe a child with advanced acute lymphoblastic leukemia who received an HLA-haploidentical cord blood transplant. The patient was transplanted in third complete remission after conditioning with fractionated total body irradiation, thiotepa and cyclophosphamide. Forty-one milliliters of cryopreserved umbilical cord blood, containing 0.15 x 10(8) nucleated cells/kg and 0.25 x 10(4) CFU-GM/kg, were infused. Cyclosporine and prednisone were administered for graft-versus-host disease (GVHD) prophylaxis. The patient received G-CSF from day +1 to day +35, but no improvement in granulocyte counts was observed. Therefore, administration of GM-CSF was started on day +36 to day +59, which resulted in a significant increase in white blood cells and granulocyte counts. Sustained myeloid engraftment was evidenced by a granulocyte count > 0.5 x 10(9)/l by day +41. The presence of donor-derived cells could be documented in the peripheral blood and bone marrow of the patient by cytogenetic analysis, HLA phenotyping and DNA studies. Forty-one days after transplant, clonogenic bone marrow assays showed the presence of low frequencies of primitive hematopoietic progenitor cells (BFU-E = 19/10(5) and CFU-GM = 8/10(5)). The chimerism was complete and no host-derived cells could be detected. However, the engraftment was restricted to the myeloid lineage whereas lymphoid and megakaryocytic engraftments were inadequate. The immunophenotype of the patient's peripheral blood showed the presence of T lymphocytes expressing an immature phenotype (CD2+ CD3-) at day +21.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of desferrioxamine and hydroxypyridones on hemopoietic progenitors and neuroectodermal tumor cells.

The iron chelator desferrioxamine (DFO) has been shown to inhibit the proliferation of hemopoietic progenitors and several tumor cell lines. We have compared the in viro hemopoietic inhibitory effect of desferrioxamine (DFO) and hydroxypyridones (HPOs) on hemopoietic progenitors and two human neuroectodermal (NE) tumor cell lines, NB 100 and SKNMC. Both DFO and HPOs showed a direct dose-related inhibitory effect on BFU-E and CFU-GM obtained from purified human non-T MNAC (T-lymphocyte-depleted nonadherent mononuclear cells) and CD34+ cells. DFO and HPOs displayed both an inhibitory and a cytotoxic effect on NE cell lines. We calculated the ratio between NE cell and hemopoietic cell growth inhibition for a range of concentrations of chelators. DFO showed the most satisfactory ratio. This suggests that DFO is still the most preferable chelating agent for the treatment of neuroblastoma, since it combines the highest antineuroblastoma effect with the lowest hematopoietic toxicity.

Umbilical cord blood stem cell transplantation.

Human umbilical cord blood as an alternative source of hematopoietic stem cells for bone marrow reconstitution, has recently been demonstrated to yield successful HLA-matched placental blood grafts in children. It has been shown that cord blood contains sufficient progenitor cells to effect hematological reconstitution. Since then, more than 25 cord blood stem cells (CBSCs) transplants have been performed worldwide for the treatment of a variety of malignant and nonmalignant diseases. The majority of the grafts performed thus far have utilized CBSCs from HLA-identical siblings. However, much of the interest in this setting is devoted to the potential use of CBSCs for HLA-mismatched and unrelated transplants. Preliminary results suggest that allorecognition and graft-versus-host disease may be less intense in CBSCs transplants than in recipients of similarly compatible bone marrow. This review summarizes the results and potential future applications of cord blood transplantation.

Expansion of cord blood progenitors and use for hemopoietic reconstitution.

A high number of stem cells migrate in fetal blood and, at birth, the number of progenitors in cord blood equals or exceeds that of adult bone marrow. Recently hemopoiesis has been successfully reconstituted with the infusion of cord blood cells. It is important to clearly define the quantity and quality of cord blood totipotent and multilineage progenitors to evaluate the possibility of their utilization in transplants. Our first aim was to study the growth characteristics of cord blood progenitors. We have evaluated the number of cycling cells with the thymidine suicide technique and the production, by phytohemagglutinin (PHA) stimulated cord blood mononuclear cells, of some cytokines involved in the proliferation of progenitor cells, such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6) and leukemia inhibitory factor (LIF). We have also studied by flow cytometry the CD34+CD33-, CD34+CD33+ cell subsets and the presence of the c-kit receptor in order to quantitate the number of earlier progenitors. Our second aim was to elucidate whether the cord blood totipotent stem cell population or the committed progenitors could be expanded in vitro. Our results showed that in cord blood the number of early progenitors, as evaluated by the number of mixed lineage colony forming units (CFU-Mix), by the CD34+CD33- subsets and the expression of the c-kit, is higher than in bone marrow. We have also demonstrated the possibility in vitro of increasing the number of progenitors by more than 30-fold by utilizing stem cell factor (SCF) in association with other cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of interleukin 6, leukemia inhibitory factor and granulocyte-macrophage colony stimulating factor by peripheral blood mononuclear cells in Fanconi's anemia.

The aim of this study was to test the in vitro cytokine production by peripheral blood mononuclear cells (PBMCs) in patients with Fanconi's anemia (FA). Phytohemagglutinin (PHA)-stimulated PBMCs from 21 patients with FA were studied for their ability to produce interleukin 6 (IL-6), leukemia inhibitory factor (LIF) and granulocyte-macrophage colony stimulating factor (GM-CSF). Enzymatic immunoassay (EIA) was used for both IL-6 and LIF, while GM-CSF was evaluated in a highly sensitive biological assay provided by GM-CSF-dependent M-07e cells. A significant decrease of IL-6 was detected in 9 out of 11 FA patients compared with five normal donors, while similar amounts of LIF were produced from 21 FA patients and 21 healthy subjects. A drastic increase of active GM-CSF was documented in PHA-stimulated PBMC-conditioned medium in all 18 FA patients tested. Since IL-6 and GM-CSF play an important role in maintaining basal hemopoiesis, our results suggest that an abnormal cytokine network may be involved in the pathogenesis of FA pancytopenia.

Human cord blood progenitors: kinetics, regulation and their use for hemopoietic reconstitution.

The high number of cord blood (CB) hematopoietic progenitors made possible CB transplantation in children with hematologic diseases. We collected and cryopreserved CB from siblings of 11 hematologic pediatric patients for possible transplant. The number of BFU-E+CFU-GEMM and CFU-GM was adequate for engraftment. However the possibility of CB transplantation in adolescents and adults is limited by the amount of CB that can be collected. We studied the possibility of expanding the pool of CB hematopoietic progenitors by preincubation of CB CD 34+ cells with several cytokines. After pre-exposition to rhIL-3, IL-1 beta, rIL-6, GM-CSF and stem cell factor (SCF) and cloning with SCF the number of hematopoietic progenitors increased 14-18 fold compared to basal values.

Effect of stem cell factor on colony growth from acquired and constitutional (Fanconi) aplastic anemia.

The aim of this study was to evaluate the effect of stem cell factor (SCF) on the in vitro growth of bone marrow hematopoietic progenitors from patients with acquired severe aplastic anemia (AA) or Fanconi's anemia (FA). For this purpose, we studied 11 patients with acquired AA (5 at diagnosis, 6 after ALG treatment), 12 patients with FA, and nine normal controls. Bone marrow cells were plated in vitro for colony-forming unit granulocyte-macrophage (CFU-GM) (in the presence of granulocyte-macrophage colony-stimulating factor [GM-CSF]), and for burst-forming unit-erythroid (BFU-E) and CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) colonies (in the presence of erythropoietin and interleukin-3 [IL-3]), with or without 20 ng/mL of SCF. In normal controls, SCF enhanced the growth of CFU-GM colonies from 103 to 263 (median), of BFU-E from 168 to 352, and of GEMM colonies from 6 to 38/10(5) cells plated. In patients with acquired AA, SCF induced a significant enhancement of BFU-E growth (8 to 29; P = .01) and allowed the formation of GEMM colonies that were not scored in baseline culture conditions (0 to 8; P = .01). CFU-GM growth was enhanced (4 to 20), but not significantly (P = .3). This was true both for patients at diagnosis and after antilymphocyte globulin treatment. By contrast, 10 of 12 FA patients grew no CFU-GM, BFU-E, or CFU-GEMM colonies, with or without SCF. In two FA patients (one transfusion-dependent and one transfusion-independent), an enhancement of CFU-GM and/or BFU-E was observed. The lack of response of hematopoietic progenitor cells from FA patients to GM-CSF+SCF or IL-3+SCF was not dependent on a defective expression of cytokine receptor messenger RNAs. Northern blot analysis showed in marrow cells from acquired AA and FA patients the presence of normal transcripts for alpha- and beta-chains of GM-CSF/IL-3 receptor and for c-kit protein. In conclusion, SCF promotes the in vitro growth of hematopoietic progenitors in patients with acquired AA, but not in patients with FA, pointing out the intrinsic nature of the defect in the latter disorder.

In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte-conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation.