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Biomedical subjects

A Grande

Publications and source records attributed to A Grande.

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

Interleukin-9 in human myeloid leukemia cells.

Here we review our recent data addressing the role of recombinant human (rh) interleukin 9 (IL-9) in acute myeloblastic leukemia (AML). We first evaluated the proliferative response of 3 leukemic cell lines and 32 primary samples from AML patients to IL-9 alone and combined with rh-IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF, c-kit ligand). The colony forming ability of leukemic cells was assessed by a clonogenic assay in methylcellulose, whereas the cell cycle characteristics of the same samples were determined by the acridine-orange (AO) flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the terminal deoxynucleotidyl transferase Assay (TDTA) and standard analysis of DNA cleavage by gel electrophoresis were used to evaluate induction or prevention of apoptosis by IL-9. IL-9, used as a single cytokine, at various concentrations stimulated the colony formation of the 3 myeloid cell lines under serum-containing and serum-free conditions and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL-9 resulted in the increase of the blast colony formation in all the cases studied and was the most effective CSF for promoting leukemic cell growth among those tested in this study including SCF, IL-3, and GM-CSF. The addition of SCF to IL-9 demonstrated an additive or synergistic effect of the 2 cytokines in 5 out of 8 AML cases tested for their CFU-L growth (187 +/- 79 colonies in comparison with 107 +/- 32 CFU-L; p = 0.05). Positive interaction was also observed when IL-9 was combined with IL-3 and GM-CSF. Studies of cell cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leukemic cells in S-phase in the majority of the cases evaluated. IL-9 and SCF in combination resulted in a remarkable decrease of the G0 cell fraction (38.2 +/- 24% compared to 58.6 +/- 22% of control cultures; p < 0.05) and induced an increase of G1 and S-phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. Furthermore, in this study, reverse transcriptase-polymerase chain reaction amplification (RT-PCR) did not show the constitutive expression of IL-9 mRNA in the cell lines and the AML samples studied at diagnosis. In summary, IL-9 may play a role in the development of acute myeloid leukemia by stimulating the proliferation of leukemic cells perhaps through a paracrine growth loop.

Apoptosis↗
PubMed1996

Interleukin-9 stimulates the proliferation of human myeloid leukemic cells.

Human interleukin-9 (IL-9) stimulates the proliferation of primitive hematopoietic erythroid and pluripotent progenitor cells, as well as the growth of selected colony-stimulating factor (CSF)-dependent myeloid cell lines. To further address the role of IL-9 in the development of acute leukemia, we evaluated the proliferative response of three leukemic cell lines and 32 primary samples from acute myeloblastic leukemia (AML) patients to recombinant human (rh)-IL-9 alone and combined with rh-IL-3, granulocyte-macrophage CSF (GM-CSF), and stem cell factor ([SCF] c-kit ligand). The colony-forming ability of HL60, K562, and KG1 cells and fresh AML cell populations upon IL-9 stimulation was assessed by a clonogenic assay in methylcellulose, whereas the cell-cycle characteristics of leukemic samples were determined by the acridine-orange flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the terminal deoxynucleotidyl transferase assay (TDTA) and standard analysis of DNA cleavage by gel electrophoresis were used to evaluate induction of prevention of apoptosis by IL-9. Il-9, as a single cytokine, at various concentrations stimulated the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions, and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL-9 resulted in an increase of blast colony formation in all the cases studied (mean +/- SEM: 19 +/- 10 colony-forming unit-leukemic [CFU-L]/10(5) cells plated in control cultures v 107 +/- 32 in IL-9-supplemented dishes, P < .02). IL-9 stimulated 36.8% of CFU-L induced by phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM), and it was the most effective CSF for promoting leukemic cell growth among those tested in this study (i.e., SCF, IL-3, and GM-CSF). The proliferative activity of IL-9 was also observed when T-cell-depleted AML specimens were incubated with increasing concentrations of the cytokine. Addition of SCF to IL-9 had an additive or synergistic effect of the two cytokines in five of eight AML cases tested for CFU-L growth (187 +/- 79 colonies v 107 +/- 32 CFU-L, P = .05). Positive interaction was also observed when IL-9 was combined with IL-3 and GM-CSF. Studies of cell-cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leukemic cells in S-phase in the majority of cases evaluated. IL-9 and SCF in combination resulted in a remarkable decrease of the G0 cell fraction (38.2% +/- 24% v 58.6% +/- 22% of control cultures, P < .05) and induced an increase of G1- and S-phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. In summary, our results indicate that IL-9 may play a role in the development of AML by stimulating leukemic cells to enter the S-phase rather than preventing cell death. Moreover, IL-9 acts synergistically with SCF for recruiting quiescent leukemic cells in cell cycle.

Adolescent↗
PubMed1995

Interleukin-11 (IL-11) acts as a synergistic factor for the proliferation of human myeloid leukaemic cells.

Interleukin-11 is a stromal cells derived cytokine which stimulates the proliferation of primitive haemopoietic progenitor cells. For this paper we have studied the constitutive expression of IL-11 mRNA in a panel of wellknown leukaemic cell lines and samples from AML patients at diagnosis. Moreover, the same cellular populations were evaluated for their proliferative response to recombinant-human-(r-hu). IL-11 alone and combined with r-hu-IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and stem cell factor (SCF, c-kit ligand). The colony-forming ability of HL60, K562, KG1 cells and eight fresh AML cell populations was assessed by a clonogenic assay in methylcellulose. In eight additional AML cases the number of S-phase leukaemic cells induced by IL-11 was determined by the bromodeoxyuridine (BRDU) incorporation assay after 3d of liquid culture. IL-11, as single cytokine, did not stimulate the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions. In contrast, the proliferation of the leukaemic cells in response to IL-3, GM-CSF and SCF was enhanced by co-incubation with IL-11, and this effect was reversed in blocking experiments by the anti-IL-11 Moab. When tested on primary AML samples, IL-11 alone showed little, if any, proliferative activity. However, it increased the IL-3-dependent blast colony formation in eight out of eight cases and GM-CSF in seven cases. IL-11 also augmented synergistically the number of CFU-L stimulated by SCF in seven cases. A combination of three factors (IL-11, SCF and IL-3) yielded optimal colony formation. The BRDU studies showed the significant increase of AML cells in S-phase when IL-11 was combined with SCF, whereas the two CSF had no activity on their own. Positive interaction was also observed when IL-11 was added to IL-3 supplemented cultures in five out of eight cases tested. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) demonstrated the constitutive expression of IL-11 mRNA in all the cell lines and 11/12 AML samples studied at diagnosis. These results indicate that IL-11 is expressed in leukaemic myeloid cells and that their proliferation is regulated by the cytokine which acts as a synergistic factor.

Adolescent↗
PubMed1995

Role of c-fes protooncogene in myeloid differentiation.

The main purpose of this report is to provide a review of the present knowledge on the structure, function, and possible regulatory role of c-fes in the genetic programs underlying the proliferation and differentiation of hematopoietic myeloid cells. Fes encodes a non-receptor tyrosine kinase that is highly expressed in immature and differentiated cells of the granulocytic and mono-macrophagic lineages. It is therefore possible that c-fes is involved in the signal transduction of myeloid cell differentiation, even if the specific substrates phosphorylated by this protooncogene are only poorly characterised. Several experimental models have been established to evaluate the role of c-fes in myeloid differentiation, in particular: the differentiation capacity of HL60 cells lacking the p92(c-fes) protein, the transfection of c-fes gene into K562 cells and transgenic animals overexpressing c-fes. The results obtained point to the importance of c-fes in myeloid cells, since it appears to be involved in granulocytic maturation as an antiapoptotic gene, and in macrophagic maturation as a regulatory gene.

Journal Article↗
PubMed1995

All-trans-retinoic acid induces simultaneously granulocytic differentiation and expression of inflammatory cytokines in HL-60 cells.

All trans-retinoic acid (ATRA) can induce granulocytic differentiation both in vitro and in vivo, and its activity is mediated by the retinoic acid receptor-alpha (RAR-alpha). In the present study, we evaluated the ability of this inducer in HL-60 cells, to stimulate simultaneously granulocytic differentiation and the expression of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-3, IL-6, tumor necrosis factor-alpha (TNF-alpha), and stem cell factor (SCF). The level of expression of these cytokines in ATRA-treated HL-60 cells was compared with that observed in normal and lipopolysaccharide (LPS)-treated peripheral granulocytes. The results indicate that the expression of these cytokines is enhanced during differentiation so that the pattern observed in ATRA-treated HL-60 cells is close to that of LPS-stimulated normal granulocytes. In addition, tetra phorbol acetate (TPA)-treated HL-60 cells express several of the above listed cytokines. It is concluded that ATRA not only induces granulocytic differentiation of HL-60 cells, but also activation of these terminally differentiated cells. The activating cytokine expression in these cells appears related to the progress of the differentiation program induced by ATRA since normal granulocytes do not respond to this inducer by activation of the expression of these genes. Furthermore, the cytokine activation is a specific effect of ATRA, since DMSO does not have any stimulatory effect.

Base Sequence↗
PubMed1994

Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes.

Keratinocytes, a key cellular component both for homeostasis and pathophysiologic processes of the skin, secrete a number of cytokines and are stimulated by several growth factors. Nerve growth factor (NGF) is synthesized in the skin and basal keratinocytes express the low-affinity nerve growth factor receptor (NGF-R). We present evidence that normal human keratinocytes in culture express the low- and the high-affinity NGF-R both at the mRNA level, as determined by reverse-transcription polymerase chain reaction and at the protein level, as shown by cytofluorimetric analysis. NGF significantly stimulates the proliferation of normal human keratinocytes in culture in a dose-dependent manner. This effect can be prevented by the addition of both an anti-NGF neutralizing antibody and a high-affinity NGF-R (trk) specific inhibitor, the natural alkaloid K252a. By contrast, keratinocyte proliferation is not inhibited by an anti-low-affinity NGF-R monoclonal antibody, thus suggesting that NGF effect on human keratinocytes is mediated by the high-affinity NGF-R. Moreover, NGF mRNA is expressed in normal human keratinocytes and NGF is secreted by keratinocytes in increasing amounts during growth, as detected by enzyme-linked immunosorbent assay. These results suggest that NGF could act as a cytokine in human skin and take part in disorders of keratinocyte proliferation.

Antibodies, Monoclonal↗
PubMed1994

Expression and functional role of c-kit ligand (SCF) in human multiple myeloma cells.

In this study we investigated the proliferation of three well-documented MM lines and 10 bone marrow samples from myeloma patients in response to rh-SCF alone and combined with Interleukin-6 (IL-6), IL-3 and IL-3/GM-CSF fusion protein PIXY 321. Neoplastic plasma cells were highly purified (> 90%) by immunomagnetic depletion of T, myeloid, monocytoid and NK cells. The number of S-phase cells was evaluated after 3 and 7 d of liquid culture by the bromodeoxyuridine (BRDU) incorporation assay. The proliferation of RPMI 8226 and U266 cell lines was also assessed by a clonogenic assay. All the experiments were performed in serum-free conditions. RPMI 8226 cell line was not stimulated by SCF which also did not augment the proliferative activity of IL-6, IL-3 and PIXY-321. Conversely, SCF addition resulted in 2.4-fold increase of the number of U266 colonies and in a higher number of U266 and MT3 cells in S-phase (24.5 +/- 2% SEM v 14.5 +/- 1% SEM and 32 +/- 3% SEM v 21 +/- 4% SEM, respectively; P < 0.05). The c-kit ligand also enhanced the proliferation of MT3 and U266 cells mediated by the other cytokines. Anti-SCF polyclonal antibodies completely abrogated the proliferative response of MT3 cells to exogenous SCF and markedly reduced the spontaneous growth of the same cell line. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) did detect SCF mRNA in MT3 and RPMI 8226 cells. Moreover, secreted SCF was found, in a biologically active form, in the supernatant of the two cell lines by the MO7e proliferation assay. When tested on fresh myeloma samples, SCF increased the number of S-phase plasma cells (4.7 +/- 1.6% v 3.4 +/- 1.3% in control cultures: P = 0.02). Significant proliferation was also induced by IL-6 (7 +/- 2.3% of BRDU+ cells; P = 0.006), IL-3 (5.3 +/- 1.3%; P = 0.01) and PIXY-321 (5.4 +/- 1.6%; P = 0.02). The addition of SCF significantly enhanced the proliferation of myeloma cells responsive to IL-6. In summary, our results indicate that SCF is expressed in MM cells and stimulates the proliferation of neoplastic plasma cells.

Aged↗
PubMed1994

Local production and action of follistatin in human placenta.

The aim of the present study was to investigate the possible production, localization, and action of follistatin in human placenta, fetal membranes (amnion, chorion), and maternal decidua. Four different experimental approaches were used: 1) Southern blot analysis following reverse polymerase chain reaction to identify follistatin messenger RNA (mRNA) in tissue homogenates; 2) immunohistochemistry to localize immunoreactive (ir-) follistatin in the various intrauterine tissues; 3) measurement by RIA of ir-follistatin levels in culture medium of placental cells; and 4) possible action of follistatin on human CG (hCG) and progesterone release from cultured placental cells. Placental and decidual cells collected during first trimester or at term gestation express follistatin mRNA; fetal membranes (amnion, chorion) at term also express follistatin mRNA. Immunoreactive follistatin is localized in syncytial cells of placental villi at term as well as in large decidual cells, in amnion epithelium, and in chorionic cells. The placental secretion of follistatin has been confirmed by the evidence of measurable levels of ir-follistatin in the medium of cultured placental cells at term; the release is time dependent and is not modified by the addition of forskolin or progesterone. The addition of increasing doses of recombinant human follistatin does not significantly influence the release of hCG or progesterone from cultured placental cells, whereas the activin A-induced hCG and progesterone release are completely reversed. The present data showed that 1) human placenta, fetal membranes, and decidua express follistatin mRNA; 2) ir-follistatin is localized and released from placental cells at term; and 3) follistatin has a functional role in the local control system regulating placental hormone production.

Base Sequence↗
PubMed1994

beta-Carotene supplementation results in an increased serum and colonic mucosal concentration of beta-carotene and a decrease in alpha-tocopherol concentration in patients with colonic neoplasia.

The aim of this study was to evaluate the colonic mucosal beta-carotene (BC) concentration following supplementation with BC and to determine if an increase in BC concentration influences vitamin E (alpha-tocopherol) status. The concentration of BC and alpha-tocopherol was assessed in serum and colonic tissue obtained from subjects with a history of colonic polyps or resected cancer (Dukes A, B1, or B2). Serum and mucosal biopsy samples were obtained prior to and following 3 months daily p.o. supplementation with 30 mg of BC or placebo. The concentration of BC was significantly increased in serum and colonic mucosa from both polyp and cancer subjects following supplementation as compared to presupplementation values and values from subjects receiving a placebo. The concentration of alpha-tocopherol in serum from cancer subjects was significantly decreased in samples obtained at the end of 3 months of BC supplementation as compared to placebo-matched controls. In BC-supplemented polyp subjects the tissue concentration of alpha-tocopherol was also significantly decreased relative to presupplementation values. The results indicate that BC supplementation does result in a significant accumulation of BC in the colonic mucosa but that the alpha-tocopherol concentration in both serum and colonic tissue may be compromised by an increased intake of BC. The mechanism for the decrease in alpha-tocopherol in conjunction with the increase in BC will require further study in order to develop strategies which will prevent vitamin E deficiency in BC-supplemented individuals.

Adult↗
PubMed1994

[Study of cutaneous microcirculation using the laser-Doppler method in syndrome X].

Aim of the study was to verify the relationship between syndrome X and generalized microangiopathy. Three groups of patients were selected: Group A, 11 patients with typical syndrome X; Group B, 8 patients with atypical chest pain; Group C, 12 control normal subjects. Microvascular circulation was investigated with a laser Doppler Periflux PF3 at rest (15 min); during ischemia (3 min); during post-ischemic hyperemia on the plantar side of the big toe (where arterovenous anastomoses are particularly abundant) and on the dorsum of the foot (where these anastomoses are very scanty). During each recording we calculated: the mean flux value (FV); the mean concentration of moving blood cells (CMBC); the FV/CMBC ratio (which is related to the flow velocity); the mean percent increase of the three parameters during hyperemia. Patients with syndrome X as compared to the other subjects showed: 1) a significantly higher FV/CMBC ratio at rest and during hyperemia both on the plantar side of the big toe (respectively p = 0.01 and 0.015) and on the dorsum of the foot (respectively p = 0.011 and 0.006). These findings suggest a higher flow velocity in microvascular circulation; significantly lower percent increase of FV and CMBC during hyperemia in the plantar side of the big toe (respectively p = 0.04 and 0.014) but not on the dorsum. The increase of flow velocity at rest and low vasodilatatory reserve after ischemia in an area which is rich of arterovenous anastomoses in patients with syndrome X might be explained by the presence of a basal arterovenous hyperstomia.

Adult↗
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

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↗
PubMed1993

Expression of interleukins 1, 3, 6, stem cell factor and their receptors in acute leukemia blast cells and in normal peripheral lymphocytes and monocytes.

Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) and Southern blot analysis were used to evaluate ligand and receptor expression of interleukin 1 alpha (IL-1 alpha), interleukin 3 (IL-3), interleukin 6 (IL-6) and stem cell factor (SCF) in peripheral blood lymphocytes and monocytes and in several acute leukemia blast cell populations. Resting peripheral lymphocytes and monocytes expressed both ligand and receptor of the four cytokines at considerable levels. The leukemic blast cells of the M1-M4 phenotypes are characterized by almost complete lack of expression of IL-1 alpha, IL-3 and IL-6 and the constant and usually high expression of SCF. On the other hand, these myeloid blast cells express generally high levels of the four cytokine receptors. The data suggest that the regulation of the expression of IL-1 alpha, IL-3 and IL-6, at least in our limited number of leukemic cell populations studied, is independent of that of SCF. The results indicate that, at least in most of the leukemic myeloid blasts cells, the expression of SCF and its receptor, the c-kit oncogene, may permit an autocrine regulation of cell cycling.

Acute Disease↗
PubMed1993

Overexpression of c-kit in a leukemic cell population carrying a trisomy 4 and its relationship with the proliferative capacity.

The expression of c-kit and its ligand, the stem cell factor (SCF), was studied in five cases of acute myeloid leukemia. One of these had a trisomy of chromosome 4, where the c-kit oncogene is located. In this case, the c-kit oncogene was overexpressed, but matched by a low expression of its ligand, SCF. The molecular evaluation of the growth rate by c-myc and the histone H3 expression indicated that the growth fraction of this cell population was very low. In one of the other leukemic cell populations studied, characterized by a low expression of c-kit and an elevated expression of the SCF, the growth fraction was also very low. Our results suggest that at least for some receptor oncogenes, the simple overexpression cannot be taken as an indication that the oncogene is involved in the deregulation of cell proliferation.

Base Sequence↗
PubMed1993

[Myocardial contusion with and without coronary artery involvement].

We present the first case of myocardial contusion secondary to blunt chest trauma with injury in two different areas, with and without coronary lesions. The akinesis of the anterolateral wall was secondary to the obstruction of the left anterior descending coronary artery and the severe hypokinesis of the posterior wall, without lesion of the right coronary artery, was probably related with the compression between the sternum and vertebral column.

Accidents, Traffic↗