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Biomedical subjects

F Felici

Publications and source records attributed to F Felici.

At least 19 recordsLinked to original sources
PubMed1999

Uptake and intracellular fate of phage display vectors in mammalian cells.

Receptor-mediated endocytosis is exploited in experimental systems for selective delivery of genes and drugs into specific cells. To improve targeting efficiency of delivery vectors, we have used phage display technology to isolate novel ligands for endocytosed receptors. We show here that phage vectors internalized by mammalian cells via integrin-mediated endocytosis can be rescued by cell lysis and quantitated by infection of bacteria. Immediately following uptake, phage enter an intracellular compartment where they remain intact, with phage titer unaffected by the addition of chloroquine. Phage are then translocated to a second intracellular compartment in which they are inactivated and their titer affected by chloroquine. Immunofluorescence microscopy showed an association of the second compartment with supranuclear organelles. The ability to recover internalized phage in an infectious form from two distinctive intracellular compartments provides a means to select novel ligands from phage libraries for targeted delivery of macromolecules into mammalian cells.

Amino Acid Sequence↗
PubMed1999

Targeted delivery of multivalent phage display vectors into mammalian cells.

Novel peptide motives targeting endocytosing receptors were isolated from phage display libraries of random peptides by recovering internalized phage from mammalian cells. The peptide-presenting phage selected by internalization in HEp-2 and ECV304 human cells were taken up 1000- to 100,000-fold more efficiently than their parent libraries, and from 10 to 100 times faster than phage particles displaying integrin-binding peptides. A high degree of selectivity of phage uptake was observed in these cells: phage selected in ECV304 cells were internalized approximately 100-fold more efficiently in ECV304 cells than in HEp-2 cells. Likewise, phage selected in HEp-2 cells were subsequently taken up approximately 40-fold more efficiently by HEp-2 cells than by ECV304 cells. In multiple independent trials using a cyclic peptide library, an identical peptide sequence displayed on phage was internalized by and recovered from ECV304 cells. These findings indicate that the internalization process is highly selective, and is capable of capturing a specific peptide from 2 x 10(7) peptide variants. Immunofluorescence microscopy showed juxtanuclear localization of internalized phage. These results demonstrate the feasibility of using multivalent phage-display libraries to identify new targeting ligands for the intracellular delivery of macromolecules.

Amino Acid Sequence↗
PubMed1999

Force generation performance and motor unit recruitment strategy in muscles of contralateral limbs.

The purpose of the present study was to determine whether the motor unit (MU) recruitment strategy of the agonist and antagonist muscles in the dominant arm differs from that in the non-dominant arm. The median frequency (MF) of the power density spectrum (PDS) of the electromyogram (EMG) was used as a tracking parameter to describe the MU recruitment. In 8 subjects the EMG was recorded from the biceps brachii and triceps brachii of each limb during isometric elbow flexion performed in a ramp fashion. Force was increased from 0 to 100% of the maximum voluntary contraction (MVC) in 3 s following a track displayed on an oscilloscope. When comparing the dominant versus non-dominant arm we found no statistical difference in the MU recruitment pattern of the biceps brachii and the triceps. Because the dominant arm was not always the better performing arm, we grouped the data according to the ability of the subjects to track the ramp signal. In this case we found a statistically significant difference between the better and worse performing arm in the full MU recruitment of the biceps. A more precise and accurate control of the increase in force was obtained when the central nervous system selected a slower and prolonged recruitment of MUs in the agonist muscle.

Adult↗
PubMed1999

Cost of walking and locomotor impairment.

The aim of the present study was to evaluate the importance and the necessity of metabolic measurements to quantify locomotor impairment in a clinical context. Oxygen consumption, heart rate, pulmonary ventilation and walking speed were measured during locomotion in 14 normal subjects, used as a control group, and 82 patients with different pathologies [hemiparetic, paraparetic, tetraparetic, orthopaedic and paraplegic patients, who walked using a reciprocating gait orthosis (RGO)]. The subjects were characterized on the basis of a cumulative impairment score (CIS), based on clinical scales commonly used to evaluate impairment and disability in locomotion. Appropriate indices of energy, cardiac and ventilatory costs expressed per metre walked, globally called physiological costs, were obtained. It resulted that the most comfortable speed (MCS) of normal subjects was significantly higher than that of each group of patients. Normal subjects' physiological costs were found to be significantly lower than those of patients who needed either a device or the help of a person to walk. All measured parameters correlated significantly with each other. The MCS was found to be the most correlated parameter with the CIS (r = 0.8), and therefore it must be considered the best single measurement, if only one is to be used. Measurements more precise than MCS, such as the physiological costs, may be necessary in clinical trials.

Adult↗
PubMed1997

Induction of anti-carbohydrate antibodies by phage library-selected peptide mimics.

One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T-independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage-displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O-antigen (O-Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage-displayed nonapeptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme-linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3-specific) and p115 (interacting with both mIgA) were able to induce anti-O-Ag antibodies. The immune response was specific for the O-Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPLGALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti-polysaccharide vaccines.

Animals↗
PubMed1997

Rehabilitation of walking for paraplegic patients by means of a treadmill.

The present study was aimed at investigating the use of a treadmill for ambulation training of paraplegic subjects. To investigate the likely effectiveness of this modality of rehabilitation, six paraplegic patients (three male and three female) were studied, using new generation reciprocating gait orthoses (RGO and ARGO), in a treadmill training program. Oxygen consumption, heart rate, and pulmonary ventilation were measured when the subjects were walking at their most comfortable speed on the treadmill and on the open field. These measurements were carried out at the beginning of the study and after two and six months of treadmill training. The following findings were significant: the treadmill walking required 30% less energy than open field ambulation prior to training (P < 0.001) and 50% after training (P < 0.05). The most comfortable walking speed was faster on the treadmill than on the open field by 18% prior to training (P < 0.05) and 42% afterwards (P < 0.05). The energy cost was 50% less after 2 months training on the treadmill (P < 0.05) showing that treadmill training can improve the efficiency of over ground ambulation. It can be concluded that the treadmill training improves the RGO/ARGO walking capability, probably both the walking efficiency (short term adaptation) and physical fitness (long term adaptation).

Adult↗
PubMed1997

Is the Olympic boardsailor an endurance athlete?

The present study was carried out in order to describe the physiological profile of top Olympic boa rdsailors of both genders and to measure the energy cost during actual boardsailing with particular attention to the most demanding conditions. Fourteen elite Olympic boardsailors (7 males and 7 females) volunteered to participate in the study. Each subject underwent a maximal cycle ergometer test in orderto measure VO2peak and ventilatory threshold (Tvent). Additionally, anthropometric measurements including body fat percentage were taken. The cardiorespiratory demand and the energy cost of actual boardsailing were assessed by means of a very light telemetric device (K2 Cosmed) which allowed the measurements of VO2, VE and HR. VO2peak was 63.6 +/- 2.3 ml x kg(-1) x min(-1) (Tvent 70% of maximum) and 49.2 +/- 4.1 (Tvent 60% of maximum) in males and females, respectively. The data recorded during actual boardsailing show that this sport activity can be classified as aerobic, the VO2 values being above or very close to those of Tvent values (75% and 60% of the maximum in males and females). Furthermore the mean blood lactate values obtained at the end of each regatta testing were 6 +/- 2 mMol x l(-1) in males and 5 +/- 1.5 mMol x l(-1) in females indicating a partial involvement of anaerobic metabolism, which in some regatta phases could represent a limiting factor for performance.

Adipose Tissue↗
PubMed1997

Lymphoblastic lymphoma of the pre-B phenotype with cutaneous presentation.

Lymphoblastic lymphoma (LBL) is a neoplasm of lymphoid precursors presenting usually as acute leukemia with bone marrow and peripheral blood involvement. Primary cutaneous involvement of LBL with a pre-B phenotype has to be considered an extremely uncommon occurrence, accounting for less than 1% of all non-Hodgkin lymphomas. A child with an LBL involving a single cutaneous manifestation of 6 months duration is presented. At the time of presentation, the lesion consisted of a rapidly enlarging deeply infiltrated tumor on the upper arm. Immunophenotypic analysis performed an paraffin-embedded and frozen tissue sections revealed 2 pre-B phenotype of the tumor cells. Similar results were obtained from lymph node and bone marrow biopsy specimens. After 26 months of polychemotherapy, the patient is currently in complete remission. We wish to add this case to the current literature of LBL with cutaneous involvement, emphasizing the importance of a correct diagnosis and the excellent response to the therapeutic regimen.

B-Lymphocytes↗
PubMed1997

A new immunohistochemical methodology for the specific detection of MDR1-P-glycoprotein in human tissues based on phage-displayed peptides mimicking the MM4.17 epitope.

It is not rare that controversial indications about the presence or the expression level of multidrug-resistant (MDR) proteins come out from different laboratories upon examination of identical tumor specimens. Distinct aspects, including the use of weakly discriminating monoclonal antibodies (MAbs) and/or unsuitable techniques and procedures, contribute in generating differences in the MDR phenotype evaluation of cancer cells. In this regard we describe here an innovative immunohistochemical approach for the determination of P-glycoprotein expression in cells and tissues. The method is based on the ability of phage-displayed peptides to mimic antibody epitopes. For this purpose we utilized the phage clone #55, which was affinity-purified from a phage-displayed random-peptide library using the MAb MM4.17 (specific for MDR1-P-glycoprotein) as previously described. This clone has been chosen since it clearly and undoubtedly reacts with its cognate MAb, as was determined by ELISA and dot blot tests. Inhibition of the MAb MM4.17 binding to MDR1-P-glycoprotein-expressing cells could be performed by adding a calibrated concentration of phage clone #55 particles, which mimic MDR1-P-glycoprotein antigen. This methodology can eliminate misleading interpretations concerning the presence and expression level of MDR1-P-glycoprotein and might well contribute in routine clinical determinations of MDR in tumor specimens, thus contributing to our understanding of the basis of the mechanisms of tumor cell resistance to drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Low intensity physical training in older subjects.

BACKGROUND: This study was designed to evaluate the effects of a low intensity general training program (< 50% of heart rate reserve) on physical fitness of healthy older subjects, by comparing maximal and submaximal indices of training response. METHODS: Twenty-two volunteers over 60 years of age participated in the present study. The sample was randomly divided in an experimental group of 13 older subjects (3 men and 10 women, mean age 63.5 +/- 3 years) while the remaining 9 subjects (3 men and 6 women, mean age 64.2 +/- 4 years) served as inactive control group. After medical screening all participants were evaluated before and after 12 weeks in which the experimental subjects underwent a low intensity training. Each subjects-either inactive or active-performed two treadmill tests at two-days interval, to measure maximal and submaximal responses to exercise, respectively. Heart rate (HR), oxygen uptake (VO2) and pulmonary ventilation (VE) were measured using a telemetric apparatus. RESULTS: The major finding of the study was the significant improvement in submaximal response to exercise of experimental subjects, expressed by the reduction in HR, VO2 VE while VO2 max did not change. CONCLUSIONS: Thus, it appears that a low intensity general training similar to that followed in the present study may represent a good means to improve physical fitness in healthy elderly people. Similarly, this study supports the effectiveness of evaluation tests based on submaximal responses to exercise in this population.

Aging↗
PubMed1996

Topology of MDR1-P-glycoprotein as indicated by epitope mapping of monoclonal antibodies to human MDR cells.

The MDR1-P-glycoprotein binding sites of three different murine monoclonal antibodies (MM4.17, MM6.15 and MC57), directed towards living, intact human multidrug-resistant cells were investigated in order to study P-glycoprotein topology. By using synthetic peptide scanning, we demonstrated that well-defined regions localized on the predicted first, fourth and sixth extracellular loops are external. On the basis of the structure of MM6.15 epitope, which is distributed on the above three different extracellular loops (and thus is discontinuous), P-glycoprotein molecules result to be differently organized in the lipid bilayer. Moreover, the outcome of the MC57 and MM4.17 epitopes localization experiments, obtained through the use of phage-displayed peptide libraries, represent an additional challenge to the classical 12-transmembrane domain model of P-glycoprotein, since they agree with the novel topography of the molecule (10-transmembrane domain), which was recently proposed on the basis of biochemical and expression studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Selection of biologically active peptides by phage display of random peptide libraries.

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein-protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.

Animals↗
PubMed1995

Isolation of antigenic mimics of MDR1-P-glycoprotein by phage-displayed peptide libraries.

To identify an MC57 epitope which is more efficiently expressed on inactivated forms of P-glycoprotein we utilized peptide libraries displayed on filamentous phage. Using this technology, we selected specific phage clones blocking the binding of the murine monoclonal (MAb) MC57 with live human multi-drug-resistant (MDR) cells, and sequenced and analyzed their DNA. The results we obtained indicate that MAb MC57 epitope could be formed by 2 regions localized on the predicted fourth and sixth extracellular loops of the current 12-transmembrane-domain model predicted for MDR1-P-glycoprotein. Surprisingly, a third region, defined by residues 800-807 of the MDR1-P-glycoprotein sequence and postulated to be intracellular, was also identified as a putative part of the MC57 epitope. This finding adds weight to the interesting hypothesis that a P-glycoprotein structure different from the current model may exist.

ATP Binding Cassette Transporter, Subfamily B, Mem↗