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N Gibelli

Publications and source records attributed to N Gibelli.

At least 19 recordsLinked to original sources
PubMed2003

Cytotoxic chemotherapy preceding apheresis of peripheral blood progenitor cells can affect the early reconstitution phase of naive T cells after autologous transplantation.

Transient T cell immunodeficiency is a common complication following hematopoietic stem cell transplantation. In breast cancer patients transplanted with autologous peripheral blood progenitor cells (PBPC) harvested after cytotoxic treatment with either cyclophosphamide or epirubicin plus paclitaxel, we evaluated T cells infused in grafts and in peripheral blood during the early reconstitution phase. We found that PBPC grafts harvested after treatment with epirubicin plus paclitaxel contained substantially larger numbers of T cells with less altered composition than after cyclophosphamide. Three months after high-dose cytotoxic chemotherapy, the numbers and the kinetics of circulating naive T cells, but not of memory and CD28- T cells, correlated positively with the number of naive T cells infused PBPC grafts. Finally, retrospective analysis of two cohorts of patients transplanted in different clinical settings with PBPC grafts harvested following cyclophosphamide or epirubicin plus paclitaxel showed apparently different susceptibilities to develop endogenous varicella zoster virus reactivation in the first year after high-dose cytotoxic chemotherapy. On the whole, these data indicate that number and composition of T cells in PBPC grafts vary according to the former cytotoxic therapy, and suggest that autologous transfer of T cells may accelerate the early T cell reconstitution phase and possibly ameliorate immune competence in patients rendered lymphopenic by high-dose chemotherapy.

Antigens, CD↗
PubMed2001

Circulating CD33+ large mononuclear cells contain three distinct populations with phenotype of putative antigen-presenting cells including myeloid dendritic cells and CD14+ monocytes with their CD16+ subset.

BACKGROUND: In peripheral blood, myeloid markers identify a heterogeneous mixture of cells in transit from the bone marrow to peripheral tissues. Similarly, HLA-class II DR expression usually identifies mononuclear cells with the potential for developing antigen-presenting activity. We gathered putative antigen presenting cells bearing myeloid markers (My-APC) to study their composition by cell surface phenotype. METHODS: To gather and dissect My-APC phenotype while excluding lymphocytes and granulocytes, we developed a strategy based on staining red cell-lysed peripheral blood and gating cells bearing myeloid markers and physical parameters of large mononuclear cells. RESULTS: Phenotypic analysis within the My-APC gate showed three distinct populations. The largest fraction was constituted by CD14+ monocytes that extended into the other two populations, each expressing gradually lower levels of CD14 surface antigen along with increasing levels of CD16 and CD2, respectively. The CD16 and CD2 expression patterns extended from CD16+CD14+ or CD2+CD14+ double- positive intermediate cells toward each single positive subset, but they were reciprocally exclusive. Interestingly, CD2+CD14- cells within the My-APC gate were equivalent to myeloid dendritic cell precursors (pre-DC) defined previously by the absence of lineage markers and expression of HLA-DR and myeloid markers. Phenotypic analysis of each population revealed differences in the expression of costimulatory molecules and CD62L. CONCLUSIONS: This novel analytical approach allowed us to distinguish circulating My-APC in three subsets and to identify relationships between monocytes and other related myeloid populations including DC.

Antigen-Presenting Cells↗
PubMed2000

Minimal tumor contamination of hematopoietic harvests from breast cancer patients can be easily detected by liquid culture assay.

BACKGROUND: Recurrence after PBSC transplantation in breast cancer (BC) patients may be related to the reinfusion of tumor cells contaminating the graft. We have developed a liquid culture (LC) method for the identification of viable epithelial tumor cells in PBSC collections. METHODS: Mononuclear fraction from PBSC harvests of BC patients undergoing high dose chemotherapy (HDC) (adjuvant setting n = 60, metastatic disease n = 30) were seeded in petri dishes containing round cover slips. Cells were cultured for 3 weeks, then cover slips were stained with the pan-cytokeratin A45-B/B3 mAb and scored under a light microscope. Samples were considered positive when more than one adherent cell or a cluster of cells staining bright red was present. Results were compared with those obtained on cytospins prepared directly from the PBSC harvest. Specificity of the method was tested on lymphoma patients, collections: all were negative. The sensitivity, evaluated by serial dilutions of CG5 BC cell line, was 1 epithelial cell in 10(6) mononuclear cells. RESULTS: The percentage of positivity was superimposable in the two groups (adjuvant 25%, metastatic 24%). However, a significantly higher proportion of positive samples from metastatic vs adjuvant patients has shown the presence of tumor clusters (86% vs 33%, p = 0.063). In 21% of all samples a discrepancy with the results obtained by immunocytochemical analysis (ICC) was found, mostly due to liquid-culture-positive/ICC-negative PBSCs. DISCUSSION: Our data suggest that LC assay may enhance the identification of viable disseminated epithelial tumor cells in PBSC grafts and might provide insights about their growth capacity.

Breast Neoplasms↗
PubMed2000

Mitomycin C as an alternative to irradiation to inhibit the feeder layer growth in long-term culture assays.

BACKGROUND: Mitomycin C (MMC), an antitumoral antibiotic, has been described inhibiting the proliferation of different cell types in vitro. Since irradiation is commonly used to stop the cell growth of adherent cells in several experimental models, we aimed to define the optimal dose and incubation time of MMC capable of inhibiting the growth of murine fibroblasts, used as an adherent feeder layer in long-term hematopoietic culture assay. METHODS: M2 10B4 (both parental and engineered to produce human IL-3 and G-CSF) and Sl/Sl (engineered to produce human IL-3 and steel factor) murine fibroblast cell-lines, frequently used in LTC-IC assay, were incubated with increasing doses of MMC for either a short (3 h) or a long (16 h) period. The efficiency of MMC in stopping the cell growth was evaluated for 5 days following MMC removal. The effects of MMC treatment on human hematopoietic cells were studied using both LTC-IC and limiting dilution (CAFC) assays. RESULTS: The growth of M2 10B4 cells was stopped at 3 and 16 h in the presence of 20 microg/mL and 2 microg/mL of MMC, respectively while Sl/Sl fibroblasts required a lower dose of drug (2 and 0.2 microg/mL, respectively). No significant difference was found between the number of LTC-IC or CAFC obtained from cultures containing irradiated or MMC-treated feeder cells. DISCUSSION: MMC inhibits the growth of murine fibroblasts used as adherent feeder cells in long-term culture assays, without interfering with the subsequent growth of co-cultured hemopoietic cells. Different cell types might present a different sensitivity to MMC and therefore a dose-response curve to MMC has to be obtained for each cell type of interest.

Animals↗
PubMed1999

Efficacy of epirubicin/paclitaxel combination in mobilizing large amounts of hematopoietic progenitor cells in patients with metastatic breast cancer showing optimal response to the same chemotherapy regimen.

BACKGROUND AND OBJECTIVE: Based on our preliminary experience, we have further evaluated the capacity of the paclitaxel/epirubicin combination (at the dose of 175 and 90 mg/m(2), respectively) plus G-CSF to mobilize hematopoietic progenitors into the circulation. DESIGN AND METHODS: The study was conducted in a homogeneous cohort of 25 stage IV breast cancer patients showing response to three cycles of the same chemotherapy regimen and who were included in a high-dose chemotherapy program. RESULTS: In most cases (68%) more than 5_10(6) CD34+ cells/kg b.w. (the threshold fixed in our study) were collected by a single leukapheresis, 28% and 4% of patients requiring 2 and 3 procedures, respectively. Based on the CD34+ cell count in the peripheral blood, most of the leukaphereses (53%) were performed on day 11 after chemotherapy. More than 50 CD34+ cells/mL along with a preleukapheresis WBC count between 10 and 20_10(9)/L predicted that only a single harvest would be required in 100% of cases. The evaluation of the clonogenic potential of collected cells showed that a large number of committed colony-forming cells (CFCs) and more primitive long-term culture-initiating cell (LTC-IC) hematopoietic progenitors were present in 20 harvests studied. INTERPRETATION AND CONCLUSIONS: These data demonstrate that the epirubicin/paclitaxel combination followed by G-CSF, besides being a very active regimen in MBC, is effective in releasing large amounts of progenitor cells into the circulation which can then be safely employed to support myeloablative regimens.

Adult↗
PubMed1998

Hematopoietic progenitor cell collection and neoplastic cell contamination in breast cancer patients receiving chemotherapy plus granulocyte-colony stimulating factor (G-CSF) or G-CSF alone for mobilization.

BACKGROUND: We compared hematopoietic progenitor cell (HPC) collection and neoplastic cell contamination in breast cancer patients given cyclophosphamide (CTX) plus granulocyte-colony stimulating factor (G-CSF) or G-CSF alone for mobilization. PATIENTS AND METHODS: In 57 stage II-III breast cancer patients, CD34+ cells, colony-forming units-granulocyte macrophage (CFU-GM), early HPC and breast cancer cells were counted in HPC collections obtained after CTX plus G-CSF (n = 27) or G-CSF-alone mobilization (n = 30). RESULTS: The CD34+ cell collection was about two-fold greater after CTX plus G-CSF mobilization (11.0 +/- 7.9 vs. 5.8 +/- 3.5 x 10(6)/kg, P < 0.001). Similarly, the total number of CFU-GM, CD34+CD38- cells and of week-5 cobblestone area forming cells (CAFC) collected was significantly higher in patients mobilized with CTX plus G-CSF. Breast cancer cells were found in the apheresis products of 22% of patients mobilized with CTX plus G-CSF and in 10% of patients mobilized with G-CSF alone (P = 0.36). Of seven patients who failed G-CSF-alone mobilization and eventually underwent chemotherapy plus G-CSF mobilization, none had cytokeratin-positive cells after G-CSF mobilization, whereas four out of seven had cytokeratin-positive cells after chemotherapy plus G-CSF (P = 0.07 by chi 2 test). CONCLUSION: The CTX plus G-CSF mobilization protocol was associated with a significantly higher HPC collection. However, this benefit was not accompanied by a reduction in the incidence of tumor-contaminated HPC graft.

Adult↗
PubMed1998

Epithelial tumour cell detection and the unsolved problems of nested RT-PCR: a new sensitive one step method without false positive results.

Sensitive detection of circulating epithelial cancer cells might have important therapeutic and prognostic implications in patients with breast cancer (BC) receiving high-dose chemotherapy and PBSC support. We have compared the specificity and sensitivity of the recently developed 'one tube' reverse transcriptase PCR (RT-PCR) assay with the more widely used nested RT-PCR method for detection of cytokeratin 19 (CK19)-positive cells. The analysis of 30 control samples provides evidence that one tube RT-PCR is highly specific in contrast to the nested method which showed 23% false positive results. The sensitivity of both techniques to detect tumour contamination was 10(-6). PBSC harvests from 45 BC patients were tested with both RT-PCR methods and the results were compared with immunocytochemistry (ICC). The five samples found positive by ICC were also positive by one tube RT-PCR; in addition, 11 more samples were positive by one tube RT-PCR analysis. The greater number of PBSC found positive by one tube RT-PCR might be due to the larger number of cells analysed. We conclude that one tube RT-PCR is sensitive and reveals no false positive results. This method is less time consuming than the nested one, technically simpler and should be considered for tumour cell detection.

Biomarkers, Tumor↗
PubMed1998

Stem cell enumeration in cord blood vs bone marrow and peripheral blood.

Recent reports have suggested that the total number of autologous or allogeneic hematopoietic stem cell (HSC) infused after high-dose chemotherapy might predict survival, post-transplant morbidity and rate of hematopoietic engraftment. However, HSC capable of long-term multilineage potential are still poorly defined, and tools for accurate and reproducible HSC enumeration are highly warranted.

ADP-ribosyl Cyclase↗
PubMed1998

Autologous platelet transfusion in patients receiving high-dose chemotherapy and circulating progenitor cell transplantation for stage II/III breast cancer.

BACKGROUND AND OBJECTIVE: Concerns about the risk of transfusion therapy are driving towards new strategies which are designed to minimize exposure to allogeneic blood products. We aimed to find out whether it is possible to support the phase of thrombocytopenia following high-dose chemotherapy (HDC) and circulating progenitor cells (CPC) transplantation by autologous platelet concentrates (PC). DESIGN AND METHODS: PC were collected from 32 patients undergoing HDC and CPC transplantation for stage II/III breast cancer. A single plateletpheresis was performed at rebound after high-dose cyclophosphamide, when platelet count exceeded 250 x 10(9)/L. PC were cryopreserved in 5% DMSO after controlled-rate freezing and stored in liquid nitrogen. In vitro studies of cryopreserved platelets (aggregation, ATP release and change of mean platelet volume induced by EDTA) were performed. When platelet counts dropped below 20 x 10(9)/L following HDC (thiotepa 600 mg/m2, L-PAM 160 mg/m2) and CPC transplant (CD34+ cells > 5 x 10(6)/kg), PC were thawed in a 37 degrees C water bath, centrifuged to remove DMSO, resuspended in autologous plasma and reinfused within one hour. RESULTS: Large quantities of platelets were harvested in all patients (median 6.6 x 10(11), range 4.8-12.2). In vitro studies showed preserved platelet function as compared to both fresh platelets and standard PC. Twenty-eight out of 32 patients received autologous PC. At the time of transfusion most of the patients were febrile (> 38 degrees C) and had mucositis > G2. The median number of platelets reinfused was 3.8 x 10(11) (range 2.0-8.1) with a median loss during the freeze-thaw-wash procedure of 37%. Autotransfusion was able to maintain platelet count above 20 x 10(9)/L in most patients, with a corrected count increment > 7.5 in 20 cases. Four patients required one additional allogeneic transfusion, two because of a poor increment and two due to a late-occurring epistaxis. No side effects related to PC infusion were recorded. Sixteen control patients who received the same HDC and a similar number of CD34+ cells required a total of 17 allogeneic PC units (1 patient did not require platelet transfusion). INTERPRETATION AND CONCLUSIONS: Our data demonstrate that large doses of autologous platelets can easily be collected and safely administered to support the period of thrombocytopenia in patients undergoing HDC and CPC transplantation. Autologous PC in these patients can abrogate the risks deriving from allogeneic platelet transfusion.

Adult↗
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

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↗
PubMed1994

Effects of granulocyte-macrophage colony-stimulating factor and interleukin-3 on small cell lung cancer cells.

Nonhematopoietic malignant cells may express receptors for hematopoietic growth factors and respond to these peptides. The aim of the present study was to investigate whether small cell lung cancer (SCLC) cells may be stimulated to proliferate by hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), which are currently used in clinical trials in combination with cytotoxic chemotherapy. We studied two SCLC cell lines, H69 and N417. The effects of GM-CSF and IL-3 were evaluated by studying clonal growth, 3H-thymidine incorporation, BUDR/DNA bivariate flow cytometry, c-myc and N-myc oncogene expression, and myeloid surface markers. Our experiments show that both GM-CSF and IL-3 can increase 3H-thymidine incorporation and cloning efficiency and reduce DNA synthesis time of H69 and, to a lesser extent, N417 cells, supporting the hypothesis that hematopoietic growth factors can stimulate the growth of some malignant nonhematopoietic cells in vitro. Further in vitro and in vivo studies are needed to determine whether clinical trials applying these factors for bone marrow recovery after chemotherapy of solid tumors may be hazardous by potentially stimulating growth of remaining tumor tissue.

Carcinoma, Small Cell↗
PubMed1993

Cell cycle kinetic effects of tamoxifen on human breast cancer cells. Flow cytometric analyses of DNA content, BrdU labeling, Ki-67, PCNA, and statin expression.

Tamoxifen is known to inhibit the growth of some human mammary carcinoma cells; this effect is accompanied by a decrease in the proportion of cells synthesizing DNA. In this work, flow cytometry of DNA and of bromodeoxyuridine labeling and the evaluation of the cell cycle-related antigens Ki-67, PCNA, and statin were used to investigate the changes in the proliferation kinetics of MCF-7 cells before and after treatment with 10(-7) M TAM. The treatment with TAM induced a significant decrease in the fraction of S-phase cells and an increase in those with a DNA content typical of G0/1 phase. The TAM-induced block in G0/1 is paralleled by a decrease in the frequency of cells expressing Ki-67 and PCNA, and by an increase in statin-positive (G0) cells. These results confirmed that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide the first experimental evidence that two main mechanisms are operating: the accumulation of cells in G1, before the onset of S-phase, and the exit of some cells from the cycling compartment.

Biomarkers, Tumor↗
PubMed1991

A study on the biological behavior of human brain tumors. Part II: Steroid receptors and arachidonic acid metabolism.

The significance of steroid receptors (SR) in human brain tumors is presently a field of intense investigation in order to clarify some aspects of the biological behavior of these neoplasms. We studied the relationship between the presence of steroid receptors and the production of metabolites of the arachidonic acid cascade which have been reported to have a role in the biological behavior of some human tumors. We found that some metabolites of arachidonic acid are produced in different amounts in brain tumors which either did or did not express some steroid receptors. In particular the PGE2 were higher in estrogen receptors (ER) positive meningiomas than in ER negative ones and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, is significantly higher in androgen receptors (AR) negative meningiomas than in AR positive ones. In neuroepithelial tumors the glucocorticoid receptors (GR) positive cases synthesized more TxB2 and less PGE2 than the GR negative ones. Our data seem to suggest that some correlations exist between the presence of some steroid receptors and arachidonic acid metabolite production.

Arachidonic Acid↗
PubMed1990

Characteristics and biological role of steroid hormone receptors in neuroepithelial tumors.

Tissue samples from 57 patients with neuroepithelial tumors (25 glioblastomas, 18 anaplastic astrocytomas, and 14 astrocytomas) were analyzed in order to evaluate the presence of estrogen, progesterone, glucocorticoid, and androgen receptors. Glucocorticoid- and androgen-specific binding proteins were present in 38.6% and 21.6% of the cases, respectively. Only a few tumors showed estrogen or progesterone receptors. A correlation was found between grade of anaplasia, patient's sex and age, and presence of glucocorticoid and androgen receptors. The biological role of these two receptors was investigated in 10 primary cell cultures derived from neuroepithelial tumors. For this purpose, dexamethasone and testosterone were added to culture medium at different concentrations (from 50 to 0.016 micrograms/ml). A significant stimulation of the cell growth was observed in four of five glucocorticoid receptor-positive cultures when dexamethasone in doses ranging from 2 to 0.016 microgram/ml was added to the culture. No modulation of the growth was observed in glucocorticoid receptor-negative cultures at the same doses. Higher dexamethasone doses induced a significant decrease of the growth index independently from the glucocorticoid receptor status. All of the cultures tested for testosterone activity were negative for androgen receptors. This hormone induced an inhibition of the growth index at doses ranging from 50 to 0.4 micrograms/ml. The data suggest that neuroepithelial tumors contain specific glucocorticoid and androgen binding proteins. Glucocorticoid receptors modulate the growth of cultured neuroepithelial tumors in the presence of different concentrations of dexamethasone.

Adult↗
PubMed1990

[Qualitative and quantitative histochemical evaluation of the dehydrogenase activity in human glioblastoma cells treated with beta-interferon].

In an attempt to clarify the role of beta interferon in vitro cell systems, the metabolic expression of dihydrofolate reductase (DHFR), succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), by histochemical methods was studied. DHFR was also quantified by flow cytometry. Glioblastoma cell line with or without beta interferon was used. Three days after the treatment the DHFR reaction was much less intense than in the control. Quantitative data confirmed these results. Immediately afterwards, most cells exhibited much more intense reaction. These facts underlined that, in this biological model in vitro, beta interferon could reduce the synthesis of these enzymes only for a short period.

Brain Neoplasms↗