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Biomedical subjects

S Ferrari

Publications and source records attributed to S Ferrari.

At least 217 records · Page 12Linked to original sources
PubMed1994

Selenium status, fatty acids, vitamins A and E, and aging: the Nove Study.

To investigate the relationships between aging and selenium status, vitamins A and E, and plasma and erythrocyte fatty acids, we studied 105 healthy subjects (53 women, 52 men) living in Nove, a village near Vicenza (Veneto Region, northern Italy). The subjects were distributed equally for age and sex into four groups: group 1, 20-39 y; group 2, 40-59 y; group 3, 60-75 y; and group 4, > 75 y. A careful selection of subjects to exclude those with chronic or acute diseases was obtained with the collaboration of the three general practitioners operating in Nove. Aging was associated with a progressive decrease in selenium status and in the ratio of plasma and erythrocyte polyunsaturated to saturated fatty acids (P:S). Stepwise multiple linear analysis revealed age, vitamin A, and n-6 polyunsaturated fatty acids (PUFAs) as useful predictors of a substantial proportion of the selenium variability (R = 0.618, R2 = 0.382; P < 0.001) and age and erythrocyte oleic acid as predictors of erythrocyte glutathione peroxidase variability (R = 0.413, R2 = 0.17; P < 0.001).

Adult↗
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 of B7 costimulatory molecule in cultured human epidermal Langerhans cells is regulated at the mRNA level.

Langerhans cells (LC) belong to the dendritic cell lineage and are the principal antigen-presenting cells of squamous epithelia. Short-term cultured LC (cLC) exhibit a marked augmented capacity to stimulate allogeneic T cells and acquire the ability to activate naive T cells, probably in relation to enhanced expression of accessory signals. In this study, we evaluated the expression of B7 costimulatory molecule (CD80) in human freshly isolated (fLC) and cLC at both the protein and mRNA level. Staining of frozen skin sections did not reveal any epidermal dendritic cell reactive with either of two different anti-B7 monoclonal antibodies. fLC in suspension did not exhibit any B7 staining as evaluated by two-color flow-cytometry analysis and immunoelectron microscopy. In contrast, LC that were cultured for 24-72 h displayed strong surface B7 reactivity with a characteristic patchy pattern. Treatment with dispase and trypsin did not reduce B7 staining of cLC. Following warming to 37 degrees C, cLC tagged with anti-B7 monoclonal antibody and gold-conjugated secondary antibody could internalize surface B7 by using the organelles of receptor-mediated endocytosis. B7 mRNA, detected by the reverse-transcriptase polymerase chain reaction technique, was expressed at a low level in purified (> 90% HLA-DR+) fLC but not in LC-depleted epidermal cells, and was markedly upregulated in purified cLC. The results indicate that 1) fLC do not express B7 protein on their surface, but acquire B7 during culture, 2) surface B7 is not sensitive to trypsin, 3) B7 expression is regulated primarily at the mRNA level, and 4) membrane B7 can be internalized within cLC. B7 molecule on CLC may be relevant to their increased antigen-presenting cell potency and ability to stimulate naive T lymphocytes.

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

High-dose methotrexate treatment and liver function in patients with osteosarcoma.

OBJECTIVES: To study the effects of short-term high-dose methotrexate therapy on liver function in patients with osteosarcoma. DESIGN: Open prospective study. SETTING: Department of Internal Medicine and Oncology, Istituto Ortopedico Rizzoli, Bologna, Italy. SUBJECTS: Fourteen patients with osteosarcoma, with no evidence of previous or actual liver disease at the time of diagnosis. INTERVENTIONS: All patients received a cumulative dose of 30-57 gm-2 of methotrexate within 6 months as neo-adjuvant chemotherapy (pre- and post-surgery). Each course of chemotherapy included methotrexate at a dose of 8-12 gm-2 and, in addition, adriamycin and cisplatinum. MAIN OUTCOME MEASURES: Galactose elimination capacity and antipyrine clearance were measured at baseline, after the first course of chemotherapy, at the end of the pre-operative period and at the end of chemotherapy. In each case they were carried out after transaminase levels had returned to normal. RESULTS: Galactose elimination capacity decreased from 2.45 (+/- 0.48) mM min-1 to 2.04 (+/- 0.60) mM min-1 after the five planned courses of chemotherapy (P = 0.013, Wilcoxon signed-rank test), without any change in routine liver function tests. No differences in antipyrine clearance and half-life were demonstrated (n = 8). CONCLUSIONS: The data are consistent with a decreased reserve capacity of the liver after short-term, high-dose methotrexate. Long-term survivors deserve monitoring of liver function for safer methotrexate use, in the light of progressive dosage increments to improve prognosis in neoplastic diseases.

Adolescent↗
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

Specific binding to vitamin D response elements of chicken intestinal DNA-binding activity is not related to the vitamin D receptor.

In this report we confirm that the putative vitamin D response element (VDRE), located between -320 and -306 in the chicken calbindin-D28K gene, is not a binding site for the vitamin D3 receptor (VDR). In examining the ability of chicken intestinal nuclear extracts (CINE) to bind known VDREs, we observed a specific VDRE-binding activity, which is distinct from VDR. In fact, VDR-depleted CINE retains the ability to bind the rat osteocalcin VDRE. The VDRE-binding activity binds DNA with high affinity and contacts it at the same guanine residues as VDR. Its specificity in binding structural variants of the AGGTCA repeat is broader than that of VDR, as direct repeats spaced by 3, 4, and 5 base pairs are almost equally effective competitors when added to the probe in molar excess. Palindromic arrangements of the same motif are lower affinity competitors. The retinoid-X receptor is involved in the binding complex, as incubation of CINE with antibody to retinoid-X receptor results in a quantitative supershift. Antibodies to retinoic acid receptors (RAR alpha and -beta), T3 receptor, or chicken ovalbumin up-stream promoter-transcription factor had no apparent effect. These data suggest that species specificity is a relevant aspect of VDR/VDRE recognition, and that a novel factor(s), different from VDR, might be involved in the effect of vitamin D on gene expression.

Animals↗
PubMed1994

Differential response of embryonic and fetal myoblasts to TGF beta: a possible regulatory mechanism of skeletal muscle histogenesis.

Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGF beta. Under the conditions employed, TGF beta inhibited differentiation of fetal but not of embryonic myoblasts. To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers first appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Co-culture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGF beta) can undergo differentiation, while fetal myoblasts are inhibited by TGF beta. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Treatment recommendations for osteosarcoma and adult soft tissue sarcomas.

During the past 20 years, dramatic improvements have been obtained in the treatment of localised osteosarcoma of the extremities, both in the rates of disease-free survival and in quality of life. Twenty years ago 80 to 90% of the patients died, in spite of mutilating surgery, but now about 75% survive and avoid the necessity of amputation. This is due to the introduction of very effective combined treatments, mostly also using preoperative chemotherapy. One of the major issues is that of intensive preoperative chemotherapy, which improves both limb salvage and survival. A multidisciplinary approach is necessary to obtain good results. When the role of adjuvant or neoadjuvant chemotherapy is not accurately defined for soft tissue sarcomas, particular emphasis is given to the staging of the diseases and to the important role of local treatment in the survival of these patients. A combination of radiation therapy and surgery is strongly recommended.

Antineoplastic Agents↗
PubMed1994

S6 phosphorylation and the p70s6k/p85s6k.

Activation of cell growth leads to the multiple phosphorylation of 40S ribosomal protein S6. The kinase responsible for controling this event is termed p70s6k/p85s6k. Both isoforms of the kinase are derived from a common gene activated by a complex set of phosphorylation events; each resides in a unique cellular compartment: the p70s6k in the cytoplasm and the p85s6k in the nucleus. Although p70s6k/p85s6k represent the first mitogen-activated serine/threonine kinase described, the signaling pathway leading to activation of both isoforms remains obscure. Recent studies have shown that this pathway is distinct from that of p21ras and the p42mapk/p44mapk, and that bifurcation of these pathways takes place at the level of the receptor. Experiments with point mutants of the PDGF receptor and inhibitors of phosphatidyl-inositol-3-OH kinase have implicated the latter molecule in this signaling event, but more recent findings suggest an alternative route may be employed. The p70s6k signaling pathway can also be ablated by the immunosuppressant rapamycin, which blocks p70s6k activation and S6 phosphorylation without affecting the other kinases whose activation is triggered by mitogen treatment. In parallel, rapamycin suppresses the translation of a family of mRNAs that contain a polypyrimidine tract at their 5' transcriptional start site. The implication is that this event is mediated by the phosphorylated form of S6 that may either (1) directly interact with the polypyrimidine tract or (2) alter the affinity of the 40S ribosome mRNA binding site for polypyrimidine tract mRNAs, or (3) recognize proteins that directly bind to the polypyrimidine tract.

Animals↗
PubMed1994

[Nonmetastatic osteosarcoma of the limbs treated with neoadjuvant chemotherapy: correlation of tumor volume evaluated by CT and prognosis].

A relationship between tumor volume and prognosis has been demonstrated in extraosseaous solid tumors, soft tissue tumors and Ewing's sarcoma. Fifty-five osteosarcoma patients, treated according to IOR-OS/NEO 3 protocol, have been studied in an attempt to verify if tumor volume is also a significant prognostic factor for the osteosarcoma of the extremities. Tumor volume was measured by CT-Scan. A trend towards a better prognosis for smaller tumors was observed, but no statistically significant differences were demonstrated according to the tumor size in the population studied. The authors conclude that for patients with osteosarcoma of the extremities, tumor volume can not be considered an adequate prognostic factor on the basis of which to tailor the chemotherapy treatment, as been recently proposed by other authors.

Adolescent↗
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

Primary chemotherapy and delayed surgery for nonmetastatic osteosarcoma of the extremities. Results in 164 patients preoperatively treated with high doses of methotrexate followed by cisplatin and doxorubicin.

BACKGROUND: Neoadjuvant chemotherapy is the most accepted treatment for localized osteosarcoma. This has led to a great improvement in limb-sparing surgery and in disease-free survival. Patients with a good response to preoperative chemotherapy showed a higher disease-free survival rate. Current studies examine the possibility of patients whose limbs could be rescued with a poor necrosis and a reduction of the side effects related to aggressive treatments. METHODS: Between September 1986 and December 1989, 164 patients entered the second neoadjuvant study conducted at the Rizzoli Institute, Bologna, Italy, for non-metastatic osteosarcoma of the extremities. Preoperative chemotherapy consisted of two cycles of high-dose methotrexate intravenously (i.v.) followed by cisplatin intraarterially and doxorubicin i.v. After surgery, patients classified as good responders (> 90% tumor necrosis) received three more cycles of these drugs, whereas poor responders (< 90% tumor necrosis) had more chemotherapy, which included ifosfamide and etoposide in addition to the other three drugs. RESULTS: Limb salvage was performed in 83% of cases. At an average follow-up of 54 months (36-76), 109 patients (66%) were continuously disease-free, 2 died from doxorubicin cardiotoxicity, and 52 experienced metastases and 3 had local recurrence. In two of these three patients, metastases followed local recurrence. The 5-year actuarial continuously disease-free survival rate was 63%, with no differences between good and poor responders. Excluding 20 patients who had major protocol violations, the projected continuous disease-free survival rate was 71%. CONCLUSIONS: With an aggressive neoadjuvant chemotherapy, it is possible to cure more than 60% of nonmetastatic osteosarcoma of the extremities, avoiding amputation in most cases. Ifosfamide and etoposide seem to be effective in patients who did not respond to preoperative chemotherapy.

Adolescent↗
PubMed1993

The immunosuppressant rapamycin induces inactivation of p70s6k through dephosphorylation of a novel set of sites.

The immunosuppressant rapamycin selectively abolishes phosphorylation and activation of p70s6k/p85s6k at concentrations that either block or suppress cell growth. The four sites of phosphorylation associated with p70s6k/p85s6k activation all display Ser/Thr-Pro motifs and are closely clustered within a putative autoinhibitory domain of the enzyme. To produce a constitutively active, rapamycin-resistant form of the kinase, these four sites were converted to either Asp or Glu. When overexpressed in human 293 cells, the activity of the mutant is similar to that of the parent enzyme, under conditions where the parent is phosphorylated and active. Unexpectedly, however, the mutant remains sensitive to rapamycin and is inactivated in vitro by protein phosphatase 2A. Peptide maps reveal that rapamycin abolishes the activity of the overexpressed p70s6k through the dephosphorylation of a novel set of sites distinct from those associated with mitogenic activation.

Amino Acid Sequence↗
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

Active baculovirus recombinant p70s6k and p85s6k produced as a function of the infectious response.

Rat p70s6k and p85s6k have been expressed in baculovirus recombinants propagated in Sf9 insect cells. Surprisingly, both recombinant isoforms were active without coinfection of other kinases which lie upstream in the signaling pathway. Treatment of either recombinant form with phosphatase 2A leads to immediate inactivation in the absence of phosphatase inhibitors. Further studies show that the same four major Ser/Thr-Pro sites associated with p70s6k activation following mitogenic stimulation in vivo are also the four major sites phosphorylated in both the p70s6k and p85s6k during the infection process. It is proposed that the production of phosphorylated and activated recombinant p70s6k and p85s6k is due to activation of a host cell signaling pathway which is triggered by viral infection. In support of this hypothesis, wild-type virus-, but not mock-infected cells, exhibit the multiple phosphorylation of a ribosomal protein which migrates similar to ribosomal protein S6 on two-dimensional-polyacrylamide gels and extracts from these same cells contain elevated levels of S6 kinase activity.

Animals↗
PubMed1993

Identification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum.

All of the phosphorylation sites in 40 S ribosomal protein S6 derived from serum-stimulated Swiss mouse 3T3 cells are found within a small cyanogen bromide (CNBr) peptide derived from the carboxyl terminus, Lys218-Lys249. Further cleavage of the CNBr peptide or the intact protein with endoproteinase Lys-C (endo Lys-C) generated a single phosphorylated peptide, implying that all the sites of phosphorylation resided either between Arg231 and Lys243 or between Arg231 and Lys249 if cleavage at Lys243 was blocked by phosphorylation at a nearby residue. To discern between these possibilities and to identify the phosphorylation sites, the protein was purified from serum-stimulated cells and cleaved with endo Lys-C, and the single endo Lys-C phosphorylated peptide was isolated and sequenced following conversion of all the phosphorylated serines to S-ethylcysteine. The results show that the phosphorylated peptide extends from Arg231 to Lys249 and that the sites of phosphorylation in vivo are Ser235, Ser240, Ser244, and Ser247.

3T3 Cells↗