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D Neri

Publications and source records attributed to D Neri.

At least 55 records · Page 3Linked to original sources
PubMed1999

On the concept of eugenics: preliminaries to a critical appraisal.

This paper's main issue is linked to what can be foreseen as the increasing capability of medical genetics to modify the genetic composition of the human species through direct interventions in the human genome for medical and non-medical purposes, i.e., the "risk" of a resurgence of eugenics. In current discussions on the topic (briefly presented in the first section), the "phantom of eugenics" is raised several times, but there is a great deal of confusion on what counts as eugenics, partly because of broad conceptual disagreement over the notion itself. Furthermore, according to some scholars there is no hope of overcoming this unsatisfactory conceptual uncertainty. Partly challenging this opinion, the second and third sections of this paper attempt to identify some basic features which could be seen as intrinsically linked to the notion of eugenics, with the aim of reducing the range of conceptual disagreement as a preliminary step in bringing into focus what exactly is wrong with practicing eugenics. The subsequent sections deal with the substantive issue of whether or not to practice eugenics from the point of view of the interest of future generations in the human species' genetic composition. The main moral arguments for and against eugenics are examined from the point of view of our obligations towards future generations, and the conclusion is in favor of a cautious "open-door" position.

Bioethics↗
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

Preparation of phage antibodies to the ED-A domain of human fibronectin.

The fibronectin (FN) isoform containing the alternative spliced ED-A domain is much more expressed in fetal, tumoral, and regenerating tissues than in normal adult tissues. The ED-A containing FN is up-regulated by numerous cytokines, such as TGF-beta, and, although in normal adult liver the ED-A domain is undetectable, in regenerating rat liver the expression of ED-A is increased and mediates the conversion of fat storing cells to myofibroblasts. Here we describe the selection from a phage display library and the characterization of human antibody fragments directed against the ED-A sequence of FN. As they can be easily radiolabeled with 32P, these antibodies are very highly sensitive reagents for the determination of ED-A levels in tissues and biological fluids; in fact, use of these scFv induced a more than 10-fold increase in sensitivity with respect to the murine monoclonal IST-9. The possibility of preparing a range of human engineered antibodies should facilitate the development of antibody reagents with suitable pharmacokinetics, valency, functional affinity, and effector functions and that could be useful for clinical purposes.

Animals↗
PubMed1998

Affinity reagents against tumour-associated extracellular molecules and newforming vessels.

Here we report some recent results of tumour targeting using extracellular matrix components of tumour stroma as targets. The possibility of using human recombinant antibodies in tumour targeting is also described. Preliminary results indicate that neovasculature markers can be targeted by recombinant antibodies and that they allow long residence time in tumours, thus exploiting the avidity properties of multivalent recombinant fragments.

Journal Article↗
PubMed1998

Cerebral aspergillosis in a liver transplant recipient: a case report of long-term survival after combined treatment with liposomal amphotericin B and surgery.

Cerebral aspergillosis is a life-threatening complication in liver transplant recipients, with mortality rates approaching 100%; treatment with amphotericin B is of limited efficacy because of its poor distribution in the cerebrospinal fluid and its systemic side effects. We report the case of a liver transplant recipient who developed recurrent cerebral Aspergillus fumigatus infection, and was successfully treated by combined surgical excision of the lesion and administration of liposomal amphotericin B. This first report of long-term complication-free survival in a liver transplant recipient suggests that therapy with liposomal amphotericin B may reduce the risk of recurrence of cerebral aspergillosis in these immunocompromised patients.

Amphotericin B↗
PubMed1998

[Bioethics: history and problems].

This paper aims at describing the circumstances of the birth of bioethics. In the first section, the paper stresses some historical data supporting the widely diffused opinion according to which bioethics arose in the USA at the beginning of the Seventies. In the second, two of the most important factors in the birth of bioethics are discussed: namely, the possibilities opened by genetic engineering and the changes in the medical enterprise and health care ethics. In the third section the paper discusses one of the most important practical realization of bioethics: the creation of ethical committees.

Bioethics↗
PubMed1997

Tumor targeting potential of the monoclonal antibody BC-1 against oncofetal fibronectin in nude mice bearing human tumor implants.

BACKGROUND: The immunoglobulin G1 (IgG1) monoclonal antibody (MoAb) BC-1 detects human oncofetal fibronectin, which has extremely restricted distribution in normal adult tissues and is highly expressed in fetal and tumor tissues. METHODS: We studied the biodistribution of 125I-labeled MoAb BC-1 in nude mice bearing subcutaneous human tumor implants of U87MG high-grade astrocytoma and SKMel28 melanoma. 125I-BC-1 was injected either intraperitoneally (i.p.) or intravenously (i.v.), and biodistribution was measured up to 144 hours after injection. In animals bearing SKMel28 implants, tumor targeting was also evaluated by in vivo imaging of the whole mouse by using a dedicated device based on transmitted light excitation after i.v. injection of MoAb BC-1 conjugated with the infrared fluorophore, CY7-bis(N-hydroxy-succinimido)-ester. RESULTS: 125I-BC-1 showed favorable uptake in the human tumor implants, reaching a maximum of 5.27 +/- 0.48% ID/g in the U87MG astrocytoma (72 hours after i.p. injection). The highest uptake in the SKMel28 melanoma implants was 3.49 +/- 0.25% ID/g (24 hours after i.v. injection). Microautoradiography of tumor specimens obtained after administration of 125I-BC-1 clearly showed radioactivity uptake within the two tumors replicating the same pattern of distribution as that of the oncofetal fibronectin shown by immunohistochemistry with MoAb BC-1. Nonspecific uptake of 125I-BC-1 in the bone marrow and skeletal muscle was much lower than in the tumors. In vivo imaging with the fluorophore-labeled MoAb clearly visualized the tumor implants 72-120 hours after i.v. injection. CONCLUSIONS: The experimental results obtained in this study demonstrate the favorable tumor targeting potential in vivo of the radiolabeled MoAb BC-1, a useful marker of neo angiogenesis induced by cancer.

Animals↗
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↗
PubMed1997

Epstein-Barr virus-associated post-transplant lympho-proliferative disease of donor origin in liver transplant recipients.

BACKGROUND/AIMS: Post-transplant lymphoproliferative disease, a potential complication of solid organ transplantation, occurs in about 3% of orthotopic liver transplant recipients. We report the genetic and virological characterization of two cases of post-transplant lymphoproliferative disease that occurred early (4 and 6 months) after orthotopic liver transplant as large-cell non-Hodgkin's lymphomas located at the hepatic hilum. METHODS: Lymphomatous tissues were analyzed for clonality and presence of Epstein-Barr virus (EBV) sequences by Southern blot, polymerase chain reaction, and in situ hybridization techniques. RESULTS: The tumors in both cases were sustained by a clonal proliferation of B lymphocytes containing type A EBV DNA. Moreover, in situ hybridization with a digoxigenin-labeled EBV-specific probe evidenced a strong nuclear signal in most of the neoplastic cells. DNA microsatellite analysis at three different loci detected alleles of donor origin in both tumor samples, suggesting that the neoplastic B cells were of donor origin. CONCLUSIONS: EBV-infected donor B lymphocytes might be responsible for intragraft post-transplant lymphoproliferative disease in orthotopic liver transplant recipients. As 20 to 30% of post-transplant lymphomas involve the graft itself, donor-derived post-transplant lymphoproliferative disease might be more frequent than presently appreciated. Prospective studies are needed to assess its real incidence and identify possible risk factors.

B-Lymphocytes↗
PubMed1997

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

Amino Acid Sequence↗
PubMed1996

Phage antibodies with pan-species recognition of the oncofoetal angiogenesis marker fibronectin ED-B domain.

Fibronectin (FN) exists in several polymorphic forms due to alternative splicing. The B-FN isoform (with ED-B domain inserted by splicing) is present in the stroma of foetal and neoplastic tissues and in adult and neoplastic blood vessels during angiogenesis but is undetectable in mature vessels. This isoform, therefore, represents a promising marker for angiogenesis, as already shown using the mouse monoclonal antibody (MAb) BC-1 directed against an epitope on human B-FN. However, this MAb does not directly recognise the human ED-B domain nor does it recognise B-FN of other species; therefore, it cannot be used as a marker of angiogenesis in animal models. In principle, antibodies directed against the human ED-B domain should provide pan-species markers for angiogenesis as the sequence of this domain is highly conserved in different species (and identical in humans and mice). As it has proved difficult to obtain such antibodies by hybridoma technology, we used phage display technology. Here, we describe the isolation of human antibody fragments against the human ED-B domain that bind to human, mouse and chicken B-FN. As shown by immunohistochemistry, the antibody fragments stain human neoplastic tissues and the human, mouse and chicken neovasculature.

Adult↗
PubMed1996

Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement.

Calmodulin is a calcium-binding protein that regulates a wide range of enzymes. It is also one of the few examples of a small protein capable of binding to peptides with very high affinity, and is therefore an interesting candidate for biotechnological applications and a good model system for studying how proteins associate. We have synthesized a complete series of peptides derived from the recognition sequence of skeletal muscle myosin light-chain kinase, corresponding to single-point amino acid mutations to alanine. These peptides bind to calmodulin with a biphasic kinetic: a fast association step followed by a slow intramolecular isomerisation. We have measured the isomerisation rate (k(isom)) of these peptides for calmodulin by stopped-flow analysis, and their association and dissociation kinetic constants (k(on) and k(off)) by real-time interaction analysis using surface plasmon resonance detection. In addition, k(off) constants were measured by competition experiments using a high-sensitivity luminescence analyser and native polyacrylamide gels. We have observed that all the alanine-scanning peptides bound to calmodulin with better affinity than the wild-type. In one case, a Asn-->Ala substitution resulted in a 1000-fold improvement in affinity, owing to a slower off-rate. Our results indicate that naturally occurring calmodulin binders may have evolved to have high affinities, but far from the maximum. Our affinity data are in contrast with recently published predictions of interactions responsible for high-affinity calmodulin binding based on modelling and energy calculations.

Alanine↗
PubMed1996

Biophysical methods for the determination of antibody-antigen affinities.

Binding specificities against virtually any antigen can be isolated from antibody libraries displayed on filamentous phage. The determination of antibody-antigen affinity constants and binding kinetics is an important part of antibody characterization, and may be predictive of antibody performance in biotechnological applications. This article, intended as a guideline for the scientist who isolates a novel antibody and wishes to characterize its binding properties, presents the authors' view on widely used methodologies for the quantitative determination of antibody-antigen interactions.

Antibody Affinity↗
PubMed1996

Radioactive labeling of recombinant antibody fragments by phosphorylation using human casein kinase II and [gamma-32P]-ATP.

A wide range of antibody fragments can be expressed in bacteria and detected immunochemically via peptide tags. Using specially designed tags, we have developed a strategy for radiolabeling antibody fragments secreted from bacteria. Tagged antibody fragments were secreted either into the bacterial periplasm or the culture medium. The tag was not subject to proteolysis either in the broth or in human plasma. After affinity purification the antibody fragments were phosphorylated with [gamma-32P]ATP and casein kinase II. The labeled fragments were used in a gel band-shift assay to measure antigen binding affinities. In contrast to non site-specific methods such as radioiodination, antibodies labeled with casein kinase II retain full immunoreactivity. Radioactively phosphorylated antibody fragments may have many other applications, including radioimmunoassays and radioimmunotherapy.

Adenosine Triphosphate↗
PubMed1996

Recombinant anti-human melanoma antibodies are versatile molecules.

The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

Amino Acid Sequence↗