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F Bertolini

Publications and source records attributed to F Bertolini.

At least 55 records · Page 3Linked to original sources
PubMed1997

Multilineage long-term engraftment potential of drug-resistant hematopoietic progenitors.

Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil, stem cell factor, and interleukin-3, 88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost, but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure, most of the progenitors displayed a CD34+, CD38-, multidrug-resistance (MDR+), Rhodamine 123 low, Hoechst 33342 low phenotype, and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals, the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts.

Animals↗
PubMed1997

Megakaryocytic progenitors can be generated ex vivo and safely administered to autologous peripheral blood progenitor cell transplant recipients.

We evaluated different culture conditions to obtain a lineage-selected proliferation of clonogenic megakaryocytic progenitors (MP). In low-density (LD) or CD34+ cell cultures, the best results were obtained in serum-free medium in the presence of megakaryocyte growth and development factor, stem cell factor, interleukin-3 (IL-3), IL-6, IL-11, FLT-ligand, and macrophage inflammatory protein-1alpha. In paired studies, expansion of LD cells was less effective than expansion of CD34+ cells, and pre-enrichment of CD34+ cells using negative depletion of lineage-positive cells produced significantly larger quantities of MP than pre-enrichment using positive selection. MP proliferation peaked on day 7 in culture, and an 8- +/- 5-fold expansion of CD34+/CD61+ cells, a 17- +/- 5-fold expansion of colony-forming units-megakaryocytes, and a 58- +/- 14-fold expansion of the total number of CD61+ cells was obtained. In a feasibility clinical study, 10 cancer patients (8 with breast cancer and 2 with non-Hodgkin's lymphoma) undergoing autologous peripheral blood progenitor cell (PBPC) transplant received MP generated ex vivo (range, 1 to 21 x 10(5)/kg CD61 cells) together with unmanipulated PBPC. Eight patients received a single allogeneic platelet transfusion, whereas platelet transfusion support was not needed in 2 of the 4 patients receiving the highest doses of cultured MP. This result compares favorably with a retrospective control group of 14 patients, all requiring platelet transfusion support. Adverse reactions or bacterial contamination of cell cultures have not been observed. In conclusion, MP can be expanded ex vivo and safely administered to autologous transplant recipients. Further clinical trials will indicate the reinfusion schedule able to consistently abrogate the need for allogeneic platelet transfusion support in autologous transplantation.

Cells, Cultured↗
PubMed1997

Collection of circulating progenitor cells after epirubicin, paclitaxel and filgrastim in patients with metastatic breast cancer.

The efficacy of high-dose chemotherapy (HDC) and circulating progenitor cell (CPC) transplantation in metastatic breast cancer (MBC) relies mainly on giving this treatment after a response to conventional induction chemotherapy has been achieved. For this reason an optimal mobilization regimen should be therapeutically effective while minimizing the number of leucaphereses required to support the myeloablative therapy. The combination of an anthracycline and paclitaxel in chemotherapy-untreated MBC has produced impressive response rates. We evaluated the CPC-mobilizing capacity of the combination epirubicin (90 mg m(-2)) and paclitaxel (135 mg m(-2)) followed by filgrastim (5 microg kg(-1) day(-1)) starting 48 h after chemotherapy administration in ten patients with MBC who were eligible for an HDC and CPC transplantation programme. Leucaphereses were performed by processing at least two blood volumes per procedure at recovery from neutrophil nadir when CD34+ cells in the peripheral blood exceeded 20 microl(-1). In most patients (six out of 10) more than 2.5 x 10(6) CD34+ cells kg(-1), a threshold considered to be sufficient for haematopoietic reconstitution, were collected with a single apheresis. In the remaining four patients an additional procedure, performed the following day, was enough to reach the required number of progenitors. These data suggest that the epirubicin-paclitaxel combination, besides being a very active regimen in MBC, is effective in releasing large amounts of progenitor cells into circulation.

Adult↗
PubMed1997

A new 'two step' procedure for 4.5 log depletion of T and B cells in allogeneic transplantation and of neoplastic cells in autologous transplantation.

To evaluate a new 'two step' method for purging T, B and neoplastic cells from hematopoietic progenitor cells (PC), PCs were collected by apheresis and some suspensions were deliberately contaminated with 2-5% breast cancer cells. PCs were first processed through CellPro columns for positive selection of cells that express CD34. After this first step, the mean CD34+ cell recovery was 68 +/- 12%, and CD34+ cell purity was 61 +/- 11%; CD3+ and neoplastic cell depletion were 2.1 +/- 0.4 and 1.9 +/0 0.4 logs, respectively. Cells were further processed through the StemSep device for direct depletion of T and B cells or of breast cancer cells. After the first and the second step, overall CD34+ cell recovery was 50 +/- 7%, T and B cell removal was 4.7 +/- 0.4 log and neoplastic cell purging was 4.4 +/- 0.3 log, ie significantly superior to methods described in the past.

Antigens, CD34↗
PubMed1997

Role of tumor cells contaminating the graft in breast cancer recurrence after high-dose chemotherapy.

One of the possible drawbacks to autologous stem cell transplantation in breast cancer (BC) patients is the potential for tumor contamination in the transplanted product. We present a patient with advanced disease who received high-dose chemotherapy (HDC) and PBPC support as consolidation therapy after achieving complete remission with standard-dose first-line treatment, and suffered recurrence of the disease 6 months after transplantation. Retrospective analysis revealed the presence of contaminating cells in the leukapheretic product, and clinical evidence suggested a role for these cells in the tumor relapse.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1997

"Stem cell candidates" purified by liquid culture in the presence of Steel factor, IL-3, and 5FU are strictly stroma-dependent and have myeloid, lymphoid, and megakaryocytic potential.

Berardi et al. (Science 1995; 267:105) reported recently that combined cytokine stimulation and antimetabolite treatment were able to isolate cells with characteristics of hemopoietic stem cells. Bone marrow (BM) low-density cells were cultured for 1 week in the presence of Steel factor (SF), IL-3, and the antimetabolite 5-FU. Following this approach one in 10(5) BM cells were purified. These cells showed no clonogenic potential in soft gel assays but presented a striking myeloid-lymphoid potential as long-term culture-initiating cells (LTC-ICs). We investigated this new "stem cell candidate." following a similar approach we purified one in 55,000/130,000 cells from cord blood and peripheral blood in mobilized cancer patients. These cells displayed no clonogenic potential in methylcellulose assays in the presence of different cytokine combinations and generated very few or no clonogenic progenitors in liquid cultures in the presence of cytokines. When seeded on layers derived from the murine BM stromal cell line M2-10B4, 38-86% of the cells purified using this approach generated multilineage progenitors acting as LTC-lCs. In a different series of studies, after 5-week culture on human BM stromal cell line L87/4 layers, cells were forced to selected lineage differentiation by culture in the presence of low concentrations of SF and high concentrations of lineage-specific cytokines such as Flt3-ligand (myeloid and pre-B cell differentiation), Tpo (megakaryocytic differentiation). IL-7, and IL-2 (pre-B and NK differentiation). After 12-day culture under these conditions, generation of myeloid, pre-B, megakaryocytic, and NK progenitors was assessed by immunohistochemistry, flow cytometry, and mRNA expression of CD7, 14, 19, 41b, S6, and 61. We conclude that this procedure for multilineage progenitor cell purification is simple and effective and could have major implications for gene transfer and stem cell transplantation.

Cell Lineage↗
PubMed1996

Engineered stromal layers and continuous flow culture enhance multidrug resistance gene transfer in hematopoietic progenitors.

We recently reported that in stroma-free cultures 11-33% of clonogenic cells derived from a bulk long-term culture [long-term culture-clonogenic cells (LTC-CC)] could be transduced by supernatant exposure or coculture of human CD34+ progenitors with MDR retroviral producer line A12M1. We reasoned that a stromal cell layer may generate niches in which LTC-CC could enter in the S-phase, thus becoming a more accessible target for gene delivery. In static culture studies in flasks, human engineered stromal cell line L87/4 or stromal murine M2-10B4 cells were used as feeder after irradiation, and CD34+ cells from either cord blood or peripheral blood of mobilized cancer patients were exposed to MDR supernatant for 7 consecutive days before 5-week culture for LTC-CC evaluation. In continuous flow perfusion culture studies, CD34+ cells were seeded over irradiated stromal murine M2-1OB4 cells and exposed to MDR supernatant for 7 days before LTC-CC evaluation. In mock-transduced controls, <5% of LTC-CC were found to he viable after exposure to 10 ng/ml Taxol. In cells exposed to MDR supernatant in static stroma cultures, 68 +/- 4% of seeded LTC-CC were found to be drug resistant and express MDR mRNA as evaluated by reverse transcription-PCR analysis of single colonies. The addition of cytokines did not further enhance transfer efficiency. After MDR retroviral exposure in continuous flow cultures, 88 +/- 5% of LTC-CC were found to be drug resistant (P < 0.01 versus static stroma culture). P-glycoprotein expression in CD34+ cells was evaluated using flow cytometry and found to he higher after continuous flow versus static cultures. Finally, very high levels of P-glycoprotein expression after MDR supernatant exposure in the presence of stroma were confirmed by APAAP staining of cultured cells. We conclude that engineered stromal cell layers and continuous flow culture conditions can significantly enhance retroviral-mediated gene transfer into human hematopoietic progenitor cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Functional and morphological characterization of immunomagnetically selected CD34+ hematopoietic progenitor cells.

We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony-forming cells and their precursors, detected as long-term culture-initiating cells (LTC-IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8-fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC-IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34-enriched fractions. In five cases CD34+ selected cells grown in a stroma-free long-term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM-CSF every 48 h, showed a 15 (+/- 15) and 31 (+/- 21) mean colony forming unit-granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well-differentiated myeloid cells (MPO+), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37 degrees C, the majority of the cells showed no iron particles, but about 30% of the cells were iron-labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short-term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.

Adult↗
PubMed1996

A multicenter inspection of the swirling phenomenon in platelet concentrates prepared in routine practice. Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion.

BACKGROUND: In a previous multicenter study, the absence of the swirling phenomenon correlated well with platelet disc-to-sphere transformation, a morphology feature associated in the past with poor platelet viability. STUDY DESIGN AND METHODS: In 13 centers, 5366 platelet concentrates (PCs) were prepared and stored under routine conditions and evaluated for the presence of swirling. PCs found not to have swirling were evaluated for pH and platelet and white cell counts in parallel with an equal number of PCs with swirling that were studied as controls. RESULTS: In 13 participating centers, swirling was reported to be absent after 3 and 5 days of storage in 0 to 3.8 percent and in 0 to 18 percent of PCs, respectively. In 94 percent of units with swirling, pH was 6.7 to 7.5; this is a range of values associated with adequate in vivo survival. In 69 percent of PCs without swirling, pH was lower than 6.4 or higher than 7.6; these values are associated with reduced platelet viability. Three-quarters of the cases in which the absence of swirling was associated with pH values between 6.7 and 7.5 were reported from 2 out of the 13 participating centers. CONCLUSION: In PCs prepared and stored under routine conditions, the presence of swirling correlated well with pH values associated with adequate platelet in vivo viability. In about 20 percent of PCs without swirling, this was not due to a low or high pH value, and it would be useful to investigate whether this lack of swirling is associated with poor platelet viability. These data support the possibility that the evaluation of swirling by visual inspection might replace invasive and more cumbersome assays currently used for routine PC quality control.

Blood Platelets↗
PubMed1996

Gene transfer-mediated generation of drug-resistant hemopoiesis.

Autologous- or allogeneic-bone marrow transplantation are increasingly used to overcome the myelosuppressive effects of high dose chemotherapy administered to cancer patients. Transfer of the multidrug resistance (MDR) gene in hemopoietic progenitors has been proposed as a tool to administer higher and possibly more curative doses of chemotherapy. Murine models have demonstrated that retrovirus-mediated MDR transfer in bone marrow cells can render animals resistant to myeloablative doses of Taxol, and in vitro studies have shown that MDR-transduced human CD34+ cells can generate drug-resistant multipotential hemopoietic progenitors such as long term culture-initiating cells. Given these results, phase I clinical trials are currently under way to evaluate feasibility and treatment-related toxicity of MDR gene transfer in cancer patients by means of safe retroviral vectors. Finally, Taxol treatment of MDR transduced mice and human CD34+ cells have indicated that MDR is a dominant selectable marker in vitro and in vivo, and vectors carrying both MDR and non selectable genes such as beta-globin or glucocerebrosidase could be used in the next future for gene therapy of inherited disorders like thalassemia or Gaucher disease.

Animals↗
PubMed1996

Unrelated mismatched cord blood transplantation in an adult with secondary AML.

Umbilical cord blood (CB) has been widely used for related and unrelated transplants in pediatric patients. We present the case of an adult with secondary AML who received an unrelated, one-antigen mismatched CB transplant due to the lack of a matched donor. The patient was a 26-year-old female (35 kg/bw) who had received an autologous bone marrow transplant for Hodgkin's disease in April 1994 and, 6 months later, developed secondary MDS (RAEB, 46, XX, -7, +mar), which slowly evolved into acute myelogenous leukemia. In May 1995, she was transplanted with a 165 ml CB unit containing a total of 1.6 x 10(9) nucleated cells, 11 x 10(6) CD34+ cells and 7.2 x 10(5) CFU-GM. GVHD prophylaxis consisted of standard CsA and methotrexate. Myeloid engraftment occurred on day +28 (PMN > 500) and full donor chimerism was confirmed twice (on days +33 and +56) by means of cytogenetics and DNA microsatellite analysis. Erythroid and megakaryocytic engraftment was documented by immunohistochemical analysis of a bone marrow biopsy on day +40, showing the presence of erythroblastic islands and isolated CD61+ immature cells. The patient did not develop GVHD but died on day +56 from idiopathic interstitial pneumonia and multiorgan failure. To our knowledge, this is one of the first case reports of unrelated mismatched CB transplantation in an adult.

Adult↗
PubMed1996

A new method for placental/cord blood processing in the collection bag. I. Analysis of factors involved in red blood cell removal.

We describe a new procedure for large-scale CB processing in the collection bag, thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C, the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal, the cell pellet was resuspended and the percent recovery of total WBC, CD34+ progenitor cells, CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300, 600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC, CFU, CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy, effective and particularly suitable for large-scale banking under GMP conditions.

Blood Component Removal↗
PubMed1996

Hematopoietic and immune recovery after transplantation of cord blood progenitor cells in children.

Matched related cord blood transplantation (CBT) has been successfully used to rescue patients undergoing myeloablative therapy. However, few data are available on the kinetics of hematological and immunological reconstitution of CBT recipients. We have investigated the hematological engraftment and immune recovery following related CBT in three patients, with acute lymphoblastic leukemia, aged 10, 9 and 7 years and with a body weight of 31, 40 and 25 kg, respectively. All patients engrafted and none experienced acute or chronic graft-versus-host disease. The time needed to achieve granulocyte recovery was 13, 26 and 29 days, respectively and platelet recovery occurred in 28, 49 and 51 days. All patients presented a marked increase of HbF, the values observed being much greater than those documented in patients given marrow transplantation and comparable with those observed in normal children in the first year of age. The recovery of T cell immunity, as well as that of natural killer subpopulations, mimicked that described in BMT recipients, a quicker return of CD8+ T cells determining the characteristic inversion of CD4/CD8 ratio. An impressive increase in the percentage and absolute number of B lymphocytes, apparently not related to viral infections, was demonstrable in all three cases. These data suggest that CBT recipients can experience a slight delay in hematological recovery when compared with patients given BMT. The reconstitution of erythropoiesis seems to recapitulate the ontogenetic pattern and the kinetics of recovery of the immune system reproduce that observed after BMT with the peculiarity of B cell expansion in peripheral blood.

Antibody Formation↗
PubMed1995

Comparative study of different procedures for the collection and banking of umbilical cord blood.

Different procedures for umbilical cord blood (CB) collection, separation, and cryopreservation were compared to evaluate the feasibility of large-scale CB banking for unrelated transplant. CB collection using an open system was associated with a 12.5% rate of bacterial contamination, whereas this rate fell to 3.3% using a closed collection system. When the umbilical cord was clamped within 30 s after delivery, on 378 occasions it was possible to collect 77 +/- 23 ml of CB without risk to mother or infant. Considering that engraftment can be obtained in recipients of 20 x 10(3) CFU-GM/kg, 86% and 28% of CB samples with a volume larger than 50 ml were found to contain a sufficient number of CFU-GM to engraft patients of 20 and 70 kg, respectively. Both Ficoll and gelatin purification procedures were associated with a recovery of 86-92% of CFU-GM, BFU-E, CFU-GEMM, and HPP-CFC, but the gelatin method appears to be more suitable for large-scale CB banking in vials. After cryopreservation, the recovery of clonogenic progenitors was similar for both CB samples stored as whole blood or as mononuclear cells separated using Ficoll or gelatin. In conclusion, large-scale CB banking seems feasible, and CB cell separation could allow storage of a large number of CB samples in a limited liquid nitrogen space.

Blood Banks↗
PubMed1995

The effect of interleukin-12 in ex-vivo expansion of human haemopoietic progenitors.

We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL-12. In cultures of low density cells, cytokine combinations including IL-12 were associated to a greater proliferation (up to 6.7 +/- 2.5 CFU-GM fold expansion). However, in cultures of purified CD34+ cells the more efficient cytokine combination (147 +/- 49 CFU-GM fold expansion) was SCF, IL-3, IL-11 and MIP-1 alpha, and the addition of IL-12 did not further enhance expansion of progenitors. These results indicate that accessory cells, lost in CD34+ cell purification, could be in part responsible for IL-12 effect on progenitor cell proliferation. In CD34+ cell cultures the addition of IL-12 led to CD19 mRNA generation, suggesting that IL-12 acts on haemopoietic cells with both myeloid and lymphoid potential.

Antigens, CD↗