Marco Vitale - Academia.edu (original) (raw)

Papers by Marco Vitale

Research paper thumbnail of Hydrogen sulfide prevents apoptosis of human PMN via inhibition of p38 and caspase 3

Laboratory Investigation, 2006

Hydrogen sulfide, together with carbon monoxide and nitric oxide, is now considered a gasotransmi... more Hydrogen sulfide, together with carbon monoxide and nitric oxide, is now considered a gasotransmitter able to induce specific cellular responses. As hydrogen sulfide is a component of several natural compounds known to be effective in many inflammatory pathologies, particularly of the respiratory tract, we studied its effects in vitro on the survival and bactericidal activity of purified human neutrophils. We found that (1) HS À ions promote the survival of granulocytes, but not that of lymphocytes or eosinophils, cultured in serum-free medium; (2) the pro-survival effect of HS À is due to inhibition of caspase-3 cleavage and p38 MAP kinase phosphorylation; (3) the bactericidal activity of neutrophils is not impaired by hydrogen sulfide. We conclude that HS À promotes the short-term survival of neutrophils potentially accelerating the resolution of inflammatory processes and preventing the occurrence of new ones.

Research paper thumbnail of All-trans retinoic acid shows multiple effects on the survival, proliferation and differentiation of human fetal CD34 + haemopoietic progenitor cells

British Journal of Haematology, 1995

Summary. To evaluate the effect of all-trans retinoic acid (RA) on fetal haemopoiesis, we perform... more Summary. To evaluate the effect of all-trans retinoic acid (RA) on fetal haemopoiesis, we performed serum-free liquid and semisolid cultures using CD34+ cells purified from mid-trimester human fetal blood samples. RA, at both physiological (10-n and 10-12M) and pharmacological (10-6 and l(r7M) concentrations, significantly (P<0.01) promoted the survival of fetal CD34+ cells in liquid cultures from day 3 onwards, by suppressing apoptosis induced by serum and growth factor deprivation. On the other hand, RA alone had no significant effect on the proliferation and differentiation of fetal haemopoietic progenitors.In the presence of optimal concentrations of recombinant interleukin-3 (IL-3), stem cell factor (SCF), granulocyte/ macrophage-colony stimulating factor (GM-CSF), and erythropoietin (Epo), low and high doses of RA induced striking differential effects on CD34+ cell proliferation in liquid cultures and colony formation in semisolid assays. In fact, 1CTU M and 1CT12M RA were able to: (i) significantly (P<0.05) increase 3H-thymidine uptake by fetal CD34+ cells in liquid cultures, and (ii) variably promote the growth of pluripotent (CFU-GEMM, P<0.05), early (BFU-meg) and late (CFU-meg, P<0.01) megakaryocyte, granulocyte/macrophage (CFU-GM. P<001) and erythroid (BFU-E) progenitors in semisolid cultures. On the contrary, 10-6 and 10-7 M RA induced: (i) an overall inhibition (P<0.01) of CD34+ cell growth in liquid cultures; (ii) a marked suppression of BFU-E colony formation (P<0.01) at all Epo concentrations examined (0-002-4IU/ml); and (iii) a significant (P<0.()1) stimulation of CFU-GM with a shift from mixed granulocyte/ macrophage to pure granulocyte colonies, whereas it had little effect on the growth of CFU-GEMM, BFU-meg and CFU-meg.Our data, as a whole, demonstrate that RA has direct complex effects on the survival, growth and clonal expansion of fetal haemopoietic progenitor cells, mainly depending on the presence of recombinant cytokines, the type of progenitor and the concentrations of RA.

Research paper thumbnail of High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy

Chromosoma, 1994

HeLa metaphase chromosomes were examined by means of &amp;amp;quot;in lens&amp;amp;quot; ... more HeLa metaphase chromosomes were examined by means of &amp;amp;quot;in lens&amp;amp;quot; field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.

Research paper thumbnail of In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes

To investigate the fate of human megakaryocytes, CD34 " deoxynucleotidyl transferase (TdT)-mediat... more To investigate the fate of human megakaryocytes, CD34 " deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin hematopoietic progenitor cells were purified from the penick end-labeling technique (TUNEL) and uptake of propidripheral blood or bone marrow of healthy donors and seeded ium iodide. In other experiments, primary a IIb b3 " megakaryin serum-free chemically defined suspension cultures. In the ocytic cells were directly purified from the bone marrow presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived aspirates of normal donors and seeded in serum-free suscells showed an eightfold numerical expansion and a propension cultures. In the absence of cytokines, a IIb b3 " megagressive maturation along the megakaryocytic lineage. Megakaryocytes progressively underwent apoptotic cell death. karyocyte maturation was characterized ultrastructurally by

Research paper thumbnail of Natural killer function in flow cytometryI. Evaluation of NK lytic activity on K562 cell line

Journal of Immunological Methods, 1988

Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These ... more Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These cells show a perpendicular light scatter (PLS) characteristically different from lymphocytes. Other physical parameters, such as forward light scatter (FLS), do not discriminate between the two populations, since dead K562 cells display similar FLS characteristics as effector cells. Killed cells are stained with propidium iodide and followed by flow analysis. It was advisable to add the dye in the medium so that, as long as the target cells are killed they will also be stained. Moreover the flow cytometric and trypan blue evaluation of target cell death rate showed a stronger correlation than did either test with the conventional 51Cr release assay, the first two methods both being based on the same biological mechanism.

Research paper thumbnail of Flow cytometric analysis of isolated rat liver nuclei during growth

Cytometry, 1987

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytomet... more The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.

Research paper thumbnail of Nuclear pores in the apoptotic cell

Histochemical Journal, 1994

During apoptosis, nuclear pores undergo strong modifications, which are described here in five di... more During apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.

Research paper thumbnail of Comparison between different laboratory tests for the detection and prevention of heparin-induced thrombocytopenia

Cytometry, 2001

Background: Heparin-induced thrombocytopenia (HIT) is a possible complication of heparin therapy ... more Background: Heparin-induced thrombocytopenia (HIT) is a possible complication of heparin therapy that can evolve with life-threatening thromboembolism, for which early diagnosis is essential. However, the specific laboratory approach to the diagnosis of HIT is still controversial. Methods: Sera from 13 patients with HIT, from 15 patients with non-HIT thrombocytopenia, and from 10 normal subjects were used to compare nonfunctional and functional methods to detect anti-heparin:PF-4 antibodies and platelet activation. We used three enzyme-linked immunosorbent assays (ELISAs) and the particle gel immunoassay as nonfunctional tests, and platelet aggregometry, CD62p (p-selectin) phenotypical expression, and Annexin V binding as functional assays. Results: CD62p expression was positive in 85% of the cases and Annexin V was positive in 40% of the HIT cases examined. Aggregometry gave variable results that depend strongly on the donor. Conclusion: Functional tests for platelet activation are more reliable for HIT diagnosis than the nonfunctional tests. We conclude that the phenotypical expression of p-selectin detected by flow cytometry on activated platelets appears to be a good functional marker for the diagnosis of HIT and its possible thromboembolic complications. Cytometry (Comm. Clin. Cytometry) 46:290–295, 2001. © 2001 Wiley-Liss, Inc.

Research paper thumbnail of Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry

Cytometry, 1998

The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation... more The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokinestimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties. Cytometry 32:280-285, 1998.

Research paper thumbnail of Mouse peritoneal cells as a reservoir of late dendritic cell progenitors

British Journal of Haematology, 1999

Despite the key role played by dendritic cells (DCs) in the physiology of immunity and related di... more Despite the key role played by dendritic cells (DCs) in the physiology of immunity and related diseases, their differentiation pathway has not yet been fully elucidated. In this study we demonstrated that cells obtained from mouse peritoneal cavity lavage can be induced to differentiate in vitro along the dendritic lineage by the addition of optimal concentrations of murine recombinant GM-CSF (50 U/ml) for 6 d. At morphological analysis, GM-CSF-treated peritoneal cells appeared loosely adherent to plastic and showed cytoplasmic protrusions and veils typical of DCs. A de novo expression of the DC phenotypic markers MIDC8, DEC205, CD11c and relB with up-regulation of surface MHC-II and complete down-regulation of non-specific esterase (NSE) was also observed in peritoneal cells upon GM-CSF treatment. Functionally, GM-CSF-treated peritoneal cells were highly stimulatory in a mixed lymphocyte reaction, showed a reduced phagocytosis of latex particles and enhanced pinocytic activity. Moreover, tumour necrosis factor (TNF)-a ᭧ 1999

Research paper thumbnail of TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis

The impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hem... more The impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hematopoietic development was investigated using adult peripheral blood CD34 ؉ hematopoietic progenitor cells, induced to differentiate along the erythroid, megakaryocytic, granulocytic, and monocytic lineages by the addition of specific cytokine cocktails.

Research paper thumbnail of Flow cytometry detection of serotonin content and release in resting and activated platelets

British Journal of Haematology, 2003

Early detection of platelet activation is important for the diagnosis and follow-up of several pa... more Early detection of platelet activation is important for the diagnosis and follow-up of several pathological conditions that primarily or secondarily involve platelets in their pathogenesis. The golden standard assay to detect thrombocyte activation is represented by the release of serotonin, classically performed by demanding methodologies, such as high-performance liquid chromatography, 14 C-labelling and enzyme-linked immunosorbent assay (ELISA). We developed a non-radioactive method, based on individual cells, for the detection of serotonin content in activated and resting platelets by flow cytometry. The assay was standardized on cells activated by Ca 2+ ionophore or by sera from patients with heparin-induced thrombocytopenia (HIT). Cells were identified by CD41a surface staining and their serotonin content measured by specific antiserotonin intracytoplasmic staining, while their activation was independently shown by annexin V binding. Cellular degranulation was detected by flow cytometry in all the cases that were also positive by standard ELISA. Moreover, multiparametric flow cytometry analysis revealed that, although virtually all activated cells bind annexin V, serotonin was released only by the platelet subset that downmodulates surface CD41a.

Research paper thumbnail of HLA Class I Antigen and Transporter Associated with Antigen Processing (TAPI and TAP2) Down-Regulation in High-Grade Primary Breast Carcinoma Lesions1

Five specimens of normal mammary tissue and 53 primary breast carcinoma lesions were tested for e... more Five specimens of normal mammary tissue and 53 primary breast carcinoma lesions were tested for expression of HLA antigens and com ponents of the antigen-processing machinery by immunohistochemical staining. The expression of transporter associated with antigen processing (TAP) l, TAP2, and HLA class I antigens in breast carcinoma lesions was significantly associated with tumor grading. Like normal mammary tis sue, the 16 low-grade (Gl) breast carcinoma lesions showed strong stain ing for TAPI, TAP2, and HLA class I antigens. In contrast, only 12 (32%) of 37 high-grade (G2 and G3) breast carcinoma lesions displayed the normal staining pattern. In 14 (38%) of 37 high-grade lesions, HLA class I antigen down-regulation was observed without loss of low molecular mass polypeptide and/or TAP staining. Congruent down-regulation of HLA class I antigen and TAPI or TAP2 was found in 8 (22%) of 37 high-grade lesions. Complete loss of HLA class I antigens, TAPI, and TAP2 was observed in 3 (8%) of 37 high-grade lesions. No lesion was negative for TAPI and/or TAP2 staining while positive for HLA class I antigen staining. These data demonstrate an association of HLA class I antigen and TAP down-regulation with tumor progression in breast car cinoma. This association suggests that loss of HLA and/or TAP may represent an escape from the host's immune pressure or may reflect the accumulation of abnormalities associated with neoplastic progression. This accumulation of defects in antigen processing and presentation may in turn be responsible for reduced recognition of malignant cells by putative clinically relevant tumor-specific T cells.

Research paper thumbnail of Antiangiogenic Activity of the MDM2 Antagonist Nutlin-3

Circulation Research, 2007

Nutlin-3, a nongenotoxic activator of the p53 pathway, dose-dependently (range 0.1 to 10 mol/L) i... more Nutlin-3, a nongenotoxic activator of the p53 pathway, dose-dependently (range 0.1 to 10 mol/L) inhibited the formation of capillaries in an in vivo matrigel assay, as well as the formation of capillary-like structures in an in vitro coculture system composed of endothelial cells surrounded by fibroblasts. In contrast to the chemotherapeutic agent doxorubicin, nutlin-3 showed no induction of apoptosis in vitro either in the cocultures or in isolated vascular endothelial cells, even when used at the highest concentration (10 mol/L). However, treatment with pharmacological inhibitors of the nuclear factor B and phosphatidylinositol 3-kinase/Akt pathways sensitized endothelial cells to nutlin-3-induced apoptosis. Although nutlin-3 and doxorubicin induced a comparable p53 accumulation in endothelial cells, nutlin-3 was significantly more efficient than doxorubicin in upregulating the p53 target genes CDKN1A/p21, MDM2, and GDF-15, as well as in inhibiting cell cycle progression. However, the predominant in vitro effect of nutlin-3 was its strong antimigratory activity observed at concentrations significantly lower (0.1 mol/L) than those required to inhibit endothelial cell cycle progression. Taken together, our data suggest that the antiangiogenic activity of nutlin-3 observed in vivo was mainly attributable to inhibition of endothelial cell migration, to some extent attributable to cell cycle arrest, and to a lesser extent attributable to induction of apoptosis. (Circ Res. 2007;100:0-0.)

Research paper thumbnail of Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation

Cytometry, 1996

Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. ... more Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods: Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes.

Research paper thumbnail of Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes

The Anatomical Record, 2000

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultur... more The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6-8 days, virtually all of the CD34-derived cells expressed the early megakaryocytic CD61 antigen, while, after 15-16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15-16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing.

Research paper thumbnail of Supravital exposure to propidium iodide identifies apoptosis on adherent cells

Cytometry, 2001

Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. ... more Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods: Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes.

Research paper thumbnail of A Subset of Human CD34' Hematopoietic Progenitors Express Low Levels of CD4, the High-Affinity Receptor for Human Immunodeficiency Virus-Type 1

Research paper thumbnail of Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation

Cytometry, 1996

Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. ... more Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods: Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes.

Research paper thumbnail of The impairment of natural killer function in the healthy aged is due to a postbinding deficient mechanism

Cellular Immunology, 1992

In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolyti... more In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolytic activity which occurs during human aging, we examined by multiparametric flow cytometry the binding and lytic activities of human natural killer cells. The flow analysis revealed a striking increase of the CD16Y subset, together with a significant decrease of CD@'"@" cells and total T cells (CD3+). Aging had no influence on the CDS"'" subset. The total lytic activity expressed by PBL as well as their binding efficiency to K562 targets were moderately but not significantly increased in the elderly. In contrast, the cytotoxicity of the single target-bound natural killer cell (i.e., iytic efficiency) was deeply impaired in aged subjects, suggesting that the NK functional impairment observed in aging is located at postbinding level. 8

Research paper thumbnail of Hydrogen sulfide prevents apoptosis of human PMN via inhibition of p38 and caspase 3

Laboratory Investigation, 2006

Hydrogen sulfide, together with carbon monoxide and nitric oxide, is now considered a gasotransmi... more Hydrogen sulfide, together with carbon monoxide and nitric oxide, is now considered a gasotransmitter able to induce specific cellular responses. As hydrogen sulfide is a component of several natural compounds known to be effective in many inflammatory pathologies, particularly of the respiratory tract, we studied its effects in vitro on the survival and bactericidal activity of purified human neutrophils. We found that (1) HS À ions promote the survival of granulocytes, but not that of lymphocytes or eosinophils, cultured in serum-free medium; (2) the pro-survival effect of HS À is due to inhibition of caspase-3 cleavage and p38 MAP kinase phosphorylation; (3) the bactericidal activity of neutrophils is not impaired by hydrogen sulfide. We conclude that HS À promotes the short-term survival of neutrophils potentially accelerating the resolution of inflammatory processes and preventing the occurrence of new ones.

Research paper thumbnail of All-trans retinoic acid shows multiple effects on the survival, proliferation and differentiation of human fetal CD34 + haemopoietic progenitor cells

British Journal of Haematology, 1995

Summary. To evaluate the effect of all-trans retinoic acid (RA) on fetal haemopoiesis, we perform... more Summary. To evaluate the effect of all-trans retinoic acid (RA) on fetal haemopoiesis, we performed serum-free liquid and semisolid cultures using CD34+ cells purified from mid-trimester human fetal blood samples. RA, at both physiological (10-n and 10-12M) and pharmacological (10-6 and l(r7M) concentrations, significantly (P<0.01) promoted the survival of fetal CD34+ cells in liquid cultures from day 3 onwards, by suppressing apoptosis induced by serum and growth factor deprivation. On the other hand, RA alone had no significant effect on the proliferation and differentiation of fetal haemopoietic progenitors.In the presence of optimal concentrations of recombinant interleukin-3 (IL-3), stem cell factor (SCF), granulocyte/ macrophage-colony stimulating factor (GM-CSF), and erythropoietin (Epo), low and high doses of RA induced striking differential effects on CD34+ cell proliferation in liquid cultures and colony formation in semisolid assays. In fact, 1CTU M and 1CT12M RA were able to: (i) significantly (P<0.05) increase 3H-thymidine uptake by fetal CD34+ cells in liquid cultures, and (ii) variably promote the growth of pluripotent (CFU-GEMM, P<0.05), early (BFU-meg) and late (CFU-meg, P<0.01) megakaryocyte, granulocyte/macrophage (CFU-GM. P<001) and erythroid (BFU-E) progenitors in semisolid cultures. On the contrary, 10-6 and 10-7 M RA induced: (i) an overall inhibition (P<0.01) of CD34+ cell growth in liquid cultures; (ii) a marked suppression of BFU-E colony formation (P<0.01) at all Epo concentrations examined (0-002-4IU/ml); and (iii) a significant (P<0.()1) stimulation of CFU-GM with a shift from mixed granulocyte/ macrophage to pure granulocyte colonies, whereas it had little effect on the growth of CFU-GEMM, BFU-meg and CFU-meg.Our data, as a whole, demonstrate that RA has direct complex effects on the survival, growth and clonal expansion of fetal haemopoietic progenitor cells, mainly depending on the presence of recombinant cytokines, the type of progenitor and the concentrations of RA.

Research paper thumbnail of High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy

Chromosoma, 1994

HeLa metaphase chromosomes were examined by means of &amp;amp;quot;in lens&amp;amp;quot; ... more HeLa metaphase chromosomes were examined by means of &amp;amp;quot;in lens&amp;amp;quot; field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.

Research paper thumbnail of In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes

To investigate the fate of human megakaryocytes, CD34 " deoxynucleotidyl transferase (TdT)-mediat... more To investigate the fate of human megakaryocytes, CD34 " deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin hematopoietic progenitor cells were purified from the penick end-labeling technique (TUNEL) and uptake of propidripheral blood or bone marrow of healthy donors and seeded ium iodide. In other experiments, primary a IIb b3 " megakaryin serum-free chemically defined suspension cultures. In the ocytic cells were directly purified from the bone marrow presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived aspirates of normal donors and seeded in serum-free suscells showed an eightfold numerical expansion and a propension cultures. In the absence of cytokines, a IIb b3 " megagressive maturation along the megakaryocytic lineage. Megakaryocytes progressively underwent apoptotic cell death. karyocyte maturation was characterized ultrastructurally by

Research paper thumbnail of Natural killer function in flow cytometryI. Evaluation of NK lytic activity on K562 cell line

Journal of Immunological Methods, 1988

Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These ... more Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These cells show a perpendicular light scatter (PLS) characteristically different from lymphocytes. Other physical parameters, such as forward light scatter (FLS), do not discriminate between the two populations, since dead K562 cells display similar FLS characteristics as effector cells. Killed cells are stained with propidium iodide and followed by flow analysis. It was advisable to add the dye in the medium so that, as long as the target cells are killed they will also be stained. Moreover the flow cytometric and trypan blue evaluation of target cell death rate showed a stronger correlation than did either test with the conventional 51Cr release assay, the first two methods both being based on the same biological mechanism.

Research paper thumbnail of Flow cytometric analysis of isolated rat liver nuclei during growth

Cytometry, 1987

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytomet... more The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.

Research paper thumbnail of Nuclear pores in the apoptotic cell

Histochemical Journal, 1994

During apoptosis, nuclear pores undergo strong modifications, which are described here in five di... more During apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.

Research paper thumbnail of Comparison between different laboratory tests for the detection and prevention of heparin-induced thrombocytopenia

Cytometry, 2001

Background: Heparin-induced thrombocytopenia (HIT) is a possible complication of heparin therapy ... more Background: Heparin-induced thrombocytopenia (HIT) is a possible complication of heparin therapy that can evolve with life-threatening thromboembolism, for which early diagnosis is essential. However, the specific laboratory approach to the diagnosis of HIT is still controversial. Methods: Sera from 13 patients with HIT, from 15 patients with non-HIT thrombocytopenia, and from 10 normal subjects were used to compare nonfunctional and functional methods to detect anti-heparin:PF-4 antibodies and platelet activation. We used three enzyme-linked immunosorbent assays (ELISAs) and the particle gel immunoassay as nonfunctional tests, and platelet aggregometry, CD62p (p-selectin) phenotypical expression, and Annexin V binding as functional assays. Results: CD62p expression was positive in 85% of the cases and Annexin V was positive in 40% of the HIT cases examined. Aggregometry gave variable results that depend strongly on the donor. Conclusion: Functional tests for platelet activation are more reliable for HIT diagnosis than the nonfunctional tests. We conclude that the phenotypical expression of p-selectin detected by flow cytometry on activated platelets appears to be a good functional marker for the diagnosis of HIT and its possible thromboembolic complications. Cytometry (Comm. Clin. Cytometry) 46:290–295, 2001. © 2001 Wiley-Liss, Inc.

Research paper thumbnail of Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry

Cytometry, 1998

The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation... more The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokinestimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties. Cytometry 32:280-285, 1998.

Research paper thumbnail of Mouse peritoneal cells as a reservoir of late dendritic cell progenitors

British Journal of Haematology, 1999

Despite the key role played by dendritic cells (DCs) in the physiology of immunity and related di... more Despite the key role played by dendritic cells (DCs) in the physiology of immunity and related diseases, their differentiation pathway has not yet been fully elucidated. In this study we demonstrated that cells obtained from mouse peritoneal cavity lavage can be induced to differentiate in vitro along the dendritic lineage by the addition of optimal concentrations of murine recombinant GM-CSF (50 U/ml) for 6 d. At morphological analysis, GM-CSF-treated peritoneal cells appeared loosely adherent to plastic and showed cytoplasmic protrusions and veils typical of DCs. A de novo expression of the DC phenotypic markers MIDC8, DEC205, CD11c and relB with up-regulation of surface MHC-II and complete down-regulation of non-specific esterase (NSE) was also observed in peritoneal cells upon GM-CSF treatment. Functionally, GM-CSF-treated peritoneal cells were highly stimulatory in a mixed lymphocyte reaction, showed a reduced phagocytosis of latex particles and enhanced pinocytic activity. Moreover, tumour necrosis factor (TNF)-a ᭧ 1999

Research paper thumbnail of TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis

The impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hem... more The impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hematopoietic development was investigated using adult peripheral blood CD34 ؉ hematopoietic progenitor cells, induced to differentiate along the erythroid, megakaryocytic, granulocytic, and monocytic lineages by the addition of specific cytokine cocktails.

Research paper thumbnail of Flow cytometry detection of serotonin content and release in resting and activated platelets

British Journal of Haematology, 2003

Early detection of platelet activation is important for the diagnosis and follow-up of several pa... more Early detection of platelet activation is important for the diagnosis and follow-up of several pathological conditions that primarily or secondarily involve platelets in their pathogenesis. The golden standard assay to detect thrombocyte activation is represented by the release of serotonin, classically performed by demanding methodologies, such as high-performance liquid chromatography, 14 C-labelling and enzyme-linked immunosorbent assay (ELISA). We developed a non-radioactive method, based on individual cells, for the detection of serotonin content in activated and resting platelets by flow cytometry. The assay was standardized on cells activated by Ca 2+ ionophore or by sera from patients with heparin-induced thrombocytopenia (HIT). Cells were identified by CD41a surface staining and their serotonin content measured by specific antiserotonin intracytoplasmic staining, while their activation was independently shown by annexin V binding. Cellular degranulation was detected by flow cytometry in all the cases that were also positive by standard ELISA. Moreover, multiparametric flow cytometry analysis revealed that, although virtually all activated cells bind annexin V, serotonin was released only by the platelet subset that downmodulates surface CD41a.

Research paper thumbnail of HLA Class I Antigen and Transporter Associated with Antigen Processing (TAPI and TAP2) Down-Regulation in High-Grade Primary Breast Carcinoma Lesions1

Five specimens of normal mammary tissue and 53 primary breast carcinoma lesions were tested for e... more Five specimens of normal mammary tissue and 53 primary breast carcinoma lesions were tested for expression of HLA antigens and com ponents of the antigen-processing machinery by immunohistochemical staining. The expression of transporter associated with antigen processing (TAP) l, TAP2, and HLA class I antigens in breast carcinoma lesions was significantly associated with tumor grading. Like normal mammary tis sue, the 16 low-grade (Gl) breast carcinoma lesions showed strong stain ing for TAPI, TAP2, and HLA class I antigens. In contrast, only 12 (32%) of 37 high-grade (G2 and G3) breast carcinoma lesions displayed the normal staining pattern. In 14 (38%) of 37 high-grade lesions, HLA class I antigen down-regulation was observed without loss of low molecular mass polypeptide and/or TAP staining. Congruent down-regulation of HLA class I antigen and TAPI or TAP2 was found in 8 (22%) of 37 high-grade lesions. Complete loss of HLA class I antigens, TAPI, and TAP2 was observed in 3 (8%) of 37 high-grade lesions. No lesion was negative for TAPI and/or TAP2 staining while positive for HLA class I antigen staining. These data demonstrate an association of HLA class I antigen and TAP down-regulation with tumor progression in breast car cinoma. This association suggests that loss of HLA and/or TAP may represent an escape from the host's immune pressure or may reflect the accumulation of abnormalities associated with neoplastic progression. This accumulation of defects in antigen processing and presentation may in turn be responsible for reduced recognition of malignant cells by putative clinically relevant tumor-specific T cells.

Research paper thumbnail of Antiangiogenic Activity of the MDM2 Antagonist Nutlin-3

Circulation Research, 2007

Nutlin-3, a nongenotoxic activator of the p53 pathway, dose-dependently (range 0.1 to 10 mol/L) i... more Nutlin-3, a nongenotoxic activator of the p53 pathway, dose-dependently (range 0.1 to 10 mol/L) inhibited the formation of capillaries in an in vivo matrigel assay, as well as the formation of capillary-like structures in an in vitro coculture system composed of endothelial cells surrounded by fibroblasts. In contrast to the chemotherapeutic agent doxorubicin, nutlin-3 showed no induction of apoptosis in vitro either in the cocultures or in isolated vascular endothelial cells, even when used at the highest concentration (10 mol/L). However, treatment with pharmacological inhibitors of the nuclear factor B and phosphatidylinositol 3-kinase/Akt pathways sensitized endothelial cells to nutlin-3-induced apoptosis. Although nutlin-3 and doxorubicin induced a comparable p53 accumulation in endothelial cells, nutlin-3 was significantly more efficient than doxorubicin in upregulating the p53 target genes CDKN1A/p21, MDM2, and GDF-15, as well as in inhibiting cell cycle progression. However, the predominant in vitro effect of nutlin-3 was its strong antimigratory activity observed at concentrations significantly lower (0.1 mol/L) than those required to inhibit endothelial cell cycle progression. Taken together, our data suggest that the antiangiogenic activity of nutlin-3 observed in vivo was mainly attributable to inhibition of endothelial cell migration, to some extent attributable to cell cycle arrest, and to a lesser extent attributable to induction of apoptosis. (Circ Res. 2007;100:0-0.)

Research paper thumbnail of Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation

Cytometry, 1996

Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. ... more Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods: Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes.

Research paper thumbnail of Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes

The Anatomical Record, 2000

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultur... more The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6-8 days, virtually all of the CD34-derived cells expressed the early megakaryocytic CD61 antigen, while, after 15-16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15-16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing.

Research paper thumbnail of Supravital exposure to propidium iodide identifies apoptosis on adherent cells

Cytometry, 2001

Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. ... more Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods: Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes.

Research paper thumbnail of A Subset of Human CD34' Hematopoietic Progenitors Express Low Levels of CD4, the High-Affinity Receptor for Human Immunodeficiency Virus-Type 1

Research paper thumbnail of Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation

Cytometry, 1996

Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. ... more Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods: Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes.

Research paper thumbnail of The impairment of natural killer function in the healthy aged is due to a postbinding deficient mechanism

Cellular Immunology, 1992

In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolyti... more In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolytic activity which occurs during human aging, we examined by multiparametric flow cytometry the binding and lytic activities of human natural killer cells. The flow analysis revealed a striking increase of the CD16Y subset, together with a significant decrease of CD@'"@" cells and total T cells (CD3+). Aging had no influence on the CDS"'" subset. The total lytic activity expressed by PBL as well as their binding efficiency to K562 targets were moderately but not significantly increased in the elderly. In contrast, the cytotoxicity of the single target-bound natural killer cell (i.e., iytic efficiency) was deeply impaired in aged subjects, suggesting that the NK functional impairment observed in aging is located at postbinding level. 8