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V Rizzoli

Publications and source records attributed to V Rizzoli.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Adult↗
PubMed1998

Reverse transcription polymerase chain reaction is a reliable assay for detecting leukemic colonies generated by chronic myelogenous leukemia cells.

Single-colony karyotyping (SCK) and reverse-transcription polymerase chain reaction (RT-PCR) are two increasingly used techniques for the quantification of leukemic colonies generated by chronic myelogenous leukemia (CML) cell fractions purged or selected in vitro. Recently, the existence of Philadelphia (Ph) chromosome positive progenitors with a silent BCR-ABL gene has been reported, thus raising concerns on the use of RT-PCR for detecting BCR-ABL positive progenitors. In order to investigate this issue further, colonies (n = 204) generated by mononuclear (MNC) or CD34+ CML cells were individually harvested, divided into two aliquots and analyzed both at the cytogenetic level to detect the Ph chromosome, and the molecular level to detect BCR-ABL transcripts. The mean (+/- s.d.) percentages of colonies analyzable by either SCK or RT-PCR were 74 +/- 16% and 86 +/- 16%, respectively. A significant percentage of colonies (67 +/- 19%) could be successfully analyzed by both SCK and RT-PCR. Although the majority of these colonies (97 +/- 5%) were Ph-positive and BCR-ABL-positive, a negligible percentage (4%) of progenitors were Ph-positive but BCR-ABL-negative. In order to test the influence of colony size on the outcome of molecular analysis, the efficiency of our RT-PCR assay in detecting BCR-ABL transcripts was investigated by means of experiments in which the number of cells used to start RNA extraction was serially reduced. These experiments showed that at least 150 cells were necessary to achieve a reproducible amplification of BCR-ABL transcripts. By correlating the size of harvested colonies with the outcome of molecular analysis, it was evident that BCR-ABL-negative but Ph-positive colonies represented false negative results occurring when a number of leukemic cells below the detection limit of our RT-PCR assay was analyzed. In conclusion, our data demonstrate that individual CML colonies grown in semisolid culture assays can be indifferently analyzed by SCK or RT-PCR, and support an extensive use of a carefully standardized RT-PCR assay to estimate the leukemic burden within samples which have been purged and selected in vitro.

Adult↗
PubMed1998

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.

Adolescent↗
PubMed1997

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.

Adolescent↗
PubMed1997

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.

Antigens, CD34↗
PubMed1997

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.

Antigens, CD34↗
PubMed1997

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.

Adolescent↗
PubMed1997

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.

Acute Disease↗
PubMed1996

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.

Adult↗
PubMed1996

Horseradish peroxidase-catalyzed sulfoxidation of promethazine and properties of promethazine sulfoxide.

Promethazine sulfoxide was obtained with a quantitative yield in a horse radish peroxidase-catalyzed reaction of promethazine and hydrogen peroxide and was also prepared by direct chemical synthesis. The enzymatic sulfoxidation of promethazine was studied in vitro as a function of pH, promethazine, and hydrogen peroxide concentration. Promethazine sulfoxide inhibits with an apparent K(i) of 59.7 microM at pH 5.5 the enzymatic reaction, followed spectrophotometrically, polarographically, potentiometrically, and luminometrically. The reaction was also inhibited by ascorbic acid (K(i) 26.8 microM) and glutathione (K(i) 41.8 microM). The spectrophotometric techniques employed, together with ESR spectrometry, allowed the identification of at least three radical species formed in the course of the reaction. Promethazine sulfoxide is devoid of the antioxidant effect exhibited by promethazine on rat brain synaptosomes. The sulfoxide also lacks photosensitizing action, while retaining the neuroleptic effect of the parent compound.

Animals↗
PubMed1996

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.

Bone Marrow Transplantation↗
PubMed1996

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.

Cell Division↗
PubMed1996

Density separation and cryopreservation of umbilical cord blood cells: evaluation of recovery in short-and long-term cultures.

The clonogenic capacity of human umbilical cord blood (UCB) has been evaluated in several studies, which have shown high numbers of primitive hematopoietic progenitor cells. Recently, UCB progenitor cells were demonstrated to possess significant advantages over bone marrow (BM) in terms of proliferative capacity and immunologic reactivity. Therefore, UCB has been considered an attractive source of hematopoietic stem cells for both research and clinical applications. 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 UCB samples with minimal loss of progenitor cells. We compared separation over three different densities of Percoll (1.069, 1.077 and 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 RBCs (hematocrit of the final cellular suspension 0.4 +/- 0.1%), while maintaining a high recovery of CD34+ cells (85.3%) and total recovery of 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 after 5 weeks of longterm culture. Moreover, the presence of Stem cell factor significantly increased CFU-GEMM (14 +/- 4 vs. 49 +/- 5, p < or = 0.0005) and CFU-GM (112 +/- 18 vs. 178 +/- 19, p < or = 0.025), but not B FU-E (42 +/- 7 vs. 53 +/- 7, p < or = 0.375) growth. In conclusion, RBC depletion of UCB can be accomplished with minimal loss of committed and primitive hematopoietic progenitors. This procedure may have important implications in the large-scale banking of UCB and in ex vivo expansion/gene therapy protocols.

Antigens, CD34↗
PubMed1996

BCL2 oncogene protein expression in human hematopoietic precursors during fetal life.

BCL2 proto-oncogene encodes a 25-kD protein that is characteristically localized in the inner mitochondrial membrane of the cell. It has been reported that BCL2 protein has the unique functional role of blocking programmed cells death without affecting proliferation. We have analyzed the expression of the BCL2 protein in fetal hematopoietic tissues from the 10th week of gestational age onward. Fetal thymus, liver, and bone marrow and cord blood were investigated. The experiments were performed by the alkaline-antialkaline phosphatase (APAAP) technique by staining air-dried acetone-fixed cytospins and by dual-color immunofluorescent assay by staining mononuclear cell suspensions with monoclonal antibodies detecting BCL2 protein and antigens expressed by different hematopoietic subsets. Flow cytometric analyses were performed on FACSort's Comsort 32 (Becton Dickinson, San Jose, CA). The results have shown that the BCL2 protein is expressed in human fetal ontogenesis at the earliest stages examined. The major conceptual aspects of the results are 1) BCL2 is largely expressed in the hematopoietic cells during ontogenesis. BCL2+ cells include both immature and more differentiated subsets. Moreover, the 25-kD protein is expression in cell subsets well known to be high proliferating. This behavior suggests that BCL2 could have more complex functions than those previously described. 2) The expression in the major part of CD34+ cells suggests that BCL2 could play a role in stem cell survival. 3) BCL2 is expressed in not only medullary but also cortical thymocytes, where it could cooperate in positive selection processes. 4) The involvement of BCL2 in the immunosurveillance is indicated not only by its role in B and T cell lineages but also by its expression in particular subsets like that of the cytoplasmic CD3+ fetal liver NK cells. 5) The discrepancy observed between the results of transgenic mice analysis and in vitro inhibition experiments by antisense oligonucleotides performed for understanding BCL2 functions must stress the importance of the direct immunologic analysis of BCL2 in human hematopoietic cells.

Bone Marrow↗