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Biomedical subjects

L Silvestro

Publications and source records attributed to L Silvestro.

At least 55 records · Page 3Linked to original sources
PubMed1997

Paclitaxel plus doxorubicin in breast cancer: an Italian experience.

Based on preclinical data, phase I/II clinical trials were performed at Istituto Oncologico Romagnolo (IOR) Operative Units (Medical Oncology Departments of Forlì, Rimini, and Ravenna, Italy) to determine the efficacy and toxicity of sequential administration of doxorubicin followed by paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in the treatment of patients with advanced breast cancer that either had been previously untreated or that had relapsed after adjuvant therapy. In the phase I trial, 19 patients received bolus doxorubicin (50 mg/m2) followed after a 16-hour interval by paclitaxel (given at dose levels ranging from 130 to 250 mg/m2) by 3-hour infusion every 3 weeks, for a maximum of eight cycles. Paclitaxel doses were escalated in 30-mg/m2 increments if the maximum tolerated dose had not been reached in the previous dose level. Analysis of the 128 cycles assessable for toxicity demonstrated neutropenia (<500/microL) in 26 courses (20.3%), with no significant clinical events. No relevant clinical cardiotoxicity was observed. The paclitaxel maximum tolerated dose was not reached at the 250-mg/m2 dose level (no grade 3 or 4 extramedullary toxicity). In the IOR phase II trial, 13 patients were treated with fixed doses of both drugs (doxorubicin 50 mg/m2 and paclitaxel 220 mg/m2). Grade 4 neutropenia occurred in 39 of the 95 cycles, but was complicated by fever in only eight cycles (8.4%); three cycles required granulocyte colony-stimulating factor support. Peripheral neurotoxicity was the most common extramedullary side effect noted. Overall clinical responses in the IOR trials included 10 complete responses (31.3%) and 15 partial responses (46.9%), with an objective response rate of 78.1%. Comparison of these results with those obtained from a phase I trial using the opposite drug sequence showed comparable overall response rates, but IOR's sequence was associated with a higher complete response rate, as well as less frequent and less severe nonhematologic toxicity.

Adult↗
PubMed1996

Cytogenetics of pediatric central nervous system tumors.

A cytogenetic analysis was performed on short-term cultures of 43 previously untreated childhood central nervous system neoplasms of various histology. The cells were obtained from pediatric patients, none of whom had received therapy before karyotypic evaluation. Successful chromosome studies were performed on 24 tumors. The most commonly detected structural abnormalities involved chromosomes 1 and 17. Other structural chromosome abnormalities involved chromosomes 3, 6, 8, 9, 11, 12, and 20.

Adolescent↗
PubMed1996

Determination of 1-O-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, platelet-activating factor and related phospholipids in biological samples by high-performance liquid chromatography--tandem mass spectrometry.

Combining normal-phase HPLC separation and tandem mass spectrometric detection, using an ion-spray HPLC-MS interface, a quantitative method for acyl-platelet activating factor (acyl-PAF), platelet-activating factor (PAF) and related phospholipids was developed. Mass spectra, positive ions, showed intense [M+H]+ ions; collision-induced dissociation of protonated molecular ions gave characteristic daughter ions corresponding to the polar head. Detection limits of 0.1-0.3 ng injected were obtained by multiple reaction monitoring. Samples of human endothelial cells treated with compounds modulating the levels of acyl-PAF and PAF have been analyzed by the present technique, proving that this approach is suitable for biochemical studies.

Cells, Cultured↗
PubMed1996

Plasmin promotes an endothelium-dependent adhesion of neutrophils. Involvement of platelet activating factor and P-selectin.

BACKGROUND: The adhesion of leukocytes to the endothelium and the edema of vessel wall may cause vascular reocclusion after thrombolytic therapy. The aim of this study was to evaluate the role of platelet activating factor (PAF) and P-selectin on the adherence of polymorphonuclear neutrophils (PMN) to the endothelium and of PAF on the increased vascular permeability induced by tissue-type plasminogen activator, streptokinase, and plasmin. METHODS AND RESULTS: We studied (1) the adhesion of 111Inlabeled PMN to human umbilical cord vein-derived cultured endothelial cells (HUVEC), (2) the transfer of 125I-labeled albumin across HUVEC monolayers, and (3) the adhesion of PMN to isolated bovine coronary arteries under flow conditions. It was found that the adhesion of PMN, induced by tissue-type plasminogen activator, streptokinase, and plasmin, correlated with the synthesis of PAF by HUVEC and was inhibited by WEB 2170, a PAF receptor antagonist. The adhesion of PMN was also inhibited by the treatment of HUVEC with anti-P-selectin antibodies or of PMN with soluble P-selectin or with anti-CD18 monoclonal antibodies. Plasmin also increased the permeability of HUVEC monolayers, an effect that was partially prevented by WEB 2170. Moreover, plasmin promoted the synthesis of PAF from isolated bovine coronary arteries and the adherence of PMN to the endothelium under flow conditions. The pretreatment of PMN with WEB 2170 or with soluble P-selectin prevented adhesion. CONCLUSIONS: The synthesis of PAF by endothelial cells at the site of plasmin generation and the endothelial expression of P-selectin may render the endothelial cell surface proadhesive for neutrophils and may favor a local increase in vascular permeability.

Animals↗
PubMed1996

A confirmatory HPLC-MS/MS method for ten synthetic corticosteroids in bovine urines.

In the present study, an HPLC-MS/MS method to confirm, in bovine urine, the most common synthetic corticosteroids illegally used as growth promoters in livestock breeding will be presented. An API III-Plus (PE-Sciex) triple quadrupole mass spectrometer, interfaced by means of an atmospheric pressure chemical ionization source to the HPLC system, was used. Urine samples were treated with a sulfatase-glucuronidase mixture to cleave the drug-conjugates and then extracted on C18 disposable columns. LC separations were performed on a reversed-phase C18 column with ammonium acetate 0.1 M/acetonitrile (60/40, v/v) as mobile phase. Detection was performed in multiple reaction monitoring mode, negative ions, selecting fragmentations characteristic of 10 corticosteroids used more frequently. Good results, in terms of sensitivity and specificity have been obtained for nine corticosteroids that can be analyzed in the same HPLC run; the limits of sensitivity achieved were 0.05-1.0 ng/ml in urine. Only a more polar corticosteroid, required a different HPLC separation. Practical applications of this technique to real samples proved that it is an effective method to confirm the illegal use of corticosteroids as growth promoter in animal. In comparison with the chemical GC-MS methods the simpler sample preparation and the faster time of analysis permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity.

Adrenal Cortex Hormones↗
PubMed1996

High-performance liquid chromatography/tandem mass spectrometry identification of salmon calcitonin degradation products in aqueous solution preparations.

Degradation impurities have been investigated, by means of high-performance liquid chromatography (HPLC)/ mass spectrometry, in commercial ampoules of salmon calcitonin, aqueous solution, stored for up to 2 years. Oxidation derivatives of the cysteine residues in positions 1 and 7, with cleavage of the disulfide bridge, were observed in all samples. Using our reversed-phase separation, these oxidation derivatives coeluted and presented an HPLC behavior identical to the derivative of salmon calcitonin with the disulfide bridge reduced. In a previous study this last compound was tentatively identified, without confirmation by mass spectrometry, as a major degradation product of salmon calcitonin in aqueous solution.

Amino Acid Sequence↗
PubMed1995

Adherence of human monocytes to haemodialysis membranes: LFA 1 (CD11a/CD18) CR1 (CD35) and CR3 (CD11b/CD18) triggering promotes the biosynthesis of platelet-activating factor and adherence.

BACKGROUND: Platelet-activating factor is a mediator of inflammation involved in the blood-membrane interaction. We report that selective stimulation of complement receptors (CR1 and CR3) triggers PAF synthesis and monocyte adherence to complement-activating membranes. METHODS: The synthesis of PAF was studied after stimulation of normal human adherent monocytes with F(ab)2 and Fab fragments of monoclonal antibodies specific to CR1 and CR3. CD11a, CD11b, CD18, and CD35 was studied by flow cytometry on neutrophils and monocytes. The molecular species of PAF from stimulated monocytes were identified by reverse-phase high-performance liquid chromatography coupled with mass spectrometry. RESULTS: Anti-CR1 and anti-CR3 monoclonal antibodies induced a dose-dependent C-16 but not C-18 PAF production. The latter occurred also with monovalent Fab fragments of both anti-CR1 and anti-CR3 monoclonal antibodies, that were not internalized as seen by immunofluorescence. Adherence of monocytes to Cuprophan membranes was markedly higher (P < 0.01) in membranes pretreated with fresh than with heat-inactivated normal plasma. However, the high adherence to fresh plasma-treated membranes was completely abrogated by coincubating the cells with Web 2170, a specific PAF receptor antagonist. This was not due to downregulation of adhesion molecules expression on leukocytes. CONCLUSIONS: These studies implicate a crucial role of PAF in blood interaction with haemodialysis membranes that fix complement activated products.

Antibodies, Monoclonal↗
PubMed1994

A novel dimeric fluoropyrimidine molecule behaves as a remote precursor of 5-fluoro-2'-deoxyuridine in human erythrocytes.

A new dimeric fluoropyrimidine molecule (5-fluoro-2'-deoxyuridilyl-(5'-->3')-5-fluoro-2'-deoxy-5'-uridylic acid, Compound 1) was chemically synthesized from two separately deblocked 5-fluoro-2'-deoxyuridine mononucleotide moieties. Other structurally related nucleotides, 5-fluoro-2'-deoxyuridine-5'-diphosphate (FdUDP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine-3',5'-bisphosphate were also synthesized. The structures of all synthesized molecules were verified by mass spectrometric analyses and were consistent with expected molecular mass values. The metabolic patterns of conversion of Compound 1 were investigated both in human erythrocyte lysates and in intact erythrocytes previously loaded with this molecule according to a highly conservative encapsulation procedure. In hemolysates, Compound 1 was transformed to 5-fluoro-2'-deoxyuridine (FUdR) and to 5-fluorouracil (FU) through the intermediate formation of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). In intact red cells, Compound 1 still generated FUdR (and to a lesser extent FU), that was then released outside. The conversion pathway involves a phosphodiesterase-catalysed hydrolysis of Compound 1 into two FdUMP molecules, followed by further dephosphorylation to FUdR and by partial conversion to FU. Unlike hemolysates, Compound 1-loaded intact erythrocytes featured transient formation of FdUDP and FdUTP, both metabolites representing storage compounds for the final and sustained production of FUdR and FU. Therefore, human erythrocytes can behave as bioreactors ensuring the time-controlled production and delivery of the two powerful antitumor drugs FUdR and FU from encapsulated Compound 1. This new molecule and other compounds as well (e.g. FdUDP and FdUTP) can be viewed as useful pre-prodrugs, exploitable for intraerythrocytic bioconversion reactions.

Erythrocytes↗
PubMed1994

Involvement of a serine protease in the synthesis of platelet-activating factor by endothelial cells stimulated by tumor necrosis factor-alpha or interleukin-1 alpha.

It has been shown that production of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells (EC) stimulated with tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha requires the synthesis of new proteins and is regulated by anti-proteinases. Here, we demonstrate that TNF-alpha and IL-1 alpha induce the expression by EC of a 34-kDa diisopropyl fluorophosphate-binding protein immunoprecipitated by an anti-human elastase antibody. This protein is released in the medium and cleaves the chromogenic substrate N-methoxysuccinyl- Ala-Ala-Pro-Val p-anilide, which is specific for elastase. The generation of this elastase-like protein seems to be important for the synthesis of PAF induced by TNF-alpha and IL-1 alpha, as suggested by the following observations: (a) it precedes the synthesis of PAF; (b) the inhibitors of serine protease and anti-human elastase antibody prevent the synthesis of PAF and the activation of 1-O-alkyl-2-lyso-glycerophosphocholine acetyl-CoA: acetyltransferase, which is a key enzyme of the PAF remodelling pathway; (c) elastase, at concentrations similar to that detectable in the medium of cytokine-activated EC, elicits a rapid synthesis of PAF by EC. High-performance liquid chromatography-tandem mass spectrometric analysis of bioactive PAF demonstrates that the molecular species produced after stimulation of EC with TNF-alpha, IL-1 alpha or elastase are similar, with a predominant synthesis of the alkyl species. These results indicate that TNF-alpha and IL-1 alpha stimulate the production of a serine protease which is critical in the activation of enzymes involved in PAF synthesis, suggesting the potential involvement of this mechanism in the regulation of EC functions.

Acetyltransferases↗
PubMed1994

Preparation and characterization of deuterium-labeled glycosaminoglycans.

Heparin, NAcHep, DS, and CS were labeled with deuterium by N-reacetylating, with the deuterated acetic anhydride (CD3CO)2O, GAGs previously N-deacetylated (by hydrazinolysis) to the desired extent. Degrees of deuteration of the present preparations, as determined by 2H- and 1H-NMR were 15%, 51%, 49%, and 79% for heparin, NAcHep, DS, and CS, respectively. The NMR analysis (including the 13C spectra) of the labeled products indicated that deuterium labeling did not involve any substantial modification of the GAG structures. Also NMR signals associated with specific sequences of heparin for antithrombin and of DS for heparin cofactor II were essentially the same in the unlabeled and in the deuterated GAGs. The substantial retention of the original structure was confirmed by data on the degree of sulfation (by conductimetry) and on the electrophoretic mobility in acid buffer. On the other hand, HPLC/SEC data indicated some depolymerization of heparin and DS in the N-deacetylation step of the labeling reactions. HPLC/MS spectrometry permitted a clear identification of disaccharide and tetrasaccharide fragments obtained from deuterated GAGs by enzymic (heparinase, chondroitinase ABC) or chemical depolymerization (deaminative cleavage, Smith degradation), opening new prospects for studies of human pharmacokinetics, with differentiation of exogenous from endogenous GAGs.

Acetic Anhydrides↗
PubMed1994

Platelet-activating factor-induced endothelial cell expression of adhesion molecules and modulation of surface glycocalyx, evaluated by electron spectroscopy chemical analysis.

PAF synthesized by or added to the endothelial cell monolayer has as first target the endothelium itself, inducing the expression of GMP-140 on the cell surface and qualitative alterations in the composition of glycocalyx. As seen by ESCA, sulfated chemical groups were significantly reduced after PAF stimulation. This reduction depended on selective loss of sulfated proteoglycans that were released into the supernatant. The alteration of glycocalyx may favor functional exposure of GMP-140 and of PAF on the surface of endothelial cells and may reduce charge repulsive forces between cells. PAF associated with endothelial cell surface has as second target PMNs and stimulates in cooperation with GMP-140, the functional activation of PMN CD11-CD18 adhesion molecules.

Antibodies, Monoclonal↗
PubMed1994

Effects of heparin and its desulfated derivatives on leukocyte-endothelial adhesion.

The effect of heparin and partially desulfated heparin derivatives on thrombin and PAF-induced adhesion of PMNs to the endothelium was studied either by a fluorescence image analysis or by 111In-labeled PMNs. The results observed with these two techniques consistently indicated that heparin and O-des-heparin inhibited PMN adhesion in a dose-dependent manner. Moreover, N-des-Hep and N-O-des-Hep, even if less effective, also inhibited the adhesion of PMNs when used at high concentrations. The effect of heparin and heparin derivatives was not directed to endothelial cells but rather to PMNs, as shown by the absence of inhibitory effects, when heparins were preincubated with endothelium.

Cell Adhesion↗
PubMed1994

Human pharmacokinetics of glycosaminoglycans using deuterium-labeled and unlabeled substances: evidence for oral absorption.

In the present study, the pharmacokinetics of extractive GAGs used as therapeutic agents have been studied after intravenous and oral administration on volunteers. The use of native or deuterium-labeled compounds, followed by HPLC/MS detection, allowed the quantitation of exogenous heparin and DS as major disaccharide fragments, obtained either by enzymatic or chemical depolymerization. In particular the high level of labeling reached in DS allowed its differentiation from structurally related endogenous species. The estimated plasmatic bioavailability was about 18% for DS. Notwithstanding the impossibility of evaluating the same parameters for heparin species, due to the interferences of endogenous GAGs, the results obtained provided clear evidence of oral availability of heparin and DS through detection and quantitation of structures specifically related to these GAGs. Due to the selectivity of the lyases used, the enzymatic degradation specifically allowed the detection of both DS and heparin species still retaining the original sulfation pattern. Additionally, the chemical degradation could detect the main metabolites of the drugs, consisting of partially to totally desulfated GAGs showing a more or less marked reduction in their molecular weight.

Administration, Oral↗
PubMed1993

Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion and non-enzymic binding to membrane proteins.

We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.

Adenine Nucleotides↗