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Biomedical subjects

D Neri

Publications and source records attributed to D Neri.

At least 19 recordsLinked to original sources
PubMed2006

Percutaneous radiofrequency thermal ablation for hepatocellular carcinoma.

BACKGROUND: Radiofrequency thermal ablation is the first therapeutic option in percutaneous treatment of hepatocellular carcinoma but data on its long-term efficacy and safety are not conclusive. AIM: This study reports a prospective survey on radiofrequency thermal ablation in north-east Italy. METHODS: Data were collected on 401 patients with hepatocellular carcinoma (males 301, mean age: 68 years) treated by radiofrequency thermal ablation in 13 centres. Indication to treatment was: single nodule not eligible for surgery in 77% of patients, 2-3 nodes in 18% and multiple lesions in 5%. Mean size was 3 cm (1-8 cm). Treatment response was assessed at 1 month by spiral computerized tomography and then with ultrasound examination and new spiral computerized tomography. RESULTS: Complete response was obtained in 67% of patients and in 27% response was 75-99%. Complete response raised to 77% in lesions smaller than 3 cm. The morbidity rate was 34%; the mortality was 0.5%, seeding was observed in four patients. Ten patients presented an unexpected rapid disease progression. CONCLUSION: The above data show that by radiofrequency thermal ablation, complete response can be achieved only in about two-third of the cases, clearly less than expected, and that, beyond seeding, unexpected progression can be observed.

Aged↗
PubMed2006

Vascular tumor targeting.

The growth of solid tumors is dependent on their capacity to acquire blood supply. Therefore, much effort has been directed towards the development of anti-angiogenic agents which inhibit the process of neovascularization. More recently, it has become apparent that the targeted destruction of the established tumor vasculature represents a complementary avenue for growing therapeutic opportunities. This review provides evidence that vascular tumor targeting is an effective anti-tumor strategy in animal models. It further describes strategies for the identification of putative tumor vascular targets and discusses future prospects for vascular targeting applications in the clinical setting.

Drug Delivery Systems↗
PubMed2004

Liver transplant: adenosine metabolism and apoptosis.

Apoptosis and necrosis coexist in ischemia-reperfusion (I/R) injury following organ transplant. During experimental liver transplant we evidenced a deep alteration in energy and antioxidant status. The activity of purine catabolic enzymes was also altered. Caspase-3 (C-3), protein tyrosine phosphatase (PTP) showed significative alterations that lead to DNA fragmentation. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

Adenosine↗
PubMed2003

Single-chain antibody and its derivatives directed against vascular endothelial growth factor: application for antiangiogenic gene therapy.

Single-chain antibodies (scFv) have an enormous potential for clinical application. However, rapid blood clearance and difficulties in large-scale production of active scFvs have limited the practical use of these antibody fragments. Recently, an anti-vascular endothelial growth factor (VEGF) scFv (scFv V65) was selected in our laboratory from a human antibody phage-display library. This antibody was able to reduce tumor growth in mice by approximately 50%. Here, we employ a gene therapy strategy for sustained in vivo expression of scFv V65 and its derivatives. scFv fusion proteins containing parts of the constant IgG1 region were generated (minibody and scFv V65-Fc) to increase the serum half-life of the scFv V65. Systemic administration of recombinant adenovirus encoding scFv V65 resulted in substantial tumor inhibition. This effect could be improved by multiple virus injections. We found that the efficacy of different scFv V65 formats was dependent on the mode of administration: whereas scFv V65-Fc was the most efficient when expressed locally, scFv V65 was superior when delivered systemically. Our results show that therapeutic levels of scFv V65 can be obtained by systemic injection of recombinant adenoviruses. Therefore, therapeutic gene delivery of scFv is a feasible strategy that overcomes several limitations of conventional antibody therapy.

Adenoviridae↗
PubMed2002

Cytotoxic targeting of F9 teratocarcinoma tumours with anti-ED-B fibronectin scFv antibody modified liposomes.

We prepared small unilamellar liposomes derivatised with single chain antibody fragments specific for the ED-B domain of B-fibronectin. This extracellular matrix associated protein is expressed around newly forming blood vessels in the vicinity of many types of tumours. The single chain antibody fragments were functionalised by introduction of C-terminal cysteines and linked to liposomes via maleimide groups located at the terminal ends of poly(ethylene glycol) modified phospholipids. The properties of these anti-ED-B single chain antibody fragments-liposomes were analysed in vitro on ED-B fibronectin expressing Caco-2 cells and in vivo by studying their biodistribution and their therapeutic potential in mice bearing subcutanous F9 teratocarcinoma tumours. Radioactively labelled ((114m)Indium) single chain antibody fragments-liposomes accumulated in the tumours at 2-3-fold higher concentrations during the first 2 h after i.v. injection compared to unmodified liposomes. After 6-24 h both liposome types were found in similar amounts (8-10% injected dose g(-1)) in the tumours. Animals treated i.v. with single chain antibody fragments-liposomes containing the new cytotoxic agent 2'-deoxy-5-fluorouridylyl-N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine (30 mg kg(-1) per dose, five times every 24 h) showed a reduction of tumour growth by 62-90% determined on days 5 and 8, respectively, compared to animals receiving control liposomes. Histological analysis revealed a marked reduction of F9 tumour cells and excessive deposition of fibronectin in the extracellular matrix after treatment with single chain antibody fragments-2-dioxy-5-fluorouridylyl-N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine-liposomes. Single chain antibody fragments-liposomes targeted to ED-B fibronectin positive tumours therefore represent a promising and versatile novel drug delivery system for the treatment of tumours.

Animals↗
PubMed2002

Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature.

Interleukin-12 (IL-12) is a heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. Its clinical applications are limited, however, by severe side-effects. Here we report that an IL-12 fusion protein, consisting of IL-12 fused to a human antibody fragment specific to the oncofetal ED-B domain of fibronectin, markedly enhances the antitumor activity of this cytokine, as demonstrated in a mouse lung-metastasis model and in two models of mice bearing different aggressive murine tumors. The residual small tumor masses seen in the treated mice were infiltrated with lymphocytes, macrophages, and natural killer cells and had elevated interferon gamma (IFN-gamma). These results are of therapeutic relevance as the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is not detectable in normal vessels and tissues.

Animals↗
PubMed2001

A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold.

We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5x10(7) independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coli. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications.

Amino Acid Sequence↗
PubMed2001

Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening.

We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration. Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse. Detection of antigen binding on the second filter allows the recovery of a number of E.coli cells, including those expressing the binding specificity of interest, which can be submitted to a second round of screening for the isolation of specific monoclonal antibodies. We tested the methodology using as antigen the ED-B domain of fibronectin, a marker of angiogenesis. From an antibody library of 7 x 10(8) clones, we recovered a number of specifically-binding antibodies of different aminoacid sequence. The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised. Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose. ME4C binds to the ED-B domain with a dissociation constant K:(d) = 1 x 10(-7) M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.

Amino Acid Sequence↗
PubMed2001

Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice.

The selective thrombosis of tumor blood vessels, leading to the starvation and subsequent death of tumor cells, is an attractive anticancer strategy. Here we report that a fusion protein, consisting of an antibody fragment specific for the oncofoetal ED-B domain of fibronectin fused to the extracellular domain of tissue factor, selectively targets tumor blood vessels in vivo. Furthermore, this fusion protein mediates the complete and selective infarction of three different types of solid tumors in mice. At the highest doses administered, complete tumor eradication was observed in 30% of the mice treated without apparent side effects. These results are of therapeutic relevance because the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is undetectable in normal vessels and tissues.

Animals↗
PubMed2001

Lack of specificity of endoglin expression for tumor blood vessels.

A monoclonal antibody (MAb; A11) has been raised following mouse immunization with cultured human microvascular endothelial cells. The MAb showed a strong positivity within tumor vessels in glioblastoma and breast carcinoma samples, and the distribution was consistent with antigen association with vascular endothelial cells. A purification procedure of the antigen was developed starting from DG-RSV-LT-2, an immortalized human endothelial cell line. Molecular mass, N-terminal sequence of the purified antigen and localization on endothelial cell surface allowed identification with human endoglin (CD105). Flow cytometry analysis of a group of normal and transformed cell lines showed that, besides endothelial cells and myelocytic leukemia cells already shown to be positive, fetal fibroblasts, choriocarcinoma, fibrosarcoma and rhabdomyosarcoma cell lines were also positive for this antigen. Immunohistochemic analysis of several normal adult tissues revealed a more extensive presence of the antigen in normal vessels compared to that described with previously characterized antibodies. In fact, even though the staining was weaker than in tumor tissues, all tissues were found to be positive, at least in microvessels, except for normal breast. Moreover, in some tissues (glands and reproductive tract) a positive reaction was observed in the stroma. Since endoglin has been proposed as a possible target for antiangiogenic therapy in tumor patients and our data demonstrate a sizable amount of endoglin in normal vessels and stroma, its clinical use should be carefully reevaluated.

Animals↗
PubMed2001

Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin.

Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse.

Animals↗
PubMed2001

Selection of catalytically active biotin ligase and trypsin mutants by phage display.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.

Amino Acid Sequence↗
PubMed2001

Antibody-based targeting of angiogenesis.

The selective targeting of neovasculature opens new avenues for the diagnosis and therapy of angiogenesis-related diseases such as cancer, blinding ocular disorders, and rheumatoid arthritis. Here we review recent advances in the identification of markers of angiogenesis as well as in the isolation and use of antibodies (and their derivatives) for the in vivo targeting of both tumoral and nontumoral neovasculature.

Animals↗
PubMed2001

Antibody-Based targeting of angiogenesis.

Angiogenesis-the sprouting of new blood vessels from pre-existing ones-is a characteristic feature of relevant diseases such as cancer, some blinding ocular disorders, rheumatoid arthritis, and psoriasis. This article reviews recent progress in the generation of monoclonal antibodies, which recognize and target new blood vessels in vivo, while sparing mature vessels and healthy tissues. The use of such antibodies as selective "molecular vehicles" is likely to open new important diagnostic and therapeutic opportunities.

Antibodies↗
PubMed2001

Comparison of pig, human and rat hepatocytes as a source of liver specific metabolic functions in culture systems--implications for use in bioartificial liver devices.

The limited availability of human hepatocytes results in the use of animal cells in most bioartificial liver support devices. In the present work, clinically relevant liver specific metabolic activities were compared in rat, pig and human hepatocytes cultured on liver-derived biomatrix to optimize the expression of differentiated functions. Pig hepatocytes showed higher rates of diazepam metabolism (2.549+/-0.821 microg/h/million cells vs. 0.474+/-0.079 microg/h/million cells rats, p<0.005, and vs. 0.704+/-0.171 microg/h/million cells in man, p<0.005) and of bilirubin conjugation (21.60116+/-8.433237 micromoles/l/24 h vs. 6.786809+/-2.983758 in man, p<0.001 and vs. 9.956538+/-1.781016 micromoles/l/24 h in rats, p<0.005). Urea synthesis was similar in pig and in human hepatocytes (150+/-46.3 vs. 144.8+/-21.46 nmoles/h/million cells) and it was lower in rats (84.38+/-35.2; p<0.001 vs. man, p<0.02 vs. pig). High liver specific metabolic activities in cultured pig hepatocytes further support their use as a substitue for human cells in bioartificial liver devices.

Animals↗
PubMed2001

A new simplified method for preparation of a synthetic phage antibody with practically acceptable detection sensitivity on immunoblots.

We produced a library of phage clones displaying a synthetic repertoire of 1x10(8) scFv antibody fragments by combinatorial mutagenesis of several amino acid residues in complementarity determining region 3 (CDR3) of a single antibody sequence used as a template. Phage antibodies specific to a cerebellum-specific protein, MEGF1/fat2, were successfully isolated from clones in this library, but their affinity in binding to MEGF1/fat2 was too low for use in immunoblotting applications. In an effort to obtain a practically useful phage antibody from these primary phage antibodies, a new simplified method based on PCR using unequally degenerate oligonucleotides was devised and applied. A secondary phage antibody showing 5-fold slower dissociation from the targeted antigen than the parental one was successfully obtained from a mutant library consisting of about 10(7) clones through only two rounds of panning. This affinity maturation followed by fusion with bacterial alkaline phosphatase improved sensitivity in immunoblot assays, with the detection limit lowered to one nanogram level. The results indicate that this method offers a straightforward route to generate a recombinant phage antibody with practically acceptable immunoblot sensitivity from a medium-size single-pot library, from which specific and high-affinity primary phage antibodies are difficult to isolate directly.

Amino Acid Sequence↗