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G Bellone

Publications and source records attributed to G Bellone.

At least 55 records · Page 3Linked to original sources
PubMed1989

Microplate selection technique (MPST). A new method for selecting mouse transfectants expressing human gene products.

This paper examines the analytical power of fluorescence activated cell sorting and immunorosetting technique as compared with the newly devised microplate selection technique in identifying transfected murine L cells expressing human surface molecules. The microplate selection technique relies on the mechanical transfer of transfected cells to a terasaki microplate, where an indirect immunofluorescence assay is carried out. It is a simple procedure not requiring costly equipment and with a detection capacity equivalent to that of the fluorescence activated cell sorter. The microplate selection technique proved to be sensitive enough to detect all the transfectants produced during the present study.

Animals↗
PubMed1989

Identification of a new epitope of the 4F2/44D7 molecular complex present on sarcolemma and isolated cardiac fibers.

The murine monoclonal antibody (mAb) CB43, raised against the K-562 erythroleukemia line, reacts with monocytes, tissue macrophages, thymocytes and with all the human lines tested but not with resting lymphocytes, large granular lymphocytes, granulocytes and erythrocytes. However, activated lymphocytes and natural killer cells express the CB43 antigen. Embryonic and fetal fibroblasts are positive, while adult fibroblasts are negative. Proximal convoluted tubules in kidney, epithelial cells in esophagus and breast, and sarcolemma in skeletal muscle are reactive with mAb CB43. This antibody can also bind to isolated guinea pig cardiac myocytes, and, furthermore, can induce a transient inotropic effect on isolated atria. The reactivity with different cell and tissue types and the functional effects of the CB43 mAb were reminiscent of the 4F2/44D7 antibodies, shown previously to block Na+/Ca2+ exchange in heart and skeletal muscle. Co-immunoprecipitation studies with CB43 and 44D7 mAb, using radiolabeled Daudi cells, revealed co-migration of polypeptides of 87 and 38 kDa. However, the epitope recognized by CB43 is not present on the human heavy chain which bears the 44D7/4F2 epitope as demonstrated by the lack of reactivity of CB43 mAb with mouse L cells transfected with the 4F2 heavy chain gene. Thus, CB43 represents a newly described epitope present on the human light chain or dependent on the conformation of the human dimer.

Adult↗
PubMed1989

Generation and selection of monoclonal antibodies identifying surface molecules discriminating between fetal and adult fibroblasts.

The present report describes the reactivity of two murine monoclonal antibodies, i.e., CB11 and CB43, the first obtained by using embryonic material as immunizer and the second elicited against a human cell line. These reagents recognize two distinct molecules marking a peculiar differentiation stage on fetal/embryonic fibroblasts. The first structure is a single chain of 14.5 kd, the second one a heterodimer of 40-80 kd. Even if these reagents are not strictly fibroblast restricted, translineage expression does not diminish the medical relevance of the reagents; in fact, they may have application in prenatal diagnostics and amniocentesis testing, in which it is necessary to discriminate between fetal and maternal cells.

Adult↗
PubMed1989

The inhibitory effect of serum from hairy-cell leukaemia patients on normal progenitor cells may disappear following prolonged treatment with alpha-interferon.

The possibility that serum from hairy-cell leukaemia (HCL) patients at diagnosis may show an inhibitory effect on the in vitro colony growth of normal haemopoietic progenitor cells has been suggested. Several studies have documented the efficacy of alpha-Interferon (alpha-IFN) in inducing a complete restoration of peripheral blood values and, in some cases, a complete clinical remission. In this study we have evaluated the regulatory effect of serum, collected before and after 3 and 12 months of alpha-IFN treatment, from 10 patients with untreated HCL, on the in vitro growth of normal bone marrow CFU-GM, BFU-E and CFU-MK. The effect of conditioned media, prepared from enriched hairy cells (HC) cultured in synthetic medium, on the growth of normal haemopoietic progenitors was also investigated. The results obtained confirm that sera from untreated HCL patients display a variable degree of inhibitory activity in the progenitor cell compartments analysed. Disappearance of the inhibitory activity, particularly evident for the erythroid compartment, was found only in patients who displayed a disappearance of circulating HC and a good haematological response after prolonged (12 months) treatment with alpha-IFN. The possibility that the serum of patients with HCL may contain a haemopoietic inhibitory factor, released by the neoplastic HC population, is suggested.

Adult↗
PubMed1989

Granulocyte-macrophage colony-stimulating factor requires interaction with accessory cells or granulocyte-colony stimulating factor for full stimulation of human myeloid progenitors.

Human recombinant GM-CSF (rGM-CSF) was tested on highly purified and fractionated CFU-GM subsets. The fractionation was performed with the DS1-1 monoclonal antibody (MoAb), which distinguishes early and late CFU-GM. On whole bone marrow cells, rGM-CSF had a colony-stimulating activity comparable to that of known sources of CSFs, ie, the supernatant (SN) of TPA 30-1 or 5637 cell lines, used as control. A greatly reduced activity was observed when CFU-GM were depleted of phagocytizing and E rosetting cells (colony growth of 27% as compared with control). On fractionated CFU-GM, the rGM-CSF activity was even more reduced on both early and late progenitors (18% and 6% of colony growth, respectively). However, when rGM-CSF was used together with rG-CSF at suboptimal concentrations, the colony growth reached values analogous to that of control cultures. A synergistic interaction between rGM-CSF and rG-CSF in stimulating either early or late myeloid progenitors was observed. The results suggest that the activity of rGM-CSF on CFU-GM is mainly exerted through cooperation with accessory cells. r-G-CSF is one of the factors that can synergistically cooperate with r-GM-CSF in the myelopoietic stimulation.

Bone Marrow↗
PubMed1989

The human myeloma cell line LP-1: a versatile model in which to study early plasma-cell differentiation and c-myc activation.

The characteristics of a human cell line (LP-1) derived from the peripheral blood of a patient with IgG-lambda myeloma in leukemic transformation are described. The cells resemble immature plasma cells in that they exhibit a membrane phenotype that is intermediate between late B lymphocytes and plasma cells, even though they secrete IgG-lambda chains. Treatment of LP-1 cells with 12-0 tetradecanoylphorbol-13-acetate (TPA) or pokeweek mitogen (PWM) induces the appearance of surface markers and ultrastructural features typical of mature plasma cells but does not affect their proliferative activity. Molecular analysis of the cell line showed an increased expression of the c-myc protooncogene and the presence of abnormally sized transcripts. Conventional cytogenetics and pulsed-field gel electrophoresis showed no structural rearrangements of the c-myc gene, suggesting that the abnormal c-myc expression may be due to point mutations or small deletions within the gene. The LP-1 cell line is a useful model in which to study the process of B-cell maturation; such study may lead to the uncovering of unusual mechanisms of c-myc activation. Furthermore, the LP-1 cell is a potential partner in the generation of human hybridomas.

B-Lymphocytes↗
PubMed1988

Soluble factor(s) released by the PF-382 T-cell line enhances the stimulatory effect of monocytes on the BFU-E growth.

PF-382 is a human T-cell line that has been shown to elaborate factors that modulate normal hemopoiesis in vitro. In the present study we report that this cell line constitutively releases in both serum-containing and serum-free supernatants a potent enhancer of BFU-E growth. The factor(s), partially purified by gel filtration, is a heat-stable molecule(s) degradable by trypsin and 2-mercaptoethanol treatments, equally active on bone marrow and peripheral blood erythroid progenitor cells, but not on CFU-GM. Unlike other sources of BPA, this stimulatory factor(s) exerts its effect in the presence of mononuclear adherent cells. In fact, the addition of conditioned medium obtained by 48 hr preincubation of isolated monocytes with 10% PF-382 supernatant (M-CM2) or the concomitant addition of supernatant from PF-382 cells (PF-382-CM) and from unstimulated monocytes (M-CM1) are capable of fully replacing the presence of monocytes in the BFU-E assay. Since the independent addition of PF-382-CM or of M-CM1 is devoid of stimulatory function, we suggest that the PF-382 derived BFU-E growth inducer, which differs from IL-1, IL-3, IL-4, GM and G-CSF, exerts its activity "via" a synergistic mechanism with a monokine.

Cell Division↗
PubMed1988

Generation of non-MHC restricted killing in cultures stimulated with B cells from chronic lymphocytic leukaemia patients: phenotypic characterization of the precursor and effector cells.

Freshly isolated B cells from chronic lymphocytic leukaemia patients (B-CLL) have been previously shown to induce a strong proliferative response and high levels of NK-like activity in lymphocytes from healthy donors. The present paper deals with the origin, mitotic state, target spectrum and cell surface phenotype of the NK-like effectors generated after stimulation with B-CLL. Experiments using large granular lymphocytes (LGL) and T cells as responders demonstrated that most of the precursors of the newly generated NK-like effectors express the CD3 antigen. The induction of NK-like activity paralleled cell activation, as judged by blast transformation, thymidine uptake and appearance of cell surface activation markers. The newly generated NK-like effectors displayed a T cell phenotype and a broader target repertoire than native NK cells.

Antigens, Surface↗
PubMed1988

Immunological separation of two CFU-GM subsets showing different responsiveness to T-cell derived growth factors.

Among human myeloid precursors two different subpopulations can be distinguished: type-2 CFU-GM, more differentiated, reacting to the monoclonal antibody DSl-1 and type-1 CFU-GM, more immature, negative to the DSl-1. Using this monoclonal antibody, type-1 and type-2 CFU-GM were fractionated and separately grown in the presence of T-cell derived CSFs, i.e., conditioned medium of PHA-stimulated T-lymphocytes (PHA-Ly) and the supernatant of Il-2-dependent normal T-cell lines (TC-SN). The supernatants of two neoplastic non-T cell lines, TPA-30-1 and GCT, were used as control. PHA-Ly and TC-SN had a very weak activity on type-2 CFU-GM (18 and 14% of colony growth, compared to control). However, they potently stimulated type-1 CFU-GM (84 and 68.5% of colony growth, compared to control). The restricted activity of T-cell derived CSFs on early myeloid progenitors is therefore demonstrated. Our results also show that CFU-GM with different responsiveness to growth factors can be physically separated on the basis of their reactivity to monoclonal antibody DSl-1.

Antibodies, Monoclonal↗
PubMed1988

Effect of cortisol on the native and in vitro induced non-MHC restricted cytotoxicity of large granular lymphocytes.

Natural Killer (NK) activity has been shown to be depressed under stressful conditions. Glucocorticoids, which are known to increase during stress, seem to negatively regulate the activity of NK cells. In the present study we have explored the effect of cortisol (hydrocortisone, HC) on NK activity. A significant inhibitory effect could be observed as early as 6 hr after the addition of HC at the concentration of 0.5 microM, corresponding to the upper physiological circulating level. Both the lysis and the binding of the K562 target cells were affected by HC, indicating that the hormone acts on the target recognition phase. The HC-mediated inhibition of the NK activity was fully reversed after 6 hr incubation in a HC-free medium. The observation of comparable levels of NK-inhibition using unseparated PBL or purified LGL, show that HC acts directly on LGL to inhibit their cytotoxic function. The effect of HC on the responsiveness of NK cells to the modifiers beta-interferon (beta-IFN) and recombinant interleukin 2 (rIL2) was also studied. Pre-incubation with HC did not alter the enhancement of the activity induced by beta-IFN, demonstrating that the HC- and beta-IFN-mediated effects occur in separate NK subsets. By contrast the increase of NK cytotoxicity induced by rIL2 was lower in the HC-treated compared to the untreated cell cultures (35.8 +/- 6.2 and 20.7 +/- 4.3% respectively, p less than 0.05) which could indicate that a portion of the cells triggered by rIL2 belong to the HC-sensitive NK subpopulation.

Cell Line, Transformed↗
PubMed1988

Recognition by monoclonal antibody CB02 of a surface molecule shared by B lymphocytes and a discrete large granular lymphocyte subset with cytotoxic activity.

Here we present the characterization of a new antigen called CB02 which is present on B cells and on a subset of CD16+ large granular lymphocytes. The pattern of reactivity of this molecule on normal cells, B cell lines, leukemic cells and tissue sections suggests that the CB02 molecule is expressed by B cells before isotype switch and is lost during terminal differentiation. Furthermore, the CB02+ population encompassed natural killer (NK) cells exerting significant cytotoxic activity: in fact, peripheral blood lymphocytes depleted of the CB02+ subset display a marked reduction in NK activity.

Animals↗
PubMed1987

Role of human serum and cellular factors on the growth of peripheral blood CFU-gm.

The effects of fetal calf serum (FCS), autologous serum (AS) and pooled human serum (PS) on granulocyte-macrophage progenitors (CFU-gm) from normal human peripheral blood mononuclear non-adherent cells (MNAC) were studied to determine optimal growth conditions. PS provided fewer variations and better growth conditions than AS and FCS. Moreover, serum inhibitors affecting both autologous and heterologous CFU-gm were detected in a small group (10%) of donors. Since CFU-gm from different donors display a relatively uniform cell-cycle status and membrane phenotype, under optimal growth conditions the number of CFU-gm probably reflects the size of the circulating myeloid progenitor cell compartment.

Adult↗
PubMed1987

Inhibitory effects of anti-HLA-A, B, C heavy chain and anti-beta 2 microglobulin monoclonal antibodies on alloantigen and microbial antigen-induced immune responses in vitro.

The role of HLA class I subunits in class II-restricted immune responses was investigated by means of a panel of monoclonal antibodies (MoAb) recognizing HLA-A,B,C heavy chain and different beta 2 microglobulin (beta 2m) epitopes. MoAb against either class I subunit strongly inhibited mixed lymphocyte cultures, generation of cytotoxic T lymphocyte cultures, generation of cytotoxic T lymphocytes or natural killer-like activity, and lymphoproliferation in response to soluble or particulate microbial antigens derived from Candida albicans. In general, anti-beta 2m MoAb were more efficient inhibitors than anti-HLA-A,B,C heavy chain MoAb. The inhibitory effects were specific, in that the parental myeloma ascitic fluid or a low-affinity MoAb against beta 2m, or MoAb directed against non-HLA surface structures did not affect any of the immune responses studied. The MoAb-induced inhibition could not be attributed to nonspecific toxic effects, since PHA-induced blastogenesis and IL-2-dependent proliferation of mixed lymphocyte culture (MLC) blasts were not inhibited. Furthermore, exogenous IL-2 did not reverse the block of MLC and microbial antigen-induced proliferative responses by MoAb. Taken together, these data suggest an involvement of both subunits of class I antigens in class II-restricted immune responses.

Antibodies, Monoclonal↗
PubMed1987

A human leukemic T cell line (PF-382) inhibits the growth of myeloid and erythroid progenitor cells.

We have recently described a human T cell line, named PF-382, obtained from the pleural effusion of a child with T-acute lymphoblastic leukemia (T-ALL), which expresses phenotypic and functional features of suppression. In this study we report that PF-382 spontaneously releases a factor which inhibits the in vitro growth of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells. The same effect is obtained when irradiated PF-382 cells are co-cultured with the hemopoietic precursors. In both instances, maximal inhibitory activity is exerted on day 14 CFU-GM and BFU-E obtained from the light density nonadherent fraction of normal human bone marrow and peripheral blood; this finding suggests that the target of the inhibition is represented by the more immature elements within the progenitor cell compartment. Progressive depletion of monocytes, T, B lymphocytes, and NK cells as well as recloning experiments indicate that the inhibitory effect is directly exerted on the target cell and not via an intermediate population of accessory cells. Partial purification by gel filtration and by subsequent high performance liquid chromatography demonstrates that this factor is a protein with a molecular weight of 47 kd. The physicochemical characterization and the specific functional properties suggest that the PF-382 inhibitory factor represents a lymphokine which differs from those so far reported. The PF-382 cell line provides a useful model toward a better understanding of the interrelations between T cell subsets and other hemopoietic compartments.

Colony-Forming Units Assay↗
PubMed1986

Modulation of in vitro myelopoiesis by LGL: different effects on early and late progenitor cells.

We describe here the modulatory activity of human peripheral blood natural killer (NK) cells on the growth and differentiation of myeloid progenitor cells at different stages of maturation. NK-enriched cell fractions containing 54 to 75% large granular lymphocytes (LGL) and displaying high levels of NK activity significantly inhibited the growth of late (7 day) granulocyte-macrophage colony-forming cells (CFU-GM) from about 50% of normal human bone marrow samples. However, the same fractions strongly enhanced the growth of early (14 day) stem cells from peripheral blood. Enhancing activity on early CFU-GM from blood was greater in highly purified NK cell preparations containing 96% LGL than in NK-depleted T cell preparations from the same donors. Analogous to the results when using the NK-enriched fractions, the NK-purified preparations inhibited late CFU-GM and stimulated the early ones. We conclude from these observations that human LGL have a modulatory effect on myelopoiesis depending on the maturation stage of the progenitor cell.

Bone Marrow Cells↗
PubMed1986

Interaction between leukocytes and serum plasminogen: an essential mechanism in peripheral blood fibrinolytic activity.

Intense fibrinolytic activity (FA) by granulocytes, mononuclear adherent cells, and natural killer (NK) cells was demonstrated by using 125I-fibrin-coated wells. This FA was greatly dependent on serum plasminogen, provided that serum inhibitors were destroyed by heating at 56 degrees C. Using monoclonal antibodies, it was also observed that these leukocytes produce urokinase and that this enzyme is secreted during adhesion by mononuclear adherent cells and during the cytotoxicity reaction against neoplastic cells by NK cells. Since cell intactness is necessary for leukocyte-serum interaction, stress is placed on the membrane function. We conclude that leukocytes are essential in peripheral blood FA and that transport of urokinase by circulating cells, inside their cytoplasm, could offset its lack of affinity for fibrin and thus avoid undesirable systemic effects.

Cell Adhesion↗
PubMed1986

Definition by CB12 monoclonal antibody of a differentiation marker specific for human monocytes and their bone marrow precursors.

The CB12 monoclonal antibody, which reacts with a molecule expressed on monocytes, was characterized using human embryonic material as immunizer. Analysis of the monoclonal antibody at the phenotypic, molecular, and functional levels indicates that its reactivity is restricted to circulating monocytes and their precursors in the bone marrow, whereas it is undetectable on tissue macrophages. CB12 displays a pattern of reactivity compatible with that of a marker of monocyte differentiation. Preliminary data indicate a possible receptor role for the CB12 molecule.

Antibodies, Monoclonal↗