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Biomedical subjects

F Falzetti

Publications and source records attributed to F Falzetti.

At least 19 recordsLinked to original sources
PubMed2000

Limited engraftment capacity of bone marrow-derived mesenchymal cells following T-cell-depleted hematopoietic stem cell transplantation.

The engraftment capacity of bone marrow-derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell-depleted allograft from human leukocyte antigen (HLA)-matched or -mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit-F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 +/- 0.13 x 10(4)/kg vs 1. 19 +/- 0.19 x 10(4)/kg; P >/=.97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell-depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

Adult↗
PubMed2000

Peripheral blood progenitor cell mobilization in healthy donors receiving recombinant human granulocyte colony-stimulating factor.

OBJECTIVE: We analyzed the incidence of primitive (LTC-IC) and committed (CFU-mix, BFU-E, CFU-GM) hematopoietic progenitors detected under steady-state conditions and upon progenitor cell mobilization in a cohort of healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF). MATERIALS AND METHODS: Healthy donors (n = 30) of HLA-mismatched or -matched stem cell transplants were mobilized with rhG-CSF (8 microg/Kg body weight subcutaneously twice daily until completion of leukapheresis). PBPC collections were started after 4 days of rhG-CSF therapy. RESULTS: Steady-state incidence of bone marrow LTC-IC, but not committed progenitors, significantly correlated with the numbers of mobilized CD34+ cells (r = 0.6, p = 0.004), CFU-GM (r = 0.79, p = 0.0005) and CFC (r = 0.76, p = 0.001) detected after 4 days of rhG-CSF therapy. Statistically significant correlations were also found between steady-state blood CFU-GM and peak numbers of CD341 cells (r = 0.68, p = 0.001), numbers of day 4 CD341 cells (r = 0.52, p = 0.005), CFU-GM (r = 0.63, p = 0.002), and CFC (r = 0.61, p = 0.003). CONCLUSION: Our data show that in normal volunteers baseline marrow LTC-IC and blood CFU-GM correlate with rhG-CSF-mobilized PBPC. The potential clinical relevance of these findings in the identification of poor mobilizers will be tested in a prospective study.

Adult↗
PubMed2000

T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection.

Generation of an efficient graft-versus-leukemia (GVL) effect in patients with hematological malignancies who relapse after allogeneic bone marrow transplantation depends in part upon the number of infused T lymphocytes. Currently, a GVL reaction cannot be achieved without inducing concomitant graft-versus-host disease (GVHD); thus, one strategy is to try to modulate this GVL/GVHD ratio. We engineered human T lymphocytes with herpes simplex virus-thymidine kinase and neomycin resistance genes, with an LXSN-derived vector that confers a ganciclovir-specific sensitivity to the transduced T cells. We analyzed proliferation, interleukin-2 production, alloreactivity in a mixed lymphocyte culture, and clonogenicity during the different stages of retroviral infection and G418 selection. Our results confirm that a sufficient number of transduced T lymphocytes can be obtained after selection for clinical studies. Their proliferative activity, alloresponsiveness, and ability to produce and respond to interleukin-2 were retained. Compared with control populations, their clonogenicity, as assessed by limiting dilution assays, was reduced after retroviral infection and G418 selection by 1.6 and 2.9 logs, respectively, with both viral supernatant incubation and coculture procedures. This study shows that infection and selection with the thymidine kinase-neomycin resistance gene retroviral vector significantly reduces the number of functional T lymphocytes. This finding should be taken into account when establishing the dose of T lymphocytes necessary to trigger a modulated GVL/GVHD effect.

Anti-Bacterial Agents↗
PubMed1999

T lymphocyte transduction with herpes simplex virus-thymidine kinase (HSV-tk) gene: comparison of four different infection protocols.

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping. tk gene expression was the same in all transduced selected populations, as indicated by gcv sensitivity. Transduction efficiency was evaluated by semiquantitative PCR. Using the standard supernatant infection method, the rate of infection was extremely low (<5%). After adding the centrifugation step or performing supernatant infection on fibronectin fragment-coated wells, PCR analysis showed a 30%-40% rate of transduced cells. After infection by cocultivation, the rate of transduced cells was 30%-40%. These results demonstrate that supernatant infection plus centrifugation, supernatant infection on fibronectin fragment-coated wells, and cocultivation methods provide equivalent rates of transduced cells. The lack of reproducibility and safety indicates that cocultivation is not suitable for clinical studies. In our view, supernatant infection plus centrifugation is easier to perform than using fibronectin fragments, and it is currently the optimal method for clinical studies when large quantities of T lymphocytes are being processed.

Cell Division↗
PubMed1998

Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype.

BACKGROUND: In this study we tried to achieve successful transplantation in patients with acute leukemia with the use of hematopoietic stem cells from donors who shared only one HLA haplotype with the recipient (a "full-haplotype mismatch"). To prevent graft failure, large doses of T-cell-depleted hematopoietic stem cells were transplanted after a conditioning regimen of enhanced myeloablation and immunosuppression was administered to the recipient. METHODS: Forty-three patients with high-risk acute leukemia who were scheduled for transplantation received total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. The graft consisted of peripheral-blood progenitor cells that had been mobilized in the donor with recombinant granulocyte colony-stimulating factor and also, in 28 cases, bone marrow. Bone marrow from the donor was depleted of T lymphocytes by processing with soybean agglutinin and E-rosetting. T-cell depletion of peripheral-blood mononuclear cells was achieved by E-rosetting followed by positive selection of CD34+ cells. No post-transplantation prophylaxis against graft-versus-host disease (GVHD) was administered. RESULTS: In all the patients, full donor-type engraftment was achieved. In none of the patients who could be evaluated did acute or chronic GVHD develop. Regimen-related toxicity was minimal. Eleven of the 23 patients with acute lymphoblastic leukemia had a relapse, as did 2 of the 20 patients with acute myeloid leukemia. Transplantation-related mortality was 40 percent. After a median follow-up of 18 months (range, 8 to 30), 12 of the 43 patients were alive and free of disease. All surviving patients had a good quality of life. CONCLUSIONS: The main limitations of transplantation of bone marrow from donors who are matched with the recipient for only one HLA haplotype GVHD and graft failure - can be overcome. Since most patients have a relative with one haplotype mismatch, advances in this method will increase the availability of hematopoietic-cell transplantation as curative therapy for acute leukemia.

Adolescent↗
PubMed1998

Mismatched T cell-depleted hematopoietic stem cell transplantation for children with high-risk acute leukemia.

The aim of this study was to extend allogeneic hematopoietic stem cell transplantation to leukemia patients without a matched donor. To prevent graft failure, large doses of T cell-depleted hematopoietic stem cells were transplanted following a highly myeloablative and immunosuppressive conditioning regimen. Fifteen children with high-risk acute leukemia received T cell-depleted hematopoietic stem cells from full-haplotype mismatched family members after a conditioning regimen that included single-dose TBI, thiotepa, ATG and fludarabine. To prevent GVHD, marrow cells were T-depleted by soybean agglutinin and E-rosetting, peripheral blood cells by E-rosetting followed by positive selection of the CD34+ cells. No post-transplant prophylaxis for GVHD was administered. In all patients full donor-type engraftment was achieved. None of the evaluable patients developed either acute or chronic GVHD. Regimen-related toxicity was minimal. Five patients are alive and event-free at a median follow-up of 18 months (range 13-28). All surviving patients have a good quality of life. Seven patients have relapsed. This study shows that GVHD and graft failure, which limited the use of full-haplotype mismatched bone marrow transplants, have been overcome. Since almost all children have a mismatched relative, advances in this area should make mismatched transplants a routine consideration for patients with high-risk leukemia without a matched related or unrelated donor.

Adolescent↗
PubMed1997

Distribution of active alpha- and beta-subunits of beta-N-acetylhexosaminidase as a function of leukaemic cell types.

beta-N-Acetylhexosaminidase isoenzymes, and the distribution of the alpha- and beta-subunits forming the enzyme in a representative series of fresh leukaemic cells and in established leukaemic cell lines, were obtained by using a combination of DEAE-cellulose chromatography and assay with the fluorogenic substrates 4-methylumbelliferyl-beta-N-acetylglucosaminide hydrolyzed by both alpha and beta subunits, and 4-methylumbelliferyl-beta-N-acetylglucosaminide-6-SO4 hydrolyzed only by hexosaminidase isoenzymes containing alpha-subunits. The presence of hexosaminidase S (the alpha alpha dimer), was found in all the leukaemic cell populations we surveyed, but not in normal human cells. The presence of this isoenzyme can therefore be considered as an additional marker of leukaemic cells. A prevalence of hexosaminidase A and A-like intermediate forms (alpha beta structure), characterize leukaemic cells of myeloid origin, whereas greater amounts of hexosaminidase B and B-like intermediate forms (beta beta structure), were consistent attributes of leukaemic cells of lymphoid origin. An over-expression of beta-subunits in blasts might be related to their undifferentiated status. These changes in the isoenzymes of hexosaminidase may prove informative about a variety of changes in the biology of leukaemic cells that could range from chromosomal alterations to changes in the proteolytic processing and glycosylation.

Hexosaminidase A↗
PubMed1997

Stem cell mobilization in normal donors.

We studied peripheral blood and apheresis samples from 39 consecutive normal donors who were parents or siblings of patients who received matched or mismatched bone marrow transplants using a combination of rhG-CSF-mobilized peripheral blood stem cells (PBSCs) and bone marrow (BM). BM was harvested from donors 1-7 days before starting rhG-CSF treatment: 12 micrograms/kg/day rhG-CSF was administered by continuous s.c. infusion for 4-7 days. Peripheral blood progenitor cells were harvested by leukapheresis using an automated continuous-flow blood cell separator, beginning on day 4 of rhG/CSF, for 1-4 consecutive days. Peak peripheral blood CD34+ cell and CFU-GM levels were reached simultaneously on day 5 or 6 of rhG-CSF administration. Median peak levels were 1.65% for CD34+ cells (range 0.34%-4.7%) and 142 CFU-GM/10(5) plated cells (range 16-700). The greatest numbers of CD34+ cells and CFU-GM, expressed per liter of blood volume processed, were harvested during the second and third leukapheresis: CD34+ cells 37.77 +/- 25.48 x 10(6) and CFU-GM 3.32 +/- 2.51 x 10(6) during the second leukapheresis, and CD34+ cells 37.01 +/- 16.33 x 10(6) and CFU-GM 3.82 +/- 4.36 x 10(6) during the third. The number of CD34+ cells and CFU-GM did not correlate with the sex, age, or body weight of the donors. This study indicates that this protocol for administration of rhG-CSF mobilizes large numbers of hematopoietic progenitor cells into the peripheral blood and that bone marrow harvesting before G-CSF administration does not impair stem cell mobilization.

Adult↗
PubMed1994

Successful engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum.

Patients who undergo transplantation with haploidentical "three-loci" mismatched T-cell-depleted bone marrow (BM) are at high risk for graft failure. To overcome the host-versus-graft barrier, we increased the size of the graft inoculum, which has been shown to be a major factor in controlling both immune rejection and stem cell competition in murine models. Seventeen patients (mean age, 23.2 years; range, 6 to 51 years) with end-stage chemoresistant leukemia were received transplants of a combination of BM with recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells from HLA-haploidentical "three-loci" incompatible family members. The average concentration of colony-forming unit-granulocyte-macrophage in the final inoculum was sevenfold to 10-fold greater than that found in BM alone. The sole graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion of the graft by the soybean agglutination and E-rosetting technique. The conditioning regimen included total body irradiation in a single fraction at a fast dose rate, antithymocyte globulin, cyclophosphamide and thiotepa to provide both immunosuppression and myeloablation. One patient rejected the graft and the other 16 had early and sustained full donor-type engraftment. One patient who received a much greater quantity of T lymphocytes than any other patient died from grade IV acute GVHD. There were no other cases of GVHD > or = grade II. Nine patients died from transplant-related toxicity, 2 relapsed, and 6 patients are alive and event-free at a median follow-up of 230 days (range, 100 to 485 days). Our results show that a highly immunosuppressive and myeloablative conditioning followed by transplantation of a large number of stem cells depleted of T lymphocytes by soybean agglutination and E-rosetting technique has made transplantation of three HLA-antigen disparate grafts possible, with only rare cases of GVHD.

Adolescent↗
PubMed1993

Sustained complete hematological remission in essential thrombocythemia after discontinuation of long-term alpha-IFN treatment.

In essential thrombocythemia patients alpha-IFN rapidly reduces platelet count, and it is also able to maintain a low count during long-term treatment. In order to verify if long-term IFN treatment can produce sustained remission in selected patients, we decided to suspend IFN treatment in two subsets of 21 patients on long-term alpha-IFN treatment: (a) all six patients who had shown a platelet count below 450 x 10(9)/l for at least 2 months with 3 MU once a week; (b) three patients who had shown the same platelet count for at least 2 months with 3 MU three times a week. After withdrawal of alpha-IFN treatment, a rapid increase in the platelet count was observed in all three patients requiring 3 MU three times a week. Three of the six patients treated with 3 MU once a week are still free of symptoms and have been in complete hematological remission (platelet count below 450 x 10(9)/l) for 9+, 13+, and 14+ months, respectively. As far as the three remaining cases are concerned, one was not assessable because of loss to follow-up, while the other two relapsed after 1 and 2 months. We believe that the three cases of sustained remission might be the result of a long-term tumor load reduction produced by the alpha-IFN treatment. Finally, the factor best able to predict sustained, unmaintained remission seems to be the clinical response to a low dose of alpha-IFN during the maintenance phase, rather than disease features prior to treatment.

Humans↗
PubMed1991

Lambda-like and V pre-B genes expression: an early B-lineage marker of human leukemias.

V pre-B and lambda-like genes are selectively expressed in human pre-B cells and encode polypeptide chains that associate in a mu-pseudolight chain complex that may regulate some crucial steps of early B-cell differentiation. We have followed by polymerase chain reaction and Northern blot analysis the expression of these "pre-B-specific" genes in correlation with the status (rearranged v germline) of Ig gene loci (H, kappa, lambda) in a panel of 32 leukemias pertaining mostly to the B lineage and including a number of ambiguously characterized samples. All cells that had rearranged the H locus only expressed V pre-B and lambda-like transcripts, in agreement with a pre-B status. In this group, some biphenotypic leukemias (mostly My/B) might, in fact, be already engaged in the B lineage. Rearrangement of V kappa or V lambda loci correlated with the disappearance of the pre-B gene products. In a pre-B acute lymphoblastic leukemia cell line that was induced to mature to the B-cell stage in culture upon kappa gene rearrangement, the mu-pseudolight chain complex was actually replaced by the classical mu-kappa molecule. Finally, V pre-B and lambda-like genes were found expressed in two leukemic cells that had retained all Ig loci in germline configuration. This finding raises the possibility of having an early pro-B progenitor in which V pre-B and lambda-like products associate with a H chain surrogate in a complex that would trigger an early event of B-cell differentiation such as the H locus rearrangements.

Antigens, CD↗
PubMed1991

Interferon alpha-2b in the long-term treatment of essential thrombocythemia.

We treated 35 patients affected with essential thrombocythemia (ET) with interferon (IFN) alpha-2b. Our treatment scheme consisted of (a) a 6-month induction phase and (b) a continuous maintenance phase. During the induction phase, our results showed that using 21 million units (mu) of IFN weekly platelet counts fell below 600 x 10(9)/1 in about 90% of patients. These data demonstrate that well-tolerated doses of IFN can rapidly correct excessive thrombocytosis. During the continuous maintenance phase, 61% of patients required 3 mu three times a week, 15% once a week, and 24% daily. Thus the minimal IFN doses able to maintain platelet count below 600 x 10(9)/l varied between 3 and 21 mu per week. During long-term treatment, subjective side effects were tolerable, especially using 3 mu three times a week. We conclude that IFN alpha-2b is an effective drug in the long-term treatment of ET.

Adult↗
PubMed1991

Activation of the granulocyte-monocyte colony stimulating factor gene in acute myeloid leukaemia cells is not related to gene rearrangement.

Several reports have documented that leukaemic blasts produce a number of cytokines among them the granulocyte-monocyte colony stimulating factor (GM-CSF). We analysed the structure of the gene that codes for GM-CSF in 44 acute myeloid leukaemia (AML) cases in an attempt to establish whether the autocrine production of GM-CSF was due to a structural gene alteration. No structural alteration was detected in the GM-CSF gene in any of the 44 cases studied. We, therefore, conclude that the autocrine production of GM-CSF by leukaemia blasts is not dependent on gene rearrangement.

Gene Expression Regulation, Neoplastic↗