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A Stacchini

Publications and source records attributed to A Stacchini.

At least 19 recordsLinked to original sources
PubMed2003

Flow cytometry in the bone marrow staging of mature B-cell neoplasms.

BACKGROUND: Even though flow cytometric (FC) analysis of bone marrow aspirates is often performed in hematolymphoid disorders at diagnosis and during disease monitoring, its role has not been defined during the staging of B-non-Hodgkin's lymphoma (B-NHL) and B-cell lymphoproliferative diseases. The goal of this study was to provide an objective evaluation of how FC might help in the detection of bone marrow involvement by the different types of B-cell malignant neoplasms. METHODS: Fifty-four staging and 156 restaging bone marrow biopsies and bone marrow aspirates, obtained from 185 consecutive patients, were analyzed retrospectively. The results of the morphologic examination and FC were reviewed independently, and their ability to detect bone marrow involvement was compared. RESULTS: FC and morphology agreed in 176 cases (83.8%), i.e., both showed 77 positive cases and 99 negative ones. Discrepant results were obtained in 30 cases (14.2%) in which morphologic examination showed 25 (11.9%) positive cases, whereas FC showed no evidence of disease. FC detected involvement in five cases (2.4%) in the presence of a histologically negative bone marrow biopsy. All morphologically undetermined bone marrow cases (four) were negative by FC. CONCLUSIONS: Neither morphologic examination nor FC alone is adequate for the detection of all cases of B-lymphoid neoplasm bone marrow involvement. FC failed to detect bone marrow involvement in those B-NHL cases having focal paratrabecular infiltration, but proved to be more sensitive than histology in detecting small clonal B-cells in B-NHL, which demonstrated fewer than 5% neoplastic infiltrates. The clinical relevance of minimal disease detected by FC alone needs further evaluation because staging of lymphomas currently is based only on morphologic data.

Biopsy↗
PubMed2001

Evaluation of rapid methods for the determination of okadaic acid in mussels.

AIMS: Two different screening methods, a Buffalo Green Monkey cytotoxicity test and a biosensor test, have been considered to replace the official mouse bioassay in monitoring for okadaic acid (OA) levels in mussels. METHODS AND RESULTS: Diarrhoetic shellfish poison-contaminated mussels from the Adriatic Sea were assayed in parallel by means of the mouse bioassay and both alternative methods. Both the cytotoxicity test and the biosensor test showed high sensitivity (OA 0.01 mg g-1 hepatopancreas and 0.002 mg g-1 hepatopancreas, respectively) and a high correlation with the mouse bioassay (r=0.932, P < 0.001 and r=- 0.850, P < 0.001, respectively). CONCLUSION: Both methods are efficacious, quick, inexpensive and provide data on the amount of toxin present in mussels. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods, besides allowing the simultaneous assay of a great number of samples, comply with the ethical need to reduce the use of animals in the laboratory.

Acid Phosphatase↗
PubMed2001

Detection of Vibrionaceae in mussels and in their seawater growing area.

AIMS: The seasonal trend and frequency of detection of Vibrionaceae in seawater samples and in molluscs collected in the Adriatic Sea was measured. METHODS AND RESULTS: Over a 2-year period, 726 bacterial strains were isolated, of which 46.9% belonged to the Vibrio genus, 29.8% to the Aeromonas genus and the remaining 23.3% was made up of the Pseudomonas, Flavobacterium, Pasteurella, Agrobacterium and Ochrobacterium genera. Many of the isolated strains were shown to produce toxins. CONCLUSION: The Vibrio genus, which was isolated more often than the other genera, was particularly prevalent in summer (54.4% of the total number of bacteria isolated during this season), while it was scarce in the winter months.

Aeromonas↗
PubMed1999

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation.

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

Amino Acid Sequence↗
PubMed1997

A rapid tissue culture assay for the detection of okadaic acid and related compounds in mussels.

Okadaic acid, the main toxin of the diarrhoetic shellfish poisoning (DSP) group, shows high cytotoxicity to Buffalo green monkey (BGM) kidney cell cultures after different times according to concentration. A rapid method of detecting the presence of okadaic acid and related compounds in contaminated mussels by means of a cytotoxicity test is presented. The method is based on direct microscopic observation of toxin-induced morphological changes in BGM cell cultures. A high correlation (r = 0.950; P < 0.001) was found between the official mouse bioassay and the cytotoxicity test conducted on naturally contaminated samples of Mytilus galloprovincialis.

Animals↗
PubMed1997

Fludarabine ability to down-regulate Bcl-2 gene product in CD5+ leukaemic B cells: in vitro/in vivo correlations.

CD5+ B-chronic lymphocytic leukaemia (B-CLL) and mantle cell lymphoma (MCL) in leukaemic phase are characterized by defects in cell death induction that primarily involves the Bcl-2 family of genes. Fludarabine (9-beta-D-arabinofuranosyl-2-fluoradenine, F-ara-A) is a potent inducer of apoptosis in CLL cells. This study aimed to determine whether F-ara-A-induced apoptosis might be related to Bcl-2 modifications and to evaluate in vitro/in vivo correlations. Peripheral blood lymphocytes from eight B-CLL and four leukaemic MCL were cultured in the presence of different concentrations of F-ara-A +/- methylprednisolone (MP). F-ara-A down-regulated the expression of Bcl-2 in 5/12 cases. mRNA down-regulation was maximal at 48 h; protein down-regulation was prominent after 48 h. Both events were dose-dependent. The amount of apoptosis was significantly higher in the samples treated with F-ara-A than in those exposed to MP alone. In the seven remaining cases, no Bcl-2 down-regulation was observed after exposure to F-ara-A and the degree of F-ara-A-induced apoptosis overlapped that induced by MP. The in vivo outcome after treatment with three to six courses of F-ara-A was evaluable in 10 patients: 4/5 cases, whose cells had shown in vitro Bcl-2 down-regulation and prominent apoptosis after exposure to F-ara-A, had a complete response (CR) and a partial response (PR) was observed in the remaining patient. Of the five patients whose cells had shown no in vitro Bcl-2 modulation after exposure to F-ara-A, two had a PR, but the other three did not show any in vivo clinical response.

Aged↗
PubMed1996

Flow cytometric detection and quantitative analysis of the GM-CSF receptor in human granulocytes and comparison with the radioligand binding assay.

A flow cytometric method to quantify the Granulocyte-macrophage colony-stimulating factor receptor (GM-CSFr) on human cells is described. The number of GM-CSFr binding sites on human neutrophils was assessed by using different bead standards. Results were compared with those from conventional receptor quantification, which was performed by using the radioligand binding assay. A high degree of correlation was found between the two methods, although quantitative evaluation of GM-CSFr expression on neutrophils performed by flow cytometry revealed a somewhat higher number of receptor molecules per cell than with that determined by Scatchard analysis. By the flow quantitative approach, we measured the GM-CSFr on mobilized CD34-positive cells and obtained results similar to those of previously published data. Our data suggest that flow cytometric analysis is a simple and reproducible method to detect and quantify the presence of GM-CSFr per cell, thus allowing the study of receptor expression on different populations selected by gating on the basis of the scatter parameters and surface markers. This assay offers the possibility to quantify the presence of GM-CSFr on different subsets of normal and pathological cells even if samples are too small (such as CD34-positive progenitor cells) for measurement with the radioligand binding assay.

Antigens, CD34↗
PubMed1996

Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion.

Several cytokines have been suggested to play a regulatory action on the neoplastic clone of patients with B-cell chronic lymphocytic leukemia (B-CLL) by interfering in the differentiation, proliferation, or death/survival pathways. Interleukin-8 (IL-8) is a chemoattractant protein constitutively expressed at the mRNA level and released by B-CLL cells. In view of the presence of the IL-8 receptor mRNA and of specific IL-8 binding, confirmed also by Scatchard analysis using 125I-IL-8, the study was extended to evaluate the possible regulatory role of this cytokine on B-CLL cells. IL-8 failed to show any in vitro proliferative effect on leukemic B-CLL cells. By contrast, the propidium iodide (PI) staining of the DNA content showed that IL-8 could prolong the survival of resting B-CLL cells in 11 of 16 cases studied. In the remaining 5 cases, 90.6% +/- 4.39% SD of the cells after culture remained viable and IL-8 could exert a significant death protection action after pretreatment with 10(-4) mol/L hydrocortisone, which reduced the percentage of viable B-CLL cells. The dose range of IL-8 capable of inducing the prolonging survival effect is comparable with the levels of IL-8 released constitutively by B-CLL cells, indicating that the death protection action is exerted at physiologic doses. The in vitro rescue from death induced by IL-8 is reflected by an increased expression of bcl-2 mRNA in B-CLL cases incubated in the presence of IL-8. These findings were further confirmed at the protein level, because in B-CLL cells that displayed a bimodal bcl-2 intracytoplasmatic protein expression IL-8 was capable of upmodulating the bcl-2high expression peak. The potential autocrine regulatory action exerted by IL-8 is supported by the evidence that exogenous IL-8 can upregulate IL-8 mRNA in B-CLL cells. These results, together with the demonstration that antibody-mediated neutralization of endogenous IL-8 could induce a significant in vitro reduction in the number of living cells, further support the hypothesis that, in B-CLL, the physiologic doses of IL-8 released constitutively by the leukemic clone may play an autocrine role in the process of cell accumulation characteristic of this disease.

Antigens, CD↗
PubMed1996

In leukaemic CD5+ B cells the expression of BCL-2 gene family is shifted toward protection from apoptosis.

This study further investigated the mechanisms that control apoptosis in leukaemic CD5+ B cells, and focused on the Bcl-2 gene family. The pattern of expression of Bcl-2, Bcl-xL, Bcl-xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5+ B-cell chronic lymphoid leukaemias. Cells from 34 patients with chronic lymphoid leukaemia of B-cell type (23 B-chronic lymphocytic leukaemia (B-CLL) and 11 mantle cell lymphoma (MCL) in leukaemic phase) were investigated. High levels of Bcl-2 mRNA were observed by Northern blot and high levels of Bcl-2 protein were detected by cytofluorograph analysis with a specific monoclonal antibody (MAb) in all cases. Strong Bax expression was detected by RT-PCR in 20/23 B-CLL cases; Bax was also observed in 8/11 MCL in leukaemic phase with variable degree of intensity. In both B-CLL and MCL samples the presence of Bax protein was confirmed by cytofluorograph analysis. RT-PCR detected high levels of Bcl-xL in 16/23 B-CLL and in 8/11 MCL in leukaemic phase, whereas Bcl-xS was detectable in low to trace amounts respectively in 13/23 B-CLL and in 6/11 MCL in leukaemic phase. According to the functional role of Bcl-2, Bcl-xL, Bcl-xS and Bax, these data indicate that the pattern of Bcl-2 family genes expression in leukaemic CD5+ B cells is skewed toward prevention of apoptosis and may thus favour the relentless accumulation of CD5+ leukaemic B cells.

Apoptosis↗
PubMed1996

The effects of human FLT3 ligand on in vitro human megakaryocytopoiesis.

The human homolog of the murine flt3/flk2 gene product is a tyrosine kinase receptor that plays a role in regulating the proliferation and differentiation of cells in the hematopoietic system. Using a plasma-clot clonal assay and a long-term bone marrow culture (LTBMC) system, we studied the effects of the recently cloned human flt3 ligand (FL) alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or stem cell factor (c-kit ligand [KL]) on human megakaryocytopoiesis. The effects of FL on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. FL alone had no megakaryocytic colony-stimulating activity (MK-CSA), but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. FL synergized with IL-3 at the level of both CFU-MK and BFU-MK and with GM-CSF and KL at the level of CFU-MK. Although FL alone exhibited a limited potential in sustaining long-term megakaryocytopoiesis in vitro, it synergistically augmented the ability of IL-3 and KL, alone or in association, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that FL plays a significant role in this process by amplifying the MK-CSA of GM-CSF, IL-3, and KL.

Bone Marrow Cells↗
PubMed1996

Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts.

The stem cell tyrosine kinase 1 (STK1) protein is the human homologue of the murine FLT3 gene product, a receptor belonging to the FMS/KIT family. FLT3 and KIT with their ligands control the growth and differentiation of early human hemopoietic cells. In the present study, 16 cases of acute myeloid leukemia (AML) were examined by flow cytometry for cell surface expression of FLT3 and KIT receptors. All cases were also tested for their proliferative response to human FLT3 ligand (FL) and KIT ligand (KL) and for colony formation in the presence of single or associated cytokines. Among 16 AML cases tested, 10/16 expressed FLT3 receptor and 12/16 expressed KIT receptor, without any correlation with FAB subtype. FL and KL stimulated the proliferation of leukemic blasts in 11/16 AML cases (including five FLT3 or KIT receptor-negative cases), with an additive effect when added simultaneously. By contrast, some receptor-expressing AMLs did not display significant proliferative responses to their respective ligands. FL and KL as single factors induced or significantly increased the colony formation by clonogenic precursor cells respectively in eight and six of 13 cases tested. In some cases growth factor association significantly enhanced colony growth. Taken together these observations provide evidence that the pattern of FLT3 and KIT receptor expression is extremely variable among the AMLs and that receptor presence is not necessarily combined with proliferative and clonogenic response or vice versa.

Blast Crisis↗
PubMed1995

Effects of human FLT3 ligand on myeloid leukemia cell growth: heterogeneity in response and synergy with other hematopoietic growth factors.

A novel hematopoietic growth factor for primitive hematopoietic progenitor cells, the ligand for the flt3/flk2 receptor, (FL), has been recently purified and its gene has been cloned. In the present study, we investigated the effects of FL on the proliferation and differentiation of normal and leukemic myeloid progenitor cells. We demonstrate that FL is a potent stimulator of the in vitro growth of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or G-CSF-dependent granulocyte-macrophage committed precursors from Lin- CD34+ bone marrow cells of normal donors. By contrast, FL does not affect the growth of erythroid-committed progenitors even in the presence of erythropoietin. The effect of FL on the proliferation and on the in vitro growth of clonogenic leukemic precursor cells was studied in 54 acute myeloid leukemia (AML) cases. Fresh leukemia blasts from 36 of 45 patients with AML significantly responded to FL without any relation to the French-American-British (FAB) subtype. FL stimulated the proliferation of leukemic blasts in a dose-dependent fashion. Synergistic activities were seen when FL was combined with G-CSF, GM-CSF, IL-3, or stem cell factor (SCF). FL as a single factor induced or increased significantly colony formation by clonogenic precursor cells from 21 of 24 patients with AML. In the presence of suboptimal and optimal concentrations of G-CSF, GM-CSF, IL3, SCF, or a combination of all factors, FL strongly enhanced the number of leukemic colonies (up to 18-fold). We also evaluated the induction of tyrosine phosphorylated protein on FL stimulation in fresh AML cells. We demonstrate that, on FL stimulation, a band of phosphorylated protein(s) of about 90 kD can be detected in FL-responsive, but not in FL-unresponsive cases. This study suggests that FL may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor with other cytokines.

Cell Division↗
PubMed1995

Influence of the extraction procedure on recovery of okadaic acid from experimentally contaminated mussels.

Hepatopancreas samples from mussels (Mytilus galloprovincialis) experimentally contaminated with okadaic acid were analysed with Yasumoto's mouse bioassay and HPLC. A likely effect of some components of the hepatopancreas on the results (matrix effect) was evaluated, and a possible loss of toxin during the extraction phase was quantified. Experiments were conducted by comparing two different extraction procedures. Under our experimental conditions, the results obtained from mouse bioassay showed no matrix effect with either procedure. A certain quantity of the actual amount of okadaic acid contained in the sample was found to be lost after the extraction, i.e. 10.2-17.0% in samples extracted with acetone alone and 9.8-18.5% in samples extracted with acetone and ether.

Acetone↗
PubMed1995

Detection of diarrhoetic shellfish toxins in mussels from Italy by ionspray liquid chromatography-mass spectrometry.

Direct detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and some of their related compounds in toxic mussels (Mytilus galloprovincialis) is reported using ionspray liquid chromatography-mass spectrometry (LC-ISP-MS). This was employed to analyse diarrhoetic shellfish poisoning (DSP) toxins in mussels collected from coastal areas of the northern and southern Adriatic Sea. DTX-1 was found in some samples from both the northern and southern Adriatic and this is the first report of the unambiguous identification of this toxin in Italian mussels. The low levels found indicate that this toxin did not play a significant role in toxicity in these samples. Okadaic acid was found in all the mussels examined, although its concentration was not always sufficient to account for DSP toxicity. Furthermore, two related compounds of OA were detected in all the samples and one related DTX-1 compound was observed in some samples from the northern Adriatic. All three compounds are still to be identified, but it is possible that these substances are involved in mussel DSP toxicity in the Adriatic Sea.

Animals↗
PubMed1995

In vivo effect of human granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophil GM-CSF receptors.

The effect of in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophils GM-CSF receptor, was investigated in patients with neoplastic diseases and normal hematopoiesis. Patients were divided into two groups. Group A received a single dose of rhGM-CSF (5 micrograms/kg/day) and receptor studies were performed 90 min and 48 h after treatment. Group B received three doses, administered subcutaneously every 24 h and receptor studies were performed 90 min after first injection and 24 h after the last. Before treatment neutrophils only displayed high-affinity receptors (KD 85 +/- 53 pM; number of receptors/cell 1318 +/- 567). The first injection of rhGM-CSF produced a transient leucopenia and the internalization of GM-CSF receptor on neutrophils in both groups of patients: 90 min after s.c. administration receptors could not be detected with conventional binding studies. In group A patients, 48 h after a single dose of rhGM-CSF, receptors, albeit with a decreased affinity (KD = 240 +/- 131 pM; number of receptors/cell 783 +/- 494) were again expressed. In group B patients, 24 h after the last rhGM-CSF injection, low intermediate affinity receptors not present before treatment appeared (KD 720 +/- 175 pM; number of receptor/cell 1222 +/- 179). They were associated with a low number of high affinity receptors (KD = 9 +/- 4 pM; number of receptors/cell 106 +/- 44). These observations indicate that more than one type of GM-CSF receptor may exist on neutrophils. It may be suggested that in vivo the regulation of the GM-CSF receptor is different from that in vitro and is related to the presence of the cytokine in patient blood.

Dose-Response Relationship, Drug↗
PubMed1995

Blood cell redistribution in the lung after administration of recombinant human granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), in addition to being a haematopoietic growth factor, has been shown to stimulate in vitro the production of interleukins 1, 6 and 8 (IL-1, IL-6 and IL-8), tumour necrosis factor-alpha (TNF-alpha) and GM-CSF by polymorphonuclear cells (PMNs), alveolar macrophages (AMs), fibroblasts and endothelial cells of the lung, and the growth and differentiation of resident alveolar macrophages. The aim of this study was to establish whether recombinant GM-CSF (rhGM-CSF), administered subcutaneously at a dose of 5 micrograms.kg-1 for 3 days in five patients with unresectable non-small cell lung cancer before starting chemotherapy, induces an increase in the alveolar cell count, and whether these cellular lung variations may be related to increases in the above-mentioned cytokines. In the bronchoalveolar lavage fluid (BALF) total cell count, polymorphonuclear cells, neutrophils, and alveolar macrophages increased significantly in comparison with the baseline, and the extent of variation of the BAL cell count was considerably greater than that of the circulating leucocytes. The mean levels of all the cytokines increased, but a significant difference with respect to the basal condition was observed only for IL-6 and IL-8. After rhGM-CSF treatment, significant correlations were found between neutrophil counts and the levels of IL-6 and IL-8. In conclusion, rhGM-CSF administration induces a cellular expansion in the lung, and the neutrophil increase appears to be related to increased levels of IL-8.

Aged↗