Clonogenic potential and phenotypic analysis of CD34+ cells mobilized by different chemotherapy regimens.

BACKGROUND AND OBJECTIVE: Since limited data concerning quantitative and qualitative differences of CD34+ cells collected after different mobilization schedules are available, we investigated phenotype, proliferative capacity and primitive progenitor cell content of CD34+ cells mobilized with four different regimens. DESIGN AND METHODS: The number, phenotype, and progenitor cell content of CD34+ cells were investigated in 46 patients mobilized with cyclophosphamide (CY) 7 g/m(2) plus granulocyte colony-stimulating factor (G-CSF, 5 microg/kg) (CY7+G-CSF) (n=16), CY 4 g/m(2) plus G-CSF (CY4+G-CSF) (n=8), IVE [ifosphamide (2.5 g/m(2) for 3 d), etoposide (150 mg/m(2) for 3 d), epirubicin (100 mg/m(2) on day 1)] plus G-CSF (IVE+G-CSF) (n=9), or G-CSF (10 microg/kg) alone (n=13). RESULTS: The number of CD34+ cells collected per liter of processed blood was significantly higher in the CY7+G-CSF group than in the CY4+G-CSF and G-CSF groups (p

Primitive hematopoietic progenitors within mobilized blood are spared by uncontrolled rate freezing.

Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at -140 degrees C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (+/- s.e.m.) storage time of cryovials and bags was 344+/-40 and 299+57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3x10(9) vs. 0.2x10(9), P< or =0.02; bags: 14x10(9) vs. 11x10(9), P< or =0.0003), the growth of committed progenitors was substantially unaffected by the freezing-thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60+/- 29% to 134+/-15%. Mean recoveries of LTC-IC from cryovials and bags were 262+/-101% and 155+/-27% (P< or =0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre-and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.

VACOP-B versus VACOP-B plus autologous bone marrow transplantation for advanced diffuse non-Hodgkin's lymphoma: results of a prospective randomized trial by the non-Hodgkin's Lymphoma Cooperative Study Group.

PURPOSE: The aim of this multicenter randomized study was to compare conventional therapy with conventional plus high-dose therapy (HDT) and autologous bone marrow transplantation (ABMT) as front-line treatment for poor-prognosis non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Between October 1991 and June 1995, 124 patients, aged 15 to 60 years, with diffuse intermediate- to high-grade NHL (Working Formulation criteria), stages II bulky (> or = 10 cm), III, or IV were enrolled. Sixty-one patients were randomized to receive etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (VACOP-B) for 12 weeks and cisplatin, cytarabine, and dexamethasone (DHAP) as a salvage regimen (arm A), and 63 to receive VACOP-B for 12 weeks plus HDT and ABMT (Arm B). RESULTS: There was no significant difference in terms of complete remissions (CRS) in the two groups: 75% in arm A, and 73% in arm B. The median follow-up observation time was 42 months. The 6-year survival probability was 65% in both arms. There was no difference in disease-free survival (DFS) or progression-free survival (PFS) between the two groups. DFS was 60% and 80% (P = .1) and PFS was 48% and 60% (P = .4) for arms A and B, respectively. Procedure feasibility was the major problem. In arm B, 29% of enrolled patients did not undergo HDT and ABMT. A statistical improvement in terms of DFS (P = .008) and a favorable trend in terms of PFS (P = .08) for intermediate-/high- plus high-risk group patients assigned to HDT and ABMT was observed. CONCLUSION: In this study, conventional chemotherapy followed by HDT and ABMT as front-line therapy seems no more successful than conventional treatment in terms of overall results. However, our results suggest that controlled studies of HDT plus ABMT should be proposed for higher risk patients.

CD34+ cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) are functionally different from CD34+ cells mobilized by G-CSF.

Mobilized peripheral blood progenitor cells (PBPC) are increasingly used as an alternative to bone marrow for autografting procedures. Currently, cyclophosphamide (CY) followed by granulocyte colony-stimulating factor (G-CSF) or G-CSF alone are the most commonly used PBPC mobilization schedules. In an attempt to investigate whether the use of these two mobilization regimens could result in the collection of functionally different CD34+ cells, we analyzed nucleated cells (NC), CD34+ cells, committed progenitor cells and long-term culture initiating-cells (LTC-IC) in 52 leukaphereses from 26 patients with lymphoid malignancies, mobilized either by CY+G-CSF (n=16) or G-CSF alone (n=10). Thirty-four aphereses from the CY+G-CSF group and 18 aphereses from the G-CSF group were investigated. According to the study design, leukaphereses were carried out until an average number of 7 x 10(6) CD34+ cells/kg body weight were collected. The mean (+/-s.e.m.) numbers of CD34+ cells mobilized per apheresis by CY+G-CSF and G-CSF were not significantly different (2.76+/-0.6 x 10(8) vs 2.53+/-0.4 x 10(8), P < or = 0.7). This resulted from a mean number of NC that was significantly lower in the CY+G-CSF products than in the G-CSF products (12.4+/-1.7 x 10(9) vs 32+/-5.4 x 10(9), P < or = 0.0001) and a mean incidence of CD34+ cells that was significantly higher in the CY+G-CSF products than in the G-CSF products (2.9+/-0.6% vs 0.9+/-0.2%, P < or = 0.0018). The mean (+/-s.e.m.) number of CFU-GM collected per apheresis was significantly higher in the CY+G-CSF group than in the G-CSF group (37+/-7 x 10(6) vs 14+/-2 x 10(6), P < or = 0.03). Interestingly, CY+G-CSF-mobilized CD34+ cells had a significantly higher plating efficiency than G-CSF-mobilized CD34+ cells (25.5+/-2.9% vs 10.8+/-1.9%, P < or = 0.0006). In addition, the mean number of LTC-IC was significantly higher in the CY+G-CSF products than in the G-CSF products (6.3+/-1 x 10[6] vs 3.3+/-0.3 x 10[6], P < or = 0.05). In conclusion, our data provide evidence that CY+G-CSF and G-CSF induce the mobilization of CD34+ cells with different clonogenic potential. As mobilized PBPC containing large numbers of progenitors lead to safer transplantation, this issue may have implications for planning mobilization strategies.

Clonogenic capacity and ex vivo expansion potential of umbilical cord blood progenitor cells are not impaired by cryopreservation.

Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been recently considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it was the aim of this study to evaluate whether cryopreservation procedures might heavily impair the clonogenic capacity, the feasibility of CD34+ selection and the ex vivo expansion potential of UCB progenitor cells. UCB samples were collected and cryopreserved as unseparated (n = 21) or mononuclear (MNC) cells (n = 15) within 12 h from delivery, and evaluated for viability, immunophenotype, cell and progenitor numbers after a minimum stay in liquid nitrogen of 6 months (range 6-14 months). Viability was always > 97% and no statistically significant difference was detected by flow cytometric analysis. Clonogenic recovery from unseparated cells was 80-87% for HPP-CFC, CFU-GEMM, BFU-E and CFU-GM, and from MNC cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, BFU-E and CFU-GM. CD34+ selection (n = 8) was performed on fresh and cryopreserved MNC cells using the MiniMACS immunomagnetic separation device, showing no difference in yield (68 +/- 7% vs 57 +/- 4%, P < or = 0.4) or in purity (89 +/- 2% vs 81 +/- 6%, < or = 0.4), for fresh in comparison to cryopreserved MNC cells. After 14 days of liquid culture in the presence of different combinations of SCF, IL-3, IL-6 and G-CSF no statistically significant difference was detected in CFC fold-expansion for fresh or cryopreserved MNC cells and for CD34+ cells, either selected and cultured from fresh or cryopreserved MNC cells. In conclusion we can state that UCB is a potential source of primitive progenitor cells that can be cryopreserved unmanipulated or after physical separation without major losses in clonogenic capacity and immunophenotypic composition. Moreover, CD34+ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the engraftment of adult patients.

In vitro growth of mobilized peripheral blood progenitor cells is significantly enhanced by stem cell factor.

The existence of primitive hematopoietic progenitors in mobilized peripheral blood is suggested by clinical, phenotypic and in vitro cell culture evidences. In order to quantify primitive progenitors, 32 leukaphereses from 15 patients with lymphoid malignancies were investigated for the growth of multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) in the absence or presence of recombinant stem cell factor (SCF), a cytokine which selectively controls stem cell self-renewal, proliferation and differentiation. Primitive progenitors were also quantitated by means of a long-term assay which allows the growth of cells capable of initiating and sustaining hematopoiesis in long-term culture (LTC-IC). Addition of SCF (50 ng/ml) to methyl-cellulose cultures stimulated with maximal concentrations of G-CSF, GM-CSF, interleukin 3 and erythropoietin significantly increased the growth (mean +/- SE) of CFU-Mix (7.7 +/- 1.7 versus 2.4 +/- 0.6, p < or = 0.0001), BFU-E (47 +/- 10 versus 32 +/- 6, p < or = 0.002) and CFU-GM (173 +/- 31 versus 112 +/- 20, p < or = 0.0001). Mean (+/- SE) percentages of SCF-dependent CFU-Mix, BFU-E and CFU-GM were 60 +/- 5%, 19 +/- 5%, and 33 +/- 4%, respectively. Mean (+/- SE) LTC-IC growth per 2 x 10(6) nucleated cells was 221 +/- 53 (range, 2 to 704). Linear regression analysis demonstrated a statistically significant correlation (r = .87; p < or = 0.0001) between LTC-IC and SCF-dependent progenitors. In conclusion, our data suggest that: A) the optimal quantification of mobilized progenitors requires supplementation of methylcellulose cultures with SCF, and B) in vitro detection of SCF-dependent progenitors might represent a reliable and technically simple method to assess the primitive progenitor cell content of blood cell autografts. Such in vitro evaluation of immature hematopoietic progenitors might be clinically relevant for predicting the reconstituting potential of autografts.

Assessment of the efficacy of a last-generation polyvalent immunoglobulin in the treatment of idiopathic thrombocytopenic purpura.

High-dose intravenous immunogammaglobulin (h.d.IgG) has been proposed as a treatment of idiopathic thrombocytopenic purpura (ITP), but the clinical effect is usually short and adverse reactions have been reported in clinical studies using different immunoglobulin (Ig) preparations. In this study, the efficacy of a last-generation polyvalent immunoglobulin in the treatment of ITP in adults and the incidences of adverse reactions of this therapy were evaluated. The reported data were based on various clinical and laboratory parameters evaluated before, during and after therapy, with a follow-up of 6 months. The data showed administration of 400 mg/kg d of intravenous polyvalent intact IgG for 5 days significantly increased the platelet count in all 15 patients, the maximum level occurring on Day 10 and being maintained in some patients for 6 months. Its very rapid onset of action suggests it may be useful for correcting life-threatening thrombocytopenia where bleeding complicates the clinical course, and for severe ITP in seriously immunosuppressed or infected patients in whom corticosteroids or immunosuppressive agents cannot be safely administered. The treatment was also well tolerated. In conclusion, polyvalent Ig may be useful in ITP steroid-refractory patients; further studies are required to evaluate clinical-laboratory parameters related to the long-term response of patients.

High-dose therapy in acute leukemia.

The use of intensive induction chemotherapy, primarily with combinations of an anthracycline and cytarabine, allows complete remission rates of greater than 70% in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). However, with currently available standard-dose therapy, only 20% of young adults are cured. In order to substantially increase the cure rate, adequate post-remission therapeutic strategies are mandatory. Three different therapeutic options are currently available: (i) dose-intensified chemotherapy; (ii) allogeneic stem cell transplantation; (iii) autologous stem cell transplantation. These therapeutic options should be carefully evaluated according to prognostic information, including cytogenetic and molecular abnormalities as well as phenotypic characterization. Randomized trials of intensive postremission therapy have now confirmed improved leukemia-free survival with the use of allogeneic or autologous transplantation. Autologous transplantation appears to be the most promising treatment modality in AML. Improved preparative regimens and purging techniques may be critical factors in determining the effectiveness of autologous transplantation in AML patients. In adult ALL, the role and optimal methods of stem cell transplantation are still under investigation.

Biologic and phenotypic analysis of early hematopoietic progenitor cells in umbilical cord blood.

Umbilical cord blood (UCB) is an attractive potential alternative to bone marrow (BM) as a source of hematopoietic progenitor cells since the number of progenitors in UCB is similar or even greater than that in normal BM. It was the aim of the present study to analyze the degree of immaturity of UCB progenitor cells. UCB mononuclear (MNC) and/or CD34+ cells were tested for surface antigen phenotype, expression of cytokines receptor, effect of stem cell factor (SCF) on colony growth, resistance to mafosfamide and replating potential. We have found that 34.9 +/- 3.4% and 77.9 +/- 2.6% of UCB CD34+ cells did not express CD38 and CD45RA antigens, respectively, suggesting that UCB contains a high proportion of immature progenitor cells. By means of three-color analysis, the receptor for SCF was detected on the majority of the CD34+ HLA-DR+ subpopulation; in fact, 81.8% +/- 4.3% of CD34+ HLA-DR+ cells were defined as SCF(low) and 8.1 +/- 1.5% as SCF(high). Colony growth of MNC and CD34+ cells was enhanced by the addition of SCF to methylcellulose mixture, resulting in a statistically significant increase in CFU-GM and CFU-GEMM but not in BFU-E numbers. UCB progenitor cells showed a higher resistance to mafosfamide treatment, in comparison to BM; the addition of SCF to the culture medium resulted in a statistically significant increase in mafosfamide concentration required to inhibit 95% of colony growth (P < or = 0.05). Moreover, as shown by single colony transfer assays, the presence of SCF in primary cultures promoted a significantly higher replating potential for both untreated (42 +/- 3.3% vs 21 +/- 4.6%, P < or = 0.018) and mafosfamide-treated samples (62 +/- 5.6% vs 44 +/- 6.1%, P < or = 0.018). In conclusion, UCB is a source of progenitor cells with immature characteristics in terms of surface antigen expression, distribution of SCF receptor, resistance to mafosfamide and replating potential. Therefore, UCB progenitor cells represent an ideal candidate population for experimental programs involving gene transfer and ex vivo stem cell expansion.

Selection of myeloid progenitors lacking BCR/ABL mRNA in chronic myelogenous leukemia patients after in vitro treatment with the tyrosine kinase inhibitor genistein.

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by a chimeric BCR/ABL gene giving rise to a 210-kD fusion protein with dysregulated tyrosine kinase activity. We investigated the effect of genistein, a protein tyrosine kinase inhibitor, on the in vitro growth of CML and normal marrow-derived multi-potent (colony-forming unit-mix [CFU-Mix]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) hematopoietic progenitors. Continuous exposure of CML and normal marrow to genistein induced a statistically significant and dose-dependent suppression of colony formation. Genistein doses causing 50% inhibition of CML and normal progenitors were not significantly different for CFU-Mix (27 mumol/L v 23 mumol/L), BFU-E (31 mumol/L v 29 mumol/L), and CFU-GM (40 mumol/L v 32 mumol/L v 32 mumol/L). Preincubation of CML and normal marrow with genistein (200 mumol/ L for 1 to 18 hours) induced a time-dependent suppression of progenitor cell growth, while sparing a substantial proportion of long-term culture-initiating cells (LTC-IC) from CML (range, 91% +/- 9% to 32% +/- 3%) and normal marrow (range, 85% +/- 8% to 38% +/- 9%). Analysis of individual CML colonies for the presence of the hybrid BCR/ABL mRNA by reverse transcription-polymerase chain reaction (RT-PCR) showed that genistein treatment significantly reduced the mean +/- SD percentage of marrow BCR/ABL+ progenitors both by continuous exposure (76% +/- 18% v 24% +/- 12%, P < or = .004) or preincubation (75% +/- 16% v 21% +/- 10%, P < or = .002) experiments. Preincubation with genistein reduced the percentage of leukemic LTC-IC from 87% +/- 12% to 37% +/- 12% (P < or = .003). Analysis of individual colonies by cytogenetics and RT-PCR confirmed that genistein-induced increase in the percentage of nonleukemic progenitors was not due to suppression of BCR/ABL transcription. Analysis of nuclear DNA fragmentation by DNA gel electrophoresis and terminal deoxynucleotidyl transferase assay showed that preincubation of CML mononuclear and CD34+ cells with genistein induced significant evidence of apoptosis. These observations show that genistein is capable of (1) exerting a strong antiproliferative effect on CFU-Mix, BFU-E, and CFU-GM while sparing the more primitive LTC-IC and (2) selecting benign hematopoietic progenitors from CML marrow, probably through an apoptotic mechanism.

Effect of the protein tyrosine kinase inhibitor genistein on normal and leukaemic haemopoietic progenitor cells.

Receptor and nonreceptor protein tyrosine kinases (PTKs) play a key role in the control of normal and neoplastic cell growth. The availability of PTK inhibitors prompted us to evaluate the effects of genistein, a natural inhibitor of PTKs, on in vitro colony formation by normal multilineage colony-forming units (CFU-Mix), erythroid bursts (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), long-term culture-initiating cells (LTC-IC) and acute myelogenous leukaemia colony-forming units (CFU-AML). Continuous exposure of normal marrow and blood mononuclear non-adherent cells, blood CD34+CD45RA- cells, and leukaemic blasts to increasing doses of genistein (1-100 microM) resulted in a statistically significant (P < or = 0.05) dose-dependent suppression of CFU-Mix, BFU-E, CFU-GM and CFU-AML growth. Regression analysis showed that growth inhibition was linearly related to genistein concentration. Genistein dose causing 50% inhibition (ID50) of CFU-AML was significantly lower compared to CFU-GM ID50 for marrow (19 v 32 microM, P < or = 0.017), unseparated blood (19 v 44 microM, P < or = 0.028) or CD34+CD45RA- blood (19 v 36, P < or = 0.04). Preincubation of leukaemic blasts with genistein (200 microM) for 1-2h confirmed that CFU-AML were significantly more sensitive than normal marrow and blood CFU-GM to genistein. Preincubation conditions which maximally suppressed leukaemic and normal colony growth spared a substantial percentage of marrow (29 +/- 4%) and blood (40 +/- 3%) LTC-IC. In conclusion, our data demonstrate that: (a) genistein strongly inhibits the growth of normal and leukaemic haemopoietic progenitors; (b) growth inhibition is dose- and time-dependent; (c) leukaemic progenitors are more sensitive than normal progenitors to genistein-induced growth inhibition; (d) genistein exerts a direct toxic effect on haemopoietic cells while sparing a substantial proportion of LTC-IC. The potent CFU-AML growth inhibition associated with the relative resistance of normal LTC-IC strongly supports the use of genistein for marrow purging.

Autologous bone marrow transplantation in chronic lymphocytic leukemia (CLL).

Chronic lymphocytic leukemia (CLL) is an indolent disease characterized by an abnormal proliferation of monoclonal lymphocytes in the bone marrow and lymphoid tissues. Most of the cases (> 90%) belong to the B-lymphocyte lineage and the course of the disease is variable depending on the presence of poor prognostic factors at diagnosis. Therefore optimal treatment is still questioned; presently there are no proven cures for CLL, but the natural history of the disease and the advanced age of the majority of patients makes prolongation of survival a reasonable therapeutic goal in most cases. The traditional therapeutic approach has been based on the activity of alkylating agents and corticosteroid, while patients resistant have been treated with nucleoside analogs. However, patients ultimately relapse and the choice of salvage therapy by conventional methods does not offer many chances. Particularly in younger patients, with poor prognostic factors, the therapeutic options may substantially change in the near future, based on alternative and innovative approaches aimed at achieving cure or long disease-free-survival. The results of high-dose therapy followed by reinfusion of hematopoietic stem cells, either from bone marrow or peripheral blood, will be presented and discussed.

Arachidonic acid induces c-jun gene expression in stromal cells stimulated by interleukin-1 and tumor necrosis factor-alpha: evidence for a tyrosine-kinase-dependent process.

We have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression induced by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the murine stromal cell line +/+.1-LDA 11 involves activation of phospholipase A2 (PLA2). Furthermore, induction of GM-CSF gene expression due to release of arachidonic acid as a result of PLA2 activation was mediated by the transcriptional factor c-jun. In the present study, we have investigated the potential mechanism involved in the induction of c-jun gene expression by arachidonic acid. Arachidonic acid induced transcription of c-jun mRNA. Downregulation of protein kinase C (PKC) by chronic exposure of stromal cells to the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 400 nmol/L) did not effect c-jun expression induced by arachidonate. Moreover, pretreatment of cells with the PKC inhibitor, calphostin C (1 mumol/L), caused a marked decrease of c-jun expression induced by TPA, but had no influence on c-jun expression induced by arachidonate. To explore the hypothesis that a tyrosine kinase signalling pathway, independent of PKC activation, was involved in arachidonate-induced c-jun expression, stromal cells were pretreated with the protein tyrosine kinase inhibitor, genistein, before challenge with arachidonic acid. Arachidonate 50 mumol/L)-induced c-jun expression was inhibited, in a dose- and time-dependent manner, by genistein. Genistein similarly inhibited c-jun expression in stromal cells exposed to IL-1 (500 U/mL) plus TNF-alpha (500 U/mL). The potential role of a tyrosine kinase pathway in arachidonate-mediated c-jun expression was further investigated by assaying the tyrosine kinase activity of cells challenged with arachidonic acid, IL-1, and TNF-alpha. Exposure of stromal cells to arachidonic acid induced a 2.1-fold increase in intracellular tyrosine kinase activity determined by phosphorylation of the synthetic peptide, raytide, in the presence of [gamma-32P]-ATP. Similarly, IL-1 and TNF-alpha induced 1.7- and 2.4-fold increases in tyrosine protein kinase activity, respectively. The effect of arachidonic acid on tyrosine kinase activity was inhibited by genistein and was enhanced by sodium vanadate. The increase of protein tyrosine kinase activity detected in arachidonate-stimulated cells was associated, in a dose- and time-dependent fashion, with tyrosine phosphorylation of 240-, 40-, and 29-kD substrates. These results are consistent with the hypothesis that a tyrosine phosphorylation process is triggered by arachidonate as an early event in the signalling pathway that leads to increased expression of c-jun.(ABSTRACT TRUNCATED AT 400 WORDS)

Density separation of umbilical cord blood and recovery of hemopoietic progenitor cells: implications for cord blood banking.

Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells suitable for clinical use in the transplantation setting. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving CB samples with minimal loss of progenitor cells. We have compared various separation procedures based on different density gradients in the attempt to obtain the highest depletion of red blood cells (RBC) while maintaining the highest recovery of progenitor cells. We compared three different densities of Percoll (1.069 g/ml, 1.077 g/ml, 1.084 g/ml), sedimentation over poligeline (Emagel ) and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 +/- 0.1%) while maintaining high recovery of CD34+ cells (85.3 +/- 5.6%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units (CFUs) after five weeks of long-term culture (LTC). We further evaluated the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors and on replating efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte colony-stimulating factor (G-CSF) prevents dose-limiting neutropenia in lymphoma patients receiving standard dose chemotherapy.

In this study, nine patients with non-Hodgkin's lymphoma (n = 6) and Hodgkin's disease (n = 3) receiving different cytotoxic chemotherapy regimens were given granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg/day) from 48 hours after the end of chemotherapy to 48 hours before the next chemotherapy administration. The decrease in mean absolute neutrophil counts (ANC) and in mean platelet (Plt) counts was not significant when pre-therapy counts were compared with post-therapy ones (p < 0.375 and p > 0.4, respectively). The mean actual dose intensity was 92% (range 68-100%). G-CSF treatment after chemotherapy reduces neutropenia and permits administration of the full chemotherapy program. A wash-out period between G-CSF treatment and chemotherapy administration is needed to prevent the detrimental effect of chemotherapy on leukocyte and platelet recovery when repeated cycles of cytotoxic drugs and G-CSF are administered.

Techniques for detection of minimal residual disease.

Analysis of leukemia-specific and leukemia-associated markers following standard or high-dose treatments is crucial in order to evaluate the efficacy of therapeutic strategies. During the last decade, several techniques have been proposed and used for detecting minimal residual disease (MRD). Each approach is characterized by advantages and limitations, mainly related to its sensitivity and specificity. The general limitations of such tests originates from the size of the sample which can be analysed and the heterogeneous distribution of leukemia after treatment. Clinically useful methods for detecting residual leukemia require not only sensitivity but also speed and reproducibility. The rate of false negative tests is low with polymerase chain reaction as well as flow cytometric analysis. Usually, patients without persistent cells carrying leukemia-associated markers have a lower risk of relapse. However, the detection of a persistent marker at one time point in complete remission cannot be considered a reliable indicator of MRD, whereas increase of positive signals or reappearance of leukemic markers usually precedes relapse. It is likely that one single approach will not allow the monitoring of the majority of patients and that a combination of techniques will be needed. Definitive results will be obtained only through prospective studies in patients receiving standardised therapy. Studies in which therapeutic strategies are designed according to the results provided by techniques for detecting MRD will be necessary to assess the relevance of their contribution to the treatment of leukemia.

Autologous transplant for chronic myelogenous leukemia using marrow treated ex vivo with mafosfamide.

Ten adult patients with Ph-positive chronic myelogenous leukemia (CML) received autologous transplantation using marrow treated ex vivo with mafosfamide. At transplant, 7 patients were in chronic phase (5 in first, 2 in second) and 3 in accelerated phase. The median time to achieve 500 x 10(6)/l neutrophils was 32 days (range 17-72 days). A platelet count of 20 x 10(9)/l was achieved at a median of 40 days (range 24-151 days). After transplant, cytogenetic analysis revealed 100% Ph-negative marrow metaphases in 6 of 9 analyzable patients with a median duration of Ph-negative hematopoiesis of 6.5 months. After a median follow-up of 16 months (range 3-31 months), five patients evolved into blast crisis, two died of non-hematological causes, one is Ph-negative in chronic phase at +4 and one is in chronic phase, but Ph-positive, at +22. In conclusion, this pilot study demonstrates that: (1) engraftment can occur from Ph-negative stem cells selected by mafosfamide, (2) mafosfamide purging may induce a transient period of Ph-negative hematopoiesis, and (3) modifications of the purging procedure and post-transplant manipulations of the immune-hematopoietic system are required to prolong cytogenetic remission.

Structural investigation of Ceratozamia spinosa mucilage.

The polysaccharide fraction from Ceratozamia spinosa appears to be made up mainly by a chemically homogeneous polysaccharide but with a wide range of molecular weight. By NMR and chemical degradative methods, it is shown to consist essentially of a backbone of alternate-->4)-beta-D-GlcpA-(1-->and-->2)-alpha-D-Manp-(1--> units. On the 4 position of the latter, beta-D-GlcpA residues are linked. End units of alpha-L-Araf, beta-D-Xylp, alpha-L-Rhap, and alpha-L-3-OMe-Rhap are linked to C-3 and/or C-4 positions of beta-D-GlcpA residues.