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Biomedical subjects

F Felici

Publications and source records attributed to F Felici.

At least 55 records · Page 3Linked to original sources
PubMed1995

Physical fitness testing in children operated on for tracheoesophageal fistula.

The maximal physical activity capacity of children operated on for tracheoesophageal fistula (TEF) has not been clearly defined. Eight patients (average age, 12 years) successfully operated on for TEF at birth underwent maximal exercise stress testing on a treadmill, according to the Bruce protocol, to test physical work capacity. Heart rate (HR), oxygen consumption (VO2), and pulmonary ventilation (VE) were measured by a portable lightweight telemetric device. Nine healthy children served as controls. Exercise duration was significantly lower for TEF subjects than for controls (11.6 +/- 1.7 minutes v 15.1 +/- 2.3 minutes; P < .01). Mean HR at rest and during exercise did not differ between the groups. All children reached the maximum HR according to their age; however, for the majority of TEF patients, this occurred at an earlier stage than in the controls. No differences were seen in mean VO2 at rest and on exertion between TEF and control children. However, maximal VO2, as measured at the end of exercise, was significantly different when normalized per kilogram of body weight (VO2/kg = 52.3 +/- 5.8 v 33.3 +/- 6.6; P < .005). The physical rehabilitation of TEF children usually takes into account only respiratory and nutritional factors. However, complete assessment of their cardiac and respiratory function, at rest and on exertion, also should be performed, because this may show that some patients have reduced motor performance; evidence is now accumulating that these children can safely participate in the same physical activities of their healthy peers.

Adolescent↗
PubMed1995

Identification of biologically active peptides using random libraries displayed on phage.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Amino Acid Sequence↗
PubMed1995

Peptide and protein display on the surface of filamentous bacteriophage.

The isolation of ligands that bind biologically relevant molecules is fundamental to the understanding of biological processes and to the search for therapeutics. Filamentous phage can be used to display foreign peptides and proteins in physical association with their DNA coding sequences. Repertoires larger than 10(8) phage clones expressing different peptide sequences can be prepared using molecular genetic techniques. The strategies utilizing this technology promise to provide not only new binding and possibly catalytic activities, but also lead structures for the development of new drugs and vaccines.

Amino Acid Sequence↗
PubMed1995

Ergonomy of paraplegic patients working with a reciprocating gait orthosis.

A reciprocating gait orthosis (RGO) is, among others, the most widely adopted device to restore the standing and walking capability of paraplegic patients. The aim of the present study was the evaluation of the energy demand (VO2), and cardiopulmonary load (HR and VE) imposed on the subject by different working tasks while sitting in a wheelchair or standing using a RGO. In addition, a comparison with the performance of normal subjects was also attempted. The RGO use allowed a dramatic improvement of patients' mobility and reach space in the workplace. A further advantage provided by the use of the RGO was represented by the increased mobility of the subjects with respect to the wheelchair confined situation. The energy demand and the cardiorespiratory load imposed on the subjects by the use of the RGO were not different from those observed both in the same subjects sitting in a wheelchair and in the controls. The energy demand slightly exceeded the values typical of light work and was, thus, compatible with the normal duration of a working day. On the other hand, the cardiac load corresponded to that typical of moderate activity, thus limiting the duration of the working task to 5-8 h. Based on the ergometry test, all of the working activities considered can be classified as aerobic activities, energy demand being under the ventilatory threshold.

Adult↗
PubMed1995

The efficiency of walking of paraplegic patients using a reciprocating gait orthosis.

A number of orthoses have been designed to restore stance and walking in paraplegic subjects. Among them, the reciprocating gait orthosis (RGO) appears to be one which is most widely adopted. The great energetic cost of orthotically aided walking is the main factor limiting the walking capability of paraplegic subjects. The purpose of the present research was to give a complete biomechanical evaluation of RGO locomotion in order to provide data that can be used for further improvements of the orthosis. From the whole body kinematics the mechanical work done to move the orthosis was estimated; by measuring the actual metabolic cost of RGO locomotion the efficiency was evaluated as the ratio between mechanical work and energy expenditure. It is found that RGO locomotion is a very demanding task in terms both of energy expenditure and of the mechanical work required. Furthermore, it is evident that the major determinant of this poor efficiency of RGO locomotion is the enormous amount of work required to move the orthosis which, in turn, reflects its poor mechanical characteristics. To accomplish the demand of real mobility, devices for transferring work from the upper to the lower portion of the body must be substantially improved. This technical aspect must be parelleled by suitable training programmes especially designed to improve the aerobic power of paraplegic subjects and their body composition (ie less fat and more muscle).

Adult↗
PubMed1995

Selection of phage-displayed peptides mimicking an extracellular epitope of human MDR1-P-glycoprotein.

To study the structural conformation of the MM4.17 monoclonal antibody (mAb) epitope, twenty-six mAb MM4.17-specific phage clones were affinity-isolated and their inserts characterized for amino acid composition and homology with MDR1 gene product (MDR1-P-glycoprotein). The resulting sequence alignment shows that a unique consensus sequence, which corresponds to the previously mapped TRIDDPET linear peptide identified through synthetic peptide scanning, could not be identified. However, similarities between the inserts of positive phage clones and P-glycoprotein primary structure, consisting in two or three amino acid-long sequences, were observed. An analysis of the over-represented amino acid residues in the inserts of positive clones, and their comparison with the sequence of the antigen was also performed. The two different procedures led to the identification of four regions in which these similarities are clustered, indicating that four different antigen regions, one of which includes the TRIDDPET linear amino acid sequence, might participate in forming the structure of monoclonal antibody MM4.17 epitope.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1994

Monoclonal antibodies that recognise filamentous phage: tools for phage display technology.

We generated six hybridoma cell lines that secrete monoclonal antibodies (mAb) which specifically bind filamentous phage coat proteins. Two of these mAb recognise epitopes that include the N terminus of the coat protein III (pIII), while two others are specific for the N terminus of the major coat protein VIII (pVIII). These mAb are valuable tools to study phage assembly and structure. Furthermore, we describe two examples of how these mAb can be exploited in the construction and screening of peptide libraries displayed by the filamentous phase major coat protein. We have used one of these mAb to develop a sensitive ELISA with crude phage supernatants. This assay allows rapid screening of large numbers of clones from random peptide phage libraries. Some of the anti-phage mAb described here can interfere with wild-type phage propagation, while phage carrying modifications in their coat proteins are insensitive to growth inhibition. We have exploited this observation as a tool to favour the growth of phage displaying peptides fused to pVIII, with respect to vector phage.

Amino Acid Sequence↗
PubMed1994

Recognition by human sera and immunogenicity of HBsAg mimotopes selected from an M13 phage display library.

We used two mouse monoclonal antibodies (mAb) specific for the human hepatitis B virus surface antigen (HBsAg) to screen a random peptide library of 15 amino-acid residues displayed as a fusion to protein III of filamentous phage M13. By a combination of affinity selection, immuno-screening and ELISA techniques, we selected peptides that are recognized by the anti-HBsAg mAb and show aa similarity with the natural antigen. The selected phage-displayed epitopes (phagotopes) behave as antigenic mimics of HBsAg. One phagotope is specifically recognized by human sera from HBsAg-immunized individuals, pointing to the possible use of phagotopes as markers to detect the presence of specific Ab in the serum. The same phagotope also elicits Ab directed against HBsAg in mice, indicating that mAb-selected phagotopes can also be immunogenic mimics of the natural antigen. These findings demonstrate that it is possible to identify disease-specific epitopes that can be used as diagnostic reagents and as leads for the development of acellular vaccines.

Animals↗
PubMed1994

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease-specific epitopes from phage-displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease-specific phagotopes can be identified out of this pool through an 'antigen independent' procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease-related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

Amino Acid Sequence↗
PubMed1994

Antigenic and immunogenic mimicry of the HER2/neu oncoprotein by phage-displayed peptides.

To recover peptides that antigenically and immunogenically mimic the p185HER2 oncoprotein, we selected the phage-peptide libraries pVIII-9aa and pVIII-9aa. Cys using murine monoclonal antibodies (mAb) MGr2 and MGr6, directed against two distinct epitopes of the p185HER2 extracellular domain. Phage-displayed peptides containing consensus amino acid motifs were recovered and shown to compete specifically for mAb binding on tumor cells that overexpress p185HER2. The deduced amino acid sequence of the peptides suggests that both epitopes defined by the mAb on p185HER2 are discontinuous and that hydrophobic interactions are involved in binding with the mAb. A phage clone displaying the GPLDSLFAQ peptide elicited a specific immune response against the p185HER2 in BALB/c mice, demonstrating that this phage-displayed peptide represents an immunological equivalent of the MGr2 epitope on p185HER2 and might be used as a substitute for this oncoprotein in in vitro and in vivo immunological studies.

3T3 Cells↗
PubMed1994

Epitope discovery using peptide libraries displayed on phage.

Peptides displayed on phage, which mimic continuous and discontinuous epitopes, can be selected using purified antibodies or preparations of polyclonal serum. This review describes recent advances in this field, discusses the application of phage-display technology to the diagnosis of human diseases, and presents new ideas for the preparation of vaccines directed against specific epitopes on a pathogen.

Amino Acid Sequence↗
PubMed1993

Mimicking of discontinuous epitopes by phage-displayed peptides, II. Selection of clones recognized by a protective monoclonal antibody against the Bordetella pertussis toxin from phage peptide libraries.

We have screened phage peptide libraries to establish if clones binding to a monoclonal antibody (mAb), specific for a discontinuous epitope, could be isolated and if the selected phage particles would be able to elicit an in vivo immuno-response against the original antigen. Two phage peptide libraries, consisting of 9 random amino acids inserted in the major coat protein (pVIII), were independently screened with a mAb which is capable of neutralizing the Bordetella pertussis toxin (PTX) in in vitro and in vivo assays. The epitope of PTX recognized by this and other protective mAb has been shown to be discontinuous. Six different positive phage clones were selected; their binding to the mAb could be competed for by PTX, showing that these clones bind to the antigen-binding site of the mAb. Three of the clones were used (alone or as a mixture) to immunize BALB/c mice. The sera showed a good immunoresponse both against the phage bearing the epitopes and against synthetic multiple-antigen peptides of the same sequence. The immune sera, however, showed no detectable signal against PTX and no capacity to neutralize the CHO-cell-clustering activity of the toxin. The results show that the selected recombinant phage are capable of mimicking the discontinuous epitope as far as binding to the corresponding mAb, but they are unable to elicit a detectable production of antibodies specific for the original antigen.

Amino Acid Sequence↗
PubMed1993

Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides.

We have constructed a random nonapeptide library in the N-terminal region of the major coat protein VIII of bacteriophage f1, with two cysteines flanking the insert, and preliminary data suggest that many of the clones display at least some of their peptides in cyclized form. This library was used to select oligopeptides binding to the monoclonal antibody (mAb) H107, recognising the assembled native conformation of recombinant human H-subunit ferritin (H Fer), whose three-dimensional structure is known. Comparison of the selected oligopeptides with one another allowed us to derive two consensus sequences characterized by conserved amino acid (aa) residues. Analysis of the distribution of the aa side chains exposed on the surface of H Fer reveals that most of the aa defining both consensus sequences are present either at the end of the big loop or at the end of the A helix. These two regions of the H Fer, though separated in the linear sequence, are very close in the folded molecule. Interestingly, each consensus sequence derived from the selected phage-displayed peptides is characterized by aa present both at the end of the big loop and at the end of the A helix. These two H Fer regions are good candidates for mimicry by the selected peptides and therefore for constituting part of the H107 epitope. To provide support to this hypothesis, we constructed several H Fer mutants carrying point mutations in different positions of these two regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

A database system for handling phage library-derived sequences.

We have implemented a system for creating and maintaining nucleotide and amino acid sequence databases especially suited for the handling of phage library-derived sequences. The system is currently used in our laboratory and has already proven to be useful for the comparison of sequences obtained by different investigators. We believe that the availability of this system will encourage and simplify the exchange of sequence data among different laboratories.

Amino Acid Sequence↗
PubMed1993

Hybrid Rop-pIII proteins for the display of constrained peptides on filamentous phage capsids.

In order to increase the versatility of phage display technology, it is desirable to be able to impose some structural constraints on the peptides that are presented by the phage particles. This is currently not feasible since the conformation of the capsid proteins, used to link the foreign peptide to the phage, are either unknown (pIII) or too simple (pVIII) to permit the engineering of peptide inserts into a constrained context. To reach this scope we have modified the amino-terminus of gene III by appending a well-characterized protein motif, the four-helix bundle of the bacterial protein Rop. Phage particles displaying Rop can be separated from wild-type (wt) particles by affinity purification with an antibody. Rop can be extensively modified by substituting its solvent-exposed residues and/or by inserting peptides either into the carboxy-terminal tail or into the bend region that connects the two alpha-helices of the monomer. These results open the possibility to construct peptide libraries where the peptides are constrained either into an omega-loop type conformation or an alpha-helix. Libraries formed by peptides inserted into the carboxy-terminus can also be constructed. Furthermore, the system that we have developed permits to produce large quantities of the elements of the libraries in the cytoplasm or to display them on the capsid of filamentous phages.

Bacterial Proteins↗
PubMed1992

Can evoked phonomyography be used to recognize fast and slow muscle in man?

The present study is aimed at ascertaining if muscle sound might be used as a detector of the contractile properties of individual human muscles "in vivo". In order to test this hypothesis, Soleus muscle (slow) and Vastus Lateralis Femoris muscle (fast) were investigated in three healthy subjects during electrically elicited contractions. Evoked phonomyograms were obtained from isometric single twitch contractions using a microphonic apparatus. Time and frequency domain analysis were performed. Evoked phonomyogram rising time values obtained from the two muscles are significantly different (p less than .01) and this difference is clearly due to their different mechanical properties. The power spectrum of all signals was obtained by means of harmonic analysis routine and mean frequency thus obtained was taken into account. Power spectrum values are approximately 1.5 times greater in fast muscle than in slow muscle (p less than .01). These findings lead us to the conclusion that evoked phonomyography can be considered a useful technique for the assessment of mechanical properties of individual human muscles.

Adult↗
PubMed1991

Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector.

Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.

Amino Acid Sequence↗