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D Barbieri

Publications and source records attributed to D Barbieri.

At least 37 records · Page 2Linked to original sources
PubMed1997

Antisense inhibition of c-fes proto-oncogene blocks PMA-induced macrophage differentiation in HL60 and in FDC-P1/MAC-11 cells.

To gain some insight into the role of c-fes in macrophage differentiation, we have analyzed the ability of HL60 leukemic promyelocytic cells and FDC-P1/MAC-11 murine myeloid precursor cells to differentiate in response to phorbol esters after inhibition of c-fes function. Fes inactivation has been obtained by using oligodeoxynucleotides (ODN) complementary to the 5' region of c-fes mRNA and to 5' splice junctions of c-fes primary transcript. After 5 days (d) in culture, in several separate experiments performed with different ODN preparations, a complete inhibition of c-fes expression was observed in HL60 and in FDC-P1/MAC-11 cells. No perturbation of cell growth was evident in our experimental conditions in both cell lines after c-fes inhibition. Furthermore, in HL60 cells lacking c-fes product, an almost complete downregulation of the alpha 4 beta 1 fibronectin receptor occurred. However, in both cell lines, the induction of macrophage differentiation by phorbol esters resulted in an almost complete maturation arrest as evaluated by morphological, cytochemical, immunological criteria, and by the cytofluorimetric cell cycle analysis. A loss of the adhesion capacity of both myeloid cell lines, when compared to terminally differentiated macrophages, was also observed. These results suggest that HL60 and FDC-P1/MAC-11 cells, when treated with phorbol 12-myristate 13-acetate, require c-fes protein expression to activate the genetic program underlying macrophage differentiation.

Animals↗
PubMed1997

Cell behavior and cell-matrix interactions of human palmar aponeurotic cells in vitro.

The present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren's affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren's aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low alpha 2, alpha 1, and alpha 5 integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren's patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren's patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren's contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren's disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand.

Cell Adhesion↗
PubMed1997

Modulation of cis-diamminedichloroplatinum (II) accumulation and cytotoxicity by spermine in sensitive and resistant human ovarian carcinoma cells.

The effect of spermine (Sp), a natural polycationic amine, on cisplatin (CDDP) sensitivity and accumulation of a human ovarian CDDP-sensitive cell line (2008) and its resistant variant (C13*) was investigated. Survival was also studied. The C13* cells were approximately 20-fold resistant to CDDP, yet were found to be just as sensitive to Sp as 2008 cells. When Sp was concurrently added with CDDP to the colony-forming assay, the IC50 dose was approximately 3-fold lower than that of CDDP alone. This decrease was the result of a synergistic interaction, as assessed by median effect analysis. The incubation of cells with the approximate IC50 dose of Sp for 1-8 h indicated that this synergism could be due to stimulation of CDDP accumulation, showing maximal uptake after 4 h of Sp exposure. This stimulation may be the result of a modulation of cellular membrane permeability by Sp, as assessed by the accumulation of [3H]mannitol. Exposure to Sp concentrations active on CDDP uptake also significantly increased [3H]mannitol accumulation in both cell lines. The triamine spermidine (Spd) did not significantly affect either the sensitivity of the two cell lines or CDDP and [3H]mannitol accumulation. These results suggest that Sp is a positive modulator of CDDP uptake, and thus of its cytotoxicity, even in resistant cells, where the phenotype is partly due to a CDDP accumulation defect.

Antineoplastic Agents↗
PubMed1996

c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.

In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully.

Adolescent↗
PubMed1996

CD45 isoforms expression on CD4+ and CD8+ T cells throughout life, from newborns to centenarians: implications for T cell memory.

CD4+ and CD8+ peripheral blood T lymphocytes show mutually exclusive expression of CD45RA or CD45R0, two isoforms of the common leukocyte antigen that seem to recognize so-called virgin/unprimed and memory/activated T cells. The expression of these isoforms has been studied by three colour cytofluorimetric analysis on CD4+ or CD8+ peripheral blood CD3+ cells from 22 healthy centenarians, analyzed in a context of 202 healthy donors 0-110 years old. An age-related unbalance of virgin and memory cells was found between CD4+ and CD8+ subsets. As expected, at birgh 95-99% of the CD3+ lymphocytes expressed the CD45RA isoform. A rapid increase of CD45R0+ cells was observed in the first 2-3 decades of life, this phenomenon being much more pronounced on CD4+ cells. Subsequently, the increase of the 'memory' compartment was much less rapid, so that in centenarians a consistent reservoire of CD45RA+ among CD4+ cells was still present (about 20%). In these exceptional individuals the percentage of CD45RA+ cells among CD8+ T lymphocytes was even higher (about 50%), and only slightly lower than that of young donors (about 55-60%). Thus, the main changes occurred at a different rate in CD4+ (about 20%). In these exceptional individuals the percentage of CD45RA+ cells among CD8+ T lymphocytes was even higher (about 50%), and only slightly lower than that of young donors (about 55-60%). Thus, the main changes occurred at a different rate in CD4+ and in CD8+ T cells, at an age of between 0 and 30 years, when the thymus is still functionally active. Interestingly, no difference in the usage of CD45 isoforms was observed within T cells bearing four different V beta-T cell receptor (TCR). The significance of this age-related unbalance is unknown. However, the presence of a great number of CD45RA+ T lymphocytes within the CD4+ and the CD8+ T cell subsets even in the peripheral blood of centenarians poses the problem of their origin (thymus? extrathymic sites?), of their functional role and of their lifespan. Moreover, the data on centenarians suggest that they may represent a very selected population where a slowing of immunosenescence occurs.

Adolescent↗
PubMed1996

A shift to Th0 cytokine production by CD4+ cells in human longevity: studies on two healthy centenarians.

Centenarians, particularly healthy centenarians, constitute the example of successful aging and the study of their immune status can help to define the endpoint of the changes occurring throughout life. We characterized T cell clones (TCC) of two healthy centenarians, studying their phenotypes and production of representative Th1 and Th2 cytokines (IFN-gamma and IL-4) and compared them with TCC obtained by three young normal subjects; in all 180TCC were analyzed. In young donors, 35TCC were CD4+, 56CD8+ and 2 were alpha beta +CD4-CD8- (double negative). In centenarians, we obtained 46CD4+TCC, 38CD8+, 2CD4+CD8+ (double positive) and 1 gamma delta + double negative. Of the young subjects' TCC, 71% produced IFN-gamma but no IL-4 (Th1 pattern) and this prevalence decreased to 39% in TCC from the centenarians. The number of clones showing the opposite Th2 pattern was similar in young and aged donors (3 out of 93TCC and 2 out of 87TCC, respectively). The intermediate profile of TCC producing both IL-4 and IFN-gamma (Th0) was found in 25.8% of clones from young people, but it almost doubled to 58.6% in centenarians. The analysis shows that the Th profiles of CD8+TCC is nearly superimposable in the two groups, whereas a major shift from a Th1 to a Th0 pattern is presented by CD4+TCC. The balance provided by a majority of CD4+TCC showing a Th0 pattern may ensure both humoral and cell-mediated defences. In CD8+TCC, however, a Th1 pattern still is present, possibly for efficient generation of cytotoxic responses. These findings should be extended by studying other centenarians and elderly subjects.

Aged↗
PubMed1996

Inhibition of cell growth by accumulated spermine is associated with a transient alteration of cell cycle progression.

Exposure of HL-60 cells to millimolar levels of spermine resulted in the inhibition of cell growth. Flow cytometry revealed that the addition of exogenous spermine prevented the accumulation of cells in the S and G2/M phases of the cell cycle as observed in the control cells. High intracellular levels of spermine completely suppressed the early onset of ornithine decarboxylase activity and, consequently, the intracellular increase in spermidine and putrescine. On the other hand, the addition of exogenous spermidine or putrescine also abolished ornithine decarboxylase activity, but in this case neither the growth of spermidine- or putrescine-treated cells nor the cell cycle phase distribution was affected. In the latter cells, intracellular levels of spermidine were not significantly different from control ones. These results suggest that the addition of exogenous spermine inhibits cell proliferation by hindering the increase in cellular spermidine needed to accelerate the G1 to S phase transition.

Cell Cycle↗
PubMed1996

Successful immunosenescence and the remodelling of immune responses with ageing.

In recent decades, major theoretical and technological advances have been achieved in the field of immunology. These have allowed the scientific community to analyse the immune system in a much more sophisticated manner than was possible even 20 years ago. Moreover, great theoretical changes have also occurred in gerontology-in particular, the hypothesis has been put forward that ageing and diseases are two different phenomena, and that successful ageing, i.e. ageing in good psychophysical conditions, is really possible for most humans and animals. Immunosenescence was then carefully investigated, either in selected healthy people of advanced age or in the oldest old people, such as healthy centenarians. The main results showed that most immune parameters are indeed well preserved even at this far advanced age. This paper deals with some of the most important theoretical problems of immunosenescence. An immunological tenet was that the most important phenomenon of immunosenescence is the involution of the thymus. In most textbooks and papers it is taken for granted that the thymus starts its involution immediately after puberty. When people aged 60-65 were considered old, it was not difficult to think that they could live for the rest of their life with a fully involuted thymus. The findings on centenarians challenge this tenet, as they have only a small reduction of T lymphocytes, and a relatively normal number of virgin and memory T cells, together with a functional T cell repertoire. Other observations reported here on centenarians, concerning the activity of B lymphocytes and the cytokine network, as well as those on the well-preserved innate immunity and the cells' capability of undergoing proliferation after appropriate stimuli, suggest that complex immune changes occur with age, but also indicate that we have to modify our attitude, to grasp the new scenario which is emerging. Immunosenescence can no longer be considered as a unidirectional deterioration, and this complex phenomenon is much better described by terms such as 'remodelling', 'reshaping' or 'retuning'.

Aging↗
PubMed1995

Involvement of CD45 in dexamethasone- and heat shock-induced apoptosis of rat thymocytes.

CD45 is a transmembrane tyrosine-specific phosphatase which participates in lymphoid cell signal transduction during T cell activation, as well as in intrathymic negative and positive selection. In mammals, this molecule exhibits a variety of isoforms of different molecular weight, whose roles have still to be fully elucidated. We report here that apoptosis of rat thymocytes after in vitro dexamethasone and heat shock treatment was accompanied by an early significative increase of cells expressing CD45RC, the high molecular weight isoform of CD45 molecule. The same phenomenon was observed in thymocytes derived from in vivo dexamethasone-treated rats. However, the increase of CD45RC+ cells was not apparently characteristic of cells undergoing apoptosis, as the same phenomenon was also observed in rat thymocytes induced to proliferate by Concanavalin A. On the whole, these results suggest that CD45 modulation can be added to the list of early molecular events, such as the increased expression of genes (ornithine decarboxylase), proto-oncogenes (c-fos, c-jun, c-myc) and activation of transcription factors (AP-1, NFkB), we previously demonstrated in the same experimental model to occur and to be shared by these two apparently opposite biological processes, i.e., cell proliferation and apoptosis, both likely depending on a complex balance of protein phosphorylation and dephosphorylation.

Animals↗
PubMed1995

A novel action for adenosine: apoptosis of astroglial cells in rat brain primary cultures.

2-chloro-adenosine induced apoptosis of astroglial cells in rat brain cultures, as shown by flow cytometry and morphological analysis. The adenosine analogue was far more potent than several previously characterized sugars (including 2-deoxy-D-ribose and D-ribose, the sugar moiety of 2-chloro-adenosine), which trigger apoptosis in a variety of cell-lines [8-10], suggesting that the effects of 2-chloro-adenosine are only partially dictated by its sugar moiety. Nevertheless, 2-chloro-adenosine and 2-deoxy-D-ribose attenuated each other's cell death when used in combination, suggesting the involvement of common intracellular mechanisms. It is suggested that 2-chloro-adenosine may induce apoptosis via a yet-to-be identified adenosine receptor, which may have intriguing implications for both nervous system development and brain response to trauma and ischemia.

2-Chloroadenosine↗
PubMed1995

Lack of selective V beta deletion in CD4+ or CD8+ T lymphocytes and functional integrity of T-cell repertoire during acute HIV syndrome.

OBJECTIVE: To study the V beta T-cell repertoire in peripheral blood lymphocytes (PBL) during acute HIV syndrome by using several anti-V beta monoclonal antibodies (MAb) and to analyse its functionality by stimulating PBL with superantigens (SAg) such as Staphylococcus aureus enterotoxins. METHODS: Cytofluorimetric analysis of V beta T-cell-receptor expression was performed on PBL from eight patients with symptomatic, acute HIV-1 primary infection, showing a dramatic decrease of CD4+ PBL accompanied by a marked increase in activated/memory CD8+ T cells, and on 12 age- and sex-matched healthy controls. PBL were then isolated, stimulated with different SAg, anti-CD3 MAb or phytohaemagglutinin and cultured for 3 days. PBL capability to progress through cell cycle was studied by the classic cytofluorimetric method of bromodeoxyuridine incorporation and DNA staining with propidium iodide. RESULTS: Despite the presence of a few expansions of some V beta families among CD8+ T lymphocytes, no gross alterations in T-cell repertoire were present in patients with acute HIV syndrome. Its functionality was maintained overall, as PBL responsiveness to SAg was well preserved. Interestingly, all CD8+ T cells, although bearing different V beta T-cell receptors, expressed marked signs of activation, i.e., CD45R0, CD38 and major histocompatibility complex class II molecules, and also high amounts of CD11a and CD18. CONCLUSIONS: Our data suggest, at least in the early phases and in the acute form of the infection, that HIV is not likely to act as a SAg. However, further studies are needed to analyse other sites, such as lymph nodes, where HIV could exert other, significant effects, and to study the expression of other V beta families than those investigated here.

Acquired Immunodeficiency Syndrome↗
PubMed1994

D-ribose and deoxy-D-ribose induce apoptosis in human quiescent peripheral blood mononuclear cells.

In previous papers we reported that D(-)-ribose and 2-deoxy-D-ribose, which rank at the top among reducing sugars, kill a variety of human and animal cells and cell lines. Here we demonstrate that these two sugars induce apoptosis in human quiescent peripheral blood mononuclear cells which are relatively insensitive to apoptosis. Apoptosis was assessed by morphological changes, DNA fragmentation by agarose gel electrophoresis and the appearance of an hypodiploid peak by flow cytometry. 2-deoxy-D-ribose was more potent than D(-)-ribose and apoptosis was evident from 48 hours of culture onwards. 2-deoxy-D-ribose-induced apoptosis was inhibited by N-acetyl-L-cysteine, suggesting that glutathione metabolism and/or oxidative stress are involved in this type of apoptosis. Thus, D(-)-ribose and 2-deoxy-D-ribose can be useful tools to study the cellular and molecular events of apoptosis in human quiescent lymphocytes.

Acetylcysteine↗
PubMed1994

Mitochondrial modifications during rat thymocyte apoptosis: a study at the single cell level.

Apoptosis is an active type of cell death, occurring under several physiological and pathological conditions. The role of cellular organelles such as mitochondria in this process is still an open question. We recently described a new method to measure mitochondrial membrane potential in intact cells using flow cytometry. Using this method we studied alterations of mitochondrial membrane potential in a classical model of apoptosis, i.e., dexamethasone-treated rat thymocytes. Moreover, apoptosis induced by heat shock was also studied in the same cells. Mitochondria are functionally intact during the early phases of apoptosis, when DNA fragmentation occurs, whereas early alteration in their potential and mass takes place after DNA damage. According to flow-cytometric analysis, the presence of a hypodiploid peak, an index of nuclear DNA loss, predates depolarization of mitochondrial membrane and decrease of mitochondrial mass. Loss of plasma membrane integrity, which indicates cell death and is revealed by the permeability to propidium iodide, eventually follows. All these phenomena are more evident in dexamethasone-treated cells than in heat-shocked cells. Thus, in these types of apoptosis the involvement of mitochondria is apparently not a primary event.

Animals↗
PubMed1994

Antiapoptotic effect of c-fes protooncogene during granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation. Its product, p92c-fes, exhibits a tyrosine-kinase activity and is involved in the cellular response to GM-CSF, but its role is not yet clarified. To study this problem, the c-fes protooncogene expression has been inhibited in HL60 cells and in fresh leukemic blast cells of Acute Promyelocytic Leukemia (APL) induced to differentiate with All-Trans-Retinoic Acid (ATRA). Inhibition of c-fes function was obtained by treatment of the cells with a specific antisense oligomer complementary to the 5' region of the c-fes mRNA. It was observed that the cells, rather then differentiate to granulocytes, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. Superimposable results are obtained on blast cells from APL. It is possible to conclude that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of c-fes expression and treatment with ATRA, is due to activation of programmed cell death rather than an accelerated differentiation. Our data suggest that the c-fes product is essential for the survival of myeloid cells during differentiation.

Apoptosis↗
PubMed1993

Transcription factors DNA-binding activity in rat thymocytes undergoing apoptosis after heat-shock or dexamethasone treatment.

The onset of apoptosis is generally thought to require the induction of a novel genetic program. Accordingly, we studied the kinetic of DNA-binding activity of several transcription factors in rat thymocytes undergoing apoptosis induced by dexamethasone (DEX) or heat shock (HS) treatment. Here we report that: 1) early activation of AP-1 occurred in both models of apoptosis, even if the intensity of activation and AP-1 complex composition were different and much less evident in HS-treated thymocytes; 2) early NFkB DNA-binding activity was also observed in both types of apoptosis; 3) downregulation of other transcription factors, such as OCT-1 and CREB, with high constitutive activity, was recorded in both models of apoptosis. The fact that some TFs are up-regulated while others are down-regulated suggests that apoptosis is the result of a complex combination of positive and negative signals regulating gene expression.

Animals↗
PubMed1993

Inhibition of c-fes expression by an antisense oligomer causes apoptosis of HL60 cells induced to granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.

Apoptosis↗