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Biomedical subjects

A Pini

Publications and source records attributed to A Pini.

At least 19 recordsLinked to original sources
PubMed2006

Randomized, double-blind, placebo-controlled trial of phenylbutyrate in spinal muscular atrophy.

OBJECTIVE: To assess the efficacy of phenylbutyrate (PB) in patients with spinal muscular atrophy in a randomized, double-blind, placebo-controlled trial involving 10 Italian centers. METHODS: One hundred seven children were assigned to receive PB (500 mg/kg/day) or matching placebo on an intermittent regimen (7 days on/7 days off) for 13 weeks. The Hammersmith functional motor scale (primary outcome measure), myometry, and forced vital capacity were assessed at baseline and at weeks 5 and 13. RESULTS: Between January and September 2004, 107 patients aged 30 to 154 months were enrolled. PB was well tolerated, with only one child withdrawing because of adverse events. Mean improvement in functional score was 0.60 in the PB arm and 0.73 in placebo arm (p = 0.70). Changes in the secondary endpoints were also similar in the two study arms. CONCLUSIONS: Phenylbutyrate was not effective at the regimen, schedule, and duration used in this study.

Child↗
PubMed2006

Reliability of the Hammersmith functional motor scale for spinal muscular atrophy in a multicentric study.

The aim of this study was to validate the Hammersmith functional motor scale for children with spinal muscular atrophy in a large cohort of 90 non-ambulant children with spinal muscular atrophy type 2 or 3. All had a baseline assessment (T0) and were reassessed either at 3 months (T1) (n = 66) or at 6 months (T2) (n = 24). Inter-observer reliability, tested on 13 children among 3 examiners, was > 95%. Of the 66 children examined after 3 months 4 had adverse effects in between assessments and were excluded from the analysis. Forty-two (68%) of the remaining 62 reassessed had no variation in scores between T0 and T1 and 13 (21%) were within +/- 1 point. 9 (37.5%) of the 24 children reassessed after 6 months had no variation in scores between T0 and T2 and another 9 (37.5%) had variations within +/- 1 point. Our study confirms previous observations of the reliability of the scale and helps to establish a baseline for assessing changes of functional ability over 3 and 6 month intervals. This information can be valuable in view of therapeutic trials.

Child↗
PubMed2006

High plasma creatine kinase: review of the literature and proposal for a diagnostic algorithm.

The condition of persistently high plasma CK levels is frequently encountered in asymptomatic patients with normal neurological examination. This condition may be the unique manifestation of several neuromuscular disorders, whose diagnosis is now possible using new diagnostic techniques. However, even if these patients are intensely investigated, specific diagnoses are not always forthcoming. Because of the lack of a widely accepted diagnostic protocol, hyperCKaemia in asymptomatic subjects is a potentially difficult clinical problem. In this paper we review the literature on conditions associated with variations in plasma CK levels and the literature on investigations carried out in asymptomatic persons with high CK to identify neuromuscular diseases. In the light of these data, and the deliberations of a working group of the Italian Association of Myology, we propose a diagnostic algorithm to guide the diagnostic work-up of persons presenting with persistently high levels of plasma CK. This algorithm has been discussed and approved by the Committee of the Italian Association of Myology.

Algorithms↗
PubMed2003

Experimental field trial with an immunostimulating complex (ISCOM) vaccine against contagious bovine pleuropneumonia.

The experimental field trial with an immunostimulating complex (ISCOM) vaccine has been an occasion to explore the role of a Th1 response in the pathogenesis caused by Mycoplasma mycoides subsp. mycoides small colony (MmmSC) and in immune protection. The ISCOM complex is known to promote Th1 response. Antibodies to MmmSC were detected by indirect enzyme-linked immunosorbent assay (ELISA) in the vaccinated cattle, although the levels were lower than in a previous study. No antibodies were detected by complement fixation test (CF). After the challenge infection, vaccinated animals developed CF antibody response. They showed significantly reduced mortality compared with controls. However, gross pathological and histopathological score for vaccinated animals was as high as for the non-vaccinated, characterized by a high inflammatory reaction with histopathology dominated by interlobular pneumonia with vasculitis.

Animals↗
PubMed2002

Phage display and colony filter screening for high-throughput selection of antibody libraries.

During the last 12 years, antibody combinatorial libraries have provided a new approach for the construction and production of reagents and drugs based on the human monoclonal antibodies. Studies employing antibodies or antibody mimics have become an important part of the explosive growth of proteomics. This places tremendous emphasis on the new approaches for faster library screening, improved methods of selection and evaluation of novel applications. The phage display system, together with its variants of ribosome and bacterial display, is the most extensively used method for the rapid screening of large antibody libraries. However, in the last two years the need to improve selection methods together with a complex patent situation regarding the phage display system, has also directed research towards the possibility of performing antibody selection by colony filter screening. Here, we summarise the results obtained by these different methods of selection comparing their efficacy and advantages.

Animals↗
PubMed2001

Mimotopes of the nicotinic receptor binding site selected by a combinatorial peptide library.

Peptide libraries allow selecting new molecules, defined as mimotopes, which are able to mimic the structural and functional features of a native protein. This technology can be applied for the development of new reagents, which can interfere with the action of specific ligands on their target receptors. In the present study we used a combinatorial library approach to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. On the basis of amino acid sequence comparison of different alpha-bungarotoxin binding receptors, we designed a 14 amino acid combinatorial synthetic peptide library with five invariant, four partially variant, and five totally variant positions. Peptides were synthesized using SPOT synthesis on cellulose membranes, and binding sequences were selected using biotinylated alpha-bungarotoxin. Each variant position was systematically identified, and all possible combinations of the best reacting amino acids in each variant position were tested. The best reactive sequences were identified, produced in soluble form, and tested in BIACORE to compare their kinetic constants. We identified several different peptides that can inhibit the binding of alpha-bungarotoxin to both muscle and neuronal nicotinic receptors. Peptide mimotopes have a toxin-binding affinity that is considerably higher than peptides reproducing native receptor sequences.

Amino Acid Sequence↗
PubMed2001

Evaluation of cardiac and respiratory involvement in sarcoglycanopathies.

Sarcoglycanopathies constitute a subgroup of limb-girdle recessive muscular dystrophies due to defects in sarcoglycan complex that comprises five distinct transmembrane proteins called alpha-, beta-, gamma-, delta-and epsilon-sarcoglycans. As it is well known that sarcoglycans are expressed both in heart and in skeletal muscles and a complete deficiency in delta-sarcoglycan is the cause of the Syrian hamster BIO.14 cardiomyopathy, we studied cardiac and respiratory involvement in 20 patients with sarcoglycanopathies by clinical, electrocardiographic, echocardiographic, scintigraphic and spirometric assessments. A normal heart function was found in 31.3% of all patients; a preclinical cardiomyopathy in 43.7%; an arrhythmogenic cardiomyopathy in 6.3% and initial signs of dilated cardiomyopathy in 18.7%. In one patient the data were examined retrospectively. No correlation was found between cardiac and skeletal muscle involvement. With reference to the type of sarcoglycanopathy, signs of hypoxic myocardial damage occurred in beta-, gamma- and delta-sarcoglycanopathies, while initial signs of a dilated cardiomyopathy in gamma- and delta-sarcoglycanopathies were found. A normal respiratory function was observed in 23.5% of all patients, a mild impairment in 35.4%, a moderate impairment in 29.4%, and a severe impairment in 11.7%.

Adolescent↗
PubMed2001

Mimicking the nicotinic receptor binding site by a single chain Fv selected by competitive panning from a synthetic phage library.

We have developed a novel competitive method to select from a phage display library a single chain Fv which is able to mimic the alpha-bungarotoxin binding site of the muscle nicotinic receptor. The single chain Fv was selected from a large synthetic library using alpha-bungarotoxin-coated magnetic beads. Toxin-bound phages were then eluted by competition with affinity purified nicotinic receptor. Recognition of the toxin by the anti-alpha-bungarotoxin single chain Fv was very similar to that of the receptor, such as indicated by the epitope mapping of alpha-bungarotoxin through overlapping synthetic peptides. Moreover, several positively charged residues located in the toxin second loop and in the C-terminal region were found to be critical, to a similar extent, for toxin recognition by the single chain Fv and the receptor. However, although the anti-alpha-bungarotoxin single chain Fv seems to mimic the toxin binding site of the nicotinic receptor, it does not bind other nicotinic agonists or antagonists. Our results suggest that competitive selection of anti-ligand antibody phages can allow the production of receptor-mimicking molecules directly and exclusively targeted at one specific ligand. Since physiologically and pharmacologically different ligands can produce opposite effects on receptor functions, such selective ligand decoys can have important therapeutic applications.

Animals↗
PubMed2001

PMP22 related congenital hypomyelination neuropathy.

The peripheral myelin protein 22 (PMP22) is a tetraspan membrane protein which is localised in the compact myelin of the peripheral nerves. In fibroblasts, where it was originally identified as growth arrest related factor 3 (Gas3), PMP22 has been shown to modulate cell proliferation; in the peripheral nervous system its roles are still debated. The duplication of PMP22 is the most common cause of the demyelinating form of the autosomal dominant Charcot-Marie-Tooth neuropathy (CMT1A); rarer missense mutations of PMP22 also cause CMT1A or severe dehypomyelinating neuropathies of infancy grouped under the heading of Dejerine-Sottas syndrome (DSS). Here, a sporadic patient affected with DSS is described; nerve biopsy disclosed a picture of hypomyelination/amyelination with basal laminae onion bulbs and no florid demyelination and it was consistent with congenital hypomyelination neuropathy (CHN); molecular analysis disclosed a novel point mutation of PMP22 that causes a non-conservative arginine for cysteine substitution at codon 109, in the third transmembrane domain. CHN is the rarest and severest form of DSS and it is thought to reflect dysmyelination rather than demyelination. The reported case suggests that missense point mutations may alter a putative role of PMP22 in modulating Schwann cell growth and differentiation.

Adolescent↗
PubMed2001

Slit proteins are not dominant chemorepellents for olfactory tract and spinal motor axons.

Members of the Slit family are large extracellular glycoproteins that may function as chemorepellents in axon guidance and neuronal cell migration. Their actions are mediated through members of the Robo family that act as their receptors. In vertebrates, Slit causes chemorepulsion of embryonic olfactory tract, spinal motor, hippocampal and retinal ganglion cell axons. Since Slits are expressed in the septum and floor plate during the period when these tissues cause chemorepulsion of olfactory tract and spinal motor axons respectively, it has been proposed that Slits function as guidance cues. We have tested this hypothesis in collagen gel co-cultures using soluble Robo/Fc chimeras, as competitive inhibitors, to disrupt Slit interactions. We find that the addition of soluble Robo/Fc has no effect on chemorepulsion of olfactory tract and spinal motor axons when co-cultured with septum or floor plate respectively. Thus, we conclude that although Slits are expressed in the septum and floor plate, their proteins do not contribute to the major chemorepulsive activities emanating from these tissues which cause repulsion of olfactory tract and spinal motor axons.

Animals↗
PubMed2000

Phage display of antibody fragments.

In recent years, phage display of peptides and proteins has become a very popular method in oncology, immunology, protein engineering and ligand-receptor studies among others. Antibody fragments, as Fabs or single chain Fv, have been among the first proteins to be displayed on the surface of a filamentous bacteriophage with a procedure initially described in 1990 by McCafferty et al. (Nature, 348, 552-554). From that time, molecular biology techniques have allowed the creation of large repertoires of antibody fragments from antibody V genes, bypassing hybrydoma technology and even immunisation. A large number of phage antibody libraries, from which molecules of the desired functional properties can be rapidly selected, have been built and distributed in many laboratories world-wide. Antibody fragments recovered from phage libraries generally show moderate binding strength; with different systems of biopanning binders can be obtained with dissociation constant ranged between 10-(5) to 10-(8) M. Nevertheless, antibody fragments can be furtherly modified to improve affinity or avidity, respectively by mutating crucial residues of complementarity determining regions or by increasing the number of binding sites making dimeric, trimeric or multimeric molecules. Here, we summarise the latest progress in this field, with particular reference to applications of scFv in the diagnosis and therapy of solid tumours and in the molecular mimicry of viral antigens and membrane receptors. In fact, the production of artificial protein epitopes by phage antibodies is becoming a valid system to overcome problems caused by difficult cloning and low expression of particular recombinant proteins.

Antibody Affinity↗
PubMed1999

Clinical correlations in 16 patients with total or partial laminin alpha2 deficiency characterized using antibodies against 2 fragments of the protein.

BACKGROUND: Many patients with classic congenital muscular dystrophy have been found to have partial or total deficiency of the alpha2 chain of laminin 2 (merosin). This deficiency has mostly been studied using only 1 antibody against a fragment of the protein. OBJECTIVES: To characterize the expression of laminin alpha2 in the skeletal muscle of patients with laminin alpha2 deficiency using antibodies against 2 different portions of the protein and to correlate the immunochemical findings with clinical phenotype. METHODS: We studied 4 patients with total lack of laminin alpha2 and 12 with partial laminin alpha2 deficiency with immunohistochemical techniques and Western blot analysis. We used antibodies recognizing an 80-kd fragment toward the C-terminus and a 300-kd fragment toward the amino-terminal. Patient characteristics examined were functional compromise, magnetic resonance imaging or computed tomography of the brain, electromyography, evoked potentials, and creatine kinase levels. RESULTS: In 4 patients, immunohistochemical analysis revealed no reactivity to either antibody; in 2 patients, the 300-kd fragment alone was partially expressed; in 2 patients, the 80-kd fragment alone was partially expressed; and in 8 patients, both fragments were partially expressed. Immunoblot analysis revealed bands of reduced intensity and normal molecular weight generally corresponding to the immunohistochemical findings. Absence of both fragments or of one with reduction of the other always produced a severe clinical phenotype, while a milder clinical phenotype was observed when both fragments were partially expressed. CONCLUSIONS: Extent of laminin alpha2 deficiency in most cases correlates with clinical phenotype but not with peripheral and central white matter abnormalities. Skin biopsy specimens may reveal laminin alpha2 deficiency in patients who have normal laminin alpha2 levels in muscle biopsy specimens.

Adolescent↗
PubMed1999

Prospective study of first-line vigabatrin monotherapy in childhood partial epilepsies.

This was a prospective open comparative pilot study to assess the efficacy and tolerability of first-line vigabatrin monotherapy in childhood partial epilepsies. Two groups of patients were recruited over the same period. The vigabatrin monotherapy group comprised 40 patients (18 male, 22 female; mean age at last visit 7.5 years); the comparative carbamazepine monotherapy group comprised 40 consecutive clinic patients (22 male, 18 female; mean age at last visit 7.8 years). Seizures disappeared in 82% of vigabatrin patients and in all carbamazepine patients with idiopathic partial epilepsy, and in 50% of vigabatrin patients and 55% of carbamazepine patients with symptomatic partial epilepsy. Interictal EEG abnormalities decreased in vigabatrin patients more than in carbamazepine patients (P < 0.05). Tolerability was good in vigabatrin patients, but four out of 37 showed mild irritability by the end of the trial. Persistent sedation was observed in eight of the 40 patients receiving carbamazepine. No patient had drug therapy discontinued because of side-effects. During vigabatrin long-term monotherapy, efficacy and good clinical tolerability were maintained. These results suggest that vigabatrin may be an alternative first-line treatment for childhood partial epilepsies. Further blinded comparative randomized trials are needed.

Adolescent↗
PubMed1998

Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel.

We report the construction and the use of a phage display human antibody library (>3 x 10(8) clones) based on principles of protein design. A large repertoire of functional antibodies with similar properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto the two antibody germ line segments most frequently found in human antibodies. With this strategy we concentrated sequence diversity in regions of the antibody structure that are centrally located in the antigen binding site, while leaving residues in more peripheral positions available for further mutagenesis aimed at improving the affinity of the selected antibodies. In addition, the library was tested by selecting antibodies against six biologically relevant antigens. Using only 0.3 microg of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis. These antibodies recognize the native antigen with affinities in the 10(7)-10(8) M-1 range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. The affinity of one anti-ED-B antibody was improved by 27-fold by combinatorially mutating six strategically selected residues in the heavy chain variable domain. A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (Kd = 54 pM), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.

Amino Acid Sequence↗
PubMed1998

Usefulness of transdermal clonidine in hypertensive patients undergoing minor surgical operations.

Transdermal clonidine (TTSC) treatment was evaluated in 29 patients with mild to moderate hypertension scheduled for minor surgery. Two weeks before the scheduled operation, patients underwent 24-h ambulatory blood-pressure monitoring (ABPM) to evaluate the efficacy of previous oral antihypertensive treatment, which was then substituted with TTSC, 0.1 mg/day. After 1 week, the efficacy of TTSC was clinically assessed, and the dose increased to 0.2 mg/day if needed. ABPM was repeated 2 days before the scheduled operation and 2 days after surgery. The 24-h blood pressure (BP) and heart rate (HR) profiles were smoothed by Fourier analysis. Three patients withdrew for adverse events and one for inefficacy after dose adjustment, TTSC being effective in the remaining 25 patients. Two patients who completed treatment lacked postsurgical ABPM recording. In the 23 patients with all ABPM recordings, average 24-h BP and HR obtained preoperatively during TTSC treatment were slightly reduced compared with values recorded during previous oral therapy. BP changes after surgery were negligible, whereas HR showed a moderate increase. Minor adverse events occurred in four (14%) of 29 patients. Our results demonstrate that TTSC provides adequate BP control in patients with mild to moderate hypertension undergoing minor surgery.

Administration, Cutaneous↗
PubMed1997

Hierarchical affinity maturation of a phage library derived antibody for the selective removal of cytomegalovirus from plasma.

Recombinant antibody fragments can be produced in large quantities using bacterial expression systems and could potentially be useful for the generation of biofilters for the selective removal of viral particles from fluids. A human single chain-Fv antibody library, derived from synthetic repertoires of germ line VH-gene segments rearranged in vitro and paired to a single light chain (Nissim et al., 1994, EMBO J., 13, 692-698), has recently been used to isolate hundreds of different binding specificities by panning with antigen. Antibodies from this library typically have affinities in the 10(6)-10(7) M-1 range. Occasionally, better binders are isolated but at other times the affinities recovered are poor. In the latter situation binding cannot be detected with soluble antibodies, but only by high-avidity display of multiple copies of antibodies on phage. By panning with human cytomegalovirus (HCMV)-coated immunotubes, we have isolated a number of antibody clones from this library that bound to the antigen only if displayed on the filamentous phage, but not in soluble form. One of these clones was selected for an affinity maturation procedure, achieved by combinatorial mutagenesis of the complementarity determining region 3 (CDR3) of the antibody light chain, followed by selection of the resulting library for HCMV binding. By this means, we were able to isolate a number of binders, some of which exhibited specific HCMV binding in soluble form. The clone that gave the strongest ELISA signal was expressed in bacteria, purified in solution, characterised using a novel capture methodology with surface plasmon resonance detection on a BIAcore instrument and used for the production of an immunofilter for the removal of HCMV form human serum. The filter removed more than 99% of applied HCMV in 10 min circulation time, while the amount of HCMV retained non-specifically in a cartridge derivatised with a non-specific antibody was less than 10% under similar conditions.

Antibodies, Viral↗